WorldWideScience

Sample records for surrounding intronic sequence

  1. Origin of introns by 'intronization' of exonic sequences

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2008-01-01

    The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting that i...

  2. Remarkable sequence conservation of the last intron in the PKD1 gene.

    Science.gov (United States)

    Rodova, Marianna; Islam, M Rafiq; Peterson, Kenneth R; Calvet, James P

    2003-10-01

    The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.

  3. Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

    Science.gov (United States)

    Evans, Teri; Johnson, Andrew D; Loose, Matthew

    2018-01-12

    Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

  4. Sequence features responsible for intron retention in human

    Directory of Open Access Journals (Sweden)

    Sakabe Noboru

    2007-02-01

    Full Text Available Abstract Background One of the least common types of alternative splicing is the complete retention of an intron in a mature transcript. Intron retention (IR is believed to be the result of intron, rather than exon, definition associated with failure of the recognition of weak splice sites flanking short introns. Although studies on individual retained introns have been published, few systematic surveys of large amounts of data have been conducted on the mechanisms that lead to IR. Results TTo understand how sequence features are associated with or control IR, and to produce a generalized model that could reveal previously unknown signals that regulate this type of alternative splicing, we partitioned intron retention events observed in human cDNAs into two groups based on the relative abundance of both isoforms and compared relevant features. We found that a higher frequency of IR in human is associated with individual introns that have weaker splice sites, genes with shorter intron lengths, higher expression levels and lower density of both a set of exon splicing silencers (ESSs and the intronic splicing enhancer GGG. Both groups of retained introns presented events conserved in mouse, in which the retained introns were also short and presented weaker splice sites. Conclusion Although our results confirmed that weaker splice sites are associated with IR, they showed that this feature alone cannot explain a non-negligible fraction of events. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating IR and also reveals previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of IR.

  5. Introns: The Functional Benefits of Introns in Genomes

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    Bong-Seok Jo

    2015-12-01

    Full Text Available The intron has been a big biological mystery since it was first discovered in several aspects. First, all of the completely sequenced eukaryotes harbor introns in the genomic structure, whereas no prokaryotes identified so far carry introns. Second, the amount of total introns varies in different species. Third, the length and number of introns vary in different genes, even within the same species genome. Fourth, all introns are copied into RNAs by transcription and DNAs by replication processes, but intron sequences do not participate in protein-coding sequences. The existence of introns in the genome should be a burden to some cells, because cells have to consume a great deal of energy to copy and excise them exactly at the correct positions with the help of complicated spliceosomal machineries. The existence throughout the long evolutionary history is explained, only if selective advantages of carrying introns are assumed to be given to cells to overcome the negative effect of introns. In that regard, we summarize previous research about the functional roles or benefits of introns. Additionally, several other studies strongly suggesting that introns should not be junk will be introduced.

  6. Introns: The Functional Benefits of Introns in Genomes.

    Science.gov (United States)

    Jo, Bong-Seok; Choi, Sun Shim

    2015-12-01

    The intron has been a big biological mystery since it was first discovered in several aspects. First, all of the completely sequenced eukaryotes harbor introns in the genomic structure, whereas no prokaryotes identified so far carry introns. Second, the amount of total introns varies in different species. Third, the length and number of introns vary in different genes, even within the same species genome. Fourth, all introns are copied into RNAs by transcription and DNAs by replication processes, but intron sequences do not participate in protein-coding sequences. The existence of introns in the genome should be a burden to some cells, because cells have to consume a great deal of energy to copy and excise them exactly at the correct positions with the help of complicated spliceosomal machineries. The existence throughout the long evolutionary history is explained, only if selective advantages of carrying introns are assumed to be given to cells to overcome the negative effect of introns. In that regard, we summarize previous research about the functional roles or benefits of introns. Additionally, several other studies strongly suggesting that introns should not be junk will be introduced.

  7. Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

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    Toro Nicolás

    2011-05-01

    Full Text Available Abstract Background Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ' in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron. Results In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo. Conclusions The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.

  8. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    Energy Technology Data Exchange (ETDEWEB)

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  9. Sequence comparison of the rDNA introns from six different species of Tetrahymena

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engberg, J

    1985-01-01

    model for the intron RNA of Cech et al. (Proc. Natl. Acad. Sci. U.S.A. 80, 3903 (83)). Most of the sequence variation in the four new sequences reported here is found in single stranded loops in the model. However, in four cases we found nucleotide substitutions in duplex stem regions, two of them...

  10. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

    2010-05-07

    Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  11. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Directory of Open Access Journals (Sweden)

    Tran Duc

    2010-05-01

    Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

  12. Use of a Fluorescent Aptamer RNA as an Exonic Sequence to Analyze Self-Splicing Ability of a Group I Intron from Structured RNAs

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    Airi Furukawa

    2016-11-01

    Full Text Available Group I self-splicing intron constitutes an important class of functional RNA molecules that can promote chemical transformation. Although the fundamental mechanism of the auto-excision from its precursor RNA has been established, convenient assay systems for its splicing activity are still useful for a further understanding of its detailed mechanism and of its application. Because some host RNA sequences, to which group I introns inserted form stable three-dimensional (3D structures, the effects of the 3D structures of exonic elements on the splicing efficiency of group I introns are important but not a fully investigated issue. We developed an assay system for group I intron self-splicing by employing a fluorescent aptamer RNA (spinach RNA as a model exonic sequence inserted by the Tetrahymena group I intron. We investigated self-splicing of the intron from spinach RNA, serving as a model exonic sequence with a 3D structure.

  13. Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences

    OpenAIRE

    Bunawan, Hamidun; Yen, Choong Chee; Yaakop, Salmah; Noor, Normah Mohd

    2017-01-01

    Background The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. Results Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consi...

  14. Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences.

    Science.gov (United States)

    Bunawan, Hamidun; Yen, Choong Chee; Yaakop, Salmah; Noor, Normah Mohd

    2017-01-26

    The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consisting of N. ampullaria, N. mirabilis, N. gracilis and N. rafflesiana, and another containing both intermediately distributed species (N. albomarginata and N. benstonei) and four highland species (N. sanguinea, N. macfarlanei, N. ramispina and N. alba). The trnL intron and ITS sequences proved to provide phylogenetic informative characters for deriving a phylogeny of Nepenthes species in Peninsular Malaysia. To our knowledge, this is the first molecular phylogenetic study of Nepenthes species occurring along an altitudinal gradient in Peninsular Malaysia.

  15. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Gniadkowski, M; Hemmings-Mieszczak, M; Klahre, U; Liu, H X; Filipowicz, W

    1996-02-15

    Introns of nuclear pre-mRNAs in dicotyledonous plants, unlike introns in vertebrates or yeast, are distinctly rich in A+U nucleotides and this feature is essential for their processing. In order to define more precisely sequence elements important for intron recognition in plants, we investigated the effects of short insertions, either U-rich or A-rich, on splicing of synthetic introns in transfected protoplast of Nicotiana plumbaginifolia. It was found that insertions of U-rich (sequence UUUUUAU) but not A-rich (AUAAAAA) segments can activate splicing of a GC-rich synthetic infron, and that U-rich segments, or multimers thereof, can function irrespective of the site of insertion within the intron. Insertions of multiple U-rich segments, either at the same or different locations, generally had an additive, stimulatory effect on splicing. Mutational analysis showed that replacement of one or two U residues in the UUUUUAU sequence with A or C residues had only a small effect on splicing, but replacement with G residues was strongly inhibitory. Proteins that interact with fragments of natural and synthetic pre-mRNAs in vitro were identified in nuclear extracts of N.plumbaginifolia by UV cross- linking. The profile of cross-linked plant proteins was considerably less complex than that obtained with a HeLa cell nuclear extract. Two major cross-linkable plant proteins had apparent molecular mass of 50 and 54 kDa and showed affinity for oligouridilates present in synGC introns or for poly(U).

  16. [Reconstruction of the phylogenetic position of larch (Larix sukaczewii Dylis) by sequencing data for the trnK intron of chloroplast DNA].

    Science.gov (United States)

    Bashalkhanov, S I; Konstantinov, Iu M; Verbitskiĭ, D S; Kobzev, V F

    2003-10-01

    To reconstruct the systematic relationships of larch Larix sukaczewii, we used the chloroplast trnK intron sequences of L. decidua, L. sukaczewii, L. sibirica, L. czekanovskii, and L. gmelinii. Analysis of phylogenetic trees constructed using the maximum parsimony and maximum likelihood methods showed a clear divergence of the trnK intron sequences between L. sukaczewii and L. sibirica. This divergence reaches intraspecific level, which supports a previously published hypothesis on the taxonomic isolation of L. sukaczewii.

  17. Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

    Science.gov (United States)

    Joardar, Vinita; Abrams, Natalie F; Hostetler, Jessica; Paukstelis, Paul J; Pakala, Suchitra; Pakala, Suman B; Zafar, Nikhat; Abolude, Olukemi O; Payne, Gary; Andrianopoulos, Alex; Denning, David W; Nierman, William C

    2012-12-12

    The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population

  18. Drosophila polytene chromosome bands formed by gene introns.

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    Zhimulev, I F; Boldyreva, L V; Demakova, O V; Poholkova, G V; Khoroshko, V A; Zykova, T Yu; Lavrov, S A; Belyaeva, E S

    2016-01-01

    Genetic organization of bands and interbands in polytene chromosomes has long remained a puzzle for geneticists. It has been recently demonstrated that interbands typically correspond to the 5'-ends of house-keeping genes, whereas adjacent loose bands tend to be composed of coding sequences of the genes. In the present work, we made one important step further and mapped two large introns of ubiquitously active genes on the polytene chromosome map. We show that alternative promoter regions of these genes map to interbands, whereas introns and coding sequences found between those promoters correspond to loose grey bands. Thus, a gene having its long intron "sandwiched" between to alternative promoters and a common coding sequence may occupy two interbands and one band in the context of polytene chromosomes. Loose, partially decompacted bands appear to host large introns.

  19. Strong Signature of Natural Selection within an FHIT Intron Implicated in Prostate Cancer Risk

    Science.gov (United States)

    Ding, Yan; Larson, Garrett; Rivas, Guillermo; Lundberg, Cathryn; Geller, Louis; Ouyang, Ching; Weitzel, Jeffrey; Archambeau, John; Slater, Jerry; Daly, Mary B.; Benson, Al B.; Kirkwood, John M.; O'Dwyer, Peter J.; Sutphen, Rebecca; Stewart, James A.; Johnson, David; Nordborg, Magnus; Krontiris, Theodore G.

    2008-01-01

    Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene (FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases (Pearson's χ2 = 12.34, df 1, P = 0.00045) and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers (Pearson's χ2 = 11.50, df 1, P = 0.00070). In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American (π = 0.0072, Tajima's D = 3.31, 14 SNPs) and the Japanese (π = 0.0049, Fay & Wu's H = 8.05, 14 SNPs), as well as in chimpanzees (Fay & Wu's H = 8.62, 12 SNPs). These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer. PMID:18953408

  20. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  1. Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution.

    Science.gov (United States)

    Rogozin, Igor B; Wolf, Yuri I; Sorokin, Alexander V; Mirkin, Boris G; Koonin, Eugene V

    2003-09-02

    Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.

  2. The distribution, diversity, and importance of 16S rRNA gene introns in the order Thermoproteales.

    Science.gov (United States)

    Jay, Zackary J; Inskeep, William P

    2015-07-09

    Intron sequences are common in 16S rRNA genes of specific thermophilic lineages of Archaea, specifically the Thermoproteales (phylum Crenarchaeota). Environmental sequencing (16S rRNA gene and metagenome) from geothermal habitats in Yellowstone National Park (YNP) has expanded the available datasets for investigating 16S rRNA gene introns. The objectives of this study were to characterize and curate archaeal 16S rRNA gene introns from high-temperature habitats, evaluate the conservation and distribution of archaeal 16S rRNA introns in geothermal systems, and determine which "universal" archaeal 16S rRNA gene primers are impacted by the presence of intron sequences. Several new introns were identified and their insertion loci were constrained to thirteen locations across the 16S rRNA gene. Many of these introns encode homing endonucleases, although some introns were short or partial sequences. Pyrobaculum, Thermoproteus, and Caldivirga 16S rRNA genes contained the most abundant and diverse intron sequences. Phylogenetic analysis of introns revealed that sequences within the same locus are distributed biogeographically. The most diverse set of introns were observed in a high-temperature, circumneutral (pH 6) sulfur sediment environment, which also contained the greatest diversity of different Thermoproteales phylotypes. The widespread presence of introns in the Thermoproteales indicates a high probability of misalignments using different "universal" 16S rRNA primers employed in environmental microbial community analysis.

  3. Occurrence of Can-SINEs and intron sequence evolution supports robust phylogeny of pinniped carnivores and their terrestrial relatives.

    Science.gov (United States)

    Schröder, Christiane; Bleidorn, Christoph; Hartmann, Stefanie; Tiedemann, Ralph

    2009-12-15

    Investigating the dog genome we found 178965 introns with a moderate length of 200-1000 bp. A screening of these sequences against 23 different repeat libraries to find insertions of short interspersed elements (SINEs) detected 45276 SINEs. Virtually all of these SINEs (98%) belong to the tRNA-derived Can-SINE family. Can-SINEs arose about 55 million years ago before Carnivora split into two basal groups, the Caniformia (dog-like carnivores) and the Feliformia (cat-like carnivores). Genome comparisons of dog and cat recovered 506 putatively informative SINE loci for caniformian phylogeny. In this study we show how to use such genome information of model organisms to research the phylogeny of related non-model species of interest. Investigating a dataset including representatives of all major caniformian lineages, we analysed 24 randomly chosen loci for 22 taxa. All loci were amplifiable and revealed 17 parsimony-informative SINE insertions. The screening for informative SINE insertions yields a large amount of sequence information, in particular of introns, which contain reliable phylogenetic information as well. A phylogenetic analysis of intron- and SINE sequence data provided a statistically robust phylogeny which is congruent with the absence/presence pattern of our SINE markers. This phylogeny strongly supports a sistergroup relationship of Musteloidea and Pinnipedia. Within Pinnipedia, we see strong support from bootstrapping and the presence of a SINE insertion for a sistergroup relationship of the walrus with the Otariidae.

  4. Frequent gain and loss of introns in fungal cytochrome b genes.

    Directory of Open Access Journals (Sweden)

    Liang-Fen Yin

    Full Text Available In this study, all available cytochrome b (Cyt b genes from the GOBASE database were compiled and the evolutionary dynamics of the Cyt b gene introns was assessed. Cyt b gene introns were frequently present in the fungal kingdom and some lower plants, but generally absent or rare in Chromista, Protozoa, and Animalia. Fungal Cyt b introns were found at 35 positions in Cyt b genes and the number of introns varied at individual positions from a single representative to 32 different introns at position 131, showing a wide and patchy distribution. Many homologous introns were present at the same position in distantly related species but absent in closely related species, suggesting that introns of the Cyt b genes were frequently lost. On the other hand, highly similar intron sequences were observed in some distantly related species rather than in closely related species, suggesting that these introns were gained independently, likely through lateral transfers. The intron loss-and-gain events could be mediated by transpositions that might have occurred between nuclear and mitochondria. Southern hybridization analysis confirmed that some introns contained repetitive sequences and might be transposable elements. An intron gain in Botryotinia fuckeliana prevented the development of QoI fungicide resistance, suggesting that intron loss-and-gain events were not necessarily beneficial to their host organisms.

  5. Comparative Analysis of Vertebrate Dystrophin Loci Indicate Intron Gigantism as a Common Feature

    Science.gov (United States)

    Pozzoli, Uberto; Elgar, Greg; Cagliani, Rachele; Riva, Laura; Comi, Giacomo P.; Bresolin, Nereo; Bardoni, Alessandra; Sironi, Manuela

    2003-01-01

    The human DMD gene is the largest known to date, spanning > 2000 kb on the X chromosome. The gene size is mainly accounted for by huge intronic regions. We sequenced 190 kb of Fugu rubripes (pufferfish) genomic DNA corresponding to the complete dystrophin gene (FrDMD) and provide the first report of gene structure and sequence comparison among dystrophin genomic sequences from different vertebrate organisms. Almost all intron positions and phases are conserved between FrDMD and its mammalian counterparts, and the predicted protein product of the Fugu gene displays 55% identity and 71% similarity to human dystrophin. In analogy to the human gene, FrDMD presents several-fold longer than average intronic regions. Analysis of intron sequences of the human and murine genes revealed that they are extremely conserved in size and that a similar fraction of total intron length is represented by repetitive elements; moreover, our data indicate that intron expansion through repeat accumulation in the two orthologs is the result of independent insertional events. The hypothesis that intron length might be functionally relevant to the DMD gene regulation is proposed and substantiated by the finding that dystrophin intron gigantism is common to the three vertebrate genes. [Supplemental material is available online at www.genome.org.] PMID:12727896

  6. 50/50 Expressional Odds of Retention Signifies the Distinction between Retained Introns and Constitutively Spliced Introns in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Rui Mao

    2017-10-01

    Full Text Available Intron retention, one of the most prevalent alternative splicing events in plants, can lead to introns retained in mature mRNAs. However, in comparison with constitutively spliced introns (CSIs, the relevantly distinguishable features for retained introns (RIs are still poorly understood. This work proposes a computational pipeline to discover novel RIs from multiple next-generation RNA sequencing (RNA-Seq datasets of Arabidopsis thaliana. Using this pipeline, we detected 3,472 novel RIs from 18 RNA-Seq datasets and re-confirmed 1,384 RIs which are currently annotated in the TAIR10 database. We also use the expression of intron-containing isoforms as a new feature in addition to the conventional features. Based on these features, RIs are highly distinguishable from CSIs by machine learning methods, especially when the expressional odds of retention (i.e., the expression ratio of the RI-containing isoforms relative to the isoforms without RIs for the same gene reaches to or larger than 50/50. In this case, the RIs and CSIs can be clearly separated by the Random Forest with an outstanding performance of 0.95 on AUC (the area under a receiver operating characteristics curve. The closely related characteristics to the RIs include the low strength of splice sites, high similarity with the flanking exon sequences, low occurrence percentage of YTRAY near the acceptor site, existence of putative intronic splicing silencers (ISSs, i.e., AG/GA-rich motifs and intronic splicing enhancers (ISEs, i.e., TTTT-containing motifs, and enrichment of Serine/Arginine-Rich (SR proteins and heterogeneous nuclear ribonucleoparticle proteins (hnRNPs.

  7. Recurrent loss of specific introns during angiosperm evolution.

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2014-12-01

    Full Text Available Numerous instances of presence/absence variations for introns have been documented in eukaryotes, and some cases of recurrent loss of the same intron have been suggested. However, there has been no comprehensive or phylogenetically deep analysis of recurrent intron loss. Of 883 cases of intron presence/absence variation that we detected in five sequenced grass genomes, 93 were confirmed as recurrent losses and the rest could be explained by single losses (652 or single gains (118. No case of recurrent intron gain was observed. Deep phylogenetic analysis often indicated that apparent intron gains were actually numerous independent losses of the same intron. Recurrent loss exhibited extreme non-randomness, in that some introns were removed independently in many lineages. The two larger genomes, maize and sorghum, were found to have a higher rate of both recurrent loss and overall loss and/or gain than foxtail millet, rice or Brachypodium. Adjacent introns and small introns were found to be preferentially lost. Intron loss genes exhibited a high frequency of germ line or early embryogenesis expression. In addition, flanking exon A+T-richness and intron TG/CG ratios were higher in retained introns. This last result suggests that epigenetic status, as evidenced by a loss of methylated CG dinucleotides, may play a role in the process of intron loss. This study provides the first comprehensive analysis of recurrent intron loss, makes a series of novel findings on the patterns of recurrent intron loss during the evolution of the grass family, and provides insight into the molecular mechanism(s underlying intron loss.

  8. An ancient spliceosomal intron in the ribosomal protein L7a gene (Rpl7a of Giardia lamblia

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    Gray Michael W

    2005-08-01

    Full Text Available Abstract Background Only one spliceosomal-type intron has previously been identified in the unicellular eukaryotic parasite, Giardia lamblia (a diplomonad. This intron is only 35 nucleotides in length and is unusual in possessing a non-canonical 5' intron boundary sequence, CT, instead of GT. Results We have identified a second spliceosomal-type intron in G. lamblia, in the ribosomal protein L7a gene (Rpl7a, that possesses a canonical GT 5' intron boundary sequence. A comparison of the two known Giardia intron sequences revealed extensive nucleotide identity at both the 5' and 3' intron boundaries, similar to the conserved sequence motifs recently identified at the boundaries of spliceosomal-type introns in Trichomonas vaginalis (a parabasalid. Based on these observations, we searched the partial G. lamblia genome sequence for these conserved features and identified a third spliceosomal intron, in an unassigned open reading frame. Our comprehensive analysis of the Rpl7a intron in other eukaryotic taxa demonstrates that it is evolutionarily conserved and is an ancient eukaryotic intron. Conclusion An analysis of the phylogenetic distribution and properties of the Rpl7a intron suggests its utility as a phylogenetic marker to evaluate particular eukaryotic groupings. Additionally, analysis of the G. lamblia introns has provided further insight into some of the conserved and unique features possessed by the recently identified spliceosomal introns in related organisms such as T. vaginalis and Carpediemonas membranifera.

  9. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2008-10-01

    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  10. Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

    Science.gov (United States)

    Piazza, Carol Lyn; Smith, Dorie

    2018-01-01

    Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

  11. Newly evolved introns in human retrogenes provide novel insights into their evolutionary roles

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    Kang Li-Fang

    2012-07-01

    Full Text Available Abstract Background Retrogenes generally do not contain introns. However, in some instances, retrogenes may recruit internal exonic sequences as introns, which is known as intronization. A retrogene that undergoes intronization is a good model with which to investigate the origin of introns. Nevertheless, previously, only two cases in vertebrates have been reported. Results In this study, we systematically screened the human (Homo sapiens genome for retrogenes that evolved introns and analyzed their patterns in structure, expression and origin. In total, we identified nine intron-containing retrogenes. Alignment of pairs of retrogenes and their parents indicated that, in addition to intronization (five cases, retrogenes also may have gained introns by insertion of external sequences into the genes (one case or reversal of the orientation of transcription (three cases. Interestingly, many intronizations were promoted not by base substitutions but by cryptic splice sites, which were silent in the parental genes but active in the retrogenes. We also observed that the majority of introns generated by intronization did not involve frameshifts. Conclusions Intron gains in retrogenes are not as rare as previously thought. Furthermore, diverse mechanisms may lead to intron creation in retrogenes. The activation of cryptic splice sites in the intronization of retrogenes may be triggered by the change of gene structure after retroposition. A high percentage of non-frameshift introns in retrogenes may be because non-frameshift introns do not dramatically affect host proteins. Introns generated by intronization in human retrogenes are generally young, which is consistent with previous findings for Caenorhabditis elegans. Our results provide novel insights into the evolutionary role of introns.

  12. Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

    Science.gov (United States)

    Francis, Warren R; Wörheide, Gert

    2017-06-01

    One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  13. The peculiarities of large intron splicing in animals.

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    Samuel Shepard

    Full Text Available In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These "large introns" must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5' and 3' acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing-a consecutive splicing from the 5'-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.

  14. Deep intronic GPR143 mutation in a Japanese family with ocular albinism.

    Science.gov (United States)

    Naruto, Takuya; Okamoto, Nobuhiko; Masuda, Kiyoshi; Endo, Takao; Hatsukawa, Yoshikazu; Kohmoto, Tomohiro; Imoto, Issei

    2015-06-10

    Deep intronic mutations are often ignored as possible causes of human disease. Using whole-exome sequencing, we analysed genomic DNAs of a Japanese family with two male siblings affected by ocular albinism and congenital nystagmus. Although mutations or copy number alterations of coding regions were not identified in candidate genes, the novel intronic mutation c.659-131 T > G within GPR143 intron 5 was identified as hemizygous in affected siblings and as heterozygous in the unaffected mother. This mutation was predicted to create a cryptic splice donor site within intron 5 and activate a cryptic acceptor site at 41nt upstream, causing the insertion into the coding sequence of an out-of-frame 41-bp pseudoexon with a premature stop codon in the aberrant transcript, which was confirmed by minigene experiments. This result expands the mutational spectrum of GPR143 and suggests the utility of next-generation sequencing integrated with in silico and experimental analyses for improving the molecular diagnosis of this disease.

  15. Exon-primed intron-crossing (EPIC markers for non-model teleost fishes

    Directory of Open Access Journals (Sweden)

    Riethoven Jean-Jack M

    2010-03-01

    Full Text Available Abstract Background Exon-primed intron-crossing (EPIC markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene sequences, particularly in teleost fishes. Results We introduce a bioinformatics pipeline for developing EPIC markers by comparing the whole genome sequences between two or more species. By applying this approach on five teleost fishes whose genomes were available in the Ensembl database http://www.ensembl.org, we identified 210 EPIC markers that have single-copy and conserved exon regions with identity greater than 85% among the five teleost fishes. We tested 12 randomly chosen EPIC markers in nine teleost species having a wide phylogenetic range. The success rate of amplifying and sequencing those markers varied from 44% to 100% in different species. We analyzed the exon sequences of the 12 EPIC markers from 13 teleosts. The resulting phylogeny contains many traditionally well-supported clades, indicating the usefulness of the exon portion of EPIC markers in reconstructing species phylogeny, in addition to the value of the intron portion of EPIC markers in interrogating the population history. Conclusions This study illustrated an effective approach to develop EPIC markers in a taxonomic group, where two or more genome sequences are available. The markers identified could be amplified across a broad taxonomic range of teleost

  16. Functional comparison of three transformer gene introns regulating conditional female lethality

    Science.gov (United States)

    The trasformer gene plays a critical role in the sex determination pathways of many insects. We cloned two transformer gene introns from Anastrepha suspensa, the Caribbean fruit fly. These introns have sequences that putatively have a role in sex-specific splicing patterns that affect sex determinat...

  17. Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria.

    Science.gov (United States)

    Santamaria, Monica; Vicario, Saverio; Pappadà, Graziano; Scioscia, Gaetano; Scazzocchio, Claudio; Saccone, Cecilia

    2009-06-16

    A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers. The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries. After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals. The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode

  18. SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

    International Nuclear Information System (INIS)

    Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

    2007-01-01

    Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

  19. Identification of a nuclear matrix attachment region like sequence in the last intron of PI3Kγ

    International Nuclear Information System (INIS)

    Dai Bingbing; Ying Lei; Cai Rong; Li Ying; Zhang Xingqian; Lu Jian; Qian Guanxiang

    2006-01-01

    MARs are not only the structure bases of chromatin higher order structure but also have much biological significance. In this study, the whole sequence of about 100 kb in length from BAC clone of GS1-223D4 (GI: 5931478), in which human PI3Kγ gene is localized, was analyzed by two online-based computer programs, MARFinder and SMARTest. A strong potential MAR was predicted in the last and largest intron of PI3Kγ. The predicted 2 kb MAR, we refer to PIMAR, was further analyzed through biochemical methods in vitro and in vivo. The results showed that the PIMAR could be associated with nuclear matrices from HeLa cells both in vitro and in vivo. Further reporter gene analysis showed that in the transient transfection the expression of reporter gene linked with reversed PIMAR was repressed slightly, while in stably integrated state, the luciferase reporter both linked with reversed and orientated PIMAR was enhanced greatly in NIH-3T3 and K-562. These results suggest that the PIMAR maybe has the capacity of shielding integrated heterogeneous gene from chromatin position effect. Through combination of computer program analysis with confirmation by biochemical methods, we identified, for First time, a 2 kb matrix attachment region like sequence in the last intron of human PI3Kγ

  20. Intronic L1 retrotransposons and nested genes cause transcriptional interference by inducing intron retention, exonization and cryptic polyadenylation.

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    Kristel Kaer

    Full Text Available Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown.Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals.Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression.

  1. Identification of novel intronic BRCA1 variants of uncertain ...

    Indian Academy of Sciences (India)

    in a Thai hereditary breast cancer family. Adisorn Ratanaphan, Pornpen Panomwan, Bhutorn Canyuk and Tanaphon Maipang. J. Genet. 90, 327–331. Table 1. Oligodeoxyribonucleotide primers used for PCR amplification of BRCA1 exon–intron 7 boundary sequences. Primers. Nucleotide position. Primer sequence (5 –3 ).

  2. Family of autocatalytic group I introns in bacteriophage T4

    International Nuclear Information System (INIS)

    Shub, D.A.; Xu, M.Q.; Gott, J.M.; Zeeh, A.; Wilson, L.D.

    1987-01-01

    The discovery of an intron in phage T4 encouraged the authors to look for additional group I introns in the T4 genome. Further examples would permit sequence and structural comparisons that might lend insight into their evolutionary origin. Additionally, they hoped that their locations within the T4 genome would infer a possible regulatory function in prokaryotic gene expression. They took advantage of the fact that, since G is added to the 5' end of the intron, autocatalytic group I introns could be specifically labeled in vitro for use as probes for DNA blotting experiments. If Group I introns were in more than just the td gene, multiple RNA species should be labeled when total RNA is extracted from T4-infected cells and incubated with [α- 32 P]GTP in vitro. When used as a probe for a Southern blot of T4 DNA, this RNA should hybridize to several DNA bands

  3. Patterns of intron gain and conservation in eukaryotic genes

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    Wolf Yuri I

    2007-10-01

    sharing of intron positions between eukaryotic species separated by different evolutionary distances. The results indicate that, although the contribution of parallel gains varies across the phylogenetic tree, the high level of intron position sharing is due, primarily, to evolutionary conservation. Accordingly, numerous introns appear to persist in the same position over hundreds of millions of years of evolution. This is compatible with recent observations of a negative correlation between the rate of intron gain and coding sequence evolution rate of a gene, suggesting that at least some of the introns are functionally relevant.

  4. Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

    Science.gov (United States)

    Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

    2003-08-14

    The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

  5. Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses.

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    Jean-Luc Da Lage

    Full Text Available Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.

  6. Architecture and Distribution of Introns in Core Genes of Four Fusarium Species

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    Mmatshepho M. Phasha

    2017-11-01

    Full Text Available Removal of introns from transcribed RNA represents a crucial step during the production of mRNA in eukaryotes. Available whole-genome sequences and expressed sequence tags (ESTs have increased our knowledge of this process and revealed various commonalities among eukaryotes. However, certain aspects of intron structure and diversity are taxon-specific, which can complicate the accuracy of in silico gene prediction methods. Using core genes, we evaluated the distribution and architecture of Fusarium circinatum spliceosomal introns, and linked these characteristics to the accuracy of the predicted gene models of the genome of this fungus. We also evaluated intron distribution and architecture in F. verticillioides, F. oxysporum, and F. graminearum, and made comparisons with F. circinatum. Results indicated that F. circinatum and the three other Fusarium species have canonical 5′ and 3′ splice sites, but with subtle differences that are apparently not shared with those of other fungal genera. The polypyrimidine tract of Fusarium introns was also found to be highly divergent among species and genes. Furthermore, the conserved adenosine nucleoside required during the first step of splicing is contained within unique branch site motifs in certain Fusarium introns. Data generated here show that introns of F. circinatum, as well as F. verticillioides, F. oxysporum, and F. graminearum, are characterized by a number of unique features such as the CTHAH and ACCAT motifs of the branch site. Incorporation of such information into genome annotation software will undoubtedly improve the accuracy of gene prediction methods used for Fusarium species and related fungi.

  7. Development of EST Intron-Targeting SNP Markers for Panax ginseng and Their Application to Cultivar Authentication.

    Science.gov (United States)

    Wang, Hongtao; Li, Guisheng; Kwon, Woo-Saeng; Yang, Deok-Chun

    2016-06-04

    Panax ginseng is one of the most valuable medicinal plants in the Orient. The low level of genetic variation has limited the application of molecular markers for cultivar authentication and marker-assisted selection in cultivated ginseng. To exploit DNA polymorphism within ginseng cultivars, ginseng expressed sequence tags (ESTs) were searched against the potential intron polymorphism (PIP) database to predict the positions of introns. Intron-flanking primers were then designed in conserved exon regions and used to amplify across the more variable introns. Sequencing results showed that single nucleotide polymorphisms (SNPs), as well as indels, were detected in four EST-derived introns, and SNP markers specific to "Gopoong" and "K-1" were first reported in this study. Based on cultivar-specific SNP sites, allele-specific polymerase chain reaction (PCR) was conducted and proved to be effective for the authentication of ginseng cultivars. Additionally, the combination of a simple NaOH-Tris DNA isolation method and real-time allele-specific PCR assay enabled the high throughput selection of cultivars from ginseng fields. The established real-time allele-specific PCR assay should be applied to molecular authentication and marker assisted selection of P. ginseng cultivars, and the EST intron-targeting strategy will provide a potential approach for marker development in species without whole genomic DNA sequence information.

  8. Genic regions of a large salamander genome contain long introns and novel genes

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 × 109 bp were isolated and sequenced to characterize the structure of genic regions. Results Annotation of genes within BACs showed that axolotl introns are on average 10× longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86% of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5× larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! Conclusion This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.

  9. The function of introns

    Directory of Open Access Journals (Sweden)

    Liran eCarmel

    2012-04-01

    Full Text Available The intron-exon architecture of many eukaryotic genes raises the intriguing question of whether this unique organization serves any function, or is it simply a result of the spread of functionless introns in eukaryotic genomes. In this review, we show that introns in contemporary species fulfill a broad spectrum of functions, and are involved in virtually every step of mRNA processing. We propose that this great diversity of intronic functions supports the notion that introns were indeed selfish elements in early eukaryotes, but then independently gained numerous functions in different eukaryotic lineages. We suggest a novel criterion of evolutionary conservation, dubbed intron positional conservation, which can identify functional introns.

  10. Polymorphism in Mitochondrial Group I Introns among Cryptococcus neoformans and Cryptococcus gattii Genotypes and Its Association with Drug Susceptibility

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    Felipe E. E. S. Gomes

    2018-02-01

    Full Text Available Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i they are polymorphic sequences; (ii their self-splicing is inhibited by some drugs; and (iii their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001. Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.

  11. Proliferation of group II introns in the chloroplast genome of the green alga Oedocladium carolinianum (Chlorophyceae

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    Jean-Simon Brouard

    2016-10-01

    Full Text Available Background The chloroplast genome sustained extensive changes in architecture during the evolution of the Chlorophyceae, a morphologically and ecologically diverse class of green algae belonging to the Chlorophyta; however, the forces driving these changes are poorly understood. The five orders recognized in the Chlorophyceae form two major clades: the CS clade consisting of the Chlamydomonadales and Sphaeropleales, and the OCC clade consisting of the Oedogoniales, Chaetophorales, and Chaetopeltidales. In the OCC clade, considerable variations in chloroplast DNA (cpDNA structure, size, gene order, and intron content have been observed. The large inverted repeat (IR, an ancestral feature characteristic of most green plants, is present in Oedogonium cardiacum (Oedogoniales but is lacking in the examined members of the Chaetophorales and Chaetopeltidales. Remarkably, the Oedogonium 35.5-kb IR houses genes that were putatively acquired through horizontal DNA transfer. To better understand the dynamics of chloroplast genome evolution in the Oedogoniales, we analyzed the cpDNA of a second representative of this order, Oedocladium carolinianum. Methods The Oedocladium cpDNA was sequenced and annotated. The evolutionary distances separating Oedocladium and Oedogonium cpDNAs and two other pairs of chlorophycean cpDNAs were estimated using a 61-gene data set. Phylogenetic analysis of an alignment of group IIA introns from members of the OCC clade was performed. Secondary structures and insertion sites of oedogonialean group IIA introns were analyzed. Results The 204,438-bp Oedocladium genome is 7.9 kb larger than the Oedogonium genome, but its repertoire of conserved genes is remarkably similar and gene order differs by only one reversal. Although the 23.7-kb IR is missing the putative foreign genes found in Oedogonium, it contains sequences coding for a putative phage or bacterial DNA primase and a hypothetical protein. Intergenic sequences are 1.5-fold

  12. The mitochondrial LSU rRNA group II intron of Ustilago maydis encodes an active homing endonuclease likely involved in intron mobility.

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    Anja Pfeifer

    Full Text Available BACKGROUND: The a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg²⁺-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3' overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5'-GACGGGAAGACCCT-3' and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA. CONCLUSIONS/SIGNIFICANCE: The present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.

  13. Determinism and randomness in the evolution of introns and sine inserts in mouse and human mitochondrial solute carrier and cytokine receptor genes.

    Science.gov (United States)

    Cianciulli, Antonia; Calvello, Rosa; Panaro, Maria A

    2015-04-01

    In the homologous genes studied, the exons and introns alternated in the same order in mouse and human. We studied, in both species: corresponding short segments of introns, whole corresponding introns and complete homologous genes. We considered the total number of nucleotides and the number and orientation of the SINE inserts. Comparisons of mouse and human data series showed that at the level of individual relatively short segments of intronic sequences the stochastic variability prevails in the local structuring, but at higher levels of organization a deterministic component emerges, conserved in mouse and human during the divergent evolution, despite the ample re-editing of the intronic sequences and the fact that processes such as SINE spread had taken place in an independent way in the two species. Intron conservation is negatively correlated with the SINE occupancy, suggesting that virus inserts interfere with the conservation of the sequences inherited from the common ancestor. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Euglena gracilis chloroplast DNA: analysis of a 1.6 kb intron of the psb C gene containing an open reading frame of 458 codons.

    Science.gov (United States)

    Montandon, P E; Vasserot, A; Stutz, E

    1986-01-01

    We retrieved a 1.6 kbp intron separating two exons of the psb C gene which codes for the 44 kDa reaction center protein of photosystem II. This intron is 3 to 4 times the size of all previously sequenced Euglena gracilis chloroplast introns. It contains an open reading frame of 458 codons potentially coding for a basic protein of 54 kDa of yet unknown function. The intron boundaries follow consensus sequences established for chloroplast introns related to class II and nuclear pre-mRNA introns. Its 3'-terminal segment has structural features similar to class II mitochondrial introns with an invariant base A as possible branch point for lariat formation.

  15. U12 type introns were lost at multiple occasions during evolution

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    Bartschat Sebastian

    2010-02-01

    Full Text Available Abstract Background Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups. Results U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost. Conclusions The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.

  16. Macronuclear genome structure of the ciliate Nyctotherus ovalis: Single-gene chromosomes and tiny introns

    Directory of Open Access Journals (Sweden)

    Landweber Laura F

    2008-12-01

    Full Text Available Abstract Background Nyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement. Results We examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242 and cDNAs (5,484 and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCCn, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21–29 nucleotides, and a significant fraction (1/3 of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway. Conclusion The combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

  17. An intronic deletion in the PROM1 gene leads to autosomal recessive cone-rod dystrophy.

    Science.gov (United States)

    Eidinger, Osnat; Leibu, Rina; Newman, Hadas; Rizel, Leah; Perlman, Ido; Ben-Yosef, Tamar

    2015-01-01

    To investigate the genetic basis for autosomal recessive cone-rod dystrophy (CRD) in a consanguineous Israeli Jewish family. Patients underwent a detailed ophthalmic evaluation, including eye examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potential (VEP). Genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array was performed to identify homozygous regions shared among two of the affected individuals. Mutation screening of the underlying gene was performed with direct sequencing. In silico and in vitro analyses were used to predict the effect of the identified mutation on splicing. The affected family members are three siblings who have various degrees of progressive visual deterioration, glare, color vision abnormalities, and night vision difficulties. Visual field tests revealed central scotomas of different extension. Cone and rod ERG responses were reduced, with cones more severely affected. Homozygosity mapping revealed several homozygous intervals shared among two of the affected individuals. One included the PROM1 gene. Sequence analysis of the 26 coding exons of PROM1 in one affected individual revealed no mutations in the coding sequence or in intronic splice sites. However, in intron 21, proximate to the intron-exon junction, we observed a homozygous 10 bp deletion between positions -26 and -17 (c.2281-26_-17del). The deletion was linked to a known SNP, c.2281-6C>G. The deletion cosegregated with the disease in the family, and was not detected in public databases or in 101 ethnically-matched control individuals. In silico analysis predicted that this deletion would lead to altered intron 21 splicing. Bioinformatic analysis predicted that a recognition site for the SRSF2 splicing factor is located within the deleted sequence. The in vitro splicing assay demonstrated that c.2281-26_-17del leads to complete exon 22 skipping. A novel

  18. Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Ido Yofe

    2014-06-01

    Full Text Available Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predictive intron features such as transcript folding and sequence motifs. Using only natural Saccharomyces cerevisiae introns as regulators, we demonstrate fine and accurate control over gene expression spanning a 100 fold expression range. These results broaden the engineering toolbox of synthetic gene expression systems and provide a framework in which precise and robust tuning of gene expression is accomplished.

  19. Length and GC content variability of introns among teleostean genomes in the light of the metabolic rate hypothesis.

    Science.gov (United States)

    Chaurasia, Ankita; Tarallo, Andrea; Bernà, Luisa; Yagi, Mitsuharu; Agnisola, Claudio; D'Onofrio, Giuseppe

    2014-01-01

    A comparative analysis of five teleostean genomes, namely zebrafish, medaka, three-spine stickleback, fugu and pufferfish was performed with the aim to highlight the nature of the forces driving both length and base composition of introns (i.e., bpi and GCi). An inter-genome approach using orthologous intronic sequences was carried out, analyzing independently both variables in pairwise comparisons. An average length shortening of introns was observed at increasing average GCi values. The result was not affected by masking transposable and repetitive elements harbored in the intronic sequences. The routine metabolic rate (mass specific temperature-corrected using the Boltzmann's factor) was measured for each species. A significant correlation held between average differences of metabolic rate, length and GC content, while environmental temperature of fish habitat was not correlated with bpi and GCi. Analyzing the concomitant effect of both variables, i.e., bpi and GCi, at increasing genomic GC content, a decrease of bpi and an increase of GCi was observed for the significant majority of the intronic sequences (from ∼ 40% to ∼ 90%, in each pairwise comparison). The opposite event, concomitant increase of bpi and decrease of GCi, was counter selected (from hypothesis that the metabolic rate plays a key role in shaping genome architecture and evolution of vertebrate genomes.

  20. Parallel loss of plastid introns and their maturase in the genus Cuscuta.

    Science.gov (United States)

    McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; Leebens-Mack, Jim; dePamphilis, Claude W

    2009-06-19

    Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

  1. Parallel loss of plastid introns and their maturase in the genus Cuscuta.

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    Joel R McNeal

    2009-06-01

    Full Text Available Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

  2. Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees.

    Science.gov (United States)

    Adams, Gerard C; Surve-Iyer, Rupa S; Iezzoni, Amy F

    2002-01-01

    the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

  3. Genomewide analysis of intronic microRNAs in rice and Arabidopsis

    Indian Academy of Sciences (India)

    2012-12-13

    Dec 13, 2012 ... Seventy-five miRNA stem– loop sequences for rice came from ... The A. thaliana genotype used in this study was Columbia. (Col-0) wildtype. ... sense strand of intronic regions of protein-coding gene, 40 were located in the ...

  4. Length and GC content variability of introns among teleostean genomes in the light of the metabolic rate hypothesis.

    Directory of Open Access Journals (Sweden)

    Ankita Chaurasia

    Full Text Available A comparative analysis of five teleostean genomes, namely zebrafish, medaka, three-spine stickleback, fugu and pufferfish was performed with the aim to highlight the nature of the forces driving both length and base composition of introns (i.e., bpi and GCi. An inter-genome approach using orthologous intronic sequences was carried out, analyzing independently both variables in pairwise comparisons. An average length shortening of introns was observed at increasing average GCi values. The result was not affected by masking transposable and repetitive elements harbored in the intronic sequences. The routine metabolic rate (mass specific temperature-corrected using the Boltzmann's factor was measured for each species. A significant correlation held between average differences of metabolic rate, length and GC content, while environmental temperature of fish habitat was not correlated with bpi and GCi. Analyzing the concomitant effect of both variables, i.e., bpi and GCi, at increasing genomic GC content, a decrease of bpi and an increase of GCi was observed for the significant majority of the intronic sequences (from ∼ 40% to ∼ 90%, in each pairwise comparison. The opposite event, concomitant increase of bpi and decrease of GCi, was counter selected (from <1% to ∼ 10%, in each pairwise comparison. The results further support the hypothesis that the metabolic rate plays a key role in shaping genome architecture and evolution of vertebrate genomes.

  5. The Biology of Intron Gain and Loss

    DEFF Research Database (Denmark)

    Jeffares, Daniel C; Mourier, Tobias; Penny, David

    2006-01-01

    Intron density in eukaryote genomes varies by more than three orders of magnitude, so there must have been extensive intron gain and/or intron loss during evolution. A favored and partial explanation for this range of intron densities has been that introns have accumulated stochastically in large...... on introns depending on the biology of the organism and the gene involved....

  6. Development of ent-kaurene Oxidase-Based Conserved Intron Spanning Primers for Species Identification in the Genus Poa (Poaceae; Bluegrass

    Directory of Open Access Journals (Sweden)

    Jonathan M. LaMantia

    2018-04-01

    Full Text Available Interspecific hybridization has been attempted to combine the heat and drought of Poa arachnifera Torr. with the turf quality characteristics of several Poa species. Confirmation of an F1 hybrid through morphological analysis of vegetative and flowering characteristics is often time consuming and ambiguous. Ent-kaurene oxidase (KO has been sequenced in rice, barley, and wheat. In rice, each of the five copies of KO gene has unique lengths for the first intron. Conserved intron spanning primers (CISP can be used as a DNA marker to exploit variations of intron lengths that flank conserved gene sequences. In the present study, we developed CISP to sequence partial genomic fragments of the KO gene from seven Poa species. Through sequence analysis, species-specific primers were also developed to produce co-dominant markers that can be used to identify interspecific hybrids between Texas bluegrass and six other Poa species used in the present study.

  7. Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

    Science.gov (United States)

    Geiss, K T; Abbas, G M; Makaroff, C A

    1994-04-01

    The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

  8. Evidence for intron length conservation in a set of mammalian genes associated with embryonic development

    LENUS (Irish Health Repository)

    2011-10-05

    Abstract Background We carried out an analysis of intron length conservation across a diverse group of nineteen mammalian species. Motivated by recent research suggesting a role for time delays associated with intron transcription in gene expression oscillations required for early embryonic patterning, we searched for examples of genes that showed the most extreme conservation of total intron content in mammals. Results Gene sets annotated as being involved in pattern specification in the early embryo or containing the homeobox DNA-binding domain, were significantly enriched among genes with highly conserved intron content. We used ancestral sequences reconstructed with probabilistic models that account for insertion and deletion mutations to distinguish insertion and deletion events on lineages leading to human and mouse from their last common ancestor. Using a randomization procedure, we show that genes containing the homeobox domain show less change in intron content than expected, given the number of insertion and deletion events within their introns. Conclusions Our results suggest selection for gene expression precision or the existence of additional development-associated genes for which transcriptional delay is functionally significant.

  9. Functional characterisation of an intron retaining K+ transporter of barley reveals intron-mediated alternate splicing

    KAUST Repository

    Shahzad, K.

    2015-01-01

    Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K+ transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K+ uptake restored growth in medium containing hygromycin in the presence of different concentrations of K+ and mediated hypersensitivity to Na+. HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K+ and Na+ concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

  10. [Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

    Science.gov (United States)

    Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

    2015-08-01

    In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

  11. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    Science.gov (United States)

    Gelfman, Sahar; Burstein, David; Penn, Osnat; Savchenko, Anna; Amit, Maayan; Schwartz, Schraga; Pupko, Tal; Ast, Gil

    2012-01-01

    Exon–intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon–intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3′ and 5′ splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery. PMID:21974994

  12. Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

    Science.gov (United States)

    Liu, Yanbin; Yap, Sihui Amy; Koh, Chong Mei John; Ji, Lianghui

    2016-11-25

    Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4-11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50-160 and 20-90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6-3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5-3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are

  13. Selection-driven extinction dynamics for group II introns in Enterobacteriales.

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    Sébastien Leclercq

    Full Text Available Transposable elements (TEs are one of the major driving forces of genome evolution, raising the question of the long-term dynamics underlying their evolutionary success. Some TEs were proposed to evolve under a pattern of periodic extinctions-recolonizations, in which elements recurrently invade and quickly proliferate within their host genomes, then start to disappear until total extinction. Depending on the model, TE extinction is assumed to be driven by purifying selection against colonized host genomes (Sel-DE model or by saturation of host genomes (Sat-DE model. Bacterial group II introns are suspected to follow an extinction-recolonization model of evolution, but whether they follow Sel-DE or Sat-DE dynamics is not known. Our analysis of almost 200 group II intron copies from 90 sequenced Enterobacteriales genomes confirms their extinction-recolonization dynamics: patchy element distributions among genera and even among strains within genera, acquisition of new group II introns through plasmids or other mobile genetic elements, and evidence for recent proliferations in some genomes. Distributions of recent and past proliferations and of their respective homing sites further provide strong support for the Sel-DE model, suggesting that group II introns are deleterious to their hosts. Overall, our observations emphasize the critical impact of host properties on TE dynamics.

  14. Novel intron markers to study the phylogeny of closely related mammalian species

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    Castresana Jose

    2010-11-01

    Full Text Available Abstract Background Multilocus phylogenies can be used to infer the species tree of a group of closely related species. In species trees, the nodes represent the actual separation between species, thus providing essential information about their evolutionary history. In addition, multilocus phylogenies can help in analyses of species delimitation, gene flow and genetic differentiation within species. However, few adequate markers are available for such studies. Results In order to develop nuclear markers that can be useful in multilocus studies of mammals, we analyzed the mammalian genomes of human, chimpanzee, macaque, dog and cow. Rodents were excluded due to their unusual genomic features. Introns were extracted from the mammalian genomes because of their greater genetic variability and ease of amplification from the flanking exons. To an initial set of more than 10,000 one-to-one orthologous introns we applied several filters to select introns that belong to single-copy genes, show neutral evolutionary rates and have an adequate length for their amplification. This analysis led to a final list of 224 intron markers randomly distributed along the genome. To experimentally test their validity, we amplified twelve of these introns in a panel of six mammalian species. The result was that seven of these introns gave rise to a PCR band of the expected size in all species. In addition, we sequenced these bands and analyzed the accumulation of substitutions in these introns in five pairs of closely related species. The results showed that the estimated genetic distances in the five species pairs was quite variable among introns and that this divergence cannot be directly predicted from the overall intron divergence in mammals. Conclusions We have designed a new set of 224 nuclear introns with optimal features for the phylogeny of closely related mammalian species. A large proportion of the introns tested experimentally showed a perfect amplification

  15. Multiple group I introns in the small-subunit rDNA of Botryosphaeria dothidea: implication for intraspecific genetic diversity.

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    Chao Xu

    Full Text Available Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU ribosomal DNA (rDNA sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF for encoding the homing endonuclease (HE, whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron and genotype IV (Bdo.S1199-B were each found in only one strain, whereas genotype I (Bdo.S1199-A and genotype II (Bdo.S943 and Bdo.S1506 occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.

  16. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    Energy Technology Data Exchange (ETDEWEB)

    Abbs, S.; Sandhu, S.; Bobrow, M. [Guy`s Hospital, London (United Kingdom)

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

  17. Frequency of intron loss correlates with processed pseudogene abundance: a novel strategy to test the reverse transcriptase model of intron loss.

    Science.gov (United States)

    Zhu, Tao; Niu, Deng-Ke

    2013-03-05

    Although intron loss in evolution has been described, the mechanism involved is still unclear. Three models have been proposed, the reverse transcriptase (RT) model, genomic deletion model and double-strand-break repair model. The RT model, also termed mRNA-mediated intron loss, suggests that cDNA molecules reverse transcribed from spliced mRNA recombine with genomic DNA causing intron loss. Many studies have attempted to test this model based on its predictions, such as simultaneous loss of adjacent introns, 3'-side bias of intron loss, and germline expression of intron-lost genes. Evidence either supporting or opposing the model has been reported. The mechanism of intron loss proposed in the RT model shares the process of reverse transcription with the formation of processed pseudogenes. If the RT model is correct, genes that have produced more processed pseudogenes are more likely to undergo intron loss. In the present study, we observed that the frequency of intron loss is correlated with processed pseudogene abundance by analyzing a new dataset of intron loss obtained in mice and rats. Furthermore, we found that mRNA molecules of intron-lost genes are mostly translated on free cytoplasmic ribosomes, a feature shared by mRNA molecules of the parental genes of processed pseudogenes and long interspersed elements. This feature is likely convenient for intron-lost gene mRNA molecules to be reverse transcribed. Analyses of adjacent intron loss, 3'-side bias of intron loss, and germline expression of intron-lost genes also support the RT model. Compared with previous evidence, the correlation between the abundance of processed pseudogenes and intron loss frequency more directly supports the RT model of intron loss. Exploring such a correlation is a new strategy to test the RT model in organisms with abundant processed pseudogenes.

  18. Imprecise intron losses are less frequent than precise intron losses but are not rare in plants.

    Science.gov (United States)

    Ma, Ming-Yue; Zhu, Tao; Li, Xue-Nan; Lan, Xin-Ran; Liu, Heng-Yuan; Yang, Yu-Fei; Niu, Deng-Ke

    2015-05-27

    In this study, we identified 19 intron losses, including 11 precise intron losses (PILs), six imprecise intron losses (IILs), one de-exonization, and one exon deletion in tomato and potato, and 17 IILs in Arabidopsis thaliana. Comparative analysis of related genomes confirmed that all of the IILs have been fixed during evolution. Consistent with previous studies, our results indicate that PILs are a major type of intron loss. However, at least in plants, IILs are unlikely to be as rare as previously reported. This article was reviewed by Jun Yu and Zhang Zhang. For complete reviews, see the Reviewers' Reports section.

  19. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

    Science.gov (United States)

    Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

    2014-01-13

    Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The

  20. Extensive intron gain in the ancestor of placental mammals

    Science.gov (United States)

    2011-01-01

    Background Genome-wide studies of intron dynamics in mammalian orthologous genes have found convincing evidence for loss of introns but very little for intron turnover. Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this study was to search for evidence of de novo intron gain in domesticated genes from an analysis of their exon/intron structures. Results A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. Gain of introns in domesticated genes has been reconstructed on well established mammalian, vertebrate and chordate phylogenies, and examined as to where and when the gain events occurred. The locations, sizes and amounts of de novo introns gained in the domesticated genes during the evolution of mammals and chordates has been analyzed. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Intron densities in Eutheria-specific domesticated genes and in older domesticated genes that originated early in vertebrates are lower than those for normal mammalian and vertebrate genes. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals. Conclusions This study provides the first evidence for numerous intron gains in the ancestor of placental mammals and demonstrates that adequate taxon sampling is crucial for reconstructing intron evolution. The

  1. Reenacting the birth of an intron

    Energy Technology Data Exchange (ETDEWEB)

    Hellsten, Uffe; Aspden, Julie L.; Rio, Donald C.; Rokhsar, Daniel S.

    2011-07-01

    An intron is an extended genomic feature whose function requires multiple constrained positions - donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers - that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half a billion years ago.

  2. Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution.

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    Pawandeep Dhami

    2010-08-01

    Full Text Available It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II occupancy show preferential association with exons ("exon-intron marking", linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing.

  3. BIALLELIC POLYMORPHISM IN THE INTRON REGION OF B-TUBULIN GENE OF CRYPTOSPORIDIUM PARASITES

    Science.gov (United States)

    Nucleotide sequencing of polymerase chain reaction-amplified intron region of the Cryptosporidium parvum B-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is...

  4. Functional characterisation of an intron retaining K+ transporter of barley reveals intron-mediated alternate splicing

    KAUST Repository

    Shahzad, K.; Rauf, M.; Ahmed, M.; Malik, Z. A.; Habib, I.; Ahmed, Z.; Mahmood, K.; Ali, R.; Masmoudi, K.; Lemtiri-Chlieh, Fouad; Gehring, Christoph A; Berkowitz, G. A.; Saeed, N. A.

    2015-01-01

    Intron retention in transcripts and the presence of 5 and 3 splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K+ transporter, HvHKT2;1, with stably

  5. Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.

    Science.gov (United States)

    Johnson, Cayla M; Fisher, Derek J

    2013-01-01

    Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron's substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA(-) mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.

  6. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

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    Cornelia Kilchert

    2015-12-01

    Full Text Available In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

  7. Evolution of the Exon-Intron Structure in Ciliate Genomes.

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    Vladyslav S Bondarenko

    Full Text Available A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates (Paramecium and Nyctotherus possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius (with means 48 bp, 69 bp, and 55 bp, 64 bp, respectively, but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia (with means 33-35 bp, 47-51 bp, and 78-80 bp. In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short

  8. The Long Intron 1 of Growth Hormone Gene from Reeves’ Turtle (Chinemys reevesii Correlates with Negatively Regulated GH Expression in Four Cell Lines

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    Wen-Sheng Liu

    2016-04-01

    Full Text Available Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves’ turtle (Chinemys reevesii have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp, comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS of the turtle’s GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves’ turtle might correlate with downregulated gene expression.

  9. Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA

    Science.gov (United States)

    Metz, Edward C.; Robles-Sikisaka, Refugio; Vacquier, Victor D.

    1998-01-01

    Strong positive Darwinian selection acts on two sperm fertilization proteins, lysin and 18-kDa protein, from abalone (Haliotis). To understand the phylogenetic context for this dramatic molecular evolution, we obtained sequences of mitochondrial cytochrome c oxidase subunit I (mtCOI), and genomic sequences of lysin, 18-kDa, and a G protein subunit. Based on mtDNA differentiation, four north Pacific abalone species diverged within the past 2 million years (Myr), and remaining north Pacific species diverged over a period of 4–20 Myr. Between-species nonsynonymous differences in lysin and 18-kDa exons exceed nucleotide differences in introns by 3.5- to 24-fold. Remarkably, in some comparisons nonsynonymous substitutions in lysin and 18-kDa genes exceed synonymous substitutions in mtCOI. Lysin and 18-kDa intron/exon segments were sequenced from multiple red abalone individuals collected over a 1,200-km range. Only two nucleotide changes and two sites of slippage variation were detected in a total of >29,000 nucleotides surveyed. However, polymorphism in mtCOI and a G protein intron was found in this species. This finding suggests that positive selection swept one lysin allele and one 18-kDa allele to fixation. Similarities between mtCOI and lysin gene trees indicate that rapid adaptive evolution of lysin has occurred consistently through the history of the group. Comparisons with mtCOI molecular clock calibrations suggest that nonsynonymous substitutions accumulate 2–50 times faster in lysin and 18-kDa genes than in rapidly evolving mammalian genes. PMID:9724763

  10. Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

    Energy Technology Data Exchange (ETDEWEB)

    Solera, J. (Unidades de Genetica Molecular, Madrid (Spain)); Magallon, M.; Martin-Villar, J. (Hemofilia Hospital, Madrid (Spain)); Coloma, A. (Departamento deBioquimica de la Facultad de Medicina de la Universidad Autonoma, Madrid (Spain))

    1992-02-01

    DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.

  11. Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

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    Rafael Nisa-Martínez

    Full Text Available Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP with reverse transcriptase (RT and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

  12. Analysis of ribosomal protein gene structures: implications for intron evolution.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs and mitochondrial ribosomal proteins (MRPs, which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.

  13. Inheritance of the group I rDNA intron in Tetrahymena pigmentosa

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Simon, E M; Engberg, J

    1992-01-01

    - strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing...

  14. Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

    Science.gov (United States)

    Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

    2008-08-22

    CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

  15. The ability to form full-length intron RNA circles is a general property of nuclear group I introns

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Fiskaa, Tonje; Birgisdottir, Asa Birna

    2003-01-01

    at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena...... ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility....

  16. Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability.

    Science.gov (United States)

    Bonnet, Amandine; Grosso, Ana R; Elkaoutari, Abdessamad; Coleno, Emeline; Presle, Adrien; Sridhara, Sreerama C; Janbon, Guilhem; Géli, Vincent; de Almeida, Sérgio F; Palancade, Benoit

    2017-08-17

    Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Development of ent-kaurene Oxidase-Based Conserved Intron Spanning Primers for Species Identification in the Genus Poa (Poaceae; Bluegrass)

    OpenAIRE

    Jonathan M. LaMantia; Ambika Chandra; David R. Huff

    2018-01-01

    Interspecific hybridization has been attempted to combine the heat and drought of Poa arachnifera Torr. with the turf quality characteristics of several Poa species. Confirmation of an F1 hybrid through morphological analysis of vegetative and flowering characteristics is often time consuming and ambiguous. Ent-kaurene oxidase (KO) has been sequenced in rice, barley, and wheat. In rice, each of the five copies of KO gene has unique lengths for the first intron. Conserved intron spanning prime...

  18. Archaeal rRNA operons, intron splicing and homing endonucleases, RNA polymerase operons and phylogeny

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Aagaard, Claus Sindbjerg; Andersen, Morten

    1994-01-01

    Over the past decade our laboratory has had a strong interest in defining the phylogenetic status of the archaea. This has involved determining and analysing the sequences of operons of both rRNAs and RNA polymerases and it led to the discovery of the first archaeal rRNA intron. What follows...

  19. Alteration of introns in a hyaluronan synthase 1 (HAS1 minigene convert Pre-mRNA [corrected] splicing to the aberrant pattern in multiple myeloma (MM: MM patients harbor similar changes.

    Directory of Open Access Journals (Sweden)

    Jitra Kriangkum

    Full Text Available Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1 have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.

  20. Three distinct modes of intron dynamics in the evolution of eukaryotes.

    Science.gov (United States)

    Carmel, Liran; Wolf, Yuri I; Rogozin, Igor B; Koonin, Eugene V

    2007-07-01

    Several contrasting scenarios have been proposed for the origin and evolution of spliceosomal introns, a hallmark of eukaryotic genes. A comprehensive probabilistic model to obtain a definitive reconstruction of intron evolution was developed and applied to 391 sets of conserved genes from 19 eukaryotic species. It is inferred that a relatively high intron density was reached early, i.e., the last common ancestor of eukaryotes contained >2.15 introns/kilobase, and the last common ancestor of multicellular life forms harbored approximately 3.4 introns/kilobase, a greater intron density than in most of the extant fungi and in some animals. The rates of intron gain and intron loss appear to have been dropping during the last approximately 1.3 billion years, with the decline in the gain rate being much steeper. Eukaryotic lineages exhibit three distinct modes of evolution of the intron-exon structure. The primary, balanced mode, apparently, operates in all lineages. In this mode, intron gain and loss are strongly and positively correlated, in contrast to previous reports on inverse correlation between these processes. The second mode involves an elevated rate of intron loss and is prevalent in several lineages, such as fungi and insects. The third mode, characterized by elevated rate of intron gain, is seen only in deep branches of the tree, indicating that bursts of intron invasion occurred at key points in eukaryotic evolution, such as the origin of animals. Intron dynamics could depend on multiple mechanisms, and in the balanced mode, gain and loss of introns might share common mechanistic features.

  1. Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA

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    Raúl Ortiz

    2017-07-01

    Full Text Available Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

  2. Correlation of PTPN11 polymorphism at intron 3 with gastric cancer

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    Li ZHANG

    2011-05-01

    Full Text Available Objective To explore the correlation of protein-tyrosine-phosphatase nonreceptor-type 11(PTPN11 polymorphism at intron 3 with gastric cancer in Chinese population,and the feasibility and accuracy of employing mastrix-assisted laser desorption ionization time-of-flight mass spectrogram(MALDI-TOF-MS in genotyping of this SNP.Methods Two hundred and forty-seven patients with gastric cancer,212 cancer-free patients and 160 cord blood samples were enrolled in present study.Genotypes of PTPN11 G/A polymorphism at intron 3 were determined by MALDI-TOF-MS analysis,and direct sequencing of PCR products with 20 samples of the gene locus was done for checking the accuracy of MALDI-TOF-MS.Histological examination,Helicobacter pylori culture,rapid urease test,serum anti-H.pylori antibodies(ELISA and urease colloidal gold test were performed to evaluate H.pylori infection.Results Direct sequencing of 20 random selected samples were well consistent with the MALDI-TOF-MS results.The rates of H.pylori infection were 73.68% in gastric cancer patients and 75.47% in cancer-free patients,implying no significant difference between the two groups.The distributions of genotypes were in Hardy Weinberg equilibrium in both gastric cancer patients and controls.There were no significant differences in the genotype frequencies between the 2 groups(P>0.05.Compared with the GG genotype,GA+AA genotype could not influence the risk of gastric cancer.When stratified for status,PTPN11 polymorphism was not associated with age,gender and H.pylori infection states in both cancer patients and controls.Conclusion It seems that PTPN11 G/A polymorphism at intron 3 has no affection on the risk of gastric cancer in Chinese population.

  3. Intronic microRNAs

    International Nuclear Information System (INIS)

    Ying, S.-Y.; Lin, S.-L.

    2005-01-01

    MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular mRNAs that contain partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. MiRNA was originally discovered in the intergenic regions of the Caenorhabditis elegans genome as native RNA fragments that modulate a wide range of genetic regulatory pathways during animal development. However, neither RNA promoter nor polymerase responsible for miRNA biogenesis was determined. Recent findings of intron-derived miRNA in C. elegans, mouse, and human have inevitably led to an alternative pathway for miRNA biogenesis, which relies on the coupled interaction of Pol-II-mediated pre-mRNA transcription and intron excision, occurring in certain nuclear regions proximal to genomic perichromatin fibrils

  4. Malonyl CoA decarboxylase deficiency: C to T transition in intron 2 of the MCD gene.

    Science.gov (United States)

    Surendran, S; Sacksteder, K A; Gould, S J; Coldwell, J G; Rady, P L; Tyring, S K; Matalon, R

    2001-09-15

    Malonyl CoA decarboxylase (MCD) is an enzyme involved in the metabolism of fatty acids synthesis. Based on reports of MCD deficiency, this enzyme is particular important in muscle and brain metabolism. Mutations in the MCD gene result in a deficiency of MCD activity, that lead to psychomotor retardation, cardiomyopathy and neonatal death. To date however, only a few patients have been reported with defects in MCD. We report here studies of a patient with MCD deficiency, who presented with hypotonia, cardiomyopathy and psychomotor retardation. DNA sequencing of MCD revealed a homozygous intronic mutation, specifically a -5 C to T transition near the acceptor site for exon 3. RT-PCR amplification of exons 2 and 3 revealed that although mRNA from a normal control sample yielded one major DNA band, the mutant mRNA sample resulted in two distinct DNA fragments. Sequencing of the patient's two RT-PCR products revealed that the larger molecular weight fragments contained exons 2 and 3 as well as the intervening intronic sequence. The smaller size band from the patient contained the properly spliced exons, similar to the normal control. Western blotting analysis of the expressed protein showed only a faint band in the patient sample in contrast to a robust band in the control. In addition, the enzyme activity of the mutant protein was lower than that of the control protein. The data indicate that homozygous mutation in intron 2 disrupt normal splicing of the gene, leading to lower expression of the MCD protein and MCD deficiency. Copyright 2001 Wiley-Liss, Inc.

  5. Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

    Science.gov (United States)

    Hiesel, Rudolf; Brennicke, Axel

    1983-01-01

    The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

  6. HFE gene polymorphism defined by sequence-based typing of the Brazilian population and a standardized nomenclature for HFE allele sequences.

    Science.gov (United States)

    Campos, W N; Massaro, J D; Martinelli, A L C; Halliwell, J A; Marsh, S G E; Mendes-Junior, C T; Donadi, E A

    2017-10-01

    The HFE molecule controls iron uptake from gut, and defects in the molecule have been associated with iron overload, particularly in hereditary hemochromatosis. The HFE gene including both coding and boundary intronic regions were sequenced in 304 Brazilian individuals, encompassing healthy individuals and patients exhibiting hereditary or acquired iron overload. Six sites of variation were detected: (1) H63D C>G in exon 2, (2) IVS2 (+4) T>C in intron 2, (3) a C>G transversion in intron 3, (4) C282Y G>A in exon 4, (5) IVS4 (-44) T>C in intron 4, and (6) a new guanine deletion (G>del) in intron 5, which were used for haplotype inference. Nine HFE alleles were detected and six of these were officially named on the basis of the HLA Nomenclature, defined by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System, and published via the IPD-IMGT/HLA website. Four alleles, HFE*001, *002, *003, and *004 exhibited variation within their exon sequences. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. KEJADIAN INDEL SIMULTAN PADA INTRON 7 GEN BRANCHED-CHAIN Α-KETOACID DEHYDROGENASE E1A (BCKDHA PADA SAPI MADURA

    Directory of Open Access Journals (Sweden)

    Asri Febriana

    2015-08-01

    Full Text Available Madura cattle is one of the Indonesian local cattle breeds derived from crossing between Zebu cattle (Bos indicus and banteng (Bos javanicus. Branched-chain α-ketoacid dehydrogenase (BCKDH is one of the main enzyme complexes in the inner mitochondrial membrane that metabolizes branched chain amino acid (BCAA, ie valine, leucine, and isoleucine. The diversity of the nucleotide sequences of the genes largely determine the efficiency of enzyme encoded. This paper aimed to determine the nucleotide variation contained in section intron 7, exon 8, and intron 8 genes BCKDHA on Madura cattle. This study was conducted on three Madura cattle that used as bull race (karapan, beauty contest (sonok, and beef cattle. The analysis showed that the variation in intron higher than occurred in the exon. Simultaneous indel found at base position 34 and 68 in sonok cattle. In addition, the C266T variant found in beef cattle. These variants do not cause significant changes in amino acids. There was no specific mutation in intron 7, exon 8, and intron 8 were found in Madura cattle designation. This indicated the absence of differentiation Madura cattle designation of selection pressure of BCKDHA gene.

  8. The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

    Science.gov (United States)

    Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

    2013-01-01

    Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications

  9. The mitochondrial genome of the prasinophyte Prasinoderma coloniale reveals two trans-spliced group I introns in the large subunit rRNA gene.

    Directory of Open Access Journals (Sweden)

    Jean-François Pombert

    Full Text Available Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI, we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V. This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI. Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the

  10. Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing.

    Science.gov (United States)

    Steele-Stallard, Heather B; Le Quesne Stabej, Polona; Lenassi, Eva; Luxon, Linda M; Claustres, Mireille; Roux, Anne-Francoise; Webster, Andrew R; Bitner-Glindzicz, Maria

    2013-08-08

    Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. We found that 8 of

  11. Development and utilization of novel intron length polymorphic markers in foxtail millet (Setaria italica (L.) P. Beauv.).

    Science.gov (United States)

    Gupta, Sarika; Kumari, Kajal; Das, Jyotirmoy; Lata, Charu; Puranik, Swati; Prasad, Manoj

    2011-07-01

    Introns are noncoding sequences in a gene that are transcribed to precursor mRNA but spliced out during mRNA maturation and are abundant in eukaryotic genomes. The availability of codominant molecular markers and saturated genetic linkage maps have been limited in foxtail millet (Setaria italica (L.) P. Beauv.). Here, we describe the development of 98 novel intron length polymorphic (ILP) markers in foxtail millet using sequence information of the model plant rice. A total of 575 nonredundant expressed sequence tag (EST) sequences were obtained, of which 327 and 248 unique sequences were from dehydration- and salinity-stressed suppression subtractive hybridization libraries, respectively. The BLAST analysis of 98 EST sequences suggests a nearly defined function for about 64% of them, and they were grouped into 11 different functional categories. All 98 ILP primer pairs showed a high level of cross-species amplification in two millets and two nonmillets species ranging from 90% to 100%, with a mean of ∼97%. The mean observed heterozygosity and Nei's average gene diversity 0.016 and 0.171, respectively, established the efficiency of the ILP markers for distinguishing the foxtail millet accessions. Based on 26 ILP markers, a reasonable dendrogram of 45 foxtail millet accessions was constructed, demonstrating the utility of ILP markers in germplasm characterizations and genomic relationships in millets and nonmillets species.

  12. The Half-Life of the HSV-1 1.5 kb LAT Intron is similar to the half-Life of the 2.0 kb LAT Intron

    Science.gov (United States)

    Brinkman, Kerry K.; Mishra, Prakhar; Fraser, Nigel W.

    2013-01-01

    Herpes Simplex Virus type 1 (HSV-1) establishes a latent infection in the sensory neurons of the peripheral nervous system of humans. Although about 80 genes are expressed during the lytic cycle of the virus infection, essentially only one gene is expressed during the latent cycle. This gene is known as the latency associated transcript (LAT) and it appears to play a role in the latency cycle through an anti-apoptotic function in the 5’ end of the gene and miRNA encoded along the length of the transcript which down regulate some of the viral immediate early (IE) gene products. The LAT gene is about 8.3 kb long and consists of two exons separated by an unusual intron. The intron between the exons consists of two nested introns. This arrangement of introns has been called a twintron. Furthermore, the larger (2 kb) intron has been shown to be very stable. In this study we measure the stability of the shorter 1.5 kb nested intron and find its half-life is similar to the longer intron. This was achieved by deleting the 0.5 kb overlapping intron from a plasmid construct designed to express the LAT transcript from a tet-inducible promoter, and measuring the half-life of the 1.5 kb intron in tissue culture cells. This finding supports the hypothesis that it is the common branch-point region of these nested introns that is responsible for their stability. PMID:23335177

  13. A Novel Heterozygous Intronic Mutation in the FBN1 Gene Contributes to FBN1 RNA Missplicing Events in the Marfan Syndrome

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    Mario Torrado

    2018-01-01

    Full Text Available Marfan syndrome (MFS is an autosomal dominantly inherited connective tissue disorder, mostly caused by mutations in the fibrillin-1 (FBN1 gene. We, by using targeted next-generation sequence analysis, identified a novel intronic FBN1 mutation (the c.2678-15C>A variant in a MFS patient with aortic dilatation. The computational predictions showed that the heterozygous c.2678-15C>A intronic variant might influence the splicing process by differentially affecting canonical versus cryptic splice site utilization within intron 22 of the FBN1 gene. RT-PCR and Western blot analyses, using FBN1 minigenes transfected into HeLa and COS-7 cells, revealed that the c.2678-15C>A variant disrupts normal splicing of intron 22 leading to aberrant 13-nt intron 22 inclusion, frameshift, and premature termination codon. Collectively, the results strongly suggest that the c.2678-15C>A variant could lead to haploinsufficiency of the FBN1 functional protein and structural connective tissue fragility in MFS complicated by aorta dilation, a finding that further expands on the genetic basis of aortic pathology.

  14. Introns in the genome of bacteriophage T4

    International Nuclear Information System (INIS)

    Gott, J.M.

    1987-01-01

    RNA from T4-infected cells yields multiple end-labeled species when incubated with [α- 32 P]GTP under self-splicing conditions. One of these corresponds to the previously characterized intron from the T4 td gene and, as shown in this work, the others represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to the mixed GTP-labeled T4 RNA probe. These were mapped to the unlinked genes nrdB and sunY. Cloned DNA from the nrdB region that contained the intron was shown to generate characteristic group I splice products with RNA synthesized in vivo or in vitro. The splice junction of the nrdB gene was determined and the nature of the RNA reaction products characterized. In vivo expression of the nrdB gene and the open reading frame within the intron was studied using in-frame lacZ fusions and primer extension analyses. The data suggest that expression of the intron open reading frame is highly regulated during T4 infection. Possible regulatory mechanisms are discussed

  15. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

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    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  16. Molecular evolution of Adh and LEAFY and the phylogenetic utility of their introns in Pyrus (Rosaceae).

    Science.gov (United States)

    Zheng, Xiaoyan; Hu, Chunyun; Spooner, David; Liu, Jing; Cao, Jiashu; Teng, Yuanwen

    2011-09-14

    The genus Pyrus belongs to the tribe Pyreae (the former subfamily Maloideae) of the family Rosaceae, and includes one of the most important commercial fruit crops, pear. The phylogeny of Pyrus has not been definitively reconstructed. In our previous efforts, the internal transcribed spacer region (ITS) revealed a poorly resolved phylogeny due to non-concerted evolution of nrDNA arrays. Therefore, introns of low copy nuclear genes (LCNG) are explored here for improved resolution. However, paralogs and lineage sorting are still two challenges for applying LCNGs in phylogenetic studies, and at least two independent nuclear loci should be compared. In this work the second intron of LEAFY and the alcohol dehydrogenase gene (Adh) were selected to investigate their molecular evolution and phylogenetic utility. DNA sequence analyses revealed a complex ortholog and paralog structure of Adh genes in Pyrus and Malus, the pears and apples. Comparisons between sequences from RT-PCR and genomic PCR indicate that some Adh homologs are putatively nonfunctional. A partial region of Adh1 was sequenced for 18 Pyrus species and three subparalogs representing Adh1-1 were identified. These led to poorly resolved phylogenies due to low sequence divergence and the inclusion of putative recombinants. For the second intron of LEAFY, multiple inparalogs were discovered for both LFY1int2 and LFY2int2. LFY1int2 is inadequate for phylogenetic analysis due to lineage sorting of two inparalogs. LFY2int2-N, however, showed a relatively high sequence divergence and led to the best-resolved phylogeny. This study documents the coexistence of outparalogs and inparalogs, and lineage sorting of these paralogs and orthologous copies. It reveals putative recombinants that can lead to incorrect phylogenetic inferences, and presents an improved phylogenetic resolution of Pyrus using LFY2int2-N. Our study represents the first phylogenetic analyses based on LCNGs in Pyrus. Ancient and recent duplications lead

  17. Molecular evolution of Adh and LEAFY and the phylogenetic utility of their introns in Pyrus (Rosaceae

    Directory of Open Access Journals (Sweden)

    Cao Jiashu

    2011-09-01

    Full Text Available Abstract Background The genus Pyrus belongs to the tribe Pyreae (the former subfamily Maloideae of the family Rosaceae, and includes one of the most important commercial fruit crops, pear. The phylogeny of Pyrus has not been definitively reconstructed. In our previous efforts, the internal transcribed spacer region (ITS revealed a poorly resolved phylogeny due to non-concerted evolution of nrDNA arrays. Therefore, introns of low copy nuclear genes (LCNG are explored here for improved resolution. However, paralogs and lineage sorting are still two challenges for applying LCNGs in phylogenetic studies, and at least two independent nuclear loci should be compared. In this work the second intron of LEAFY and the alcohol dehydrogenase gene (Adh were selected to investigate their molecular evolution and phylogenetic utility. Results DNA sequence analyses revealed a complex ortholog and paralog structure of Adh genes in Pyrus and Malus, the pears and apples. Comparisons between sequences from RT-PCR and genomic PCR indicate that some Adh homologs are putatively nonfunctional. A partial region of Adh1 was sequenced for 18 Pyrus species and three subparalogs representing Adh1-1 were identified. These led to poorly resolved phylogenies due to low sequence divergence and the inclusion of putative recombinants. For the second intron of LEAFY, multiple inparalogs were discovered for both LFY1int2 and LFY2int2. LFY1int2 is inadequate for phylogenetic analysis due to lineage sorting of two inparalogs. LFY2int2-N, however, showed a relatively high sequence divergence and led to the best-resolved phylogeny. This study documents the coexistence of outparalogs and inparalogs, and lineage sorting of these paralogs and orthologous copies. It reveals putative recombinants that can lead to incorrect phylogenetic inferences, and presents an improved phylogenetic resolution of Pyrus using LFY2int2-N. Conclusions Our study represents the first phylogenetic analyses based

  18. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron...

  19. Amino acid sequence surrounding the chondroitin sulfate attachment site of thrombomodulin regulates chondroitin polymerization.

    Science.gov (United States)

    Izumikawa, Tomomi; Kitagawa, Hiroshi

    2015-05-01

    Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAβ1-3Galβ1-3Galβ1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Multiple splicing defects in an intronic false exon.

    Science.gov (United States)

    Sun, H; Chasin, L A

    2000-09-01

    Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5' splice site that perfectly matches the 5' consensus combined with mutation to match the CAG/G sequence of the 3' consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3' splice site and a consensus 5' splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5' splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with beta-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

  1. An intron capture strategy used to identify and map a lysyl oxidase-like gene on chromosome 9 in the mouse

    Energy Technology Data Exchange (ETDEWEB)

    Wydner, K.S.; Passmore, H.C. [Rutgers Univ., Piscataway, NJ (United States); Kim, Houngho; Csiszar, K.; Boyd, C.D. [UMDNJ, New Brunswick, NJ (United States)

    1997-03-01

    An intron capture strategy involving use of polymerase chain reaction was used to identify and map the mouse homologue of a human lysyl oxidase-like gene (LOXL). Oligonucleotides complementary to conserved domains within exons 4 and 5 of the human lysyl oxidase-like gene were used to amplify the corresponding segment from mouse genomic DNA. Sequencing of the resulting mouse DNA fragment of approximately 1 kb revealed that the exon sequences at the ends of the amplified fragment are highly homologous (90% nucleotide identity) to exons 4 and 5 of the human lysyl oxidase-like gene. An AluI restriction site polymorphism within intron 4 was used to map the mouse lysyl oxidase-like gene (Loxl) to mouse Chromosome 9 in a region that shares linkage conservation with human chromosome 15q24, to which the LOXL was recently mapped. 22 refs., 3 figs.

  2. Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer

    Directory of Open Access Journals (Sweden)

    Tai Phillip WL

    2011-07-01

    Full Text Available Abstract Background Hundreds of genes, including muscle creatine kinase (MCK, are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. Results Modulatory region 1 (MR1 is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE containing a paired E-box/myocyte enhancer factor 2 (MEF2 regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively, but is not required for expression in fast-twitch muscle fibers (types IIb and IId. Conclusions In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

  3. The splicing of tiny introns of Paramecium is controlled by MAGO.

    Science.gov (United States)

    Contreras, Julia; Begley, Victoria; Marsella, Laura; Villalobo, Eduardo

    2018-07-15

    The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Localized Retroprocessing as a Model of Intron Loss in the Plant Mitochondrial Genome.

    Science.gov (United States)

    Cuenca, Argelia; Ross, T Gregory; Graham, Sean W; Barrett, Craig F; Davis, Jerrold I; Seberg, Ole; Petersen, Gitte

    2016-08-03

    Loss of introns in plant mitochondrial genes is commonly explained by retroprocessing. Under this model, an mRNA is reverse transcribed and integrated back into the genome, simultaneously affecting the contents of introns and edited sites. To evaluate the extent to which retroprocessing explains intron loss, we analyzed patterns of intron content and predicted RNA editing for whole mitochondrial genomes of 30 species in the monocot order Alismatales. In this group, we found an unusually high degree of variation in the intron content, even expanding the hitherto known variation among angiosperms. Some species have lost some two-third of the cis-spliced introns. We found a strong correlation between intron content and editing frequency, and detected 27 events in which intron loss is consistent with the presence of nucleotides in an edited state, supporting retroprocessing. However, we also detected seven cases of intron loss not readily being explained by retroprocession. Our analyses are also not consistent with the entire length of a fully processed cDNA copy being integrated into the genome, but instead indicate that retroprocessing usually occurs for only part of the gene. In some cases, several rounds of retroprocessing may explain intron loss in genes completely devoid of introns. A number of taxa retroprocessing seem to be very common and a possibly ongoing process. It affects the entire mitochondrial genome. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  5. Differential GC Content between Exons and Introns Establishes Distinct Strategies of Splice-Site Recognition

    Directory of Open Access Journals (Sweden)

    Maayan Amit

    2012-05-01

    Full Text Available During evolution segments of homeothermic genomes underwent a GC content increase. Our analyses reveal that two exon-intron architectures have evolved from an ancestral state of low GC content exons flanked by short introns with a lower GC content. One group underwent a GC content elevation that abolished the differential exon-intron GC content, with introns remaining short. The other group retained the overall low GC content as well as the differential exon-intron GC content, and is associated with longer introns. We show that differential exon-intron GC content regulates exon inclusion level in this group, in which disease-associated mutations often lead to exon skipping. This group's exons also display higher nucleosome occupancy compared to flanking introns and exons of the other group, thus “marking” them for spliceosomal recognition. Collectively, our results reveal that differential exon-intron GC content is a previously unidentified determinant of exon selection and argue that the two GC content architectures reflect the two mechanisms by which splicing signals are recognized: exon definition and intron definition.

  6. Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages

    International Nuclear Information System (INIS)

    Chu, F.K.; Maley, F.; Martinez, J.; Maley, G.F.

    1987-01-01

    Southern hybridization analyses of procaryotic DNA from Escherchia coli, λ bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. [α- 32 P]GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns

  7. Aberrant splicing in transgenes containing introns, exons, and V5 epitopes: lessons from developing an FSHD mouse model expressing a D4Z4 repeat with flanking genomic sequences.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD. This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV and strong viral control elements (CMV promoter, SV40 poly A to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.

  8. Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria

    Science.gov (United States)

    Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R.; Gray, Amie K.; Baluarte, H. Jorge; Econs, Michael J.

    2013-01-01

    Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6 ½-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24- hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH) levels, and elevated 1,25(OH)2D level. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440–1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63 bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH. PMID:24176905

  9. Molecular genetic analysis of cereal β-amylase genes using exon-primed intron-crossing (EPIC PCR

    Directory of Open Access Journals (Sweden)

    Stratula Olga

    2014-01-01

    Full Text Available The proteins encoded by cereal β-amylase genes Bamy1 and Bamy2 genes play an important role in seedling germination and in the brewing process. Here, we use exon-primed intron-crossing (EPIC to analyse Bamy1 and Bamy2 genetic diversity among 38 accessions belonging to six Poaceae tribes. DNA sequence alignment of multiple Poaceae species β-amylase sequences allowed design of EPIC primers that simultaneously amplify Bamy1 and Bamy2 in all the cereal species investigated. The genetic variation observed in the samples investigated is analysed and discussed, and illustrates the effectiveness of this approach for intra- and interspecific analysis in plant species.

  10. Phylogenetic relationships in the genus Leonardoxa (Leguminosae: Caesalpinioideae) inferred from chloroplast trnL intron and trnL-trnF intergenic spacer sequences.

    Science.gov (United States)

    Brouat, Carine; Gielly, Ludovic; McKey, Doyle

    2001-01-01

    The African genus LEONARDOXA: (Leguminosae: Caesalpinioideae) comprises two Congolean species and a group of four mostly allopatric subspecies principally located in Cameroon and clustered together in the L. africana complex. LEONARDOXA: provides a good opportunity to investigate the evolutionary history of ant-plant mutualisms, as it exhibits various grades of ant-plant interactions from diffuse to obligate and symbiotic associations. We present in this paper the first molecular phylogenetic study of this genus. We sequenced both the chloroplast DNA trnL intron (677 aligned base pairs [bp]) and trnL-trnF intergene spacer (598 aligned bp). Inferred phylogenetic relationships suggested first that the genus is paraphyletic. The L. africana complex is clearly separated from the two Congolean species, and the integrity of the genus is thus in question. In the L. africana complex, our data showed a lack of congruence between clades suggested by morphological and chloroplast characters. This, and the low level of molecular divergence found between subspecies, suggests gene flow and introgressive events in the L. africana complex.

  11. Class I self-splicing introns are found in the T-even bacteriophage family

    International Nuclear Information System (INIS)

    Chu, F.K.; Maley, F.; Maley, G.F.

    1987-01-01

    The thymidylate synthase gene (td) and ribonucleotide reductase B2 subunit gene (nrdB) EMBO both of bacteriophage T4 in origin, are procaryotic intron-containing protein-encoding genes. To screen for other procaryotic introns, southern hybridization analysis of several procaryotic genomes was carried out, using T4 phage td DNA restriction fragments and synthetic oligodeoxynucleotides defining strategic td exon and intron regions. Furthermore, the labeling pattern of total RNA with [α- 32 P]GTP, a typical reaction of self-splicing RNAs (class I), was examined. Experimental data implicate multiple self-splicing introns only in the T-even phages: five (1, 0.9, 0.83, 0.75 and 0.6 kb) in T4 and three (1, 0.9 and 0.75 kb) each in T2 and T6 phages. Northern hybridization analysis of total RNA extracted from T-even phage-infected cells confirms that the 1 kb RNA from each phage is in fact the excised intron segment from the precursor RNA transcribed from an intron-containing td gene in each case. This RNA cyclizes to form a contiguous circular molecule. The 0.6 kb RNA is most likely the T4 phage nrdB intron which seems to be absent from the corresponding gene in T2 and T6. The remaining RNA species are candidates for other self-splicing introns in these phages

  12. Morquio A syndrome: Cloning, sequence, and structure of the human N-acetylgalactosamine 6-sulfatase (GALNS) gene

    Energy Technology Data Exchange (ETDEWEB)

    Morris, C.P.; Guo, Xiao-Hui; Apostolou, S. [Adelaide Children`s Hospital, North Adelaide (Australia)] [and others

    1994-08-01

    Deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulfatase (GALNS;EC 3.1.6.4), results in the storage of the glycosaminoglycans, keratan sulfate and chrondroitin 6-sulfate, which leads to the lysosomal storage disorder Morquio A syndrome. Four overlapping genomic clones derived from a chromosome 16-specific gridded cosmid library containing the entire GALNS gene were isolated. The structure of the gene and the sequence of the exon/intron boundaries and the 5{prime} promoter region were determined. The GALNS gene is split into 14 exons spanning approximately 40 kb. The potential promoter for GALNS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. The GALNS gene contains an Alu repeat in intron 5 and a VNTR-like sequence in intron 6. 12 refs., 3 figs., 1 tab.

  13. Molecular characterization of a new member of the lariat capping twin-ribozyme introns

    DEFF Research Database (Denmark)

    Tang, Yunjia; Nielsen, Henrik; Masquida, Benoît

    2014-01-01

    BACKGROUND: Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Na...

  14. Characteristics of binding sites of intergenic, intronic and exonic ...

    African Journals Online (AJOL)

    user

    2013-03-06

    Mar 6, 2013 ... miR-1587). Such part of mRNA is very important for its regulation via several miRNA. Interaction of intronic miRNAs with mRNAs genes coding in-miRNA. Oncogenes (51) are host genes and target genes for in-. miRNAs. Majority of these in-miRNAs are encoded in intron. Five of the studied genes (ATF2, ...

  15. Inferring Invasion History of Red Swamp Crayfish (Procambarus clarkii) in China from Mitochondrial Control Region and Nuclear Intron Sequences

    Science.gov (United States)

    Li, Yanhe; Guo, Xianwu; Chen, Liping; Bai, Xiaohui; Wei, Xinlan; Zhou, Xiaoyun; Huang, Songqian; Wang, Weimin

    2015-01-01

    Identifying the dispersal pathways of an invasive species is useful for adopting the appropriate strategies to prevent and control its spread. However, these processes are exceedingly complex. So, it is necessary to apply new technology and collect representative samples for analysis. This study used Approximate Bayesian Computation (ABC) in combination with traditional genetic tools to examine extensive sample data and historical records to infer the invasion history of the red swamp crayfish, Procambarus clarkii, in China. The sequences of the mitochondrial control region and the proPOx intron in the nuclear genome of samples from 37 sites (35 in China and one each in Japan and the USA) were analyzed. The results of combined scenarios testing and historical records revealed a much more complex invasion history in China than previously believed. P. clarkii was most likely originally introduced into China from Japan from an unsampled source, and the species then expanded its range primarily into the middle and lower reaches and, to a lesser extent, into the upper reaches of the Changjiang River in China. No transfer was observed from the upper reaches to the middle and lower reaches of the Changjiang River. Human-mediated jump dispersal was an important dispersal pathway for P. clarkii. The results provide a better understanding of the evolutionary scenarios involved in the rapid invasion of P. clarkii in China. PMID:26132567

  16. Inferring Invasion History of Red Swamp Crayfish (Procambarus clarkii in China from Mitochondrial Control Region and Nuclear Intron Sequences

    Directory of Open Access Journals (Sweden)

    Yanhe Li

    2015-06-01

    Full Text Available Identifying the dispersal pathways of an invasive species is useful for adopting the appropriate strategies to prevent and control its spread. However, these processes are exceedingly complex. So, it is necessary to apply new technology and collect representative samples for analysis. This study used Approximate Bayesian Computation (ABC in combination with traditional genetic tools to examine extensive sample data and historical records to infer the invasion history of the red swamp crayfish, Procambarus clarkii, in China. The sequences of the mitochondrial control region and the proPOx intron in the nuclear genome of samples from 37 sites (35 in China and one each in Japan and the USA were analyzed. The results of combined scenarios testing and historical records revealed a much more complex invasion history in China than previously believed. P. clarkii was most likely originally introduced into China from Japan from an unsampled source, and the species then expanded its range primarily into the middle and lower reaches and, to a lesser extent, into the upper reaches of the Changjiang River in China. No transfer was observed from the upper reaches to the middle and lower reaches of the Changjiang River. Human-mediated jump dispersal was an important dispersal pathway for P. clarkii. The results provide a better understanding of the evolutionary scenarios involved in the rapid invasion of P. clarkii in China.

  17. Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

    Science.gov (United States)

    Shin, Dong-Ho; Webb, Barbara M; Nakao, Miki; Smith, Sylvia L

    2009-07-01

    Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1-3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent.

  18. Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

    2008-11-01

    Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

  19. Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity

    International Nuclear Information System (INIS)

    Lubahn, D.B.; Simental, J.A.; Higgs, H.N.; Wilson, E.M.; French, F.S.; Brown, T.R.; Migeon, C.J.

    1989-01-01

    Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. The authors have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46, XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development

  20. Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

    Science.gov (United States)

    Sasikumar, Ponnusamy; Paul, Eldho; Gomathi, Sivasamy; Abhishek, Albert; Sasikumar, Sundaresan; Selvam, Govindan Sadasivam

    2016-10-01

    The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real-time PCR experiment shows 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E-0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA - mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 μM) supported the growth of the culture until saturation. In conclusion, ThyA - mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. ASPIC: a novel method to predict the exon-intron structure of a gene that is optimally compatible to a set of transcript sequences

    Directory of Open Access Journals (Sweden)

    Pesole Graziano

    2005-10-01

    Full Text Available Abstract Background: Currently available methods to predict splice sites are mainly based on the independent and progressive alignment of transcript data (mostly ESTs to the genomic sequence. Apart from often being computationally expensive, this approach is vulnerable to several problems – hence the need to develop novel strategies. Results: We propose a method, based on a novel multiple genome-EST alignment algorithm, for the detection of splice sites. To avoid limitations of splice sites prediction (mainly, over-predictions due to independent single EST alignments to the genomic sequence our approach performs a multiple alignment of transcript data to the genomic sequence based on the combined analysis of all available data. We recast the problem of predicting constitutive and alternative splicing as an optimization problem, where the optimal multiple transcript alignment minimizes the number of exons and hence of splice site observations. We have implemented a splice site predictor based on this algorithm in the software tool ASPIC (Alternative Splicing PredICtion. It is distinguished from other methods based on BLAST-like tools by the incorporation of entirely new ad hoc procedures for accurate and computationally efficient transcript alignment and adopts dynamic programming for the refinement of intron boundaries. ASPIC also provides the minimal set of non-mergeable transcript isoforms compatible with the detected splicing events. The ASPIC web resource is dynamically interconnected with the Ensembl and Unigene databases and also implements an upload facility. Conclusion: Extensive bench marking shows that ASPIC outperforms other existing methods in the detection of novel splicing isoforms and in the minimization of over-predictions. ASPIC also requires a lower computation time for processing a single gene and an EST cluster. The ASPIC web resource is available at http://aspic.algo.disco.unimib.it/aspic-devel/.

  2. Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy

    Directory of Open Access Journals (Sweden)

    RODRIGO GONZÁLEZ

    2009-01-01

    Full Text Available We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivaríate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

  3. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation

    International Nuclear Information System (INIS)

    O'Hara, P.J.; Grant, F.J.; Haldeman, B.A.; Gray, C.L.; Insley, M.Y.; Hagen, F.S.; Murray, M.J.

    1987-01-01

    Activated factor VII (factor VIIa) is a vitamin K-dependent plasma serine protease that participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span about 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylylated at multiple sites but contains only one AAUAAA poly(A) signal sequence. The mRNA can undergo alternative splicing, forming one transcript containing eight segments as exons and another with an additional exon that encodes a larger prepro leader sequence. The latter transcript has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C, and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family. The comparable introns in these genes, however, are dissimilar with respect to size and sequence, with the exception of intron C in factor VII and protein C. The gene for factor VII also contains five regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats

  4. Examination of species boundaries in the Acropora cervicornis group (Scleractinia, cnidaria) using nuclear DNA sequence analyses.

    Science.gov (United States)

    Oppen, M J; Willis, B L; Vugt, H W; Miller, D J

    2000-09-01

    Although Acropora is the most species-rich genus of the scleractinian (stony) corals, only three species occur in the Caribbean: A. cervicornis, A. palmata and A. prolifera. Based on overall coral morphology, abundance and distribution patterns, it has been suggested that A. prolifera may be a hybrid between A. cervicornis and A. palmata. The species boundaries among these three morphospecies were examined using DNA sequence analyses of the nuclear Pax-C 46/47 intron and the ribosomal DNA Internal Transcribed Spacer (ITS1 and ITS2) and 5.8S regions. Moderate levels of sequence variability were observed in the ITS and 5.8S sequences (up to 5.2% overall sequence difference), but variability within species was as large as between species and all three species carried similar sequences. Since this is unlikely to represent a shared ancestral polymorphism, the data suggest that introgressive hybridization occurs among the three species. For the Pax-C intron, A. cervicornis and A. palmata had very distinct allele frequencies and A. cervicornis carried a unique allele at a frequency of 0.769 (although sequence differences between alleles were small). All A. prolifera colonies examined were heterozygous for the Pax-C intron, whereas heterozygosity was only 0.286 and 0.333 for A. cervicornis and A. palmata, respectively. These data support the hypothesis that A. prolifera is the product of hybridization between two species that have a different allelic composition for the Pax-C intron, i.e. A. cervicornis and A. palmata. We therefore suggest that A. prolifera is a hybrid between A. cervicornis and A. palmata, which backcrosses with the parental species at low frequency.

  5. A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3' untranslated region and intronic cis-elements.

    Science.gov (United States)

    Muhle, Rebecca A; Adjalley, Sophie; Falkard, Brie; Nkrumah, Louis J; Muhle, Michael E; Fidock, David A

    2009-11-01

    Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitised erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilising the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var subtelomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronised parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may result from the integrated UpsA promoter being largely silenced by the neighbouring cg6 promoter. Our analyses also revealed that the DownsA 3' untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA-promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyse promoter activity of Group A var genes, which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of var

  6. The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

    Science.gov (United States)

    Zhao, Chen; Pyle, Anna Marie

    2017-12-01

    The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Characterization of novel and uncharacterized p53 SNPs in the Chinese population--intron 2 SNP co-segregates with the common codon 72 polymorphism.

    Directory of Open Access Journals (Sweden)

    Beng Hooi Phang

    Full Text Available Multiple single nucleotide polymorphisms (SNPs have been identified in the tumor suppressor gene p53, though the relevance of many of them is unclear. Some of them are also differentially distributed in various ethnic populations, suggesting selective functionality. We have therefore sequenced all exons and flanking regions of p53 from the Singaporean Chinese population and report here the characterization of some novel and uncharacterized SNPs - four in intron 1 (nucleotide positions 8759/10361/10506/11130, three in intron 3 (11968/11969/11974 and two in the 3'UTR (19168/19514. Allelic frequencies were determined for all these and some known SNPs, and were compared in a limited scale to leukemia and lung cancer patient samples. Intron 2 (11827 and 7 (14181/14201 SNPs were found to have a high minor allele frequency of between 26-47%, in contrast to the lower frequencies found in the US population, but similar in trend to the codon 72 polymorphism (SNP12139 that shows a distribution pattern correlative with latitude. Several of the SNPs were linked, such as those in introns 1, 3 and 7. Most interestingly, we noticed the co-segregation of the intron 2 and the codon 72 SNPs, the latter which has been shown to be expressed in an allele-specific manner, suggesting possible regulatory cross-talk. Association analysis indicated that the T/G alleles in both the co-segregating intron 7 SNPs and a 4tagSNP haplotype was strongly associated increased susceptibility to lung cancer in non-smoker females [OR: 1.97 (1.32, 3.394]. These data together demonstrate high SNP diversity in p53 gene between different populations, highlighting ethnicity-based differences, and their association with cancer risk.

  8. The complete chloroplast genome sequence of Euonymus japonicus (Celastraceae).

    Science.gov (United States)

    Choi, Kyoung Su; Park, SeonJoo

    2016-09-01

    The complete chloroplast (cp) genome sequence of the Euonymus japonicus, the first sequenced of the genus Euonymus, was reported in this study. The total length was 157 637 bp, containing a pair of 26 678 bp inverted repeat region (IR), which were separated by small single copy (SSC) region and large single copy (LSC) region of 18 340 bp and 85 941 bp, respectively. This genome contains 107 unique genes, including 74 coding genes, four rRNA genes, and 29 tRNA genes. Seventeen genes contain intron of E. japonicus, of which three genes (clpP, ycf3, and rps12) include two introns. The maximum likelihood (ML) phylogenetic analysis revealed that E. japonicus was closely related to Manihot and Populus.

  9. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

    Directory of Open Access Journals (Sweden)

    Madeleine Zerbato

    Full Text Available Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

  10. Phylogenomic Resolution of the Phylogeny of Laurasiatherian Mammals: Exploring Phylogenetic Signals within Coding and Noncoding Sequences.

    Science.gov (United States)

    Chen, Meng-Yun; Liang, Dan; Zhang, Peng

    2017-08-01

    The interordinal relationships of Laurasiatherian mammals are currently one of the most controversial questions in mammalian phylogenetics. Previous studies mainly relied on coding sequences (CDS) and seldom used noncoding sequences. Here, by data mining public genome data, we compiled an intron data set of 3,638 genes (all introns from a protein-coding gene are considered as a gene) (19,055,073 bp) and a CDS data set of 10,259 genes (20,994,285 bp), covering all major lineages of Laurasiatheria (except Pholidota). We found that the intron data contained stronger and more congruent phylogenetic signals than the CDS data. In agreement with this observation, concatenation and species-tree analyses of the intron data set yielded well-resolved and identical phylogenies, whereas the CDS data set produced weakly supported and incongruent results. Further analyses showed that the phylogeny inferred from the intron data is highly robust to data subsampling and change in outgroup, but the CDS data produced unstable results under the same conditions. Interestingly, gene tree statistical results showed that the most frequently observed gene tree topologies for the CDS and intron data are identical, suggesting that the major phylogenetic signal within the CDS data is actually congruent with that within the intron data. Our final result of Laurasiatheria phylogeny is (Eulipotyphla,((Chiroptera, Perissodactyla),(Carnivora, Cetartiodactyla))), favoring a close relationship between Chiroptera and Perissodactyla. Our study 1) provides a well-supported phylogenetic framework for Laurasiatheria, representing a step towards ending the long-standing "hard" polytomy and 2) argues that intron within genome data is a promising data resource for resolving rapid radiation events across the tree of life. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

    Science.gov (United States)

    Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

    2014-01-01

    Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

  12. Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement

    International Nuclear Information System (INIS)

    Broderick, T.P.; Schaff, D.A.; Bertino, A.M.; Dush, M.K.; Tischfield, J.A.; Stambrook, P.J.

    1987-01-01

    The functional human adenine phosphoribosyltransferase (APRT) gene is <2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other housekeeping genes, lacks TATA and CCAAT boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function

  13. A var gene promoter implicated in severe malaria nucleates silencing and is regulated by 3’ untranslated region and intronic cis-elements

    Science.gov (United States)

    Muhle, Rebecca A.; Adjalley, Sophie; Falkard, Brie; Nkrumah, Louis J.; Muhle, Michael E.; Fidock, David A.

    2009-01-01

    Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitized erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilizing the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var sub-telomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronized parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may well result from the integrated UpsA promoter being largely silenced by the neighboring cg6 promoter. Our analyses also revealed that the DownsA 3’ untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyze promoter activity of Group A var genes which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of

  14. Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene.

    Science.gov (United States)

    Gonçalves, Ana; Oliveira, Jorge; Coelho, Teresa; Taipa, Ricardo; Melo-Pires, Manuel; Sousa, Mário; Santos, Rosário

    2017-10-03

    A broad mutational spectrum in the dystrophin ( DMD ) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD , adding to the diversity of mutational events that give rise to D/BMD.

  15. Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

    Science.gov (United States)

    Gonçalves, Ana; Coelho, Teresa; Melo-Pires, Manuel; Sousa, Mário

    2017-01-01

    A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD. PMID:28972564

  16. Phylogenetic relationships in Taxodiaceae and Cupressaceae sensu stricto based on matK gene, chlL gene, trnL-trnF IGS region, and trnL intron sequences.

    Science.gov (United States)

    Kusumi, J; Tsumura, Y; Yoshimaru, H; Tachida, H

    2000-10-01

    Nucleotide sequences from four chloroplast genes, the matK, chlL, intergenic spacer (IGS) region between trnL and trnF, and an intron of trnL, were determined from all species of Taxodiaceae and five species of Cupressaceae sensu stricto (s.s.). Phylogenetic trees were constructed using the maximum parsimony and the neighbor-joining methods with Cunninghamia as an outgroup. These analyses provided greater resolution of relationships among genera and higher bootstrap supports for clades compared to previous analyses. Results indicate that Taiwania diverged first, and then Athrotaxis diverged from the remaining genera. Metasequoia, Sequoia, and Sequoiadendron form a clade. Taxodium and Glyptostrobus form a clade, which is the sister to Cryptomeria. Cupressaceae s.s. are derived from within Taxodiaceae, being the most closely related to the Cryptomeria/Taxodium/Glyptostrobus clade. These relationships are consistent with previous morphological groupings and the analyses of molecular data. In addition, we found acceleration of evolutionary rates in Cupressaceae s.s. Possible causes for the acceleration are discussed.

  17. Widespread alternative and aberrant splicing revealed by lariat sequencing

    Science.gov (United States)

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  18. An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A.

    Science.gov (United States)

    Xia, Zunjing; Lin, Jie; Lu, Lingping; Kim, Chol; Yu, Ping; Qi, Ming

    2018-06-01

    : Hemophilia A is a bleeding disorder caused by coagulation factor VIII protein deficiency or dysfunction, which is classified into severe, moderate, and mild according to factor clotting activity. An overwhelming majority of missense and nonsense mutations occur in exons of F8 gene, whereas mutations in introns can also be pathogenic. This study aimed to investigate the effect of an intronic mutation, c.6430-3C>G (IVS22-3C>G), on pre-mRNA splicing of the F8 gene. We applied DNA and cDNA sequencing in a Chinese boy with hemophilia A to search if any pathogenic mutation in the F8 gene. Functional analysis was performed to investigate the effect of an intronic mutation at the transcriptional level. Human Splicing Finder and PyMol were also used to predict its effect. We found the mutation c.6430-3C>G (IVS22-3C>G) in the F8 gene in the affected boy, with his mother being a carrier. cDNA from the mother and pSPL3 splicing assay showed that the mutation IVS22-3C>G results in a two-nucleotide AG inclusion at the 3' end of intron 22 and leads to a truncated coagulation factor VIII protein, with partial loss of the C1 domain and complete loss of the C2 domain. The in-silico tool predicted that the mutation induces altered pre-mRNA splicing by using a cryptic acceptor site in intron 22. The IVS22-3C>G mutation was confirmed to affect pre-mRNA splicing and produce a truncated protein, which reduces the stability of binding between the F8 protein and von Willebrand factor carrier protein due to the loss of an interaction domain.

  19. Predicting human splicing branchpoints by combining sequence-derived features and multi-label learning methods.

    Science.gov (United States)

    Zhang, Wen; Zhu, Xiaopeng; Fu, Yu; Tsuji, Junko; Weng, Zhiping

    2017-12-01

    Alternative splicing is the critical process in a single gene coding, which removes introns and joins exons, and splicing branchpoints are indicators for the alternative splicing. Wet experiments have identified a great number of human splicing branchpoints, but many branchpoints are still unknown. In order to guide wet experiments, we develop computational methods to predict human splicing branchpoints. Considering the fact that an intron may have multiple branchpoints, we transform the branchpoint prediction as the multi-label learning problem, and attempt to predict branchpoint sites from intron sequences. First, we investigate a variety of intron sequence-derived features, such as sparse profile, dinucleotide profile, position weight matrix profile, Markov motif profile and polypyrimidine tract profile. Second, we consider several multi-label learning methods: partial least squares regression, canonical correlation analysis and regularized canonical correlation analysis, and use them as the basic classification engines. Third, we propose two ensemble learning schemes which integrate different features and different classifiers to build ensemble learning systems for the branchpoint prediction. One is the genetic algorithm-based weighted average ensemble method; the other is the logistic regression-based ensemble method. In the computational experiments, two ensemble learning methods outperform benchmark branchpoint prediction methods, and can produce high-accuracy results on the benchmark dataset.

  20. Functional understanding of the diverse exon-intron structures of human GPCR genes.

    Science.gov (United States)

    Hammond, Dorothy A; Olman, Victor; Xu, Ying

    2014-02-01

    The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762 * e(-09), assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes.

  1. Cloning, sequencing and expression of a xylanase gene from the maize pathogen Helminthosporium turcicum

    DEFF Research Database (Denmark)

    Degefu, Y.; Paulin, L.; Lübeck, Peter Stephensen

    2001-01-01

    A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and ...

  2. SinEx DB: a database for single exon coding sequences in mammalian genomes.

    Science.gov (United States)

    Jorquera, Roddy; Ortiz, Rodrigo; Ossandon, F; Cárdenas, Juan Pablo; Sepúlveda, Rene; González, Carolina; Holmes, David S

    2016-01-01

    Eukaryotic genes are typically interrupted by intragenic, noncoding sequences termed introns. However, some genes lack introns in their coding sequence (CDS) and are generally known as 'single exon genes' (SEGs). In this work, a SEG is defined as a nuclear, protein-coding gene that lacks introns in its CDS. Whereas, many public databases of Eukaryotic multi-exon genes are available, there are only two specialized databases for SEGs. The present work addresses the need for a more extensive and diverse database by creating SinEx DB, a publicly available, searchable database of predicted SEGs from 10 completely sequenced mammalian genomes including human. SinEx DB houses the DNA and protein sequence information of these SEGs and includes their functional predictions (KOG) and the relative distribution of these functions within species. The information is stored in a relational database built with My SQL Server 5.1.33 and the complete dataset of SEG sequences and their functional predictions are available for downloading. SinEx DB can be interrogated by: (i) a browsable phylogenetic schema, (ii) carrying out BLAST searches to the in-house SinEx DB of SEGs and (iii) via an advanced search mode in which the database can be searched by key words and any combination of searches by species and predicted functions. SinEx DB provides a rich source of information for advancing our understanding of the evolution and function of SEGs.Database URL: www.sinex.cl. © The Author(s) 2016. Published by Oxford University Press.

  3. Spectral entropy criteria for structural segmentation in genomic DNA sequences

    International Nuclear Information System (INIS)

    Chechetkin, V.R.; Lobzin, V.V.

    2004-01-01

    The spectral entropy is calculated with Fourier structure factors and characterizes the level of structural ordering in a sequence of symbols. It may efficiently be applied to the assessment and reconstruction of the modular structure in genomic DNA sequences. We present the relevant spectral entropy criteria for the local and non-local structural segmentation in DNA sequences. The results are illustrated with the model examples and analysis of intervening exon-intron segments in the protein-coding regions

  4. [Sequence analysis of LEAFY homologous gene from Dendrobium moniliforme and application for identification of medicinal Dendrobium].

    Science.gov (United States)

    Xing, Wen-Rui; Hou, Bei-Wei; Guan, Jing-Jiao; Luo, Jing; Ding, Xiao-Yu

    2013-04-01

    The LEAFY (LFY) homologous gene of Dendrobium moniliforme (L.) Sw. was cloned by new primers which were designed based on the conservative region of known sequences of orchid LEAFY gene. Partial LFY homologous gene was cloned by common PCR, then we got the complete LFY homologous gene Den LFY by Tail-PCR. The complete sequence of DenLFY gene was 3 575 bp which contained three exons and two introns. Using BLAST method, comparison analysis among the exon of LFY homologous gene indicted that the DenLFY gene had high identity with orchids LFY homologous, including the related fragment of PhalLFY (84%) in Phalaenopsis hybrid cultivar, LFY homologous gene in Oncidium (90%) and in other orchid (over 80%). Using MP analysis, Dendrobium is found to be the sister to Oncidium and Phalaenopsis. Homologous analysis demonstrated that the C-terminal amino acids were highly conserved. When the exons and introns were separately considered, exons and the sequence of amino acid were good markers for the function research of DenLFY gene. The second intron can be used in authentication research of Dendrobium based on the length polymorphism between Dendrobium moniliforme and Dendrobium officinale.

  5. Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

    Science.gov (United States)

    Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

    2016-01-01

    Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

  6. Evolution and dispersal of emmer wheat (Triticum sp.) from novel haplotypes of Ppd-1 (photoperiod response) genes and their surrounding DNA sequences.

    Science.gov (United States)

    Takenaka, Shotaro; Kawahara, Taihachi

    2012-09-01

    The sequence data from 5' UTR, intronic, coding and 3' UTR regions of Ppd-A1 and Ppd-B1 were investigated for a total of 158 accessions of emmer wheat landraces comprising 19 of wild emmer wheat (Triticum dicoccoides), 45 of hulled emmer wheat (T. dicoccum) and 94 of free-threshing (FT) emmer wheat (T. durum etc.). We detected some novel types of deletions in the coding regions from 22 hulled emmer accessions and 20 FT emmer accessions. Emmer wheat accessions with these deletions could produce predicted proteins likely to lack function. We also observed some novel mutations in Ppd-B1. Sixty-seven and forty-one haplotypes were found in Ppd-A1 and Ppd-B1, respectively. Some mutations found in this study have not been known, so they have potential for useful genetic resources for wheat breeding. On the basis of sequence data from the 5' UTR region, both Ppd-A1 and Ppd-B1 haplotypes were divided into two groups (Type AI/AII and Type BI/BII). Types AI and AII of Ppd-A1 suggested gene flow between wild and hulled emmer. On the other hand, Types BI and BII of Ppd-B1 suggested gene flow between wild and FT emmer. More than half of hulled emmer accessions were Type AII/BI but few FT emmer accessions were of this type. Therefore, over half of the hulled emmer did not contribute to evolution of FT emmer.

  7. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  8. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

    Directory of Open Access Journals (Sweden)

    Natasha A Hamilton

    Full Text Available Angiotensin converting enzyme (ACE is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  9. Multi-species comparative analysis of the equine ACE gene identifies a highly conserved potential transcription factor binding site in intron 16.

    Science.gov (United States)

    Hamilton, Natasha A; Tammen, Imke; Raadsma, Herman W

    2013-01-01

    Angiotensin converting enzyme (ACE) is essential for control of blood pressure. The human ACE gene contains an intronic Alu indel (I/D) polymorphism that has been associated with variation in serum enzyme levels, although the functional mechanism has not been identified. The polymorphism has also been associated with cardiovascular disease, type II diabetes, renal disease and elite athleticism. We have characterized the ACE gene in horses of breeds selected for differing physical abilities. The equine gene has a similar structure to that of all known mammalian ACE genes. Nine common single nucleotide polymorphisms (SNPs) discovered in pooled DNA were found to be inherited in nine haplotypes. Three of these SNPs were located in intron 16, homologous to that containing the Alu polymorphism in the human. A highly conserved 18 bp sequence, also within that intron, was identified as being a potential binding site for the transcription factors Oct-1, HFH-1 and HNF-3β, and lies within a larger area of higher than normal homology. This putative regulatory element may contribute to regulation of the documented inter-individual variation in human circulating enzyme levels, for which a functional mechanism is yet to be defined. Two equine SNPs occurred within the conserved area in intron 16, although neither of them disrupted the putative binding site. We propose a possible regulatory mechanism of the ACE gene in mammalian species which was previously unknown. This advance will allow further analysis leading to a better understanding of the mechanisms underpinning the associations seen between the human Alu polymorphism and enzyme levels, cardiovascular disease states and elite athleticism.

  10. A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Barkan, Alice

    2007-12-01

    The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

  11. Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

    Directory of Open Access Journals (Sweden)

    Tuhin Kumar Guha

    Full Text Available In Chaetomium thermophilum (DSM 1495 within the mitochondrial DNA (mtDNA small ribosomal subunit (rns gene a group IIA1 intron interrupts an open reading frame (ORF encoded within a group I intron (mS1247. This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase. Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2 stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2 to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

  12. Estimating the Nucleotide Diversity in Ceratodon purpureus (Ditrichaceae from 218 Conserved Exon-Primed, Intron-Spanning Nuclear Loci

    Directory of Open Access Journals (Sweden)

    Stuart F. McDaniel

    2013-04-01

    Full Text Available Premise of the study: We developed and tested primers for 218 nuclear loci for studying population genetics, phylogeography, and genome evolution in bryophytes. Methods and Results: We aligned expressed sequence tags (ESTs from Ceratodon purpureus to the Physcomitrella patens genome sequence, and designed primers that are homologous to conserved exons but span introns in the P. patens genome. We tested these primers on four isolates from New York, USA; Otavalo, Ecuador; and two laboratory isolates from Austria (WT4 and GG1. The median genome-wide nucleotide diversity was 0.008 substitutions/site, but the range was large (0–0.14, illustrating the among-locus heterogeneity in the species. Conclusions: These loci provide a valuable resource for finely resolved, genome-wide population genetic and species-level phylogenetic analyses of C. purpureus and its relatives.

  13. Plastid and mitochondrion genomic sequences from Arctic Chlorella sp. ArM0029B

    Science.gov (United States)

    2014-01-01

    Background Chorella is the representative taxon of Chlorellales in Trebouxiophyceae, and its chloroplast (cp) genomic information has been thought to depend only on studies concerning Chlorella vulgaris and GenBank information of C. variablis. Mitochondrial (mt) genomic information regarding Chlorella is currently unavailable. To elucidate the evolution of organelle genomes and genetic information of Chlorella, we have sequenced and characterized the cp and mt genomes of Arctic Chlorella sp. ArM0029B. Results The 119,989-bp cp genome lacking inverted repeats and 65,049-bp mt genome were sequenced. The ArM0029B cp genome contains 114 conserved genes, including 32 tRNA genes, 3 rRNA genes, and 79 genes encoding proteins. Chlorella cp genomes are highly rearranged except for a Chlorella-specific six-gene cluster, and the ArM0029B plastid resembles that of Chlorella variabilis except for a 15-kb gene cluster inversion. In the mt genome, 62 conserved genes, including 27 tRNA genes, 3 rRNA genes, and 32 genes encoding proteins were determined. The mt genome of ArM0029B is similar to that of the non-photosynthetic species Prototheca and Heicosporidium. The ArM0029B mt genome contains a group I intron, with an ORF containing two LAGLIDADG motifs, in cox1. The intronic ORF is shared by C. vulgaris and Prototheca. The phylogeny of the plastid genome reveals that ArM0029B showed a close relationship of Chlorella to Parachlorella and Oocystis within Chlorellales. The distribution of the cox1 intron at 721 support membership in the order Chlorellales. Mitochondrial phylogenomic analyses, however, indicated that ArM0029B shows a greater affinity to MX-AZ01 and Coccomyxa than to the Helicosporidium-Prototheca clade, although the detailed phylogenetic relationships among the three taxa remain to be resolved. Conclusions The plastid genome of ArM0029B is similar to that of C. variabilis. The mt sequence of ArM0029B is the first genome to be reported for Chlorella. Chloroplast

  14. The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

    Science.gov (United States)

    Gurusamy, Raman; Lee, Do-Hyung; Park, SeonJoo

    2016-05-01

    The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns.

  15. Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing

    Czech Academy of Sciences Publication Activity Database

    Wang, H.; Dai, K.; Xiao, J.; Yuan, C.; Zhao, R. L.; Doležel, Jaroslav; Wu, Y.; Cao, A.; Chen, P.; Zhang, S.; Wang, X.

    2017-01-01

    Roč. 18, FEB 15 (2017), č. článku 167. ISSN 1471-2164 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : triticum-aestivum l. * conferring resistance * wild relatives * mosaic-virus * plug markers * bread wheat * genome * gene * rye * improvement * Triticum aestivum * Haynaldia villosa * Molecular marker * Intron polymorphism Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 3.729, year: 2016

  16. Evidence against the energetic cost hypothesis for the short introns in highly expressed genes

    Directory of Open Access Journals (Sweden)

    Niu Deng-Ke

    2008-05-01

    Full Text Available Abstract Background In animals, the moss Physcomitrella patens and the pollen of Arabidopsis thaliana, highly expressed genes have shorter introns than weakly expressed genes. A popular explanation for this is selection for transcription efficiency, which includes two sub-hypotheses: to minimize the energetic cost or to minimize the time cost. Results In an individual human, different organs may differ up to hundreds of times in cell number (for example, a liver versus a hypothalamus. Considered at the individual level, a gene specifically expressed in a large organ is actually transcribed tens or hundreds of times more than a gene with a similar expression level (a measure of mRNA abundance per cell specifically expressed in a small organ. According to the energetic cost hypothesis, the former should have shorter introns than the latter. However, in humans and mice we have not found significant differences in intron length between large-tissue/organ-specific genes and small-tissue/organ-specific genes with similar expression levels. Qualitative estimation shows that the deleterious effect (that is, the energetic burden of long introns in highly expressed genes is too negligible to be efficiently selected against in mammals. Conclusion The short introns in highly expressed genes should not be attributed to energy constraint. We evaluated evidence for the time cost hypothesis and other alternatives.

  17. A distant cis acting intronic element induces site-selective RNA editing

    DEFF Research Database (Denmark)

    Daniel, Chammiran; Venø, Morten Trillingsgaard; Ekdahl, Ylva

    2012-01-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated...... shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce...... the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been...

  18. Nucleotide sequence of the triosephosphate isomerase gene from Macaca mulatta

    Energy Technology Data Exchange (ETDEWEB)

    Old, S.E.; Mohrenweiser, H.W. (Univ. of Michigan, Ann Arbor (USA))

    1988-09-26

    The triosephosphate isomerase gene from a rhesus monkey, Macaca mulatta, charon 34 library was sequenced. The human and chimpanzee enzymes differ from the rhesus enzyme at ASN 20 and GLU 198. The nucleotide sequence identity between rhesus and human is 97% in the coding region and >94% in the flanking regions. Comparison of the rhesus and chimp genes, including the intron and flanking sequences, does not suggest a mechanism for generating the two TPI peptides of proliferating cells from hominoids and a single peptide from the rhesus gene.

  19. Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

    Science.gov (United States)

    Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

    2012-03-01

    Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

  20. A nucleotide substitution at the 5′ splice site of intron 1 of rice HEADING DATE 1 (HD1 gene homolog in foxtail millet, broadly found in landraces from Europe and Asia

    Directory of Open Access Journals (Sweden)

    Kenji Fukunaga

    2015-12-01

    Full Text Available We investigated genetic variation of a rice HEADING DATE 1(HD1 homolog in foxtail millet. First, we searched for a rice HD1 homolog in a foxtail millet genome sequence and designed primers to amplify the entire coding sequence of the gene. We compared full HD1 gene sequences of 11 accessions (including Yugu 1, a Chinese cultivar used for genome sequencing from various regions in Europe and Asia, found a nucleotide substitution at a putative splice site of intron 1, and designated the accessions with the nucleotide substitution as carrying a splicing variant. We verified by RT-PCR that this single nucleotide substitution causes aberrant splicing of intron 1. We investigated the geographical distribution of the splicing variant in 480 accessions of foxtail millet from various regions of Europe and Asia and part of Africa by dCAPS and found that the splicing variant is broadly distributed in Europe and Asia. Differences of heading times between accessions with wild type allele of the HD1 gene and those with the splicing variant allele were unclear. We also investigated variation in 13 accessions of ssp. viridis, the wild ancestor, and the results suggested that the wild type is predominant in the wild ancestor.

  1. The strength of intron donor splice sites in human genes displays a bell-shaped pattern

    DEFF Research Database (Denmark)

    Wang, Kai; Wernersson, Rasmus; Brunak, Søren

    2011-01-01

    introns. Interestingly, when analysing the intron containing gene pool from mouse consisting of >15 000 genes, we found the convex pattern to be conserved despite >75 million years of evolutionary divergence between the two organisms. We also analysed an interesting, novel class of chimeric genes which...

  2. In Silico Genome Comparison and Distribution Analysis of Simple Sequences Repeats in Cassava

    Directory of Open Access Journals (Sweden)

    Andrea Vásquez

    2014-01-01

    Full Text Available We conducted a SSRs density analysis in different cassava genomic regions. The information obtained was useful to establish comparisons between cassava’s SSRs genomic distribution and those of poplar, flax, and Jatropha. In general, cassava has a low SSR density (~50 SSRs/Mbp and has a high proportion of pentanucleotides, (24,2 SSRs/Mbp. It was found that coding sequences have 15,5 SSRs/Mbp, introns have 82,3 SSRs/Mbp, 5′ UTRs have 196,1 SSRs/Mbp, and 3′ UTRs have 50,5 SSRs/Mbp. Through motif analysis of cassava’s genome SSRs, the most abundant motif was AT/AT while in intron sequences and UTRs regions it was AG/CT. In addition, in coding sequences the motif AAG/CTT was also found to occur most frequently; in fact, it is the third most used codon in cassava. Sequences containing SSRs were classified according to their functional annotation of Gene Ontology categories. The identified SSRs here may be a valuable addition for genetic mapping and future studies in phylogenetic analyses and genomic evolution.

  3. An intronic microRNA silences genes that are functionally antagonistic to its host gene.

    Science.gov (United States)

    Barik, Sailen

    2008-09-01

    MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

  4. Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

    Science.gov (United States)

    Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

    1991-02-15

    The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

  5. A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

    Science.gov (United States)

    Sano, R; Kuboya, E; Nakajima, T; Takahashi, Y; Takahashi, K; Kubo, R; Kominato, Y; Takeshita, H; Yamao, H; Kishida, T; Isa, K; Ogasawara, K; Uchikawa, M

    2015-04-01

    We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently. © 2014 International Society of Blood Transfusion.

  6. A DEL phenotype attributed to RHD Exon 9 sequence deletion: slipped-strand mispairing and blood group polymorphisms.

    Science.gov (United States)

    Lopez, Genghis H; Turner, Robyn M; McGowan, Eunike C; Schoeman, Elizna M; Scott, Stacy A; O'Brien, Helen; Millard, Glenda M; Roulis, Eileen V; Allen, Amanda J; Liew, Yew-Wah; Flower, Robert L; Hyland, Catherine A

    2018-03-01

    The RhD blood group antigen is extremely polymorphic and the DEL phenotype represents one such class of polymorphisms. The DEL phenotype prevalent in East Asian populations arises from a synonymous substitution defined as RHD*1227A. However, initially, based on genomic and cDNA studies, the genetic basis for a DEL phenotype in Taiwan was attributed to a deletion of RHD Exon 9 that was never verified at the genomic level by any other independent group. Here we investigate the genetic basis for a Caucasian donor with a DEL partial D phenotype and compare the genomic findings to those initial molecular studies. The 3'-region of the RHD gene was amplified by long-range polymerase chain reaction (PCR) for massively parallel sequencing. Primers were designed to encompass a deletion, flanking Exon 9, by standard PCR for Sanger sequencing. Targeted sequencing of exons and flanking introns was also performed. Genomic DNA exhibited a 1012-bp deletion spanning from Intron 8, across Exon 9 into Intron 9. The deletion breakpoints occurred between two 25-bp repeat motifs flanking Exon 9 such that one repeat sequence remained. Deletion mutations bordered by repeat sequences are a hallmark of slipped-strand mispairing (SSM) event. We propose this genetic mechanism generated the germline deletion in the Caucasian donor. Extensive studies show that the RHD*1227A is the most prevalent DEL allele in East Asian populations and may have confounded the initial molecular studies. Review of the literature revealed that the SSM model explains some of the extreme polymorphisms observed in the clinically significant RhD blood group antigen. © 2017 AABB.

  7. Genome-wide development and deployment of informative intron-spanning and intron-length polymorphism markers for genomics-assisted breeding applications in chickpea.

    Science.gov (United States)

    Srivastava, Rishi; Bajaj, Deepak; Sayal, Yogesh K; Meher, Prabina K; Upadhyaya, Hari D; Kumar, Rajendra; Tripathi, Shailesh; Bharadwaj, Chellapilla; Rao, Atmakuri R; Parida, Swarup K

    2016-11-01

    The discovery and large-scale genotyping of informative gene-based markers is essential for rapid delineation of genes/QTLs governing stress tolerance and yield component traits in order to drive genetic enhancement in chickpea. A genome-wide 119169 and 110491 ISM (intron-spanning markers) from 23129 desi and 20386 kabuli protein-coding genes and 7454 in silico InDel (insertion-deletion) (1-45-bp)-based ILP (intron-length polymorphism) markers from 3283 genes were developed that were structurally and functionally annotated on eight chromosomes and unanchored scaffolds of chickpea. A much higher amplification efficiency (83%) and intra-specific polymorphic potential (86%) detected by these markers than that of other sequence-based genetic markers among desi and kabuli chickpea accessions was apparent even by a cost-effective agarose gel-based assay. The genome-wide physically mapped 1718 ILP markers assayed a wider level of functional genetic diversity (19-81%) and well-defined phylogenetics among domesticated chickpea accessions. The gene-derived 1424 ILP markers were anchored on a high-density (inter-marker distance: 0.65cM) desi intra-specific genetic linkage map/functional transcript map (ICC 4958×ICC 2263) of chickpea. This reference genetic map identified six major genomic regions harbouring six robust QTLs mapped on five chromosomes, which explained 11-23% seed weight trait variation (7.6-10.5 LOD) in chickpea. The integration of high-resolution QTL mapping with differential expression profiling detected six including one potential serine carboxypeptidase gene with ILP markers (linked tightly to the major seed weight QTLs) exhibiting seed-specific expression as well as pronounced up-regulation especially in seeds of high (ICC 4958) as compared to low (ICC 2263) seed weight mapping parental accessions. The marker information generated in the present study was made publicly accessible through a user-friendly web-resource, "Chickpea ISM-ILP Marker Database

  8. Surrounding Moving Obstacle Detection for Autonomous Driving Using Stereo Vision

    Directory of Open Access Journals (Sweden)

    Hao Sun

    2013-06-01

    Full Text Available Detection and tracking surrounding moving obstacles such as vehicles and pedestrians are crucial for the safety of mobile robotics and autonomous vehicles. This is especially the case in urban driving scenarios. This paper presents a novel framework for surrounding moving obstacles detection using binocular stereo vision. The contributions of our work are threefold. Firstly, a multiview feature matching scheme is presented for simultaneous stereo correspondence and motion correspondence searching. Secondly, the multiview geometry constraint derived from the relative camera positions in pairs of consecutive stereo views is exploited for surrounding moving obstacles detection. Thirdly, an adaptive particle filter is proposed for tracking of multiple moving obstacles in surrounding areas. Experimental results from real-world driving sequences demonstrate the effectiveness and robustness of the proposed framework.

  9. Comparison of next generation sequencing technologies for transcriptome characterization

    Directory of Open Access Journals (Sweden)

    Soltis Douglas E

    2009-08-01

    Full Text Available Abstract Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19. We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica and the magnoliid avocado (Persea americana using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB, 119,518 (88.7% mapped exactly to known exons, while 1,117 (0.8% mapped to introns, 11,524 (8.6% spanned annotated intron/exon boundaries, and 3,066 (2.3% extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance

  10. Cell type-specific termination of transcription by transposable element sequences.

    Science.gov (United States)

    Conley, Andrew B; Jordan, I King

    2012-09-30

    Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3' UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The extent of transcription

  11. Asthma and COPD in cystic fibrosis intron-8 5T carriers. A population-based study

    DEFF Research Database (Denmark)

    Dahl, Morten; Tybjaerg-Hansen, Anne; Lange, Peter

    2005-01-01

    Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD).......Carriers of cystic fibrosis intron-8 5T alleles with high exon-9 skipping could have increased annual lung function decline and increased risk for asthma or chronic obstructive pulmonary disease (COPD)....

  12. Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations.

    Science.gov (United States)

    Etang, Josiane; Vicente, Jose L; Nwane, Philippe; Chouaibou, Mouhamadou; Morlais, Isabelle; Do Rosario, Virgilio E; Simard, Frederic; Awono-Ambene, Parfait; Toto, Jean Claude; Pinto, Joao

    2009-07-01

    Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance (kdr) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr-intron-1 haplotypes in S-form and MS3-1014F kdr-intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West-Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.

  13. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to PromoteInclusion of Protein 4.1R Alternative Exon 16

    Energy Technology Data Exchange (ETDEWEB)

    Ponthier, Julie L.; Schluepen, Christina; Chen, Weiguo; Lersch,Robert A.; Gee, Sherry L.; Hou, Victor C.; Lo, Annie J.; Short, Sarah A.; Chasis, Joel A.; Winkelmann, John C.; Conboy, John G.

    2006-03-01

    Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.

  14. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid conjugates targeting intron-exon junctions

    Directory of Open Access Journals (Sweden)

    Nielsen Peter E

    2010-06-01

    Full Text Available Abstract Background Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. Methods We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512 targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT. Results We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406 targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512 targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. Conclusion We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.

  15. Virtual Ribosome - a comprehensive DNA translation tool with support for integration of sequence feature annotation

    DEFF Research Database (Denmark)

    Wernersson, Rasmus

    2006-01-01

    of alternative start codons. ( ii) Integration of sequences feature annotation - in particular, native support for working with files containing intron/ exon structure annotation. The software is available for both download and online use at http://www.cbs.dtu.dk/services/VirtualRibosome/....

  16. An intronic LINE-1 insertion in MERTK is strongly associated with retinopathy in Swedish Vallhund dogs.

    Directory of Open Access Journals (Sweden)

    Richard Everson

    Full Text Available The domestic dog segregates a significant number of inherited progressive retinal diseases, several of which mirror human retinal diseases and which are collectively termed progressive retinal atrophy (PRA. In 2014, a novel form of PRA was reported in the Swedish Vallhund breed, and the disease was mapped to canine chromosome 17. The causal mutation was not identified, but expression analyses of the retinas of affected Vallhunds demonstrated a 6-fold increased expression of the MERTK gene compared to unaffected dogs. Using 24 retinopathy cases and 97 controls with no clinical signs of retinopathy, we replicated the chromosome 17 association in Swedish Vallhunds from the UK and aimed to elucidate the causal variant underlying this association using whole genome sequencing (WGS of an affected dog. This revealed a 6-8 kb insertion in intron 1 of MERTK that was not present in WGS of 49 dogs of other breeds. Sequencing and BLASTN analysis of the inserted segment was consistent with the insertion comprising a full-length intact LINE-1 retroelement. Testing of the LINE-1 insertion for association with retinopathy in the UK set of 24 cases and 97 controls revealed a strong statistical association (P-value 6.0 x 10-11 that was subsequently replicated in the original Finnish study set (49 cases and 89 controls (P-value 4.3 x 10-19. In a pooled analysis of both studies (73 cases and 186 controls, the LINE-1 insertion was associated with a ~20-fold increased risk of retinopathy (odds ratio 23.41, 95% confidence intervals 10.99-49.86, P-value 1.3 x 10-27. Our study adds further support for regulatory disruption of MERTK in Swedish Vallhund retinopathy; however, further work is required to establish a functional overexpression model. Future work to characterise the mechanism by which this intronic mutation disrupts gene regulation will further improve the understanding of MERTK biology and its role in retinal function.

  17. Un gene con intrones en vez de exones / Envejecimiento Prematuro de la Piel

    Directory of Open Access Journals (Sweden)

    Tobías Mojica

    1996-04-01

    Full Text Available Un gene con intrones en vez de exones. La noción de que los genes son discontinuos (compuestos de exones e intrones en forma alterna y en cuya organización los exones representan regiones presentes, por medio del código genético en las proteínas, y los intrones nadie sabe todavía que representan produjo una cierta cantidad de desasosiego entre los genetistas mayores de edad, pero hoy día es ampliamente aceptada, con poco o ningún dolor, y se ha convertido en parte del cánon científico. / Envejecimiento Prematuro de la Piel. La exposición a largo plazo de la piel a la luz ultravioleta proveniente del sol resulta en daño al colágeno de la piel y a la elastina de la matriz extracelular; se cree que este daño es responsable de la apariencia típicamente arrugadita de la piel expuesta al sol por mucho tiempo (como en los vaqueros de los comerciales de la televisión.

  18. Structural organization of glycophorin A and B genes: Glycophorin B gene evolved by homologous recombination at Alu repeat sequences

    International Nuclear Information System (INIS)

    Kudo, Shinichi; Fukuda, Minoru

    1989-01-01

    Glycophorins A (GPA) and B (GPB) are two major sialoglycoproteins of the human erythrocyte membrane. Here the authors present a comparison of the genomic structures of GPA and GPB developed by analyzing DNA clones isolated from a K562 genomic library. Nucleotide sequences of exon-intron junctions and 5' and 3' flanking sequences revealed that the GPA and GPB genes consist of 7 and 5 exons, respectively, and both genes have >95% identical sequence from the 5' flanking region to the region ∼ 1 kilobase downstream from the exon encoding the transmembrane regions. In this homologous part of the genes, GPB lacks one exon due to a point mutation at the 5' splicing site of the third intron, which inactivates the 5' cleavage event of splicing and leads to ligation of the second to the fourth exon. Following these very homologous sequences, the genomic sequences for GPA and GPB diverge significantly and no homology can be detected in their 3' end sequences. The analysis of the Alu sequences and their flanking direct repeat sequences suggest that an ancestral genomic structure has been maintained in the GPA gene, whereas the GPB gene has arisen from the acquisition of 3' sequences different from those of the GPA gene by homologous recombination at the Alu repeats during or after gene duplication

  19. Learning to live together: mutualism between self-splicing introns and their hosts

    Directory of Open Access Journals (Sweden)

    Chalamcharla Venkata R

    2011-04-01

    Full Text Available Abstract Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress.

  20. Genome-wide identification, phylogenetic classification, and exon-intron structure characterisation of the tubulin and actin genes in flax (Linum usitatissimum).

    Science.gov (United States)

    Pydiura, Nikolay; Pirko, Yaroslav; Galinousky, Dmitry; Postovoitova, Anastasiia; Yemets, Alla; Kilchevsky, Aleksandr; Blume, Yaroslav

    2018-06-08

    Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, β-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 β-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analysed the phylogenetic relationships between flax and A. thaliana tubulin and actin genes. Six α-tubulin genes are represented by 3 paralogous pairs, among 13 β-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent 7 paralogous pairs - 7 actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the β-tubulin genes and intron number variation among the α-tubulin gene: 3 or 4 introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be 2 or 3. These data will be useful to support further studies on the specificity, functioning, regulation and evolution of the flax cytoskeleton proteins. This article is protected by copyright. All rights reserved.

  1. A Conserved Splicing Silencer Dynamically Regulates O-GlcNAc Transferase Intron Retention and O-GlcNAc Homeostasis

    Directory of Open Access Journals (Sweden)

    Sung-Kyun Park

    2017-08-01

    Full Text Available Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT and O-GlcNAcase (OGA add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.

  2. A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology.

    Science.gov (United States)

    Ustek, Duran; Sirma, Sema; Gumus, Ergun; Arikan, Muzaffer; Cakiris, Aris; Abaci, Neslihan; Mathew, Jaicy; Emrence, Zeliha; Azakli, Hulya; Cosan, Fulya; Cakar, Atilla; Parlak, Mahmut; Kursun, Olcay

    2012-10-01

    One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Intron Retention and TE Exonization Events in ZRANB2

    Directory of Open Access Journals (Sweden)

    Sang-Je Park

    2012-01-01

    Full Text Available The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2, contains arginine/serine-rich (RS domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3 between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3. Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species. Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3. RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys and were expressed via intron retention (IR. Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

  4. Nuclear introns outperform mitochondrial DNA in inter-specific phylogenetic reconstruction: Lessons from horseshoe bats (Rhinolophidae: Chiroptera).

    Science.gov (United States)

    Dool, Serena E; Puechmaille, Sebastien J; Foley, Nicole M; Allegrini, Benjamin; Bastian, Anna; Mutumi, Gregory L; Maluleke, Tinyiko G; Odendaal, Lizelle J; Teeling, Emma C; Jacobs, David S

    2016-04-01

    Despite many studies illustrating the perils of utilising mitochondrial DNA in phylogenetic studies, it remains one of the most widely used genetic markers for this purpose. Over the last decade, nuclear introns have been proposed as alternative markers for phylogenetic reconstruction. However, the resolution capabilities of mtDNA and nuclear introns have rarely been quantified and compared. In the current study we generated a novel ∼5kb dataset comprising six nuclear introns and a mtDNA fragment. We assessed the relative resolution capabilities of the six intronic fragments with respect to each other, when used in various combinations together, and when compared to the traditionally used mtDNA. We focused on a major clade in the horseshoe bat family (Afro-Palaearctic clade; Rhinolophidae) as our case study. This old, widely distributed and speciose group contains a high level of conserved morphology. This morphological stasis renders the reconstruction of the phylogeny of this group with traditional morphological characters complex. We sampled multiple individuals per species to represent their geographic distributions as best as possible (122 individuals, 24 species, 68 localities). We reconstructed the species phylogeny using several complementary methods (partitioned Maximum Likelihood and Bayesian and Bayesian multispecies-coalescent) and made inferences based on consensus across these methods. We computed pairwise comparisons based on Robinson-Foulds tree distance metric between all Bayesian topologies generated (27,000) for every gene(s) and visualised the tree space using multidimensional scaling (MDS) plots. Using our supported species phylogeny we estimated the ancestral state of key traits of interest within this group, e.g. echolocation peak frequency which has been implicated in speciation. Our results revealed many potential cryptic species within this group, even in taxa where this was not suspected a priori and also found evidence for mt

  5. Mutation analysis in Duchenne and Becker muscular dystrophy patients from Bulgaria shows a peculiar distribution of breakpoints by intron

    Energy Technology Data Exchange (ETDEWEB)

    Todorova, A.; Bronzova, J.; Kremensky, I. [Univ. Hospital of Obstetrics and Gynecology, Sofia (Bulgaria)] [and others

    1996-10-02

    For the first time in Bulgaria, a deletion/duplication screening was performed on a group of 84 unrelated Duchenne/Becker muscular dystrophy patients, and the breakpoint distribution in the dystrophin gene was analyzed. Intragenic deletions were detected in 67.8% of patients, and intragenic duplications in 2.4%. A peculiar distribution of deletion breakpoints was found. Only 13.2% of the deletion breakpoints fell in the {open_quotes}classical{close_quotes} hot spot in intron 44, whereas the majority (> 54%) were located within the segment encompassing introns 45-51, which includes intron 50, the richest in breakpoints (16%) in the Bulgarian sample. Comparison with data from Greece and Turkey points at the probable existence of a deletion hot spot within intron 50, which might be a characteristic of populations of the Balkan region. 17 refs., 2 figs.

  6. Cell type-specific termination of transcription by transposable element sequences

    Directory of Open Access Journals (Sweden)

    Conley Andrew B

    2012-09-01

    Full Text Available Abstract Background Transposable elements (TEs encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Results Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3′ UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. Conclusions TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are

  7. Towards Semantic Understanding of Surrounding Vehicular Maneuvers

    DEFF Research Database (Denmark)

    Kristoffersen, Miklas Strøm; Dueholm, Jacob Velling; Satzoda, Ravi K.

    2016-01-01

    This paper proposes the use of multiple low-cost visual sensors to obtain a surround view of the ego-vehicle for semantic understanding. A multi-perspective view will assist the analysis of naturalistic driving studies (NDS), by automating the task of data reduction of the observed sequences...... into events. A user-centric vision-based framework is presented using a vehicle detector and tracker in each separate perspective. Multi-perspective trajectories are estimated and analyzed to extract 14 different events, including potential dangerous behaviors such as overtakes and cut-ins. The system...... is tested on ten sequences of real-world data collected on U. S. highways. The results show the potential use of multiple low-cost visual sensors for semantic understanding around the ego-vehicle....

  8. Porcine calbindin-D9k gene: expression in endometrium, myometrium, and placenta in the absence of a functional estrogen response element in intron A.

    Science.gov (United States)

    Krisinger, J; Jeung, E B; Simmen, R C; Leung, P C

    1995-01-01

    The expression of Calbindin-D9k (CaBP-9k) in the pig uterus and placenta was measured by Northern blot analysis and reverse transcription polymerase chain reaction (PCR), respectively. Progesterone (P4) administration to ovariectomized pigs decreased CaBP-9k mRNA levels. Expression of endometrial CaBP-9k mRNA was high on pregnancy Days 10-12 and below the detection limit on Days 15 and 18. On Day 60, expression could be detected at low levels. In myometrium and placenta, CaBP-9k mRNA expression was not detectable by Northern analysis using total RNA. Reverse-transcribed RNA from both tissues demonstrated the presence of CaBP-9k transcripts by means of PCR. The partial CaBP-9k gene was amplified by PCR and cloned to determine the sequence of intron A. In contrast to the rat CaBP-9k gene, the pig gene does not contain a functional estrogen response element (ERE) within this region. A similar ERE-like sequence located at the identical location was examined by gel retardation analysis and failed to bind the estradiol receptor. A similar disruption of this ERE-like sequence has been described in the human CaBP-9k gene, which is not expressed at any level in placenta, myometrium, or endometrium. It is concluded that the pig CaBP-9k gene is regulated in these reproductive tissues in a manner distinct from that in rat and human tissues. The regulation is probably due to a regulatory region outside of intron A, which in the rat gene contains the key cis element for uterine expression of the CaBP-9k gene.

  9. Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

    Directory of Open Access Journals (Sweden)

    D'Souza Lisa M

    2002-12-01

    Full Text Available Abstract Background Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. Results The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2. This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct. Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459 was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. Conclusions The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2 and DII(iia,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

  10. ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

    Science.gov (United States)

    Jonsson, Frida; Westin, Ida Maria; Österman, Lennart; Sandgren, Ola; Burstedt, Marie; Holmberg, Monica; Golovleva, Irina

    2018-02-20

    Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP-binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing. The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE-19, using a minigene system containing variants c.4773+3A>G and c.5461-10T>C. We showed that both ABCA4 variants, c.4773+3A>G and c.5461-10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type. Two intronic variants c.4773+3A>G and c.5461-10T>C, both predicted to affect splicing, are indeed disease-causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable. © 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  11. Naturally occuring nucleosome positioning signals in human exons and introns

    DEFF Research Database (Denmark)

    Baldi, Pierre; Brunak, Søren; Chauvin, Yves

    1996-01-01

    We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity of rou...

  12. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  13. Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

    Science.gov (United States)

    Chandrasekharappa, Settara C; Lach, Francis P; Kimble, Danielle C; Kamat, Aparna; Teer, Jamie K; Donovan, Frank X; Flynn, Elizabeth; Sen, Shurjo K; Thongthip, Supawat; Sanborn, Erica; Smogorzewska, Agata; Auerbach, Arleen D; Ostrander, Elaine A

    2013-05-30

    Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

  14. A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

    Science.gov (United States)

    Voorbij, Annemarie M. W. Y.; van Steenbeek, Frank G.; Vos-Loohuis, Manon; Martens, Ellen E. C. P.; Hanson-Nilsson, Jeanette M.; van Oost, Bernard A.; Kooistra, Hans S.; Leegwater, Peter A.

    2011-01-01

    Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism. PMID:22132174

  15. Polymorphism of the aryl-hydrocarbon receptor gene in intron 10 of human cancers

    Directory of Open Access Journals (Sweden)

    M. Rocas

    2011-11-01

    Full Text Available Polychlorinated dibenzo-p-dioxins (PCDDs and related halogenated aromatic hydrocarbons (e.g., PCDFs, often called "dioxins", are ubiquitously present environmental contaminants. Some of them, notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, are among the most toxic synthetic compounds known. The biological effects of dioxins are mediated via the aryl hydrocarbon receptor (AhR. Mutations in the AhR transactivation domain are linked to sensitivity to the acute lethality of TCDD. We present here a study of AhR gene polymorphism in normal and cancer human tissues affecting pre-mRNA splicing in the AhR gene-coding transactivation domain region (exon 10, intron 10, exon 11 region, previously shown to be associated with AhR dysfunction. We tested 126 pairs of normal and cancer tissue samples from liver, lung, stomach, kidney, mucous, breast, and pancreas of 49 males and 77 females (45-70 years of age. We used in vitro splicing assay, RT-PCR and sequencing methods. Our results showed that in an in vitro system it is possible to reconstitute cellular pre-mRNA splicing events. Tested cancer tissues did not contain mutations in the AhR transactivation domain region when the DNA sequences were compared with those from normal tissues. There were also no differences in AhR mRNA splice variants between normal and malignant breast tissues and no polymorphisms in the studied regions or cDNA.

  16. Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis.

    Science.gov (United States)

    Fu, Jianmin; Liu, Huimin; Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

    2016-01-01

    Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. 'Jinzaoshi', support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

  17. Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis.

    Directory of Open Access Journals (Sweden)

    Jianmin Fu

    Full Text Available Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp in the cp genome of D. 'Jinzaoshi', support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

  18. First intron of nestin gene regulates its expression during C2C12 myoblast ifferentiation

    Institute of Scientific and Technical Information of China (English)

    Hua Zhong; Zhigang Jin; Yongfeng Chen; Ting Zhang; Wei Bian; Xing Cui; Naihe Jing

    2008-01-01

    Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural-specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells.We localized the core enhancer activity to the 291-661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells.

  19. Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

    DEFF Research Database (Denmark)

    Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan

    2004-01-01

    an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially...... in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.......The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate...

  20. Horse domestication and conservation genetics of Przewalski's horse inferred from sex chromosomal and autosomal sequences.

    Science.gov (United States)

    Lau, Allison N; Peng, Lei; Goto, Hiroki; Chemnick, Leona; Ryder, Oliver A; Makova, Kateryna D

    2009-01-01

    Despite their ability to interbreed and produce fertile offspring, there is continued disagreement about the genetic relationship of the domestic horse (Equus caballus) to its endangered wild relative, Przewalski's horse (Equus przewalskii). Analyses have differed as to whether or not Przewalski's horse is placed phylogenetically as a separate sister group to domestic horses. Because Przewalski's horse and domestic horse are so closely related, genetic data can also be used to infer domestication-specific differences between the two. To investigate the genetic relationship of Przewalski's horse to the domestic horse and to address whether evolution of the domestic horse is driven by males or females, five homologous introns (a total of approximately 3 kb) were sequenced on the X and Y chromosomes in two Przewalski's horses and three breeds of domestic horses: Arabian horse, Mongolian domestic horse, and Dartmoor pony. Five autosomal introns (a total of approximately 6 kb) were sequenced for these horses as well. The sequences of sex chromosomal and autosomal introns were used to determine nucleotide diversity and the forces driving evolution in these species. As a result, X chromosomal and autosomal data do not place Przewalski's horses in a separate clade within phylogenetic trees for horses, suggesting a close relationship between domestic and Przewalski's horses. It was also found that there was a lack of nucleotide diversity on the Y chromosome and higher nucleotide diversity than expected on the X chromosome in domestic horses as compared with the Y chromosome and autosomes. This supports the hypothesis that very few male horses along with numerous female horses founded the various domestic horse breeds. Patterns of nucleotide diversity among different types of chromosomes were distinct for Przewalski's in contrast to domestic horses, supporting unique evolutionary histories of the two species.

  1. Sequence analyses reveal that a TPR-DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR-DP domains and prokaryotic GerD proteins.

    Science.gov (United States)

    Hernández Torres, Jorge; Papandreou, Nikolaos; Chomilier, Jacques

    2009-05-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR-DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR-DP domains.

  2. A contracted DNA repeat in LHX3 intron 5 is associated with aberrant splicing and pituitary dwarfism in German shepherd dogs.

    Directory of Open Access Journals (Sweden)

    Annemarie M W Y Voorbij

    Full Text Available Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

  3. The association between Interleukin (IL)-4 gene intron 3 VNTR polymorphism and alopecia areata (AA) in Turkish population.

    Science.gov (United States)

    Kalkan, Göknur; Karakus, Nevin; Baş, Yalçın; Takçı, Zennure; Ozuğuz, Pınar; Ateş, Omer; Yigit, Serbulent

    2013-09-25

    Alopecia areata (AA) is hypothesized to be an organ-specific autoimmune disease of hair follicles mediated by T cells. As immunological and genetic factors have been implicated in the pathogenesis of AA, the purpose of the present study was to investigate possible associations between the functional Interleukin (IL)-4 gene intron 3 VNTR polymorphism and AA susceptibility and disease progression in Turkish population. The study group consisted of 116 unrelated patients with AA and 125 unrelated healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers. No association was observed between AA patients and controls according to genotype distribution (p=0.051). The allele distribution of IL-4 gene intron 3 VNTR polymorphism was statistically different between AA patients and control group (p=0.026). The frequency of P1 allele in patients was significantly higher than that in the control group. When the P2P2 genotype was compared with P1P2+P1P1 genotypes, a statistically significant difference was observed between patients and controls (p=0.036). Intron 3 VNTR polymorphism in the IL-4 gene was found to be associated with AA susceptibility in Turkish population. The results suggest that IL-4 VNTR polymorphism in the intron 3 region may be a risk factor for the development of AA among Turkish population. This is the first to report that intron 3 VNTR polymorphism in the IL-4 gene is associated with AA susceptibility. © 2013.

  4. Deep developmental transcriptome sequencing uncovers numerous new genes and enhances gene annotation in the sponge Amphimedon queenslandica.

    Science.gov (United States)

    Fernandez-Valverde, Selene L; Calcino, Andrew D; Degnan, Bernard M

    2015-05-15

    The demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence. Amphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3' and 5' untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3' UTRs. The inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.

  5. Genome-wide generation and use of informative intron-spanning and intron-length polymorphism markers for high-throughput genetic analysis in rice

    Science.gov (United States)

    Badoni, Saurabh; Das, Sweta; Sayal, Yogesh K.; Gopalakrishnan, S.; Singh, Ashok K.; Rao, Atmakuri R.; Agarwal, Pinky; Parida, Swarup K.; Tyagi, Akhilesh K.

    2016-01-01

    We developed genome-wide 84634 ISM (intron-spanning marker) and 16510 InDel-fragment length polymorphism-based ILP (intron-length polymorphism) markers from genes physically mapped on 12 rice chromosomes. These genic markers revealed much higher amplification-efficiency (80%) and polymorphic-potential (66%) among rice accessions even by a cost-effective agarose gel-based assay. A wider level of functional molecular diversity (17–79%) and well-defined precise admixed genetic structure was assayed by 3052 genome-wide markers in a structured population of indica, japonica, aromatic and wild rice. Six major grain weight QTLs (11.9–21.6% phenotypic variation explained) were mapped on five rice chromosomes of a high-density (inter-marker distance: 0.98 cM) genetic linkage map (IR 64 x Sonasal) anchored with 2785 known/candidate gene-derived ISM and ILP markers. The designing of multiple ISM and ILP markers (2 to 4 markers/gene) in an individual gene will broaden the user-preference to select suitable primer combination for efficient assaying of functional allelic variation/diversity and realistic estimation of differential gene expression profiles among rice accessions. The genomic information generated in our study is made publicly accessible through a user-friendly web-resource, “Oryza ISM-ILP marker” database. The known/candidate gene-derived ISM and ILP markers can be enormously deployed to identify functionally relevant trait-associated molecular tags by optimal-resource expenses, leading towards genomics-assisted crop improvement in rice. PMID:27032371

  6. RANDNA: a random DNA sequence generator.

    Science.gov (United States)

    Piva, Francesco; Principato, Giovanni

    2006-01-01

    Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.

  7. The 253-kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3.

    Science.gov (United States)

    Qian, Yaping; Johnson, Judith A; Connor, Jessica A; Valencia, C Alexander; Barasa, Nathaniel; Schubert, Jeffery; Husami, Ammar; Kissell, Diane; Zhang, Ge; Weirauch, Matthew T; Filipovich, Alexandra H; Zhang, Kejian

    2014-06-01

    The mutations in UNC13D are responsible for familial hemophagocytic lymphohistiocytosis (FHL) type 3. A 253-kb inversion and two deep intronic mutations, c.118-308C > T and c.118-307G > A, in UNC13D were recently reported in European and Asian FHL3 patients. We sought to determine the prevalence of these three non-coding mutations in North American FHL patients and evaluate the significance of examining these new mutations in genetic testing. We performed DNA sequencing of UNC13D and targeted analysis of these three mutations in 1,709 North American patients with a suspected clinical diagnosis of hemophagocytic lymphohistiocytosis (HLH). The 253-kb inversion, intronic mutations c.118-308C > T and c.118-307G > A were found in 11, 15, and 4 patients, respectively, in which the genetic basis (bi-allelic mutations) explained 25 additional patients. Taken together with previously diagnosed FHL3 patients in our HLH patient registry, these three non-coding mutations were found in 31.6% (25/79) of the FHL3 patients. The 253-kb inversion, c.118-308C > T and c.118-307G > A accounted for 7.0%, 8.9%, and 1.3% of mutant alleles, respectively. Significantly, eight novel mutations in UNC13D are being reported in this study. To further evaluate the expression level of the newly reported intronic mutation c.118-307G > A, reverse transcription PCR and Western blot analysis revealed a significant reduction of both RNA and protein levels suggesting that the c.118-307G > A mutation affects transcription. These specified non-coding mutations were found in a significant number of North American patients and inclusion of them in mutation analysis will improve the molecular diagnosis of FHL3. © 2014 Wiley Periodicals, Inc.

  8. Evolution of red algal plastid genomes: ancient architectures, introns, horizontal gene transfer, and taxonomic utility of plastid markers.

    Directory of Open Access Journals (Sweden)

    Jan Janouškovec

    Full Text Available Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase - a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.

  9. Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

    Directory of Open Access Journals (Sweden)

    Neng Chen

    2014-07-01

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR gene mutation analysis has been implemented for Cystic Fibrosis (CF carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD. Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM curve analysis, allele-specific PCR (AS-PCR and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

  10. Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

    Science.gov (United States)

    Papandreou, Nikolaos; Chomilier, Jacques

    2008-01-01

    The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains. Electronic supplementary material The online version of this article (doi:10.1007/s12192-008-0083-8) contains supplementary material, which is available to authorized users. PMID:18987995

  11. The complete chloroplast genome sequence of Dendrobium officinale.

    Science.gov (United States)

    Yang, Pei; Zhou, Hong; Qian, Jun; Xu, Haibin; Shao, Qingsong; Li, Yonghua; Yao, Hui

    2016-01-01

    The complete chloroplast sequence of Dendrobium officinale, an endangered and economically important traditional Chinese medicine, was reported and characterized. The genome size is 152,018 bp, with 37.5% GC content. A pair of inverted repeats (IRs) of 26,284 bp are separated by a large single-copy region (LSC, 84,944 bp) and a small single-copy region (SSC, 14,506 bp). The complete cp DNA contains 83 protein-coding genes, 39 tRNA genes and 8 rRNA genes. Fourteen genes contained one or two introns.

  12. Developing Exon-Primed Intron-Crossing (EPIC) markers for population genetic studies in three Aedes disease vectors.

    Science.gov (United States)

    White, Vanessa Linley; Endersby, Nancy Margaret; Chan, Janice; Hoffmann, Ary Anthony; Weeks, Andrew Raymond

    2015-03-01

    Aedes aegypti, Aedes notoscriptus, and Aedes albopictus are important vectors of many arboviruses implicated in human disease such as dengue fever. Genetic markers applied across vector species can provide important information on population structure, gene flow, insecticide resistance, and taxonomy, however, robust microsatellite markers have proven difficult to develop in these species and mosquitoes generally. Here we consider the utility and transferability of 15 Ribosome protein (Rp) Exon-Primed Intron-Crossing (EPIC) markers for population genetic studies in these 3 Aedes species. Rp EPIC markers designed for Ae. aegypti also successfully amplified populations of the sister species, Ae. albopictus, as well as the distantly related species, Ae. notoscriptus. High SNP and good indel diversity in sequenced alleles plus support for amplification of the same regions across populations and species were additional benefits of these markers. These findings point to the general value of EPIC markers in mosquito population studies. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  13. A Bifunctional Intronic Element Regulates the Expression of the Arginine/Lysine Transporter Cat-1 via Mechanisms Involving the Purine-rich Element Binding Protein A (Purα)*

    Science.gov (United States)

    Huang, Charlie C.; Chiribau, Calin-Bogdan; Majumder, Mithu; Chiang, Cheng-Ming; Wek, Ronald C.; Kelm, Robert J.; Khalili, Kamel; Snider, Martin D.; Hatzoglou, Maria

    2009-01-01

    Expression of the arginine/lysine transporter Cat-1 is highly induced in proliferating and stressed cells via mechanisms that include transcriptional activation. A bifunctional INE (intronic element) within the first intron of the Cat-1 gene was identified and characterized in this study. The INE had high sequence homology to an amino acid response element and was shown to act as a transcriptional enhancer in unstressed cells by binding the transcription factor, purine-rich element binding protein A (Purα). During endoplasmic reticulum stress, binding of Purα to the INE decreased; the element acted as a positive regulator in early stress by binding of the transcription factor ATF4 and as a negative regulator in prolonged stress by binding the stress-induced C/EBP family member, CHOP. We conclude that transcriptional control of the Cat-1 gene is tightly controlled by multiple cis-DNA elements, contributing to regulation of cationic amino acid transport for cell growth and proliferation. In addition, we propose that genes may use stress-response elements such as the INE to support basal expression in the absence of stress. PMID:19720825

  14. Reconstruction of structural evolution in the trnL intron P6b loop of symbiotic Nostoc (Cyanobacteria).

    Science.gov (United States)

    Olsson, Sanna; Kaasalainen, Ulla; Rikkinen, Jouko

    2012-02-01

    In this study we reconstruct the structural evolution of the hyper-variable P6b region of the group I trnLeu intron in a monophyletic group of lichen-symbiotic Nostoc strains and establish it as a useful marker in the phylogenetic analysis of these organisms. The studied cyanobacteria occur as photosynthetic and/or nitrogen-fixing symbionts in lichen species of the diverse Nephroma guild. Phylogenetic analyses and secondary structure reconstructions are used to improve the understanding of the replication mechanisms in the P6b stem-loop and to explain the observed distribution patterns of indels. The variants of the P6b region in the Nostoc clade studied consist of different combinations of five sequence modules. The distribution of indels together with the ancestral character reconstruction performed enables the interpretation of the evolution of each sequence module. Our results indicate that the indel events are usually associated with single nucleotide changes in the P6b region and have occurred several times independently. In spite of their homoplasy, they provide phylogenetic information for closely related taxa. Thus we recognize that features of the P6b region can be used as molecular markers for species identification and phylogenetic studies involving symbiotic Nostoc cyanobacteria.

  15. Genetic diagnosis of Mendelian disorders via RNA sequencing.

    Science.gov (United States)

    Kremer, Laura S; Bader, Daniel M; Mertes, Christian; Kopajtich, Robert; Pichler, Garwin; Iuso, Arcangela; Haack, Tobias B; Graf, Elisabeth; Schwarzmayr, Thomas; Terrile, Caterina; Koňaříková, Eliška; Repp, Birgit; Kastenmüller, Gabi; Adamski, Jerzy; Lichtner, Peter; Leonhardt, Christoph; Funalot, Benoit; Donati, Alice; Tiranti, Valeria; Lombes, Anne; Jardel, Claude; Gläser, Dieter; Taylor, Robert W; Ghezzi, Daniele; Mayr, Johannes A; Rötig, Agnes; Freisinger, Peter; Distelmaier, Felix; Strom, Tim M; Meitinger, Thomas; Gagneur, Julien; Prokisch, Holger

    2017-06-12

    Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.

  16. Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity

    Directory of Open Access Journals (Sweden)

    Bolhaar Suzanne THP

    2008-11-01

    Full Text Available Abstract Background Mal d 1 is a major apple allergen causing food allergic symptoms of the oral allergy syndrome (OAS in birch-pollen sensitised patients. The Mal d 1 gene family is known to have at least 7 intron-containing and 11 intronless members that have been mapped in clusters on three linkage groups. In this study, the allelic diversity of the seven intron-containing Mal d 1 genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. Protein variant constitutions were subsequently compared with Skin Prick Test (SPT responses to study the association of deduced protein variants with allergenicity in a set of 14 cultivars. Results From the seven intron-containing Mal d 1 genes investigated, Mal d 1.01 and Mal d 1.02 were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. Mal d 1.04, Mal d 1.05 and Mal d 1.06 A, B and C were more variable, coding for three to six different protein variants. Comparison of Mal d 1 allelic composition between the high-allergenic cultivar Golden Delicious and the low-allergenic cultivars Santana and Priscilla, which are linked in pedigree, showed an association between the protein variants coded by the Mal d 1.04 and -1.06A genes (both located on linkage group 16 with allergenicity. This association was confirmed in 10 other cultivars. In addition, Mal d 1.06A allele dosage effects associated with the degree of allergenicity based on prick to prick testing. Conversely, no associations were observed for the protein variants coded by the Mal d 1.01 (on linkage group 13, -1.02, -1.06B, -1.06C genes (all on linkage group 16, nor by the Mal d 1.05 gene (on linkage group 6. Conclusion Protein variant compositions of Mal d 1.04 and -1.06A and, in case of Mal d 1.06A, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. This information

  17. Conservation patterns in different functional sequence categoriesof divergent Drosophila species

    Energy Technology Data Exchange (ETDEWEB)

    Papatsenko, Dmitri; Kislyuk, Andrey; Levine, Michael; Dubchak, Inna

    2005-10-01

    We have explored the distributions of fully conservedungapped blocks in genome-wide pairwise alignments of recently completedspecies of Drosophila: D.yakuba, D.ananassae, D.pseudoobscura, D.virilisand D.mojavensis. Based on these distributions we have found that nearlyevery functional sequence category possesses its own distinctiveconservation pattern, sometimes independent of the overall sequenceconservation level. In the coding and regulatory regions, the ungappedblocks were longer than in introns, UTRs and non-functional sequences. Atthe same time, the blocks in the coding regions carried 3N+2 signaturecharacteristic to synonymic substitutions in the 3rd codon positions.Larger block sizes in transcription regulatory regions can be explainedby the presence of conserved arrays of binding sites for transcriptionfactors. We also have shown that the longest ungapped blocks, or'ultraconserved' sequences, are associated with specific gene groups,including those encoding ion channels and components of the cytoskeleton.We discussed how restrained conservation patterns may help in mappingfunctional sequence categories and improving genomeannotation.

  18. The First Symbiont-Free Genome Sequence of Marine Red Alga, Susabi-nori (Pyropia yezoensis)

    Science.gov (United States)

    Nakamura, Yoji; Sasaki, Naobumi; Kobayashi, Masahiro; Ojima, Nobuhiko; Yasuike, Motoshige; Shigenobu, Yuya; Satomi, Masataka; Fukuma, Yoshiya; Shiwaku, Koji; Tsujimoto, Atsumi; Kobayashi, Takanori; Nakayama, Ichiro; Ito, Fuminari; Nakajima, Kazuhiro; Sano, Motohiko; Wada, Tokio; Kuhara, Satoru; Inouye, Kiyoshi; Gojobori, Takashi; Ikeo, Kazuho

    2013-01-01

    Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae. PMID:23536760

  19. The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis.

    Directory of Open Access Journals (Sweden)

    Yoji Nakamura

    Full Text Available Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb, which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35% are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

  20. Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

    Science.gov (United States)

    Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

    2008-01-01

    So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

  1. Estrogen receptor alpha regulates expression of the breast cancer 1 associated ring domain 1 (BARD1) gene through intronic DNA sequence.

    Science.gov (United States)

    Creekmore, Amy L; Ziegler, Yvonne S; Bonéy, Jamie L; Nardulli, Ann M

    2007-03-15

    We have used a chromatin immunoprecipitation (ChIP)-based cloning strategy to isolate and identify genes associated with estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. One of the gene regions isolated was a 288bp fragment from the ninth intron of the breast cancer 1 associated ring domain (BARD1) gene. We demonstrated that ERalpha associated with this region of the endogenous BARD 1 gene in MCF-7 cells, that ERalpha bound to three of five ERE half sites located in the 288bp BARD1 region, and that this 288bp BARD1 region conferred estrogen responsiveness to a heterologous promoter. Importantly, treatment of MCF-7 cells with estrogen increased BARD1 mRNA and protein levels. These findings demonstrate that ChIP cloning strategies can be utilized to successfully isolate regulatory regions that are far removed from the transcription start site and assist in identifying cis elements involved in conferring estrogen responsiveness.

  2. Roles of genes and Alu repeats in nonlinear correlations of HUMHBB DNA sequence

    International Nuclear Information System (INIS)

    Xiao Yi; Huang Yanzhao

    2004-01-01

    DNA sequences of different species and different portion of the DNA of the same species may have completely different correlation properties, but the origin of these correlations is still not very clear and is currently being investigated, especially in different particular cases. We report here a study of the DNA sequence of human beta globin region (HUMHBB) which has strong linear and nonlinear correlations. We studied the roles of two of the typical elements of DNA sequence, genes and Alu repeats, in the nonlinear correlations of HUMHBB. We find that there exist strong nonlinear correlations between the exons or introns in different genes and between the Alu repeats. They may be one of the major sources of the nonlinear correlations in HUMBHB

  3. Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

    DEFF Research Database (Denmark)

    Haugen, P; Huss, V A; Nielsen, Henrik

    1999-01-01

    on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear...

  4. Human Ro60 (SSA2) genomic organization and sequence alterations, examined in cutaneous lupus erythematosus.

    Science.gov (United States)

    Millard, T P; Ashton, G H S; Kondeatis, E; Vaughan, R W; Hughes, G R V; Khamashta, M A; Hawk, J L M; McGregor, J M; McGrath, J A

    2002-02-01

    The Ro 60 kDa protein (Ro60 or SSA2) is the major component of the Ro ribonucleoprotein (Ro RNP) complex, to which an immune response is a specific feature of several autoimmune diseases. The genomic organization and any sequence variation within the DNA encoding Ro60 are unknown. To characterize the Ro60 gene structure and to assess whether any sequence alterations might be associated with serum anti-Ro antibody in subacute cutaneous lupus erythematosus (SCLE), thus potentially providing new insight into disease pathogenesis. The cDNA sequence for Ro60 was obtained from the NCBI database and used for a BLAST search for a clone containing the entire genomic sequence. The intron-exon borders were confirmed by designing intronic primer pairs to flank each exon, which were then used to amplify genomic DNA for automated sequencing from 36 caucasian patients with SCLE (anti-Ro positive) and 49 with discoid LE (DLE, anti-Ro negative), in addition to 36 healthy caucasian controls. Heteroduplex analysis of polymerase chain reaction (PCR) products from patients and controls spanning all Ro60 exons (1-8) revealed a common bandshift in the PCR products spanning exon 7. Sequencing of the corresponding PCR products demonstrated an A > G substitution at nucleotide position 1318-7, within the consensus acceptor splice site of exon 7 (GenBank XM001901). The allele frequencies were major allele A (0.71) and minor allele G (0.29) in 72 control chromosomes, with no significant differences found between SCLE patients, DLE patients and controls. The genomic organization of the DNA encoding the Ro60 protein is described, including a common polymorphism within the consensus acceptor splice site of exon 7. Our delineation of a strategy for the genomic amplification of Ro60 forms a basis for further examination of the pathological functions of the Ro RNP in autoimmune disease.

  5. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta) along the Eastern coast of South America

    Science.gov (United States)

    2008-01-01

    Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA) of some species of the genus Porphyra (Bangiales, Rhodophyta). Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG) inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA) populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of degeneration of the HEG

  6. Group I introns and associated homing endonuclease genes reveals a clinal structure for Porphyra spiralis var. amplifolia (Bangiales, Rhodophyta along the Eastern coast of South America

    Directory of Open Access Journals (Sweden)

    Matioli Sergio R

    2008-11-01

    Full Text Available Abstract Background Group I introns are found in the nuclear small subunit ribosomal RNA gene (SSU rDNA of some species of the genus Porphyra (Bangiales, Rhodophyta. Size polymorphisms in group I introns has been interpreted as the result of the degeneration of homing endonuclease genes (HEG inserted in peripheral loops of intron paired elements. In this study, intron size polymorphisms were characterized for different Porphyra spiralis var. amplifolia (PSA populations on the Southern Brazilian coast, and were used to infer genetic relationships and genetic structure of these PSA populations, in addition to cox2-3 and rbcL-S regions. Introns of different sizes were tested qualitatively for in vitro self-splicing. Results Five intron size polymorphisms within 17 haplotypes were obtained from 80 individuals representing eight localities along the distribution of PSA in the Eastern coast of South America. In order to infer genetic structure and genetic relationships of PSA, these polymorphisms and haplotypes were used as markers for pairwise Fst analyses, Mantel's test and median joining network. The five cox2-3 haplotypes and the unique rbcL-S haplotype were used as markers for summary statistics, neutrality tests Tajima's D and Fu's Fs and for median joining network analyses. An event of demographic expansion from a population with low effective number, followed by a pattern of isolation by distance was obtained for PSA populations with the three analyses. In vitro experiments have shown that introns of different lengths were able to self-splice from pre-RNA transcripts. Conclusion The findings indicated that degenerated HEGs are reminiscent of the presence of a full-length and functional HEG, once fixed for PSA populations. The cline of HEG degeneration determined the pattern of isolation by distance. Analyses with the other markers indicated an event of demographic expansion from a population with low effective number. The different degrees of

  7. Identification of genome-wide non-canonical spliced regions and analysis of biological functions for spliced sequences using Read-Split-Fly.

    Science.gov (United States)

    Bai, Yongsheng; Kinne, Jeff; Ding, Lizhong; Rath, Ethan C; Cox, Aaron; Naidu, Siva Dharman

    2017-10-03

    It is generally thought that most canonical or non-canonical splicing events involving U2- and U12 spliceosomes occur within nuclear pre-mRNAs. However, the question of whether at least some U12-type splicing occurs in the cytoplasm is still unclear. In recent years next-generation sequencing technologies have revolutionized the field. The "Read-Split-Walk" (RSW) and "Read-Split-Run" (RSR) methods were developed to identify genome-wide non-canonical spliced regions including special events occurring in cytoplasm. As the significant amount of genome/transcriptome data such as, Encyclopedia of DNA Elements (ENCODE) project, have been generated, we have advanced a newer more memory-efficient version of the algorithm, "Read-Split-Fly" (RSF), which can detect non-canonical spliced regions with higher sensitivity and improved speed. The RSF algorithm also outputs the spliced sequences for further downstream biological function analysis. We used open access ENCODE project RNA-Seq data to search spliced intron sequences against the U12-type spliced intron sequence database to examine whether some events could occur as potential signatures of U12-type splicing. The check was performed by searching spliced sequences against 5'ss and 3'ss sequences from the well-known orthologous U12-type spliceosomal intron database U12DB. Preliminary results of searching 70 ENCODE samples indicated that the presence of 5'ss with U12-type signature is more frequent than U2-type and prevalent in non-canonical junctions reported by RSF. The selected spliced sequences have also been further studied using miRBase to elucidate their functionality. Preliminary results from 70 samples of ENCODE datasets show that several miRNAs are prevalent in studied ENCODE samples. Two of these are associated with many diseases as suggested in the literature. Specifically, hsa-miR-1273 and hsa-miR-548 are associated with many diseases and cancers. Our RSF pipeline is able to detect many possible junctions

  8. THE POLYMORPHISM OF THE SUS4 SUCROSE SYNTHASE DOMAIN SEQUENCES IN RUSSIAN, BELORUSSIAN AND KAZAKH POTATO CULTIVARS

    Directory of Open Access Journals (Sweden)

    M. A. Slugina

    2016-01-01

    Full Text Available The potato is one of the main strategic crops in the Russian Federation, Belarus and Kazakhstan. Currently, we have achieved significant advances in the understanding of metabolic mechanism of carbohydrate and interconversion «sucrose – starch» in potato tubers. Sucrose synthase (Sus is a key enzyme in the breakdown of sucrose. Sucrose synthase (Sus is catalyzing a reversible reaction of conversion sucrose and UDP into fructose and UDP-glucose. The identification and subsequent characterization of the genes encoding plant sucrose synthase is the first step towards understanding their physiological roles and metabolic mechanism involved in carbohydrate accumulation in potato tubers. In the present work the nucleotide and amino acid polymorphism of the Sus4 gene fragments containing sequences of the sucrose synthase domain were analyzed. Sus4 gene fragments (intron III – exon VI in 9 potato cultivars of Russian, Kazakh and Belarusian breeding were analyzed. The polymorphism of the Sus4 sucrose synthase domain sequences was first examined. The length of analyzed fragment varied from 977 b.p. (cultivars Favorit, Karasaiskii, Miras to 1013 b.p. (cultivars Zorochka, Manifest, Elisaveta, Bashkirskii. It was demonstrated that the examined sequences contained point mutations, as well as insertions and deletions. The common polymorphism level was 5.82%. It was shown that the examined sequences contained 58 SNPs and 4 indels. The most variable were introns IV (12.4% and V (9.18%. The most variable was exon IV. 7 allelic variants were detected. 6 different amino acid sequences specific to different varieties were also identified.

  9. Identification of β-SiC surrounded by relatable surrounding diamond ...

    Indian Academy of Sciences (India)

    β-SiC is identified in the presence of a relatable surrounding diamond medium using subtle, but discernible Raman ... Change in the nature of the surrounding material structure and its .... intensity implies very low graphite content in thin film. In.

  10. Distribution of Ds-like sequences in genomes of cereals

    International Nuclear Information System (INIS)

    Vershinin, A.V.; Salina, E.A.; Shumnii, V.K.; Svitashev, S.K.

    1986-01-01

    It has been suggested that insertions of Ds-elements may alter the effectiveness of transcription or translation of the genetic loci and the normal processing of introns and exons, and that they may impair coding frames, etc. The object of the present study was to determine the frequency of occurence of DNA sequences similar to the Ds-controlling elements of mazie (Ds-like sequences) among other representatives of cereals. The conservative feature of the primary structure of transposons from different eukaryotic species served as a basis in this investigation. By means of the ''nick-translation'' reaction with the aid of DNA-polymerase I (alpha- 32 P) dCTP or TTP was introduced into the Ds-element. The specific radioactivity of the preparations obtained was 5 x 10 7 to 1 x 10 8 cpm/gamma. From the results obtained, it is suggested that the genomes of cereals examined contain a collection of Ds-like sequences. The Ds-element may have a significant effect on gene expression in the presence of Ac-like or other sequences, which undergo transposition

  11. Speciation of a group I intron into a lariat capping ribozyme

    DEFF Research Database (Denmark)

    Meyer, Mélanie; Nielsen, Henrik; Oliéric, Vincent

    2014-01-01

    The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes ....... The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching....

  12. CLONING AND SEQUENCING OF PGIP FROM ‘JIN SERIES’ ALMOND (PRUNUS DULCIS

    Directory of Open Access Journals (Sweden)

    Yuhu Han

    2015-12-01

    Full Text Available Specific primers synthesized according to conservative regions of polygalacturonase inhibiting protein (PGIP gene were used to amplify Prunus Dulcis genomic DNA by polymerase-chain reaction (PCR. Six bands (pgip1, pgip2, pgip3, pgip4, pgip5 and pgip6 of genes were obtained and cloned into PBS-T vector. According to the length of bands, 717bp, 864bp, 796bp were A1 (pgip1, pgip2, pgip3, A2 (pgip4, A4 (pgip5, pgip6, respectively. DNA sequences showed that the fragments taken together were the gene encoding PGIP. A2 and A3 contained two exons interrupted by one intron, which has GT-AG sequence. Its DNA and amino acid sequences were highly homologies to those from Prunus Persica; Prunus Salicina; Prunus Americana; Prunus Mume, respectively. A conserved lencinerial fragment exists in the derived protein sequence.

  13. Sequence-to-Sequence Prediction of Vehicle Trajectory via LSTM Encoder-Decoder Architecture

    OpenAIRE

    Park, Seong Hyeon; Kim, ByeongDo; Kang, Chang Mook; Chung, Chung Choo; Choi, Jun Won

    2018-01-01

    In this paper, we propose a deep learning based vehicle trajectory prediction technique which can generate the future trajectory sequence of surrounding vehicles in real time. We employ the encoder-decoder architecture which analyzes the pattern underlying in the past trajectory using the long short-term memory (LSTM) based encoder and generates the future trajectory sequence using the LSTM based decoder. This structure produces the $K$ most likely trajectory candidates over occupancy grid ma...

  14. Significant association of interleukin-4 gene intron 3 VNTR polymorphism with susceptibility to knee osteoarthritis.

    Science.gov (United States)

    Yigit, Serbulent; Inanir, Ahmet; Tekcan, Akın; Tural, Ercan; Ozturk, Gokhan Tuna; Kismali, Gorkem; Karakus, Nevin

    2014-03-01

    Interleukin-4 (IL-4) is a strong chondroprotective cytokine and polymorphisms within this gene may be a risk factor for osteoarthritis (OA). We aimed to investigate genotype and allele frequencies of IL-4 gene intron 3 variable number of tandem repeats (VNTR) polymorphism in patients with knee OA in a Turkish population. The study included 202 patients with knee OA and 180 healthy controls. Genomic DNA was isolated and IL-4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers followed by restriction fragment length polymorphism (RFLP) analysis. Our result show that there was statistically significant difference between knee OA patients and control group with respect to IL-4 genotype distribution and allele frequencies (p=0.000, OR: 0.20, 95% CI: 0.10-0.41, OR: 0.22, 95% CI: 0.12-0.42, respectively). Our findings suggest that there is an association of IL-4 gene intron 3 VNTR polymorphism with susceptibility of a person for development of knee OA. As a result, IL-4 gene intron 3 VNTR polymorphism could be a genetic marker in OA in a Turkish study population. This is the first association study that evaluates the associations between IL-4 gene VNTR polymorphism and knee OA. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  15. Genome survey sequencing and genetic background characterization of Gracilariopsis lemaneiformis (Rhodophyta) based on next-generation sequencing.

    Science.gov (United States)

    Zhou, Wei; Hu, Yiyi; Sui, Zhenghong; Fu, Feng; Wang, Jinguo; Chang, Lianpeng; Guo, Weihua; Li, Binbin

    2013-01-01

    Gracilariopsis lemaneiformis has a high economic value and is one of the most important aquaculture species in China. Despite it is economic importance, it has remained largely unstudied at the genomic level. In this study, we conducted a genome survey of Gp. lemaneiformis using next-generation sequencing (NGS) technologies. In total, 18.70 Gb of high-quality sequence data with an estimated genome size of 97 Mb were obtained by HiSeq 2000 sequencing for Gp. lemaneiformis. These reads were assembled into 160,390 contigs with a N50 length of 3.64 kb, which were further assembled into 125,685 scaffolds with a total length of 81.17 Mb. Genome analysis predicted 3490 genes and a GC% content of 48%. The identified genes have an average transcript length of 1,429 bp, an average coding sequence size of 1,369 bp, 1.36 exons per gene, exon length of 1,008 bp, and intron length of 191 bp. From the initial assembled scaffold, transposable elements constituted 54.64% (44.35 Mb) of the genome, and 7737 simple sequence repeats (SSRs) were identified. Among these SSRs, the trinucleotide repeat type was the most abundant (up to 73.20% of total SSRs), followed by the di- (17.41%), tetra- (5.49%), hexa- (2.90%), and penta- (1.00%) nucleotide repeat type. These characteristics suggest that Gp. lemaneiformis is a model organism for genetic study. This is the first report of genome-wide characterization within this taxon.

  16. Genome Survey Sequencing and Genetic Background Characterization of Gracilariopsis lemaneiformis (Rhodophyta) Based on Next-Generation Sequencing

    Science.gov (United States)

    Sui, Zhenghong; Fu, Feng; Wang, Jinguo; Chang, Lianpeng; Guo, Weihua; Li, Binbin

    2013-01-01

    Gracilariopsis lemaneiformis has a high economic value and is one of the most important aquaculture species in China. Despite it is economic importance, it has remained largely unstudied at the genomic level. In this study, we conducted a genome survey of Gp. lemaneiformis using next-generation sequencing (NGS) technologies. In total, 18.70 Gb of high-quality sequence data with an estimated genome size of 97 Mb were obtained by HiSeq 2000 sequencing for Gp. lemaneiformis. These reads were assembled into 160,390 contigs with a N50 length of 3.64 kb, which were further assembled into 125,685 scaffolds with a total length of 81.17 Mb. Genome analysis predicted 3490 genes and a GC% content of 48%. The identified genes have an average transcript length of 1,429 bp, an average coding sequence size of 1,369 bp, 1.36 exons per gene, exon length of 1,008 bp, and intron length of 191 bp. From the initial assembled scaffold, transposable elements constituted 54.64% (44.35 Mb) of the genome, and 7737 simple sequence repeats (SSRs) were identified. Among these SSRs, the trinucleotide repeat type was the most abundant (up to 73.20% of total SSRs), followed by the di- (17.41%), tetra- (5.49%), hexa- (2.90%), and penta- (1.00%) nucleotide repeat type. These characteristics suggest that Gp. lemaneiformis is a model organism for genetic study. This is the first report of genome-wide characterization within this taxon. PMID:23875008

  17. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    LENUS (Irish Health Repository)

    Shieh, Grace S.

    2011-12-22

    Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3\\' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  18. H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

    Directory of Open Access Journals (Sweden)

    Shieh Grace S

    2011-12-01

    Full Text Available Abstract Background The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA. Results Here we provide evidence that H2B monoubiquitylation (H2BK123ub1 marks introns in Saccharomyces cerevisiae. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3, which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an htb-K123R mutation, leads to synthetic lethality. Conclusion These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.

  19. Conserved intron positions in FGFR genes reflect the modular structure of FGFR and reveal stepwise addition of domains to an already complex ancestral FGFR.

    Science.gov (United States)

    Rebscher, Nicole; Deichmann, Christina; Sudhop, Stefanie; Fritzenwanker, Jens Holger; Green, Stephen; Hassel, Monika

    2009-10-01

    We have analyzed the evolution of fibroblast growth factor receptor (FGFR) tyrosine kinase genes throughout a wide range of animal phyla. No evidence for an FGFR gene was found in Porifera, but we tentatively identified an FGFR gene in the placozoan Trichoplax adhaerens. The gene encodes a protein with three immunoglobulin-like domains, a single-pass transmembrane, and a split tyrosine kinase domain. By superimposing intron positions of 20 FGFR genes from Placozoa, Cnidaria, Protostomia, and Deuterostomia over the respective protein domain structure, we identified ten ancestral introns and three conserved intron groups. Our analysis shows (1) that the position of ancestral introns correlates to the modular structure of FGFRs, (2) that the acidic domain very likely evolved in the last common ancestor of triploblasts, (3) that splicing of IgIII was enabled by a triploblast-specific insertion, and (4) that IgI is subject to substantial loss or duplication particularly in quickly evolving genomes. Moreover, intron positions in the catalytic domain of FGFRs map to the borders of protein subdomains highly conserved in other serine/threonine kinases. Nevertheless, these introns were introduced in metazoan receptor tyrosine kinases exclusively. Our data support the view that protein evolution dating back to the Cambrian explosion took place in such a short time window that only subtle changes in the domain structure are detectable in extant representatives of animal phyla. We propose that the first multidomain FGFR originated in the last common ancestor of Placozoa, Cnidaria, and Bilateria. Additional domains were introduced mainly in the ancestor of triploblasts and in the Ecdysozoa.

  20. Complete chloroplast DNA sequence from a Korean endemic genus, Megaleranthis saniculifolia, and its evolutionary implications.

    Science.gov (United States)

    Kim, Young-Kyu; Park, Chong-wook; Kim, Ki-Joong

    2009-03-31

    The chloroplast DNA sequences of Megaleranthis saniculifolia, an endemic and monotypic endangered plant species, were completed in this study (GenBank FJ597983). The genome is 159,924 bp in length. It harbors a pair of IR regions consisting of 26,608 bp each. The lengths of the LSC and SSC regions are 88,326 bp and 18,382 bp, respectively. The structural organizations, gene and intron contents, gene orders, AT contents, codon usages, and transcription units of the Megaleranthis chloroplast genome are similar to those of typical land plant cp DNAs. However, the detailed features of Megaleranthis chloroplast genomes are substantially different from that of Ranunculus, which belongs to the same family, the Ranunculaceae. First, the Megaleranthis cp DNA was 4,797 bp longer than that of Ranunculus due to an expanded IR region into the SSC region and duplicated sequence elements in several spacer regions of the Megaleranthis cp genome. Second, the chloroplast genomes of Megaleranthis and Ranunculus evidence 5.6% sequence divergence in the coding regions, 8.9% sequence divergence in the intron regions, and 18.7% sequence divergence in the intergenic spacer regions, respectively. In both the coding and noncoding regions, average nucleotide substitution rates differed markedly, depending on the genome position. Our data strongly implicate the positional effects of the evolutionary modes of chloroplast genes. The genes evidencing higher levels of base substitutions also have higher incidences of indel mutations and low Ka/Ks ratios. A total of 54 simple sequence repeat loci were identified from the Megaleranthis cp genome. The existence of rich cp SSR loci in the Megaleranthis cp genome provides a rare opportunity to study the population genetic structures of this endangered species. Our phylogenetic trees based on the two independent markers, the nuclear ITS and chloroplast matK sequences, strongly support the inclusion of the Megaleranthis to the Trollius. Therefore, our

  1. Trajectories and Maneuvers of Surrounding Vehicles with Panoramic Camera Arrays

    DEFF Research Database (Denmark)

    Dueholm, Jacob Velling; Kristoffersen, Miklas Strøm; Satzoda, Ravi K.

    2016-01-01

    Vision-based research for intelligent vehicles have traditionally focused on specific regions around a vehicle, such as a front looking camera for, e.g., lane estimation. Traffic scenes are complex and vital information could be lost in unobserved regions. This paper proposes a framework that uses...... four visual sensors for a full surround view of a vehicle in order to achieve an understanding of surrounding vehicle behaviors. The framework will assist the analysis of naturalistic driving studies by automating the task of data reduction of the observed trajectories. To this end, trajectories...... are estimated using a vehicle detector together with a multiperspective optimized tracker in each view. The trajectories are transformed to a common ground plane, where they are associated between perspectives and analyzed to reveal tendencies around the ego-vehicle. The system is tested on sequences from 2.5 h...

  2. Sequence analysis and overexpression of a pectin lyase gene (pel1) from Aspergillus oryzae KBN616.

    Science.gov (United States)

    Kitamoto, N; Yoshino-Yasuda, S; Ohmiya, K; Tsukagoshi, N

    2001-01-01

    A gene (pel1) encoding pectin lyase (Pel1) was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,196 bp with a single intron. The ORF encoded 381 amino acids with a signal peptide of 20 amino acids. The deduced amino acid sequence showed high similarity to those of Aspergillus niger pectin lyases and Glomerella cingulata PnlA. The pel1 gene was successfully overexpressed under the promoter of the A. oryzae TEF1 gene. The molecular mass of the recombinant pectin lyase substantially coincided with that calculated based on nucleotide sequence.

  3. AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

    Science.gov (United States)

    Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

    2018-04-01

    The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

  4. The complete chloroplast genome sequence of Dendrobium nobile.

    Science.gov (United States)

    Yan, Wenjin; Niu, Zhitao; Zhu, Shuying; Ye, Meirong; Ding, Xiaoyu

    2016-11-01

    The complete chloroplast (cp) genome sequence of Dendrobium nobile, an endangered and traditional Chinese medicine with important economic value, is presented in this article. The total genome size is 150,793 bp, containing a large single copy (LSC) region (84,939 bp) and a small single copy region (SSC) (13,310 bp) which were separated by two inverted repeat (IRs) regions (26,272 bp). The overall GC contents of the plastid genome were 38.8%. In total, 130 unique genes were annotated and they were consisted of 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Fourteen genes contained one or two introns.

  5. quatre-quart1 is an indispensable U12 intron-containing gene that plays a crucial role in Arabidopsis development.

    Science.gov (United States)

    Kwak, Kyung Jin; Kim, Bo Mi; Lee, Kwanuk; Kang, Hunseung

    2017-05-17

    Despite increasing understanding of the importance of the splicing of U12-type introns in plant development, the key question of which U12 intron-containing genes are essential for plant development has not yet been explored. Here, we assessed the functional role of the quatre-quart1 (QQT1) gene, one of the ~230 U12 intron-containing genes in Arabidopsis thaliana. Expression of QQT1 in the U11/U12-31K small nuclear ribonucleoprotein mutant (31k) rescued the developmental-defect phenotypes of the 31k mutant, whereas the miRNA-mediated qqt1 knockdown mutants displayed severe defects in growth and development, including severely arrested stem growth, small size, and the formation of serrated leaves. The structures of the shoot apical meristems in the qqt1 mutants were abnormal and disordered. Identification of QQT1-interacting proteins via a yeast two-hybrid screening and a firefly luciferase complementation-imaging assay revealed that a variety of proteins, including many chloroplast-targeted proteins, interacted with QQT1. Importantly, the levels of chloroplast-targeted proteins in the chloroplast were reduced, and the chloroplast structure was abnormal in the qqt1 mutant. Collectively, these results provide clear evidence that QQT1 is an indispensable U12 intron-containing gene whose correct splicing is crucial for the normal development of Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  6. BanII dimorphic site located in the third intron of the human apolipoprotein AI (APOA1) gene

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, R T; Kresnak, M T; Frossard, P M

    1988-02-11

    A 0.7kb fragment generated by AvaII digestion of pBL13AI, a 0.965kb full-length human apolipoprotein AI cDNA was cloned into the EcoRI site of pBR322. The apoAI cDNA was isolated from a lambdagt10 human fetal liver cDNA library. BanII (GPuGCPyC) (International Biotechnologies, Inc.) identifies two invariant bands at 1122bp and 417bp, and a single two-allele polymorphism with bands at either 274bp or 452bp. The human apolipoprotein AI-CIII-AIV gene complex has been localized on the long arm of chromosome 11 by Southern blot analysis of human-chinese hamster cell hybrids. Co-dominant segregation has been observed in two families (13 individuals). The BanII restriction map was constructed from DNA sequence data of the human apoAI gene. The 452bp fragment is generated by the loss of a BanII dimorphic site in the third intron of the apoAI gene, between the 178bp and the 274bp fragments.

  7. Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

    Directory of Open Access Journals (Sweden)

    Mallya Meera

    2008-09-01

    Full Text Available Abstract Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD. This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC. This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C and not on their own.

  8. A heuristic model for computational prediction of human branch point sequence.

    Science.gov (United States)

    Wen, Jia; Wang, Jue; Zhang, Qing; Guo, Dianjing

    2017-10-24

    Pre-mRNA splicing is the removal of introns from precursor mRNAs (pre-mRNAs) and the concurrent ligation of the flanking exons to generate mature mRNA. This process is catalyzed by the spliceosome, where the splicing factor 1 (SF1) specifically recognizes the seven-nucleotide branch point sequence (BPS) and the U2 snRNP later displaces the SF1 and binds to the BPS. In mammals, the degeneracy of BPS motifs together with the lack of a large set of experimentally verified BPSs complicates the task of BPS prediction in silico. In this paper, we develop a simple and yet efficient heuristic model for human BPS prediction based on a novel scoring scheme, which quantifies the splicing strength of putative BPSs. The candidate BPS is restricted exclusively within a defined BPS search region to avoid the influences of other elements in the intron and therefore the prediction accuracy is improved. Moreover, using two types of relative frequencies for human BPS prediction, we demonstrate our model outperformed other current implementations on experimentally verified human introns. We propose that the binding energy contributes to the molecular recognition involved in human pre-mRNA splicing. In addition, a genome-wide human BPS prediction is carried out. The characteristics of predicted BPSs are in accordance with experimentally verified human BPSs, and branch site positions relative to the 3'ss and the 5'end of the shortened AGEZ are consistent with the results of published papers. Meanwhile, a webserver for BPS predictor is freely available at http://biocomputer.bio.cuhk.edu.hk/BPS .

  9. Domestication of self-splicing introns during eukaryogenesis : the rise of the complex spliceosomal machinery

    NARCIS (Netherlands)

    Vosseberg, Julian; Snel, Berend

    2017-01-01

    ᅟ: The spliceosome is a eukaryote-specific complex that is essential for the removal of introns from pre-mRNA. It consists of five small nuclear RNAs (snRNAs) and over a hundred proteins, making it one of the most complex molecular machineries. Most of this complexity has emerged during

  10. Duplication of the dystroglycan gene in most branches of teleost fish

    Directory of Open Access Journals (Sweden)

    Giardina Bruno

    2007-05-01

    Full Text Available Abstract Background The dystroglycan (DG complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. A single copy of the DG gene (DAG1 has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. Similarly, D. rerio (zebrafish seems to have a single copy of DAG1, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD event, many teleost fish acquired multiple copies of several genes (paralogues. Results Data mining of pufferfish (T. nigroviridis and T. rubripes and other teleost fish (O. latipes and G. aculeatus available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in T. nigroviridis. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by DAG1 in mammals and D. rerio. A similar scenario emerged also in D. labrax (sea bass, from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. Western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost Antarctic species (T. bernacchii and C. hamatus. Conclusion Our evolutionary analysis has shown that the whole-genome duplication event in the Class Actinopterygii (ray-finned fish involved also DAG1. We unravelled new important molecular genetic details

  11. Enhancing area surrounding breast carcinoma on MR mammography: comparison with pathological examination

    International Nuclear Information System (INIS)

    Goethem, M. van; Verslegers, I.; Biltjes, I.; Schepper, A. de; Schelfout, K.; Colpaert, C.; Kersschot, E.; Tjalma, W.A.; Weyler, J.

    2004-01-01

    The enhancing area surrounding breast carcinoma on MR mammography is correlated with findings from pathological examination. We studied 194 patients with breast cancer who underwent preoperative MR mammography. Of all malignant lesions presenting with an enhancing surrounding area on MR mammography, morphologic features including long spicules, a ductal pattern, diffuse enhancement or nodules were evaluated and compared with histopathological examination. A double breast coil was used; we performed a 3D FLASH sequence with contiguous coronal slices of 2 mm, before and after injection of 0.2 mmol/kg GD-DTPA, and subtraction images were obtained. In total, 297 malignant lesions were detected at MR mammography and 101 of them had one or more types of enhancing surrounding area. In 49 of the 53 cancers with long spicules and in 49 of the 55 cancers with surrounding ductal pattern of enhancement, pathological examination showed in situ and/or invasive carcinoma. Multiple nodules adjacent to the carcinoma were seen in 20 patients and corresponded with six cases of invasive and ten cases of ductal in situ carcinoma. A diffuse enhancing area next to a mass was seen in ten patients and consisted of carcinoma in all cases: seven in situ and three invasive carcinomas. Enhancing areas including long spicules, a ductal pattern, noduli, or diffuse enhancement surrounding a carcinoma corresponded with in situ or invasive extension of the carcinoma in 92.5, 89, 80 and 100% of cases, respectively. (orig.)

  12. RNA sequencing of the exercise transcriptome in equine athletes.

    Directory of Open Access Journals (Sweden)

    Stefano Capomaccio

    Full Text Available The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases, as well as "nucleic acid binding" and "signal transduction activity" functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise

  13. Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients.

    Science.gov (United States)

    Petersen, Sanne M; Dandanell, Mette; Rasmussen, Lene J; Gerdes, Anne-Marie; Krogh, Lotte N; Bernstein, Inge; Okkels, Henrik; Wikman, Friedrik; Nielsen, Finn C; Hansen, Thomas V O

    2013-10-03

    Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T). In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

  14. Sequence and Analysis of the Genome of the Pathogenic Yeast Candida orthopsilosis

    Science.gov (United States)

    Riccombeni, Alessandro; Vidanes, Genevieve; Proux-Wéra, Estelle; Wolfe, Kenneth H.; Butler, Geraldine

    2012-01-01

    Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina) with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families. PMID:22563396

  15. Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

    Science.gov (United States)

    De Rocquigny, H; Ficheux, D; Gabus, C; Allain, B; Fournie-Zaluski, M C; Darlix, J L; Roques, B P

    1993-02-25

    The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

  16. Evolutionary dynamics of an expressed MHC class IIβ locus in the Ranidae (Anura) uncovered by genome walking and high-throughput amplicon sequencing

    Science.gov (United States)

    Mulder, Kevin P.; Cortazar-Chinarro, Maria; Harris, D. James; Crottini, Angelica; Grant, Evan H. Campbell; Fleischer, Robert C.; Savage, Anna E.

    2017-01-01

    The Major Histocompatibility Complex (MHC) is a genomic region encoding immune loci that are important and frequently used markers in studies of adaptive genetic variation and disease resistance. Given the primary role of infectious diseases in contributing to global amphibian declines, we characterized the hypervariable exon 2 and flanking introns of the MHC Class IIβ chain for 17 species of frogs in the Ranidae, a speciose and cosmopolitan family facing widespread pathogen infections and declines. We find high levels of genetic variation concentrated in the Peptide Binding Region (PBR) of the exon. Ten codons are under positive selection, nine of which are located in the mammal-defined PBR. We hypothesize that the tenth codon (residue 21) is an amphibian-specific PBR site that may be important in disease resistance. Trans-species and trans-generic polymorphisms are evident from exon-based genealogies, and co-phylogenetic analyses between intron, exon and mitochondrial based reconstructions reveal incongruent topologies, likely due to different locus histories. We developed two sets of barcoded adapters that reliably amplify a single and likely functional locus in all screened species using both 454 and Illumina based sequencing methods. These primers provide a resource for multiplexing and directly sequencing hundreds of samples in a single sequencing run, avoiding the labour and chimeric sequences associated with cloning, and enabling MHC population genetic analyses. Although the primers are currently limited to the 17 species we tested, these sequences and protocols provide a useful genetic resource and can serve as a starting point for future disease, adaptation and conservation studies across a range of anuran taxa.

  17. DIVERSITY OF THE TYPE 1 INTRON-ITS REGION OF THE 18S rRNA GENE IN PSEUDOGYMNOASCUS SPECIES FROM THE RED HILLS OF KANSAS.

    Science.gov (United States)

    Chen, Xi; Crupper, Scott S

    2016-09-01

    Gypsum caves found throughout the Red Hills of Kansas have the state's most diverse and largest population of cave-roosting bats. White-nose syndrome (WNS), a disease caused by the fungus Pseudogymnoascus destructans, which threatens all temperate bat species, has not been previously detected in the gypsum caves as this disease moves westward from the eastern United States. Cave soil was obtained from the gypsum caves, and using the polymerase chain reaction, a 624-nucleotide DNA fragment specific to the Type 1 intron-internal transcribed spacer region of the 18S rRNA gene from Pseudogymnoascus species was amplified. Subsequent cloning and DNA sequencing indicated P. destructans DNA was present, along with 26 uncharacterized Pseudogymnoascus DNA variants. However, no evidence of WNS was observed in bat populations residing in these caves.

  18. Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Simon, E M; Engberg, J

    1985-01-01

    an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number...

  19. Sequence variations in the FAD2 gene in seeded pumpkins.

    Science.gov (United States)

    Ge, Y; Chang, Y; Xu, W L; Cui, C S; Qu, S P

    2015-12-21

    Seeded pumpkins are important economic crops; the seeds contain various unsaturated fatty acids, such as oleic acid and linoleic acid, which are crucial for human and animal nutrition. The fatty acid desaturase-2 (FAD2) gene encodes delta-12 desaturase, which converts oleic acid to linoleic acid. However, little is known about sequence variations in FAD2 in seeded pumpkins. Twenty-seven FAD2 clones from 27 accessions of Cucurbita moschata, Cucurbita maxima, Cucurbita pepo, and Cucurbita ficifolia were obtained (totally 1152 bp; a single gene without introns). More than 90% nucleotide identities were detected among the 27 FAD2 clones. Nucleotide substitution, rather than nucleotide insertion and deletion, led to sequence polymorphism in the 27 FAD2 clones. Furthermore, the 27 FAD2 selected clones all encoded the FAD2 enzyme (delta-12 desaturase) with amino acid sequence identities from 91.7 to 100% for 384 amino acids. The same main-function domain between 47 and 329 amino acids was identified. The four species clustered separately based on differences in the sequences that were identified using the unweighted pair group method with arithmetic mean. Geographic origin and species were found to be closely related to sequence variation in FAD2.

  20. Accumulation of Stable Full-Length Circular Group I Intron RNAs during Heat-Shock

    DEFF Research Database (Denmark)

    Andersen, Kasper L.; Beckert, Bertrand; Masquida, Benoit

    2016-01-01

    the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show...... integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility...

  1. BCL2-like 11 intron 2 deletion polymorphism is not associated with non-small cell lung cancer risk and prognosis.

    Science.gov (United States)

    Cho, Eun Na; Kim, Eun Young; Jung, Ji Ye; Kim, Arum; Oh, In Jae; Kim, Young Chul; Chang, Yoon Soo

    2015-10-01

    BCL2-Like 11(BIM), which encodes a BH3-only protein, is a major pro-apoptotic molecule that facilitates cell death. We hypothesized that a BIM intron 2 deletion polymorphism increases lung cancer risk and predicts poor prognosis in non-small lung cancer (NSCLC) patients. We prospectively recruited 450 lung cancer patients and 1:1 age, sex, and smoking status matched control subjects from February 2013 to April 2014 among patients treated at Severance, Gangnam Severance, and Chonnam Hwasoon Hospital. The presence of a 2903-bp genomic DNA deletion polymorphism of intron 2 of BIM was analyzed by PCR and validated by sequencing. Odds ratios were calculated by chi-square tests and survival analysis with Kaplan-Meier estimation. Sixty-nine out of 450 (15.3%) lung cancer patients carried the BIM deletion polymorphism, while 66 out of 450 (14.7%) control subjects carried the BIM deletion polymorphism, with an odds ratio of for lung cancer of 1.054 (95% CI; 0.731-1.519). We categorized 406 NSCLC patients according to the presence of the polymorphism and found that there were no statistically significant differences in age, sex, histologic type, or stage between subjects with and without the deletion polymorphism. The BIM deletion polymorphism did not influence overall survival (OS) or progression free survival (PFS) in our sample (OS; 37.6 vs 34.4 months (P=0.759), PFS; 49.6 vs 26.0 months (P=0.434)). These findings indicate that the BIM deletion polymorphism is common in Korean NSCLC patients but does not significantly affect the intrinsic biologic function of BH3-only protein. Furthermore, the BIM deletion polymorphism did not predict clinical outcomes in patients with NSCLC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. The complete mitochondrial genome of Porites harrisoni (Cnidaria: Scleractinia) obtained using next-generation sequencing

    KAUST Repository

    Terraneo, Tullia Isotta; Arrigoni, Roberto; Benzoni, Francesca; Forsman, Zac H.; Berumen, Michael L.

    2018-01-01

    In this study, we sequenced the complete mitochondrial genome of Porites harrisoni using ezRAD and Illumina technology. Genome length consisted of 18,630 bp, with a base composition of 25.92% A, 13.28% T, 23.06% G, and 37.73% C. Consistent with other hard corals, P. harrisoni mitogenome was arranged in 13 protein-coding genes, 2 rRNA, and 2 tRNA genes. nad5 and cox1 contained embedded Group I Introns of 11,133 bp and 965 bp, respectively.

  3. The complete mitochondrial genome of Porites harrisoni (Cnidaria: Scleractinia) obtained using next-generation sequencing

    KAUST Repository

    Terraneo, Tullia Isotta

    2018-02-24

    In this study, we sequenced the complete mitochondrial genome of Porites harrisoni using ezRAD and Illumina technology. Genome length consisted of 18,630 bp, with a base composition of 25.92% A, 13.28% T, 23.06% G, and 37.73% C. Consistent with other hard corals, P. harrisoni mitogenome was arranged in 13 protein-coding genes, 2 rRNA, and 2 tRNA genes. nad5 and cox1 contained embedded Group I Introns of 11,133 bp and 965 bp, respectively.

  4. Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

    Science.gov (United States)

    Wickramasinghe, Susiji; Yatawara, Lalani; Nagataki, Mitsuru; Agatsuma, Takeshi

    2016-10-01

    To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K m value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K m value of the mutant (Serine to Glycine) increased to 0.19 mM. The K m value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T. canis has a seven-exon/six-intron gene

  5. Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

    Science.gov (United States)

    Kretsinger, R. H.; Nakayama, S.

    1993-01-01

    In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

  6. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract

    Energy Technology Data Exchange (ETDEWEB)

    Ogg, S C; Anderson, P; Wickens, M P [Univ. of Wisconsin, Madison (USA)

    1990-01-11

    Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intro. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, the authors determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. They demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3{prime} and 5{prime} sites as are used in C. elegans. The branch point used lies in the inserted sequences. They conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

  7. DNA barcode and identification of the varieties and provenances of Taiwan's domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences.

    Science.gov (United States)

    Lee, Shih-Chieh; Wang, Chia-Hsiang; Yen, Cheng-En; Chang, Chieh

    2017-04-01

    The major aim of made tea identification is to identify the variety and provenance of the tea plant. The present experiment used 113 tea plants [Camellia sinensis (L.) O. Kuntze] housed at the Tea Research and Extension Substation, from which 113 internal transcribed spacer 2 (ITS2) fragments, 104 trnL intron, and 98 trnL-trnF intergenic sequence region DNA sequences were successfully sequenced. The similarity of the ITS2 nucleotide sequences between tea plants housed at the Tea Research and Extension Substation was 0.379-0.994. In this polymerase chain reaction-amplified noncoding region, no varieties possessed identical sequences. Compared with the trnL intron and trnL-trnF intergenic sequence fragments of chloroplast cpDNA, the proportion of ITS2 nucleotide sequence variation was large and is more suitable for establishing a DNA barcode database to identify tea plant varieties. After establishing the database, 30 imported teas and 35 domestic made teas were used in this model system to explore the feasibility of using ITS2 sequences to identify the varieties and provenances of made teas. A phylogenetic tree was constructed using ITS2 sequences with the unweighted pair group method with arithmetic mean, which indicated that the same variety of tea plant is likely to be successfully categorized into one cluster, but contamination from other tea plants was also detected. This result provides molecular evidence that the similarity between important tea varieties in Taiwan remains high. We suggest a direct, wide collection of made tea and original samples of tea plants to establish an ITS2 sequence molecular barcode identification database to identify the varieties and provenances of tea plants. The DNA barcode comparison method can satisfy the need for a rapid, low-cost, frontline differentiation of the large amount of made teas from Taiwan and abroad, and can provide molecular evidence of their varieties and provenances. Copyright © 2016. Published by Elsevier B.V.

  8. Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China

    Directory of Open Access Journals (Sweden)

    Dong Hua Cao

    2014-09-01

    Full Text Available OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband’s cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

  9. Spliced RNA of woodchuck hepatitis virus.

    Science.gov (United States)

    Ogston, C W; Razman, D G

    1992-07-01

    Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

  10. In silico Coding Sequence Analysis of Walnut GAI and PIP2 Genes and Comparison with Different Plant Species

    Directory of Open Access Journals (Sweden)

    Mahdi Mohseniazar

    2017-02-01

    Full Text Available Introduction: Dwarfism is one of the important traits in breeding of crops and horticulture plants. A dwarfing rootstock will produce trees with 15-50% of standard trees size. In modern intensive fruit tree orchards, dwarfing rootstocks are commonly used to reduce trees size, enabling high-density planting and easy management, thus achieving higher yield. Trees on dwarfing rootstocks can also exhibit other economically important traits, such as precocious flowering, increased yield and increased disease resistance. Dwarf rootstocks have been extensively studied and released in stone and pome fruits, because of presence of genetic materials and the simplicity of budding methods. Control of tree size using genetically dwarf rootstocks for achievement to higher density and mechanized orchard systems is now very important for walnut production in the world especially in Iran. Many different genes can be involved in appear of this. Mutations in GAI and PIP2 genes cause dwarf trait by two different mechanisms in some plant species. In this case, we study in silico analysis of GAI and PIP2 genes consist of conserved sequences and domains, exon and intron number, function of their proteins, targeting, secondary and tertiary structure, and post translational modification. Materials and methods: The GAI and PIP2 mRNA and protein sequences (FASTA format belonging to 17 monocotyledon and dicotyledon were downloaded from NCBI (http://www.ncbi.nlm.nih.gov accessed, on September 2014. Several online web services and software were used for analysis of GAI and PIP2 mRNA and Proteins in plants. Comparative and bioinformatics analyses of PIP2 and GAI proteins were performed online at two websites NCBI (http://www.ncbi.nih.gov and EXPASY (http://expasy.org/tools. Molecular Evolutionary Genetics Analysis (MEGA; version 4 program and CLUSTAL-W with default parameters were used for multiple alignments of sequences. The phylogenetic analysis of GAI and PIP2 protein was

  11. In silico Coding Sequence Analysis of Walnut GAI and PIP2 Genes and Comparison with Different Plant Species

    Directory of Open Access Journals (Sweden)

    Mahdi Mohseniazar

    2017-09-01

    Full Text Available Introduction: Dwarfism is one of the important traits in breeding of crops and horticulture plants. A dwarfing rootstock will produce trees with 15-50% of standard trees size. In modern intensive fruit tree orchards, dwarfing rootstocks are commonly used to reduce trees size, enabling high-density planting and easy management, thus achieving higher yield. Trees on dwarfing rootstocks can also exhibit other economically important traits, such as precocious flowering, increased yield and increased disease resistance. Dwarf rootstocks have been extensively studied and released in stone and pome fruits, because of presence of genetic materials and the simplicity of budding methods. Control of tree size using genetically dwarf rootstocks for achievement to higher density and mechanized orchard systems is now very important for walnut production in the world especially in Iran. Many different genes can be involved in appear of this. Mutations in GAI and PIP2 genes cause dwarf trait by two different mechanisms in some plant species. In this case, we study in silico analysis of GAI and PIP2 genes consist of conserved sequences and domains, exon and intron number, function of their proteins, targeting, secondary and tertiary structure, and post translational modification. Materials and methods: The GAI and PIP2 mRNA and protein sequences (FASTA format belonging to 17 monocotyledon and dicotyledon were downloaded from NCBI (http://www.ncbi.nlm.nih.gov accessed, on September 2014. Several online web services and software were used for analysis of GAI and PIP2 mRNA and Proteins in plants. Comparative and bioinformatics analyses of PIP2 and GAI proteins were performed online at two websites NCBI (http://www.ncbi.nih.gov and EXPASY (http://expasy.org/tools. Molecular Evolutionary Genetics Analysis (MEGA; version 4 program and CLUSTAL-W with default parameters were used for multiple alignments of sequences. The phylogenetic analysis of GAI and PIP2 protein was

  12. A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report

    Directory of Open Access Journals (Sweden)

    Asakawa Takeshi

    2012-03-01

    Full Text Available Abstract Background Von Hippel-Lindau disease (VHL is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH of the VHL gene is common in squamous cell carcinoma tissues of the tongue. Case Presentation We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C revealed a deletion of the mutant C allele in the carcinoma tissues. Conclusions The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

  13. Probable Diagnosis of a Patient with Niemann-Pick Disease Type C: Managing Pitfalls of Exome Sequencing.

    Science.gov (United States)

    Zeiger, William A; Jamal, Nasheed I; Scheuner, Maren T; Pittman, Patricia; Raymond, Kimiyo M; Morra, Massimo; Mishra, Shri K

    2018-02-17

    Here, we present a case of a 31-year-old man with progressive cognitive decline, ataxia, and dystonia. Extensive laboratory, radiographic, and targeted genetic studies over the course of several years failed to yield a diagnosis. Initial whole exome sequencing through a commercial laboratory identified several variants of uncertain significance; however, follow-up clinical examination and testing ruled each of these out. Eventually, repeat whole exome sequencing identified a known pathogenic intronic variant in the NPC1 gene (NM_000271.4, c.1554-1009G>A) and an additional heterozygous exonic variant of uncertain significance in the NPC1 gene (NM_000271.4, c.2524T>C). Follow-up biochemical testing was consistent with a diagnosis of probable Niemann-Pick disease Type C (NP-C). This case illustrates the potential of whole exome sequencing for diagnosing rare complex neurologic diseases. It also identifies several potential common pitfalls that must be navigated by clinicians when interpreting commercial whole exome sequencing results.

  14. mCSF1, a nucleus-encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis.

    Science.gov (United States)

    Zmudjak, Michal; Colas des Francs-Small, Catherine; Keren, Ido; Shaya, Felix; Belausov, Eduard; Small, Ian; Ostersetzer-Biran, Oren

    2013-07-01

    The coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized. CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized. Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants. Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  15. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

    Directory of Open Access Journals (Sweden)

    El Andaloussi Samir

    2011-05-01

    Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

  16. Comparative genomic survey, exon-intron annotation and phylogenetic analysis of NAT-homologous sequences in archaea, protists, fungi, viruses, and invertebrates

    Science.gov (United States)

    We have previously published extensive genomic surveys [1-3], reporting NAT-homologous sequences in hundreds of sequenced bacterial, fungal and vertebrate genomes. We present here the results of our latest search of 2445 genomes, representing 1532 (70 archaeal, 1210 bacterial, 43 protist, 97 fungal,...

  17. MRI of spondylodiscities: contribution of gadolinium-DTPA and fat suppression sequence

    International Nuclear Information System (INIS)

    Cova, M.A.; Dalla Palma, L.; Pozzi-Mucelli, R.S.; Ricci, C.

    1993-01-01

    Twenty-six patients with a clinical diagnosis of spondylodiscitis were examined with non-contrast and contrast-enhanced MRI in order to define the contribution of gadolinium-DTPA (Gd-DTPA) and different pulse sequences, including a fat suppression sequence (SPIR). Spin echo (SE) T1-weighted images before and after Gd-DTPA injection and SE T2-weighted images were obtained in all patients. Twelve patients were also examined using the SPIR sequence following Gd-DTPA injection. Signal intensity and morphological features of the disc and vertebral lesions were then evaluated. The SE T1-weighted sequence with Gd-DTPA was very effective in showing the pathological changes at the level of the disc as an area of low signal intensity surrounded by a peripheral rim of enhancement in 24 of 26 cases (92%). This feature was not visible on non-enhanced images. As regards contiguous vertebral lesions this sequence was less informative, since in 8 of 26 cases (31%) the vertebral lesions became isointense and therefore not detectable. In 12 cases there was extension into the surrounding structures (spinal canal and/or paravertebral tissues). An enhanced SE T1-weighted sequence provided good anatomical definition of the extension of the infection in the spinal canal in all cases with this type of involvement (7 of 12). Regarding the 7 cases with paravertebral extension, no extension was visible in 1 case due to the reduced contrast with the surrounding fat following Gd-DTPA injection. The enhanced SPIR sequence was very effective, particularly in detecting the lesions in the vertebral bodies, avoiding the limitation of the enhanced SE T1-weighted sequence. The SPIR sequence was also effective in showing the extension within the spinal canal and the paravertebral fat. On the basis of our results the combination of a SE T1-weighted sequence without contrast and SPIR sequence with Gd-DTPA seems to be the best approach in cases of spondylodiscitis. (orig.)

  18. Unexpected diversity in the mobilome of a Pseudomonas aeruginosa strain isolated from a dental unit waterline revealed by SMRT Sequencing.

    Science.gov (United States)

    Vincent, Antony T; Charette, Steve J; Barbeau, Jean

    2018-05-01

    The Gram-negative bacterium Pseudomonas aeruginosa is found in several habitats, both natural and human-made, and is particularly known for its recurrent presence as a pathogen in the lungs of patients suffering from cystic fibrosis, a genetic disease. Given its clinical importance, several major studies have investigated the genomic adaptation of P. aeruginosa in lungs and its transition as acute infections become chronic. However, our knowledge about the diversity and adaptation of the P. aeruginosa genome to non-clinical environments is still fragmentary, in part due to the lack of accurate reference genomes of strains from the numerous environments colonized by the bacterium. Here, we used PacBio long-read technology to sequence the genome of PPF-1, a strain of P. aeruginosa isolated from a dental unit waterline. Generating this closed genome was an opportunity to investigate genomic features that are difficult to accurately study in a draft genome (contigs state). It was possible to shed light on putative genomic islands, some shared with other reference genomes, new prophages, and the complete content of insertion sequences. In addition, four different group II introns were also found, including two characterized here and not listed in the specialized group II intron database.

  19. A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

    Science.gov (United States)

    Ben Khalaf, Noureddine; Taha, Safa; Bakhiet, Moiz; Fathallah, M Dahmani

    2016-01-01

    The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.

  20. A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

    Directory of Open Access Journals (Sweden)

    Noureddine Ben Khalaf

    Full Text Available The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV, with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416 of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.

  1. Molecular characterization, sequence analysis and tissue expression of a porcine gene – MOSPD2

    Directory of Open Access Journals (Sweden)

    Yang Jie

    2017-01-01

    Full Text Available The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2 of five species: horse (89%, human (90%, chimpanzee (89%, rhesus monkey (89% and mouse (85%; thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.

  2. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

    Lin, Dong; Shi, Y.; Miller, W.L.

    1990-01-01

    Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

  3. Smart Surroundings

    NARCIS (Netherlands)

    Havinga, Paul J.M.; Jansen, P.G.; Lijding, M.E.M.; Scholten, Johan

    2004-01-01

    Ambient systems are networked embedded systems integrated with everyday environments and supporting people in their activities. These systems will create a Smart Surrounding for people to facilitate and enrich daily life and increase productivity at work. Such systems will be quite different from

  4. A comparison of microbial communities in deep-sea polymetallic nodules and the surrounding sediments in the Pacific Ocean

    Science.gov (United States)

    Wu, Yue-Hong; Liao, Li; Wang, Chun-Sheng; Ma, Wei-Lin; Meng, Fan-Xu; Wu, Min; Xu, Xue-Wei

    2013-09-01

    Deep-sea polymetallic nodules, rich in metals such as Fe, Mn, and Ni, are potential resources for future exploitation. Early culturing and microscopy studies suggest that polymetallic nodules are at least partially biogenic. To understand the microbial communities in this environment, we compared microbial community composition and diversity inside nodules and in the surrounding sediments. Three sampling sites in the Pacific Ocean containing polymetallic nodules were used for culture-independent investigations of microbial diversity. A total of 1013 near full-length bacterial 16S rRNA gene sequences and 640 archaeal 16S rRNA gene sequences with ~650 bp from nodules and the surrounding sediments were analyzed. Bacteria showed higher diversity than archaea. Interestingly, sediments contained more diverse bacterial communities than nodules, while the opposite was detected for archaea. Bacterial communities tend to be mostly unique to sediments or nodules, with only 13.3% of sequences shared. The most abundant bacterial groups detected only in nodules were Pseudoalteromonas and Alteromonas, which were predicted to play a role in building matrix outside cells to induce or control mineralization. However, archaeal communities were mostly shared between sediments and nodules, including the most abundant OTU containing 290 sequences from marine group I Thaumarchaeota. PcoA analysis indicated that microhabitat (i.e., nodule or sediment) seemed to be a major factor influencing microbial community composition, rather than sampling locations or distances between locations.

  5. Evolution of the tRNALeu (UAA) Intron and Congruence of Genetic Markers in Lichen-Symbiotic Nostoc.

    Science.gov (United States)

    Kaasalainen, Ulla; Olsson, Sanna; Rikkinen, Jouko

    2015-01-01

    The group I intron interrupting the tRNALeu UAA gene (trnL) is present in most cyanobacterial genomes as well as in the plastids of many eukaryotic algae and all green plants. In lichen symbiotic Nostoc, the P6b stem-loop of trnL intron always involves one of two different repeat motifs, either Class I or Class II, both with unresolved evolutionary histories. Here we attempt to resolve the complex evolution of the two different trnL P6b region types. Our analysis indicates that the Class II repeat motif most likely appeared first and that independent and unidirectional shifts to the Class I motif have since taken place repeatedly. In addition, we compare our results with those obtained with other genetic markers and find strong evidence of recombination in the 16S rRNA gene, a marker widely used in phylogenetic studies on Bacteria. The congruence of the different genetic markers is successfully evaluated with the recently published software Saguaro, which has not previously been utilized in comparable studies.

  6. Evolution of the tRNALeu (UAA Intron and Congruence of Genetic Markers in Lichen-Symbiotic Nostoc.

    Directory of Open Access Journals (Sweden)

    Ulla Kaasalainen

    Full Text Available The group I intron interrupting the tRNALeu UAA gene (trnL is present in most cyanobacterial genomes as well as in the plastids of many eukaryotic algae and all green plants. In lichen symbiotic Nostoc, the P6b stem-loop of trnL intron always involves one of two different repeat motifs, either Class I or Class II, both with unresolved evolutionary histories. Here we attempt to resolve the complex evolution of the two different trnL P6b region types. Our analysis indicates that the Class II repeat motif most likely appeared first and that independent and unidirectional shifts to the Class I motif have since taken place repeatedly. In addition, we compare our results with those obtained with other genetic markers and find strong evidence of recombination in the 16S rRNA gene, a marker widely used in phylogenetic studies on Bacteria. The congruence of the different genetic markers is successfully evaluated with the recently published software Saguaro, which has not previously been utilized in comparable studies.

  7. (rpS16) intron sequences in Morus

    African Journals Online (AJOL)

    use

    2011-12-05

    Dec 5, 2011 ... morphological classification. Keywords: ... economically important plant in the sericulture industry and ..... A dictionary of flowering plants and ferns, 8th edition ... relative rates of nucleotide and insertion/deletion evolution at six.

  8. Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells

    DEFF Research Database (Denmark)

    Bruun, Gitte H; Bang, Jeanne Mv; Christensen, Lise L

    2018-01-01

    designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific...

  9. A branch point consensus from Arabidopsis found by non-circular analysis allows for better prediction of acceptor sites

    DEFF Research Database (Denmark)

    Tolstrup, Niels; Rouzé, Pierre; Brunak, Søren

    1997-01-01

    Little knowledge exists about branch points in plants; it has even been claimed that plant introns lack conserved branch point sequences similar to those found in vertebrate introns. A putative branch point consensus sequence for Arabidopsis thaliana resembling the well known metazoan consensus s...... in the recognition of true acceptor sites; the false positive rate being reduced by a factor of 2. We take this as an indication that the consensus found here is the genuine one and that the branch point does play a role in the proper recognition of the acceptor site in plants.......Little knowledge exists about branch points in plants; it has even been claimed that plant introns lack conserved branch point sequences similar to those found in vertebrate introns. A putative branch point consensus sequence for Arabidopsis thaliana resembling the well known metazoan consensus...... sequence has been proposed, but this is based on search of sequences similar to those in yeast and metazoa. Here we present a novel consensus sequence found by a non-circular approach. A hidden Markov model with a fixed A nucleotide was trained on sequences upstream of the acceptor site. The consensus...

  10. Intronic variants in the dopa decarboxylase (DDC) gene are associated with smoking behavior in European-Americans and African-Americans.

    Science.gov (United States)

    Yu, Yi; Panhuysen, Carolien; Kranzler, Henry R; Hesselbrock, Victor; Rounsaville, Bruce; Weiss, Roger; Brady, Kathleen; Farrer, Lindsay A; Gelernter, Joel

    2006-07-15

    We report here a study considering association of alleles and haplotypes at the DOPA decarboxylase (DDC) locus with the DSM-IV diagnosis of nicotine dependence (ND) or a quantitative measure for ND using the Fagerstrom Test for Nicotine Dependence (FTND). We genotyped 18 single nucleotide polymorphisms (SNPs) spanning a region of approximately 210 kb that includes DDC and the genes immediately flanking DDC in 1,590 individuals from 621 families of African-American (AA) or European-American (EA) ancestry. Evidence of association (family-based tests) was observed with several SNPs for both traits (0.0002DDC lacking exons 10-15. Haplotype analysis did not reveal any SNP combination with stronger evidence for association than rs12718541 alone. Although sequence analysis suggests that rs12718541 may be an intronic splicing enhancer, further studies are needed to determine whether a direct link exists between an alternatively spliced form of DDC and predisposition to ND. These findings confirm a previous report of association of DDC with ND, localize the causative variants to the 3' end of the coding region and extend the association to multiple population groups.

  11. Degradation of YRA1 Pre-mRNA in the cytoplasm requires translational repression, multiple modular intronic elements, Edc3p, and Mex67p.

    Directory of Open Access Journals (Sweden)

    Shuyun Dong

    2010-04-01

    Full Text Available Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron. Two of these elements target the pre-mRNA as an Edc3p substrate and the other three mediate transcript-specific translational repression. Translational repression of YRA1 pre-mRNA also requires the heterodimeric Mex67p/Mtr2p general mRNA export receptor, but not Edc3p, and serves to enhance Edc3p substrate specificity by inhibiting the susceptibility of this pre-mRNA to NMD. Collectively, our data indicate that YRA1 pre-mRNA degradation is a highly regulated process that proceeds through translational repression, substrate recognition by Edc3p, recruitment of the Dcp1p/Dcp2p decapping enzyme, and activation of decapping.

  12. Sequence Classification: 894064 [

    Lifescience Database Archive (English)

    Full Text Available al protein; intron of RPL18A pre-mRNA forms stem-loop structures that are a target for Rnt1p cleavage leading to degradation; Rpl18ap || http://www.ncbi.nlm.nih.gov/protein/6324452 ...

  13. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  14. Sequencing Intractable DNA to Close Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Hurt, Jr., Richard Ashley [ORNL; Brown, Steven D [ORNL; Podar, Mircea [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  15. Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

    Science.gov (United States)

    Li, Wencheng; You, Bei; Hoque, Mainul; Zheng, Dinghai; Luo, Wenting; Ji, Zhe; Park, Ji Yeon; Gunderson, Samuel I.; Kalsotra, Auinash; Manley, James L.; Tian, Bin

    2015-01-01

    Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors. PMID:25906188

  16. Systematic profiling of poly(A+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

    Directory of Open Access Journals (Sweden)

    Wencheng Li

    2015-04-01

    Full Text Available Alternative cleavage and polyadenylation (APA results in mRNA isoforms containing different 3' untranslated regions (3'UTRs and/or coding sequences. How core cleavage/polyadenylation (C/P factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A sites (pAs, CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

  17. cDNA, genomic sequence cloning, and overexpression of EIF1 from the giant panda (Ailuropoda Melanoleuca) and the black bear (Ursus Thibetanus Mupinensis).

    Science.gov (United States)

    Hou, Wan-ru; Tang, Yun; Hou, Yi-ling; Song, Yan; Zhang, Tian; Wu, Guang-fu

    2010-07-01

    Eukaryotic initiation factor (eIF) EIF1 is a universally conserved translation factor that is involved in translation initiation site selection. The cDNA and the genomic sequences of EIF1 were cloned successfully from the giant panda (Ailuropoda melanoleuca) and the black bear (Ursus thibetanus mupinensis) using reverse transcription polymerase chain reaction (RT-PCR) technology and touchdown-polymerase chain reaction, respectively. The cDNAs of the EIF1 cloned from the giant panda and the black bear are 418 bp in size, containing an open reading frame (ORF) of 342 bp encoding 113 amino acids. The length of the genomic sequence of the giant panda is 1909 bp, which contains four exons and three introns. The length of the genomic sequence of the black bear is 1897 bp, which also contains four exons and three introns. Sequence alignment indicates a high degree of homology to those of Homo sapiens, Mus musculus, Rattus norvegicus, and Bos Taurus at both amino acid and DNA levels. Topology prediction shows there are one N-glycosylation site, two Casein kinase II phosphorylation sites, and a Amidation site in the EIF1 protein of the giant panda and black bear. In addition, there is a protein kinase C phosphorylation site in EIF1 of the giant panda. The giant panda and the black bear EIF1 genes were overexpressed in E. coli BL21. The results indicated that the both EIF1 fusion proteins with the N-terminally His-tagged form gave rise to the accumulation of two expected 19 kDa polypeptide. The expression products obtained could be used to purify the proteins and study their function further.

  18. Use of a culture independent method to analyze the diversity of soil fungi surrounding Chroogomphus rutilus in the Beijing region of China

    DEFF Research Database (Denmark)

    Liu, Yu; Wang, Shouxian; Yin, Yonggang

    2012-01-01

    habitat to facilitate its large-scale cultivation. A culture-independent molecular approach—a powerful technology for microbiological ecology studies—was used to investigate the diversity of soil fungal communities in samples surrounding C. rutilus from the Beijing region of China. Metagenomic DNA...... was isolated from soil samples collected around C. rutilus, and an internal transcribed spacer (ITS) gene library was constructed. Subsequently, polymerase chain reaction products were digested with HinfI, HaeIII, MspI, TaqI, or MboI. Clones were selected and sequenced based on their restriction fragment...... length polymorphisms. The diversity of the fungi represented by their ITS sequences was analyzed. Our results indicate the presence of numerous fungi in the C. rutilus habitat. This study is the first demonstration of the fungal ecology surrounding C. rutilus using a culture independent method...

  19. Phylogenetics and Gene Structure Dynamics of Polygalacturonase Genes in Aspergillus and Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Jin-Sung Hong

    2013-09-01

    Full Text Available Polygalacturonase (PG gene is a typical gene family present in eukaryotes. Forty-nine PGs were mined from the genomes of Neurospora crassa and five Aspergillus species. The PGs were classified into 3 clades such as clade 1 for rhamno-PGs, clade 2 for exo-PGs and clade 3 for exo- and endo-PGs, which were further grouped into 13 sub-clades based on the polypeptide sequence similarity. In gene structure analysis, a total of 124 introns were present in 44 genes and five genes lacked introns to give an average of 2.5 introns per gene. Intron phase distribution was 64.5% for phase 0, 21.8% for phase 1, and 13.7% for phase 2, respectively. The introns varied in their sequences and their lengths ranged from 20 bp to 424 bp with an average of 65.9 bp, which is approximately half the size of introns in other fungal genes. There were 29 homologous intron blocks and 26 of those were sub-clade specific. Intron losses were counted in 18 introns in which no obvious phase preference for intron loss was observed. Eighteen introns were placed at novel positions, which is considerably higher than those of plant PGs. In an evolutionary sense both intron loss and gain must have taken place for shaping the current PGs in these fungi. Together with the small intron size, low conservation of homologous intron blocks and higher number of novel introns, PGs of fungal species seem to have recently undergone highly dynamic evolution.

  20. A deep intronic CLRN1 (USH3A) founder mutation generates an aberrant exon and underlies severe Usher syndrome on the Arabian Peninsula.

    Science.gov (United States)

    Khan, Arif O; Becirovic, Elvir; Betz, Christian; Neuhaus, Christine; Altmüller, Janine; Maria Riedmayr, Lisa; Motameny, Susanne; Nürnberg, Gudrun; Nürnberg, Peter; Bolz, Hanno J

    2017-05-03

    Deafblindness is mostly due to Usher syndrome caused by recessive mutations in the known genes. Mutation-negative patients therefore either have distinct diseases, mutations in yet unknown Usher genes or in extra-exonic parts of the known genes - to date a largely unexplored possibility. In a consanguineous Saudi family segregating Usher syndrome type 1 (USH1), NGS of genes for Usher syndrome, deafness and retinal dystrophy and subsequent whole-exome sequencing each failed to identify a mutation. Genome-wide linkage analysis revealed two small candidate regions on chromosome 3, one containing the USH3A gene CLRN1, which has never been associated with Usher syndrome in Saudi Arabia. Whole-genome sequencing (WGS) identified a homozygous deep intronic mutation, c.254-649T > G, predicted to generate a novel donor splice site. CLRN1 minigene-based analysis confirmed the splicing of an aberrant exon due to usage of this novel motif, resulting in a frameshift and a premature termination codon. We identified this mutation in an additional two of seven unrelated mutation-negative Saudi USH1 patients. Locus-specific markers indicated that c.254-649T > G CLRN1 represents a founder allele that may significantly contribute to deafblindness in this population. Our finding underlines the potential of WGS to uncover atypically localized, hidden mutations in patients who lack exonic mutations in the known disease genes.

  1. DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

    Science.gov (United States)

    Wahls, Wayne P.; Davidson, Mari K.

    2011-01-01

    Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

  2. High frequency of the IVS2-2A>G DNA sequence variation in SLC26A5, encoding the cochlear motor protein prestin, precludes its involvement in hereditary hearing loss

    Directory of Open Access Journals (Sweden)

    Pereira Fred A

    2005-08-01

    Full Text Available Abstract Background Cochlear outer hair cells change their length in response to variations in membrane potential. This capability, called electromotility, is believed to enable the sensitivity and frequency selectivity of the mammalian cochlea. Prestin is a transmembrane protein required for electromotility. Homozygous prestin knockout mice are profoundly hearing impaired. In humans, a single nucleotide change in SLC26A5, encoding prestin, has been reported in association with hearing loss. This DNA sequence variation, IVS2-2A>G, occurs in the exon 3 splice acceptor site and is expected to abolish splicing of exon 3. Methods To further explore the relationship between hearing loss and the IVS2-2A>G transition, and assess allele frequency, genomic DNA from hearing impaired and control subjects was analyzed by DNA sequencing. SLC26A5 genomic DNA sequences from human, chimp, rat, mouse, zebrafish and fruit fly were aligned and compared for evolutionary conservation of the exon 3 splice acceptor site. Alternative splice acceptor sites within intron 2 of human SLC26A5 were sought using a splice site prediction program from the Berkeley Drosophila Genome Project. Results The IVS2-2A>G variant was found in a heterozygous state in 4 of 74 hearing impaired subjects of Hispanic, Caucasian or uncertain ethnicity and 4 of 150 Hispanic or Caucasian controls (p = 0.45. The IVS2-2A>G variant was not found in 106 subjects of Asian or African American descent. No homozygous subjects were identified (n = 330. Sequence alignment of SLC26A5 orthologs demonstrated that the A nucleotide at position IVS2-2 is invariant among several eukaryotic species. Sequence analysis also revealed five potential alternative splice acceptor sites in intron 2 of human SLC26A5. Conclusion These data suggest that the IVS2-2A>G variant may not occur more frequently in hearing impaired subjects than in controls. The identification of five potential alternative splice acceptor sites in

  3. Isolation, structural determination, synthesis and quantitative determination of impurities in Intron-A, leached from a silicone tubing.

    Science.gov (United States)

    Chan, Tze-Ming; Pramanik, Birendra; Aslanian, Robert; Gullo, Vincent; Patel, Mahesh; Cronin, Bart; Boyce, Chris; McCormick, Kevin; Berlin, Mike; Zhu, Xiaohong; Buevich, Alexei; Heimark, Larry; Bartner, Peter; Chen, Guodong; Pu, Haiyan; Hegde, Vinod

    2009-02-20

    Investigation of unexpected levels of impurities in Intron product has revealed the presence of low levels of impurities leached from the silicone tubing (Rehau RAU-SIK) on the Bosch filling line. In order to investigate the effect of these compounds (1a, 1b and 2) on humans, they were isolated identified and synthesized. They were extracted from the tubing by stirring in Intron placebo at room temperature for 72 h and were enriched on a reverse phase CHP-20P column, eluting with gradient aqueous ACN and were separated by HPLC. Structural elucidation of 1a, 1b and 2 by MS and NMR studies demonstrated them to be halogenated biphenyl carboxylic acids. The structures were confirmed by independent synthesis. Levels of extractable impurities in first filled vials of actual production are estimated to be in the range of 0.01-0.55 microg/vial for each leached impurity. Potential toxicity of these extractables does not represent a risk for patients under the conditions of clinical use.

  4. Detecting change in stochastic sound sequences.

    Directory of Open Access Journals (Sweden)

    Benjamin Skerritt-Davis

    2018-05-01

    Full Text Available Our ability to parse our acoustic environment relies on the brain's capacity to extract statistical regularities from surrounding sounds. Previous work in regularity extraction has predominantly focused on the brain's sensitivity to predictable patterns in sound sequences. However, natural sound environments are rarely completely predictable, often containing some level of randomness, yet the brain is able to effectively interpret its surroundings by extracting useful information from stochastic sounds. It has been previously shown that the brain is sensitive to the marginal lower-order statistics of sound sequences (i.e., mean and variance. In this work, we investigate the brain's sensitivity to higher-order statistics describing temporal dependencies between sound events through a series of change detection experiments, where listeners are asked to detect changes in randomness in the pitch of tone sequences. Behavioral data indicate listeners collect statistical estimates to process incoming sounds, and a perceptual model based on Bayesian inference shows a capacity in the brain to track higher-order statistics. Further analysis of individual subjects' behavior indicates an important role of perceptual constraints in listeners' ability to track these sensory statistics with high fidelity. In addition, the inference model facilitates analysis of neural electroencephalography (EEG responses, anchoring the analysis relative to the statistics of each stochastic stimulus. This reveals both a deviance response and a change-related disruption in phase of the stimulus-locked response that follow the higher-order statistics. These results shed light on the brain's ability to process stochastic sound sequences.

  5. Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene

    Science.gov (United States)

    Gastaldo, Elena; Harries, Lorna W.; Rubio-Cabezas, Oscar; Castaño, Luis

    2012-01-01

    Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. PMID:22235272

  6. Molecular study in children with hemophilia A in Colombia: analysis of Intron 1 and 22 inversion using long-distance PCR technique

    Directory of Open Access Journals (Sweden)

    María Fernanda Garcés

    2017-04-01

    Conclusions: Inversions of intron 22 and 1 were found in half of this group of patients. These results are reproducible and useful to identify the two most frequent mutations in severe hemophilia A patients.

  7. Purifying selection acts on coding and non-coding sequences of paralogous genes in Arabidopsis thaliana.

    Science.gov (United States)

    Hoffmann, Robert D; Palmgren, Michael

    2016-06-13

    Whole-genome duplications in the ancestors of many diverse species provided the genetic material for evolutionary novelty. Several models explain the retention of paralogous genes. However, how these models are reflected in the evolution of coding and non-coding sequences of paralogous genes is unknown. Here, we analyzed the coding and non-coding sequences of paralogous genes in Arabidopsis thaliana and compared these sequences with those of orthologous genes in Arabidopsis lyrata. Paralogs with lower expression than their duplicate had more nonsynonymous substitutions, were more likely to fractionate, and exhibited less similar expression patterns with their orthologs in the other species. Also, lower-expressed genes had greater tissue specificity. Orthologous conserved non-coding sequences in the promoters, introns, and 3' untranslated regions were less abundant at lower-expressed genes compared to their higher-expressed paralogs. A gene ontology (GO) term enrichment analysis showed that paralogs with similar expression levels were enriched in GO terms related to ribosomes, whereas paralogs with different expression levels were enriched in terms associated with stress responses. Loss of conserved non-coding sequences in one gene of a paralogous gene pair correlates with reduced expression levels that are more tissue specific. Together with increased mutation rates in the coding sequences, this suggests that similar forces of purifying selection act on coding and non-coding sequences. We propose that coding and non-coding sequences evolve concurrently following gene duplication.

  8. Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome

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    Olatz Villate

    2018-01-01

    Full Text Available Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

  9. Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome.

    Science.gov (United States)

    Villate, Olatz; Ibarluzea, Nekane; Fraile-Bethencourt, Eugenia; Valenzuela, Alberto; Velasco, Eladio A; Grozeva, Detelina; Raymond, F L; Botella, María P; Tejada, María-Isabel

    2018-01-01

    Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD ® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.

  10. Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

    Science.gov (United States)

    Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

  11. Use of intron-exonic marker in assessment of genetic diversity of two subspecies of Thymus daenensis

    Directory of Open Access Journals (Sweden)

    Ahmad Ismaili

    2013-11-01

    Full Text Available Study of genetic diversity in medicinal plant is very important for improvement and evolutionary variations. In this study, assessment of genetic diversity in two subspecies of Thymus daenensis was evaluated, using intron-exonic markers. Thirty primers produced 633 polymorphic bands (98% polymorphism. The highest polymorphic information content (PIC included ISJ5 and ISJ9 primers and the lowest PIC also included IT15-32 primer. The highest marker index (MI produced by IT10-6 primer. Results of Analysis of Molecular Variance (AMOVA showed that intra-sub specific variation was more than inter-sub specific variation. Dendrogram obtained from Cluster analysis, using NTSYS-pc software and UPGMA method based on Dice's similarity matrix, divided accessions into 4 groups. The maximum range of genetic similarity was observed between two accessions of sub-species daenensis. Two accessions of Fars and Semnan formed a separate group. Results showed that clustering based on molecular data and principal coordinate analysis had a medium alignment. Grouping based on cluster analysis also could separate two subspecies of Thymus daenensis. Results obtained from this study showed that intron-exonic markers had an effective potential in assessment of genetic relationships between the two sub-species of daenensis.

  12. Genetic alteration with variable intron/exon organization amongst five PI-homoeologous genes in Platanus acerifolia.

    Science.gov (United States)

    Zhang, Jiaqi; Guo, Cong; Liu, Guofeng; Li, Zhineng; Li, Xiaomei; Bao, Manzhu

    2011-03-01

    Flower development has been extensively characterized in the model species Arabidopsis thaliana and Antirrhinum majus. However, there have been few studies in woody species. Here, we report the isolation and characterization of five PISTILLATA (PI) homoeologous genes (PaPI1-to-5) from the London Plane tree (Platanus acerifolia Willd). PaPI1 and PaPI2 show a similar genomic structure to other known PI homoeologs, but PaPI3/4/5 lack intron sequences. In addition, PaPI5 lacks the third, fourth and fifth exons which encode the K-domain. These altered gene copies may have originated as 'processed' retrogenes. PaPI2 appears micro-regulated by alternative splicing, displaying three splice forms (PaPI2a, PaPI2b and PaPI2c). RT-PCR analysis showed different expression profiles and transcript abundance for the five PaPI genes. PaPI transcripts encoding full-length polypeptides were expressed predominantly in male/female inflorescences and PaPI2a was the most abundant transcript (59%) indicating that PaPI2 may be the major functional PI-homoeolog in London Plane. Phenotypic characterization in a heterologous expression system demonstrated that the full-length PaPI product functions as a B class gene. By contrast the PaPI5 form, which lacks the K-domain, had no apparent effect on flower development. In vitro studies also demonstrated that the K-domain is required to form PaPI/PaAP3 heterodimers. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. A BRCA2 mutation incorrectly mapped in the original BRCA2 reference sequence, is a common West Danish founder mutation disrupting mRNA splicing

    DEFF Research Database (Denmark)

    Thomassen, Mads; Pedersen, Inge Søkilde; Vogel, Ida

    2011-01-01

    Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose carriers to breast and ovarian cancer. The authors have identified a mutation in BRCA2, 7845+1G>A (c.7617+1G>A), not previously regarded as deleterious because of incorrect mapping of the splice junction in the originally...... published genomic reference sequence. This reference sequence is generally used in many laboratories and it maps the mutation 16 base pairs inside intron 15. However, according to the recent reference sequences the mutation is located in the consensus donor splice sequence. By reverse transcriptase analysis......, loss of exon 15 in the final transcript interrupting the open reading frame was demonstrated. Furthermore, the mutation segregates with a cancer phenotype in 18 Danish families. By genetic analysis of more than 3,500 Danish breast/ovarian cancer risk families, the mutation was identified as the most...

  14. Changes in unique hues induced by chromatic surrounds.

    Science.gov (United States)

    Klauke, Susanne; Wachtler, Thomas

    2016-03-01

    A chromatic surround can have a strong influence on the perceived hue of a stimulus. We investigated whether chromatic induction has similar effects on the perception of colors that appear pure and unmixed (unique red, green, blue, and yellow) as on other colors. Subjects performed unique hue settings of stimuli in isoluminant surrounds of different chromaticities. Compared with the settings in a neutral gray surround, unique hue settings altered systematically with chromatic surrounds. The amount of induced hue shift depended on the difference between stimulus and surround hues, and was similar for unique hue settings as for settings of nonunique hues. Intraindividual variability in unique hue settings was roughly twice as high as for settings obtained in asymmetric matching experiments, which may reflect the presence of a reference stimulus in the matching task. Variabilities were also larger with chromatic surrounds than with neutral gray surrounds, for both unique hue settings and matching of nonunique hues. The results suggest that the neural representations underlying unique hue percepts are influenced by the same neural processing mechanisms as the percepts of other colors.

  15. Comparative analysis of myostatin gene and promoter sequences of Qinchuan and Red Angus cattle.

    Science.gov (United States)

    He, Y L; Wu, Y H; Quan, F S; Liu, Y G; Zhang, Y

    2013-09-04

    To better understand the function of the myostatin gene and its promoter region in bovine, we amplified and sequenced the myostatin gene and promoter from the blood of Qinchuan and Red Angus cattle by using polymerase chain reaction. The sequences of Qinchuan and Red Angus cattle were compared with those of other cattle breeds available in GenBank. Exon splice sites were confirmed by mRNA sequencing. Compared to the published sequence (GenBank accession No. AF320998), 69 single nucleotide polymorphisms (SNPs) were identified in the Qinchuan myostatin gene, only one of which was an insertion mutation in Qinchuan cattle. There was a 16-bp insertion in the first 705-bp intron in 3 Qinchuan cattle. A total of 7 SNPs were identified in exon 3, in which the mutation occurred in the third base of the codon and was synonymous. On comparing the Qinchuan myostatin gene sequence to that of Red Angus cattle, a total of 50 SNPs were identified in the first and third exons. In addition, there were 18 SNPs identified in the Qinchuan cattle promoter region compared with those of other cattle compared to the Red Angus cattle myostatin promoter region. breeds (GenBank accession No. AF348479), but only 14 SNPs when compared to the Red Angus cattle myostatin promoter region.

  16. Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

    Energy Technology Data Exchange (ETDEWEB)

    Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

    2003-06-01

    OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.

  17. Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencing during primate evolution

    Energy Technology Data Exchange (ETDEWEB)

    Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.; Bertina, R.M. (University Hospital, Leiden (Netherlands))

    1990-08-28

    The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.

  18. Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.

    Science.gov (United States)

    Pan, Tzu-Yu; Chiou, Shyh-Shin; Wang, Chun-Chi; Wu, Shou-Mei

    2014-12-01

    An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

    Directory of Open Access Journals (Sweden)

    Mattiuz Pier

    2005-10-01

    Full Text Available Abstract Background The minor histocompatibility antigens (mHags are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.

  20. The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

    Directory of Open Access Journals (Sweden)

    Lemieux Claude

    2006-02-01

    Full Text Available Abstract Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae, in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR featuring an inverted rRNA operon and a small single-copy (SSC region containing 14 genes normally found in the large single-copy (LSC region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of

  1. Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA

    International Nuclear Information System (INIS)

    Indik, Z.; Yeh, H.; Ornstein-goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J.C.; Peltonen, L.; Rosenbloom, J.

    1987-01-01

    Poly(A) + RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into λgt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: (i) the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, (ii) exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and (iii) a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population

  2. Amino acid substitutions and intron polymorphism of acetylcholinesterase1 associated with mevinphos resistance in diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    Yeh, Shih-Chia; Lin, Chia-Li; Chang, Cheng; Feng, Hai-Tung; Dai, Shu-Mei

    2014-06-01

    The diamondback moth, Plutella xylostella L., is the most destructive insect pest of Brassica crops in the world. It has developed resistance rapidly to almost every insecticide used for its control. Mevinphos, a fast degrading and slow resistance evocating organophosphorus insecticide, has been recommended for controlling P. xylostella in Taiwan for more than 40years. SHM strain of P. xylostella, with ca. 22-fold resistance to this chemical, has been established from a field SH strain by selecting with mevinphos since 1997. Three mutations, i.e., G892T, G971C, and T1156T/G leading to A298S, G324A, and F386F/V amino acid substitutions in acetylcholinesterase1 (AChE1), were identified in these two strains; along with three haplotype pairs and a polymorphic intron in AChE1 gene (ace1). Two genetically pure lines, i.e., an SHggt wild type with intron AS and an SHMTCN mutant carrying G892T, G971C, T1156T/G mutations and intron AR in ace1, were established by single pair mating and haplotype determination. The F1 of SHMTCN strain had 52-fold resistance to mevinphos in comparison with the F1 of SHggt strain. In addition, AChE1 of this SHMTCN population, which exhibited lower maximum velocity (Vmax) and affinity (Km), was less susceptible to the inhibition of mevinphos, with an I50 32-fold higher than that of the SHggt F1 population. These results imply that amino acid substitutions in AChE1 of SHMTCN strain are associated with mevinphos resistance in this insect pest, and this finding is important for insecticide resistance management of P. xylostella in the field. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Intron-mediated alternative splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B regulates cell wall thickening during fiber development in Populus species.

    Science.gov (United States)

    Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

    2014-02-01

    Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation.

  4. The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

    Directory of Open Access Journals (Sweden)

    Huei-Mei Chen

    Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

  5. Whole-Genome Sequencing of Sordaria macrospora Mutants Identifies Developmental Genes.

    Science.gov (United States)

    Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

    2012-02-01

    The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

  6. Using Behavior Sequence Analysis to Map Serial Killers' Life Histories.

    Science.gov (United States)

    Keatley, David A; Golightly, Hayley; Shephard, Rebecca; Yaksic, Enzo; Reid, Sasha

    2018-03-01

    The aim of the current research was to provide a novel method for mapping the developmental sequences of serial killers' life histories. An in-depth biographical account of serial killers' lives, from birth through to conviction, was gained and analyzed using Behavior Sequence Analysis. The analyses highlight similarities in behavioral events across the serial killers' lives, indicating not only which risk factors occur, but the temporal order of these factors. Results focused on early childhood environment, indicating the role of parental abuse; behaviors and events surrounding criminal histories of serial killers, showing that many had previous convictions and were known to police for other crimes; behaviors surrounding their murders, highlighting differences in victim choice and modus operandi; and, finally, trial pleas and convictions. The present research, therefore, provides a novel approach to synthesizing large volumes of data on criminals and presenting results in accessible, understandable outcomes.

  7. Is PPARα intron 7 G/C polymorphism associated with muscle strength characteristics in nonathletic young men?

    Science.gov (United States)

    Broos, S; Windelinckx, A; De Mars, G; Huygens, W; Peeters, M W; Aerssens, J; Vlietinck, R; Beunen, G P; Thomis, M A

    2013-08-01

    Peroxisome proliferator-activated receptor alpha (PPARα), a ligand-dependent transcription factor, regulates fatty acid metabolism in heart and skeletal muscle. The intron 7 G/C polymorphism (rs4253778) has been associated with athletic performance. The rare C-allele was predominant in power athletes, whereas the G-allele was more frequent in endurance athletes. In the present study, we investigated the association between this polymorphism and strength characteristics in nonathletic, healthy young adults (n = 500; age 24.2 ± 4.4 years). Knee torque was measured during concentric knee flexion and extension movements at 60°/s, 120°/s, and 240°/s during 3, 25, and 5 repetitions, respectively. Also, resistance to muscle fatigue (i.e. work last 20% repetitions/work first 20% repetitions *100) was calculated. Differences in knee strength phenotypes between GG homozygous individuals and C-allele carriers were analyzed. The polymorphism did not influence the ability to produce isometric or dynamic knee flexor or extensor peak torque during static or dynamic conditions in this population (0.23 < P < 0.95). Similar results were found for the endurance ratio, a measure for resistance to muscle fatigue. In conclusion, the PPARα intron 7 G/C polymorphism does not seem to influence strength characteristics in a nonathletic population. © 2011 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts.

    Science.gov (United States)

    Samuel, Stylianos; Papayiannis, Lambros C; Leroch, Michaela; Veloukas, Thomas; Hahn, Matthias; Karaoglanidis, George S

    2011-08-01

    Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005-2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real-time TaqMan PCR assay developed in the present study. QoI-resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse-grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI-resistant and QoI-sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. The results of the study suggest that a high risk for selection of QoI-resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real-time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre- and post-amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry.

  9. Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.

    Science.gov (United States)

    Noh, Ju Young; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Seo, Gi Won; Kim, Dong Hyun; Patnaik, Hongray Howrelia; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Han, Yeon Soo

    2014-01-25

    Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (molitor. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. PPARA intron polymorphism associated with power performance in 30-s anaerobic Wingate Test.

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    Miroslav Petr

    Full Text Available To date, polymorphisms in several genes have been associated with a strength/power performance including alpha 3 actinin, ciliary neurotrophic factor, vitamin D receptor, or angiotensin I converting enzyme, underlining the importance of genetic component of the multifactorial strength/power-related phenotypes. The single nucleotide variation in peroxisome proliferator-activated receptor alpha gene (PPARA intron 7 G/C (rs4253778; g.46630634G>C has been repeatedly found to play a significant role in response to different types of physical activity. We investigated the effect of PPARA intron 7 G/C polymorphism specifically on anaerobic power output in a group of 77 elite male Czech ice hockey players (18-36 y. We determined the relative peak power per body weight (Pmax.kg(-1 and relative peak power per fat free mass (W.kg(-1FFM during the 30-second Wingate Test (WT30 on bicycle ergometer (Monark 894E Peak bike, MONARK, Sweden. All WT30s were performed during the hockey season. Overall genotype frequencies were 50.6% GG homozygotes, 40.3% CG heterozygotes, and 9.1% CC homozygotes. We found statistically significant differences in Pmax.kg(-1 and marginally significant differences in Pmax.kg(-1FFM values in WT30 between carriers and non-carriers for C allele (14.6 ± 0.2 vs. 13.9 ± 0.3 W.kg(-1 and 15.8 ± 0.2 vs. 15.2 ± 0.3 W.kg(-1FFM, P = 0.036 and 0.12, respectively. Furthermore, Pmax.kg(-1FFM strongly positively correlated with the body weight only in individuals with GG genotypes (R = 0.55; p<0.001. Our results indicate that PPARA 7C carriers exhibited higher speed strength measures in WT30. We hypothesize that C allele carriers within the cohort of trained individuals may possess a metabolic advantage towards anaerobic metabolism.

  11. Forest Fragments Surrounded by Sugar Cane Are More Inhospitable to Terrestrial Amphibian Abundance Than Fragments Surrounded by Pasture

    Directory of Open Access Journals (Sweden)

    Paula Eveline Ribeiro D’Anunciação

    2013-01-01

    Full Text Available In recent years, there has been increasing interest in matrix-type influence on forest fragments. Terrestrial amphibians are good bioindicators for this kind of research because of low vagility and high philopatry. This study compared richness, abundance, and species composition of terrestrial amphibians through pitfall traps in two sets of semideciduous seasonal forest fragments in southeastern Brazil, according to the predominant surrounding matrix (sugar cane and pasture. There were no differences in richness, but fragments surrounded by sugar cane had the lowest abundance of amphibians, whereas fragments surrounded by pastures had greater abundance. The most abundant species, Rhinella ornata, showed no biometric differences between fragment groups but like many other amphibians sampled showed very low numbers of individuals in fragments dominated by sugar cane fields. Our data indicate that the sugar cane matrix negatively influences the community of amphibians present in fragments surrounded by this type of land use.

  12. The Future of Whole-Genome Sequencing for Public Health and the Clinic

    OpenAIRE

    Allard, Marc W.

    2016-01-01

    An American Society for Microbiology (ASM) conference titled the Conference on Rapid Next-Generation Sequencing and Bioinformatic Pipelines for Enhanced Molecular Epidemiological Investigation of Pathogens provided a venue for discussing how technologies surrounding whole-genome sequencing (WGS) are advancing microbiology. Several applications in microbial taxonomy, microbial forensics, and genomics for public health pathogen surveillance were presented at the meeting and are reviewed. All of...

  13. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

    International Nuclear Information System (INIS)

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J.; Goswami, Chandan

    2014-01-01

    Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys

  14. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek, E-mail: akumar@bot.uni-kiel.de [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Bhandari, Anita [Molecular Physiology, Zoological Institute, Christian-Albrechts-University at Kiel, Kiel (Germany); Sarde, Sandeep J. [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Goswami, Chandan [National Institute of Science Education and Research, Bhubaneswar, Orissa (India)

    2014-07-18

    Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

  15. The Effects of GH Transgenic Goats on the Microflora of the Intestine, Feces and Surrounding Soil.

    Directory of Open Access Journals (Sweden)

    Zekun Bao

    Full Text Available The development of genetically engineered animals has brought with it increasing concerns about biosafety issues. We therefore evaluated the risks of growth hormone from transgenic goats, including the probability of horizontal gene transfer and the impact on the microbial community of the goats' gastrointestinal tracts, feces and the surrounding soil. The results showed that neither the GH nor the neoR gene could be detected in the samples. Moreover, there was no significant change in the microbial community of the gastrointestinal tracts, feces and soil, as tested with PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing. Finally, phylogenetic analysis showed that the intestinal content, feces and soil samples all contained the same dominant group of bacteria. These results demonstrated that expression of goat growth hormone in the mammary of GH transgenic goat does not influence the microflora of the intestine, feces and surrounding soil.

  16. Molecular analysis of the androgen-receptor gene in a family with receptor-positive partial androgen insensitivity: an unusual type of intronic mutation

    NARCIS (Netherlands)

    H.T. Brüggenwirth (Hennie); A.L.M. Boehmer (Annemie); S. Ramnarain; M.C. Verleun-Mooijman; D.P.E. Satijn (David); J. Trapman (Jan); J.A. Grootegoed (Anton); A.O. Brinkmann (Albert)

    1997-01-01

    textabstractIn the coding part and the intron-exon boundaries of the androgen-receptor gene of a patient with partial androgen insensitivity, no mutation was found. The androgen receptor of this patient displayed normal ligand-binding parameters and migrated as a

  17. PEST sequences in the malaria parasite Plasmodium falciparum: a genomic study

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    Bell Angus

    2003-06-01

    Full Text Available Abstract Background Inhibitors of the protease calpain are known to have selectively toxic effects on Plasmodium falciparum. The enzyme has a natural inhibitor calpastatin and in eukaryotes is responsible for turnover of proteins containing short sequences enriched in certain amino acids (PEST sequences. The genome of P. falciparum was searched for this protease, its natural inhibitor and putative substrates. Methods The publicly available P. falciparum genome was found to have too many errors to permit reliable analysis. An earlier annotation of chromosome 2 was instead examined. PEST scores were determined for all annotated proteins. The published genome was searched for calpain and calpastatin homologs. Results Typical PEST sequences were found in 13% of the proteins on chromosome 2, including a surprising number of cell-surface proteins. The annotated calpain gene has a non-biological "intron" that appears to have been created to avoid an unrecognized frameshift. Only the catalytic domain has significant similarity with the vertebrate calpains. No calpastatin homologs were found in the published annotation. Conclusion A calpain gene is present in the genome and many putative substrates of this enzyme have been found. Calpastatin homologs may be found once the re-annotation is completed. Given the selective toxicity of calpain inhibitors, this enzyme may be worth exploring further as a potential drug target.

  18. The essential function of the Trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr

    Czech Academy of Sciences Publication Activity Database

    Lopes, R.R.S.; Silveira, G. de O.; Eitler, R.; Vidal, R.S.; Kessler, A.; Hinger, S.; Paris, Zdeněk; Alfonzo, J. D.; Polycarpo, C.

    2016-01-01

    Roč. 22, č. 8 (2016), s. 1190-1199 ISSN 1355-8382 R&D Projects: GA ČR GJ15-21450Y Institutional support: RVO:60077344 Keywords : Trypanosoma * tRNA * tRNA editing * splicing * intron Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.605, year: 2016

  19. Mutation analysis and characterization of ATR sequence variants in breast cancer cases from high-risk French Canadian breast/ovarian cancer families

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    Pichette Roxane

    2006-09-01

    Full Text Available Abstract Background Ataxia telangiectasia-mutated and Rad3-related (ATR is a member of the PIK-related family which plays, along with ATM, a central role in cell-cycle regulation. ATR has been shown to phosphorylate several tumor suppressors like BRCA1, CHEK1 and TP53. ATR appears as a good candidate breast cancer susceptibility gene and the current study was designed to screen for ATR germline mutations potentially involved in breast cancer predisposition. Methods ATR direct sequencing was performed using a fluorescent method while widely available programs were used for linkage disequilibrium (LD, haplotype analyses, and tagging SNP (tSNP identification. Expression analyses were carried out using real-time PCR. Results The complete sequence of all exons and flanking intronic sequences were analyzed in DNA samples from 54 individuals affected with breast cancer from non-BRCA1/2 high-risk French Canadian breast/ovarian families. Although no germline mutation has been identified in the coding region, we identified 41 sequence variants, including 16 coding variants, 3 of which are not reported in public databases. SNP haplotypes were established and tSNPs were identified in 73 healthy unrelated French Canadians, providing a valuable tool for further association studies involving the ATR gene, using large cohorts. Our analyses led to the identification of two novel alternative splice transcripts. In contrast to the transcript generated by an alternative splicing site in the intron 41, the one resulting from a deletion of 121 nucleotides in exon 33 is widely expressed, at significant but relatively low levels, in both normal and tumoral cells including normal breast and ovarian tissue. Conclusion Although no deleterious mutations were identified in the ATR gene, the current study provides an haplotype analysis of the ATR gene polymorphisms, which allowed the identification of a set of SNPs that could be used as tSNPs for large-scale association

  20. Unexpected allelic heterogeneity and spectrum of mutations in Fowler syndrome revealed by next-generation exome sequencing.

    Science.gov (United States)

    Lalonde, Emilie; Albrecht, Steffen; Ha, Kevin C H; Jacob, Karine; Bolduc, Nathalie; Polychronakos, Constantin; Dechelotte, Pierre; Majewski, Jacek; Jabado, Nada

    2010-08-01

    Protein coding genes constitute approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute our understanding of human diseases. We used a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform, and investigated two unrelated fetuses from nonconsanguineous families with Fowler Syndrome (FS), a stereotyped phenotype lethal disease. We report novel germline mutations in feline leukemia virus subgroup C cellular-receptor-family member 2, FLVCR2, which has recently been shown to cause FS. Using this technology, we identified three types of genetic abnormalities: point-mutations, insertions-deletions, and intronic splice-site changes (first pathogenic report using this technology), in the fetuses who both were compound heterozygotes for the disease. Although revealing a high level of allelic heterogeneity and mutational spectrum in FS, this study further illustrates the successful application of whole-exome sequencing to uncover genetic defects in rare Mendelian disorders. Of importance, we show that we can identify genes underlying rare, monogenic and recessive diseases using a limited number of patients (n=2), in the absence of shared genetic heritage and in the presence of allelic heterogeneity.

  1. Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

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    Yu ZHAO

    2017-09-01

    Full Text Available Background: Toxoplasma gondii, as a eukaryotic parasite of the phylum Apicomplexa, can infect almost all the warm-blooded animals and humans, causing toxoplasmosis. Rhoptry neck proteins (RONs play a key role in the invasion process of T. gondii and are potential vaccine candidate molecules against toxoplasmosis.Methods: The present study examined sequence variation in the rhoptry neck protein 10 (TgRON10 gene among 10 T. gondii isolates from different hosts and geographical locations from Lanzhou province during 2014, and compared with the corresponding sequences of strains ME49 and VEG obtained from the ToxoDB database, using polymerase chain reaction (PCR amplification, sequence analysis, and phylogenetic reconstruction by Bayesian inference (BI and maximum parsimony (MP. Results: Analysis of all the 12 TgRON10 genomic and cDNA sequences revealed 7 exons and 6 introns in the TgRON10 gDNA. The complete genomic sequence of the TgRON10 gene ranged from 4759 bp to 4763 bp, and sequence variation was 0-0.6% among the 12 T. gondii isolates, indicating a low sequence variation in TgRON10 gene. Phylogenetic analysis of TgRON10 sequences showed that the cluster of the 12 T. gondii isolates was not completely consistent with their respective genotypes.Conclusion: TgRON10 gene is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, worth further studies.

  2. Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations.

    Science.gov (United States)

    Zhao, Yu; Zhou, Donghui; Chen, Jia; Sun, Xiaolin

    2017-01-01

    Toxoplasma gondii, as a eukaryotic parasite of the phylum Apicomplexa, can infect almost all the warm-blooded animals and humans, causing toxoplasmosis. Rhoptry neck proteins (RONs) play a key role in the invasion process of T. gondii and are potential vaccine candidate molecules against toxoplasmosis. The present study examined sequence variation in the rhoptry neck protein 10 (TgRON10) gene among 10 T. gondii isolates from different hosts and geographical locations from Lanzhou province during 2014, and compared with the corresponding sequences of strains ME49 and VEG obtained from the ToxoDB database, using polymerase chain reaction (PCR) amplification, sequence analysis, and phylogenetic reconstruction by Bayesian inference (BI) and maximum parsimony (MP). Analysis of all the 12 TgRON10 genomic and cDNA sequences revealed 7 exons and 6 introns in the TgRON10 gDNA. The complete genomic sequence of the TgRON10 gene ranged from 4759 bp to 4763 bp, and sequence variation was 0-0.6% among the 12 T. gondii isolates, indicating a low sequence variation in TgRON10 gene. Phylogenetic analysis of TgRON10 sequences showed that the cluster of the 12 T. gondii isolates was not completely consistent with their respective genotypes. TgRON10 gene is not a suitable genetic marker for the differentiation of T. gondii isolates from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, worth further studies.

  3. Enhancement of Afterimage Colors by Surrounding Contours

    Directory of Open Access Journals (Sweden)

    Takao Sato

    2011-05-01

    Full Text Available Presenting luminance contours surrounding the adapted areas in test phase enhances color afterimages in both duration and color appearance. The presence of surrounding contour is crucial to some color phenomenon such as van Lier's afterimage, but the contour-effect itself has not been seriously examined. In this paper, we compared the contour-effect to color afterimages and to actually colored patches to examine the nature of color information subserving color-aftereffect. In the experiment, observers were adapted for 1 sec to a small colored square (red, green, yellow, or blue presented on a gray background. Then, a test field either with or without surrounding contour was presented. Observers matched the color of a test-patch located near the afterimage to the color of afterimage. It was found that the saturation of negative afterimage was almost doubled by the presence of surrounding contours. There was no effect of luminance contrast or polarity of contours. In contrast, no enhancement of saturation by surrounding contours was observed for actually colored patches even though the colors of patches were equalized to that of afterimage without contours. This dissociation in the contour-effect demonstrates the crucial difference between the color information for aftereffects and for ordinary bottom-up color perception.

  4. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  5. Molecular characterization of a novel HEXA mutation at the +3 position of intron 8 in a Tay-Sachs disease patient

    Energy Technology Data Exchange (ETDEWEB)

    Richard, M.; Triggs-Raine, B. [Univ. of Manitoba, Winnipeg (Canada); Natowicz, M. [E.K. Shriver Center, Waltham, MA (United States)

    1994-09-01

    Tay-Sachs disease is an autosomal recessive lysosomal storage disorder resulting from mutations in the HEXA gene that cause a deficiency in the activity of that enzyme {beta}-hexosaminidase A (Hex A). This deficiency leads to the build-up of G{sub M2} ganglioside, resulting in neurodegeneration and death. Biochemical analysis of a non-Jewish patient with a late-infantile form of Tay-Sachs disease revealed a substantial level of Hex A activity (38.4%) when 4-MUG was used as the substrate. However, when a substrate (4-MUGS) specific for the {alpha}-subunit of Hex A ({alpha}{beta}) was used, almost no activity was detected in the HEXA gene of the patient using SSCP analysis followed by sequencing. The first mutation, a G533A substitution in exon 5, is previously described and associated with the B1 form of Tay-Sachs disease. The second mutation is a novel a-to-g base change at the +3 position of intron 8. This was confirmed using the AIRS method, whereby a MaeIII site was created in the presence of the mutation. Normal and patient mRNA was reverse transcribed and exons 7 to 9 were PCR-amplified from the cDNA. An abnormally sized amplification product detected only in the patient cDNA was sequenced; exon 8 had been deleted and exons 7 and 9 were spliced together. A substantial level of normally-sized PCR product was also detected in the patient`s cDNA. Experiments are in progress to determine if this is produced from the allele harboring the G533A mutation. Given that previous mutations of this type have been associated with 97-100% abnormal splicing, this mutation is likely to be the cause, together with the G533A mutation, of Tay-Sachs disease in this patient.

  6. Complete chloroplast genome sequence of common bermudagrass (Cynodon dactylon (L.) Pers.) and comparative analysis within the family Poaceae.

    Science.gov (United States)

    Huang, Ya-Yi; Cho, Shu-Ting; Haryono, Mindia; Kuo, Chih-Horng

    2017-01-01

    Common bermudagrass (Cynodon dactylon (L.) Pers.) belongs to the subfamily Chloridoideae of the Poaceae family, one of the most important plant families ecologically and economically. This grass has a long connection with human culture but its systematics is relatively understudied. In this study, we sequenced and investigated the chloroplast genome of common bermudagrass, which is 134,297 bp in length with two single copy regions (LSC: 79,732 bp; SSC: 12,521 bp) and a pair of inverted repeat (IR) regions (21,022 bp). The annotation contains a total of 128 predicted genes, including 82 protein-coding, 38 tRNA, and 8 rRNA genes. Additionally, our in silico analyses identified 10 sets of repeats longer than 20 bp and predicted the presence of 36 RNA editing sites. Overall, the chloroplast genome of common bermudagrass resembles those from other Poaceae lineages. Compared to most angiosperms, the accD gene and the introns of both clpP and rpoC1 genes are missing. Additionally, the ycf1, ycf2, ycf15, and ycf68 genes are pseudogenized and two genome rearrangements exist. Our phylogenetic analysis based on 47 chloroplast protein-coding genes supported the placement of common bermudagrass within Chloridoideae. Our phylogenetic character mapping based on the parsimony principle further indicated that the loss of the accD gene and clpP introns, the pseudogenization of four ycf genes, and the two rearrangements occurred only once after the most recent common ancestor of the Poaceae diverged from other monocots, which could explain the unusual long branch leading to the Poaceae when phylogeny is inferred based on chloroplast sequences.

  7. Chromatic induction from surrounding stimuli under perceptual suppression.

    Science.gov (United States)

    Horiuchi, Koji; Kuriki, Ichiro; Tokunaga, Rumi; Matsumiya, Kazumichi; Shioiri, Satoshi

    2014-11-01

    The appearance of colors can be affected by their spatiotemporal context. The shift in color appearance according to the surrounding colors is called color induction or chromatic induction; in particular, the shift in opponent color of the surround is called chromatic contrast. To investigate whether chromatic induction occurs even when the chromatic surround is imperceptible, we measured chromatic induction during interocular suppression. A multicolor or uniform color field was presented as the surround stimulus, and a colored continuous flash suppression (CFS) stimulus was presented to the dominant eye of each subject. The subjects were asked to report the appearance of the test field only when the stationary surround stimulus is invisible by interocular suppression with CFS. The resulting shifts in color appearance due to chromatic induction were significant even under the conditions of interocular suppression for all surround stimuli. The magnitude of chromatic induction differed with the surround conditions, and this difference was preserved regardless of the viewing conditions. The chromatic induction effect was reduced by CFS, in proportion to the magnitude of chromatic induction under natural (i.e., no-CFS) viewing conditions. According to an analysis with linear model fitting, we revealed the presence of at least two kinds of subprocesses for chromatic induction that reside at higher and lower levels than the site of interocular suppression. One mechanism yields different degrees of chromatic induction based on the complexity of the surround, which is unaffected by interocular suppression, while the other mechanism changes its output with interocular suppression acting as a gain control. Our results imply that the total chromatic induction effect is achieved via a linear summation of outputs from mechanisms that reside at different levels of visual processing.

  8. A Two-Piece Derivative of a Group I Intron RNA as a Platform for Designing Self-Assembling RNA Templates to Promote Peptide Ligation

    Directory of Open Access Journals (Sweden)

    Takahiro Tanaka

    2012-01-01

    Full Text Available Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

  9. CELF1 preferentially binds to exon-intron boundary and regulates alternative splicing in HeLa cells.

    Science.gov (United States)

    Xia, Heng; Chen, Dong; Wu, Qijia; Wu, Gang; Zhou, Yanhong; Zhang, Yi; Zhang, Libin

    2017-09-01

    The current RIP-seq approach has been developed for the identification of genome-wide interaction between RNA binding protein (RBP) and the bound RNA transcripts, but still rarely for identifying its binding sites. In this study, we performed RIP-seq experiments in HeLa cells using a monoclonal antibody against CELF1. Mapping of the RIP-seq reads showed a biased distribution at the 3'UTR and intronic regions. A total of 15,285 and 1384 CELF1-specific sense and antisense peaks were identified using the ABLIRC software tool. Our bioinformatics analyses revealed that 5' and 3' splice site motifs and GU-rich motifs were highly enriched in the CELF1-bound peaks. Furthermore, transcriptome analyses revealed that alternative splicing was globally regulated by CELF1 in HeLa cells. For example, the inclusion of exon 16 of LMO7 gene, a marker gene of breast cancer, is positively regulated by CELF1. Taken together, we have shown that RIP-seq data can be used to decipher RBP binding sites and reveal an unexpected landscape of the genome-wide CELF1-RNA interactions in HeLa cells. In addition, we found that CELF1 globally regulates the alternative splicing by binding the exon-intron boundary in HeLa cells, which will deepen our understanding of the regulatory roles of CELF1 in the pre-mRNA splicing process. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Comparison of Socioeconomic Factors between Surrounding and Non-Surrounding Areas of the Qinghai–Tibet Railway before and after Its Construction

    Directory of Open Access Journals (Sweden)

    Shicheng Li

    2016-08-01

    Full Text Available As the world’s highest railway, and the longest highland railway, the Qinghai–Tibet Railway (QTR has been paid considerable attention by researchers. However, most attention has been paid to the ecological and environmental issues affecting it, and sustainable ecological, social, and economic development-related studies of the QTR are rare. In this study, by analyzing the passenger traffic, freight traffic, passenger-kilometers, and freight-kilometers of the QTR for the period 1982–2013 and the transport structure of the Tibetan Plateau (TP for 1990–2013, the evolutionary process of the transport system in the TP following the construction of the QTR has been revealed. Subsequently, by comparing Gross Domestic Product (GDP, population, industrial structure, and urbanization level at the county and 1 km scales between surrounding and non-surrounding areas of the QTR, the differences in socioeconomic performance before and after its construction were detected. The results show that (1 in the TP, the highway-dominated transport system will break up and an integrated and sustainable transport system will form; (2 at the county scale, the annual growth rates of GDP of counties surrounding the QTR were greater than those of non-surrounding counties for the period 2000–2010. At the 1 km scale, following the opening of the completed line, the GDP of surrounding areas had a greater growth rate than before; (3 analysis at the county and 1 km scales indicated that population was not aggregated into the surrounding areas of the QTR in the period 2000–2010; (4 in terms of industrial structure, the proportion of primary industry decreased continuously, while the proportion of secondary and tertiary industries increased overall in the period 1984–2012. The QTR had no obvious impact on changes in the urbanization level of its surrounding areas.

  11. An Intron 9 CYP19 Gene Variant (IVS9+5G>A), Present in an Aromatase-Deficient Girl, Affects Normal Splicing and Is Also Present in Normal Human Steroidogenic Tissues.

    Science.gov (United States)

    Saraco, Nora; Nesi-Franca, Suzana; Sainz, Romina; Marino, Roxana; Marques-Pereira, Rosana; La Pastina, Julia; Perez Garrido, Natalia; Sandrini, Romolo; Rivarola, Marco Aurelio; de Lacerda, Luiz; Belgorosky, Alicia

    2015-01-01

    Splicing CYP19 gene variants causing aromatase deficiency in 46,XX disorder of sexual development (DSD) patients have been reported in a few cases. A misbalance between normal and aberrant splicing variants was proposed to explain spontaneous pubertal breast development but an incomplete sex maturation progress. The aim of this study was to functionally characterize a novel CYP19A1 intronic homozygote mutation (IVS9+5G>A) in a 46,XX DSD girl presenting spontaneous breast development and primary amenorrhea, and to evaluate similar splicing variant expression in normal steroidogenic tissues. Genomic DNA analysis, splicing prediction programs, splicing assays, and in vitro protein expression and enzyme activity analyses were carried out. CYP19A1 mRNA expression in human steroidogenic tissues was also studied. A novel IVS9+5G>A homozygote mutation was found. In silico analysis predicts the disappearance of the splicing donor site in intron 9, confirmed by patient peripheral leukocyte cP450arom and in vitro studies. Protein analysis showed a shorter and inactive protein. The intron 9 transcript variant was also found in human steroidogenic tissues. The mutation IVS9+5G>A generates a splicing variant that includes intron 9 which is also present in normal human steroidogenic tissues, suggesting that a misbalance between normal and aberrant splicing variants might occur in target tissues, explaining the clinical phenotype in the affected patient. © 2015 S. Karger AG, Basel.

  12. Mitochondrial transcription factor A (Tfam) gene sequencing and mitochondrial evaluation in inherited retinal dysplasia in miniature schnauzer dogs.

    Science.gov (United States)

    Bauer, Bianca S; Forsyth, George W; Sandmeyer, Lynne S; Grahn, Bruce H

    2011-04-01

    Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

  13. Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

    Science.gov (United States)

    Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

    2016-02-27

    In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a

  14. Might there be a link between intron 3 VNTR polymorphism in the XRCC4 DNA repair gene and the etiopathogenesis of rheumatoid arthritis?

    Science.gov (United States)

    Pehlivan, Sacide; Balci, Sibel Oguzkan; Aydeniz, Ali; Pehlivan, Mustafa; Sever, Tugce; Gursoy, Savas

    2015-01-01

    DNA repair genes are involved in several diseases such as cancers and autoimmune diseases. Previous studies indicated that a DNA repair system was involved in the development of rheumatoid arthritis (RA). In this study, we aimed to examine whether four polymorphisms in the DNA repair genes (xeroderma pigmentosum complementation group D [XPD], X-ray repair cross-complementing group 1 [XRCC1], and X-ray repair cross-complementing group 4 [XRCC4]) were associated with RA. Sixty-five patients with RA and 70 healthy controls (HCs) were examined for XPD (A-751G), XRCC1 (A399G), and XRCC4 (intron 3 VNTR and G-1394T) polymorphisms. All polymorphisms were genotyped by PCR and/or PCR-RFLP. The association between the polymorphisms and RA was analyzed using the chi-square test and de Finetti program. The intron 3 VNTR polymorphism in the XRCC4 gene showed an association with RA patients. The DI genotype was found lower in RA patients (χ(2)=8.227; p=0.0021), while the II genotype was higher in RA patients (χ(2)=5.285; p=0.010). There were deviations from the Hardy-Weinberg Equilibrium (HWE) in both intron 3 VNTR and G-1394T polymorphisms in the XRCC4 gene and in the polymorphism in the XRCC1 gene, and the observed genotype counts deviated from those expected according to the HWE (p=0.027, 0.004, and 0.002, respectively); however, there was no deviation in the other gene polymorphisms. There is no statistical difference between the RA patients and HCs for XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms (p>0.05). Although XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms have been extensively investigated in different clinical pictures, this is the first study to evaluate the role of these polymorphisms in the genetic etiopathogenesis of RA in Turkish patients. In conclusion, we suggested that the intron 3 VNTR polymorphism in the XRCC4 gene may be associated with the etiopathogenesis of RA as a marker of immune aging.

  15. Intron-Mediated Alternative Splicing of WOOD-ASSOCIATED NAC TRANSCRIPTION FACTOR1B Regulates Cell Wall Thickening during Fiber Development in Populus Species1[W

    Science.gov (United States)

    Zhao, Yunjun; Sun, Jiayan; Xu, Peng; Zhang, Rui; Li, Laigeng

    2014-01-01

    Alternative splicing is an important mechanism involved in regulating the development of multicellular organisms. Although many genes in plants undergo alternative splicing, little is understood of its significance in regulating plant growth and development. In this study, alternative splicing of black cottonwood (Populus trichocarpa) wood-associated NAC domain transcription factor (PtrWNDs), PtrWND1B, is shown to occur exclusively in secondary xylem fiber cells. PtrWND1B is expressed with a normal short-transcript PtrWND1B-s as well as its alternative long-transcript PtrWND1B-l. The intron 2 structure of the PtrWND1B gene was identified as a critical sequence that causes PtrWND1B alternative splicing. Suppression of PtrWND1B expression specifically inhibited fiber cell wall thickening. The two PtrWND1B isoforms play antagonistic roles in regulating cell wall thickening during fiber cell differentiation in Populus spp. PtrWND1B-s overexpression enhanced fiber cell wall thickening, while overexpression of PtrWND1B-l repressed fiber cell wall thickening. Alternative splicing may enable more specific regulation of processes such as fiber cell wall thickening during wood formation. PMID:24394777

  16. An RNAi-Enhanced Logic Circuit for Cancer Specific Detection and Destruction

    Science.gov (United States)

    2013-02-01

    monomeric protein secreted by Corynebacterium diphtheriae, and pro-apoptotic members of Bcl-2 family: mBax (Mus musculus), hBax ( Homo sapiens ), and its...Gata3 mStaple. Intron- feature sequences – donor site, branch point, poly- pyrimidine tract, and acceptor site – were selected based on previously...sequences found in literature our intron features were chosen according SplicePort [4], an online analyzer that detects the likelihood of splicing to

  17. TSHR intronic polymorphisms (rs179247 and rs12885526) and their role in the susceptibility of the Brazilian population to Graves' disease and Graves' ophthalmopathy.

    Science.gov (United States)

    Bufalo, N E; Dos Santos, R B; Marcello, M A; Piai, R P; Secolin, R; Romaldini, J H; Ward, L S

    2015-05-01

    Intronic thyroid-stimulating hormone receptor polymorphisms have been associated with the risk for both Graves' disease and Graves' ophthalmopathy, but results have been inconsistent among different populations. We aimed to investigate the influence of thyroid-stimulating hormone receptor intronic polymorphisms in a large well-characterized population of GD patients. We studied 279 Graves' disease patients (231 females and 48 males, 39.80 ± 11.69 years old), including 144 with Graves' ophthalmopathy, matched to 296 healthy control individuals. Thyroid-stimulating hormone receptor genotypes of rs179247 and rs12885526 were determined by Real Time PCR TaqMan(®) SNP Genotyping. A multivariate analysis showed that the inheritance of the thyroid-stimulating hormone receptor AA genotype for rs179247 increased the risk for Graves' disease (OR = 2.821; 95 % CI 1.595-4.990; p = 0.0004), whereas the thyroid-stimulating hormone receptor GG genotype for rs12885526 increased the risk for Graves' ophthalmopathy (OR = 2.940; 95 % CI 1.320-6.548; p = 0.0083). Individuals with Graves' ophthalmopathy also presented lower mean thyrotropin receptor antibodies levels (96.3 ± 143.9 U/L) than individuals without Graves' ophthalmopathy (98.3 ± 201.9 U/L). We did not find any association between the investigated polymorphisms and patients clinical features or outcome. We demonstrate that thyroid-stimulating hormone receptor intronic polymorphisms are associated with the susceptibility to Graves' disease and Graves' ophthalmopathy in the Brazilian population, but do not appear to influence the disease course.

  18. Pseudogene of dihydrolipoyl succinyltransferase (E2k) found by PCR amplification and direct sequencing of rodent-human cell hybrid DNAs

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.; Ali, G.; Blass, J.P. [Cornell Univ. Medical College, White Plains, NY (United States); Szabo, P. [Cornell Univ. Medical College, New York, NY (United States); Tanzi, R.E. [Massachusetts General Hospital, Boston, MA (United States)

    1994-07-01

    Previous studies have indicated that the cDNA for the E2k component of the human {alpha}-ketoglutarate dehydrogenase complex (KGDHC) hybridized not only to a major locus on chromosome 14q24.3 in a region associated with familial Alzheimer`s disease and with Joseph-Machado disease, but also to another locus on chromosome 1p31. The authors now report that PCR of genomic DNA and direct sequencing indicated that the chromosome 1 locus is an intronless pseudogene. PCR of genomic DNA amplified E2k fragments from mouse-human cell hybrids containing human chromosome 1 DNA but not from hybrids containing human chromosome 14 DNA. The resulting amplicons were of comparable sizes to those when the cDNA was used to template. The direct sequencing of these amplicons confirmed the lack of introns and indicated a frame shift, which led to the presence of four termination codons early in the coding region. PCR followed by direct sequencing of the amplicons appears to be a convenient method for identifying intronless pseudogenes.

  19. Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

    Science.gov (United States)

    Collins, Kathleen; Nilsen, Timothy W

    2013-08-01

    Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

  20. Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison

    Directory of Open Access Journals (Sweden)

    Saville Barry J

    2007-09-01

    Full Text Available Abstract Background Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription. Results Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 × 521 and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs; among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database, while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis, other varieties were also evident (e.g. exon skipping. Selected instances of both alternative splicing and anti-sense transcription were independently confirmed using RT-PCR. Conclusion Through this work: 1 substantial sequence information has been provided for U. maydis genome annotation; 2 new genes were identified through the discovery of 619

  1. Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata.

    Science.gov (United States)

    Templeton, M D; Rikkerink, E H; Solon, S L; Crowhurst, R N

    1992-12-01

    The glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has been identified from a genomic DNA library prepared from the plant pathogenic fungus Glomerella cingulata. Nucleotide sequence data revealed that this gene codes for a putative 338-amino-acid protein encoded by two exons of 129 and 885 bp, separated by an intron 216 bp long. The 5' leader sequence is also spliced by an intron of 156 bp. A cDNA clone was prepared using the polymerase chain reaction, the sequence of which was used to confirm the presence of the intron in the coding sequence and the splicing of the 5' leader sequence. The transcriptional start point (tsp) was mapped at -253 nt from the site of the initiation of translation by primer extension and is adjacent to a 42-bp pyrimidine-rich region. The general structure of the 5' flanking region shows similarities to gpdA from Aspergillus nidulans. The putative protein product is 71-86% identical at the aa level to GPDs from Aspergillus nidulans, Cryphonectria parasitica, Curvularia lunata, Podospora anserina and Ustilago maydis.

  2. Targeted exome sequencing reveals novel USH2A mutations in Chinese patients with simplex Usher syndrome.

    Science.gov (United States)

    Shu, Hai-Rong; Bi, Huai; Pan, Yang-Chun; Xu, Hang-Yu; Song, Jian-Xin; Hu, Jie

    2015-09-16

    Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing impairment and vision dysfunction due to retinitis pigmentosa. Phenotypic and genetic heterogeneities of this disease make it impractical to obtain a genetic diagnosis by conventional Sanger sequencing. In this study, we applied a next-generation sequencing approach to detect genetic abnormalities in patients with USH. Two unrelated Chinese families were recruited, consisting of two USH afflicted patients and four unaffected relatives. We selected 199 genes related to inherited retinal diseases as targets for deep exome sequencing. Through systematic data analysis using an established bioinformatics pipeline, all variants that passed filter criteria were validated by Sanger sequencing and co-segregation analysis. A homozygous frameshift mutation (c.4382delA, p.T1462Lfs*2) was revealed in exon20 of gene USH2A in the F1 family. Two compound heterozygous mutations, IVS47 + 1G > A and c.13156A > T (p.I4386F), located in intron 48 and exon 63 respectively, of USH2A, were identified as causative mutations for the F2 family. Of note, the missense mutation c.13156A > T has not been reported so far. In conclusion, targeted exome sequencing precisely and rapidly identified the genetic defects in two Chinese USH families and this technique can be applied as a routine examination for these disorders with significant clinical and genetic heterogeneity.

  3. Monitoring program of surrounding of the NPP SE-EBO

    International Nuclear Information System (INIS)

    Dobis, L.; Kostial, J.

    1997-01-01

    The paper dealt with monitoring program of radiation control of surrounding of the NPP Bohunice, which has the aim: (1) to ensure the control of influence of work of the NPP Bohunice on the environment in their surrounding; (2) to ensure the back-ground for regular brief of control and supervisory organs about condition of the environment in surrounding of the NPP Bohunice; (3) to maintain the expected technical level of control of the NPP Bohunice and to exploit optimally the technical means; (4) to solicit permanently the data about the radioactivity of environment in surrounding of the NPP Bohunice for forming of files of the data; (5) to exploit purposefully the technical equipment, technical workers and to maintain their in permanent emergency and technical eligibility for the case of the breakdown; (6) to obtain permanently the files of the values for qualification of the reference levels. This program of monitoring includes the radiation control of surrounding of the NPP Bohunice, in the time of normal work of power-station's blocks, inclusively of all types of trouble-shooting and repairer works in surrounding of the NPP Bohunice, up to distance 20 km from power-station. The monitoring includes: outlets from the NPP Bohunice, monitoring of radiation characteristics in surrounding of the NPP Bohunice, (aerosols, fall-outs, soil), the links of food chains: (grass and fodder, milk, agriculture products), hydrosphere in surrounding (surface waters, drink water, bores of radiation control in complex of the NPP Bohunice, components of the hydrosphere), measurement of radiation from external sources (measurement of the dose rates, measurement of the doses [sk

  4. Sequence analysis of the ATM gene in 20 patients with RTOG grade 3 or 4 acute and/or late tissue radiation side effects

    International Nuclear Information System (INIS)

    Oppitz, Ulrich; Bernthaler, Ulrike; Schindler, Detlev; Sobeck, Alexandra; Hoehn, Holger; Platzer, Matthias; Rosenthal, Andre; Flentje, Michael

    1999-01-01

    Purpose: Patients with ataxia-telangiectasia (A-T) show greatly increased radiation sensitivity and cancer predisposition. Family studies imply that the otherwise clinically silent heterozygotes of this autosomal recessive disease run a 3.5 to 3.8 higher risk of developing cancer. In vitro studies suggest moderately increased cellular radiation sensitivity of A-T carriers. They may also show elevated clinical radiosensitivity. We retrospectively examined patients who presented with severe adverse reactions during or after standard radiation treatment for mutations in the gene responsible for A-T, ATM, considering a potential means of future identification of radiosensitive individuals prospectively to adjust dosage schedules. Material and Methods: We selected 20 cancer patients (breast, 11; rectum, 2; ENT, 2; bladder, 1; prostate, 1; anus, 1; astrocytoma, 1; Hodgkins lymphoma, 1) with Grade 3 to 4 (RTOG) acute and/or late tissue radiation side effects by reaction severity. DNA from the peripheral blood of patients was isolated. All 66 exons and adjacent intron regions of the ATM gene were PCR-amplified and examined for mutations by a combination of agarose gel electrophoresis, single-stranded conformational polymorphism (SSCP) analysis, and exon-scanning direct sequencing. Results: Only 2 of the patients revealed altogether four heteroallelic sequence variants. The latter included two single-base deletions in different introns, a single-base change causing an amino acid substitution in an exon, and a large insertion in another intron. Both the single-base deletions and the single-base change represent known polymorphisms. The large insertion was an Alu repeat, shown not to give rise to altered gene product. Conclusions: Despite high technical efforts, no unequivocal ATM mutation was detected. Nevertheless, extension of similar studies to larger and differently composed cohorts of patients suffering severe adverse effects of radiotherapy, and application of new

  5. Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

    Science.gov (United States)

    Abraham, Anup; Chandler, Douglas E

    2017-10-01

    Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

  6. Genome-wide data-mining of candidate human splice translational efficiency polymorphisms (STEPs and an online database.

    Directory of Open Access Journals (Sweden)

    Christopher A Raistrick

    2010-10-01

    Full Text Available Variation in pre-mRNA splicing is common and in some cases caused by genetic variants in intronic splicing motifs. Recent studies into the insulin gene (INS discovered a polymorphism in a 5' non-coding intron that influences the likelihood of intron retention in the final mRNA, extending the 5' untranslated region and maintaining protein quality. Retention was also associated with increased insulin levels, suggesting that such variants--splice translational efficiency polymorphisms (STEPs--may relate to disease phenotypes through differential protein expression. We set out to explore the prevalence of STEPs in the human genome and validate this new category of protein quantitative trait loci (pQTL using publicly available data.Gene transcript and variant data were collected and mined for candidate STEPs in motif regions. Sequences from transcripts containing potential STEPs were analysed for evidence of splice site recognition and an effect in expressed sequence tags (ESTs. 16 publicly released genome-wide association data sets of common diseases were searched for association to candidate polymorphisms with HapMap frequency data. Our study found 3324 candidate STEPs lying in motif sequences of 5' non-coding introns and further mining revealed 170 with transcript evidence of intron retention. 21 potential STEPs had EST evidence of intron retention or exon extension, as well as population frequency data for comparison.Results suggest that the insulin STEP was not a unique example and that many STEPs may occur genome-wide with potentially causal effects in complex disease. An online database of STEPs is freely accessible at http://dbstep.genes.org.uk/.

  7. Insights into the evolution of mitochondrial genome size from complete sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae).

    Science.gov (United States)

    Alverson, Andrew J; Wei, XiaoXin; Rice, Danny W; Stern, David B; Barry, Kerrie; Palmer, Jeffrey D

    2010-06-01

    The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

  8. Stimulus size dependence of hue changes induced by chromatic surrounds.

    Science.gov (United States)

    Kellner, Christian Johannes; Wachtler, Thomas

    2016-03-01

    A chromatic surround induces a change in the perceived hue of a stimulus. This shift in hue depends on the chromatic difference between the stimulus and the surround. We investigated how chromatic induction varies with stimulus size and whether the size dependence depends on the surround hue. Subjects performed asymmetric matching of color stimuli with different sizes in surrounds of different chromaticities. Generally, induced hue shifts decreased with increasing stimulus size. This decrease was quantitatively different for different surround hues. However, when size effects were normalized to an overall induction strength, the chromatic specificity was largely reduced. The separability of inducer chromaticity and stimulus size suggests that these effects are mediated by different neural mechanisms.

  9. Targeted next generation sequencing for molecular diagnosis of Usher syndrome.

    Science.gov (United States)

    Aparisi, María J; Aller, Elena; Fuster-García, Carla; García-García, Gema; Rodrigo, Regina; Vázquez-Manrique, Rafael P; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Françoise; Jaijo, Teresa; Millán, José M

    2014-11-18

    Usher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing. A custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations. Forty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements. Targeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.

  10. Comparative genomic analysis of the arthropod muscle myosin heavy chain genes allows ancestral gene reconstruction and reveals a new type of 'partially' processed pseudogene

    Directory of Open Access Journals (Sweden)

    Kollmar Martin

    2008-02-01

    Full Text Available Abstract Background Alternative splicing of mutually exclusive exons is an important mechanism for increasing protein diversity in eukaryotes. The insect Mhc (myosin heavy chain gene produces all different muscle myosins as a result of alternative splicing in contrast to most other organisms of the Metazoa lineage, that have a family of muscle genes with each gene coding for a protein specialized for a functional niche. Results The muscle myosin heavy chain genes of 22 species of the Arthropoda ranging from the waterflea to wasp and Drosophila have been annotated. The analysis of the gene structures allowed the reconstruction of an ancient muscle myosin heavy chain gene and showed that during evolution of the arthropods introns have mainly been lost in these genes although intron gain might have happened in a few cases. Surprisingly, the genome of Aedes aegypti contains another and that of Culex pipiens quinquefasciatus two further muscle myosin heavy chain genes, called Mhc3 and Mhc4, that contain only one variant of the corresponding alternative exons of the Mhc1 gene. Mhc3 transcription in Aedes aegypti is documented by EST data. Mhc3 and Mhc4 inserted in the Aedes and Culex genomes either by gene duplication followed by the loss of all but one variant of the alternative exons, or by incorporation of a transcript of which all other variants have been spliced out retaining the exon-intron structure. The second and more likely possibility represents a new type of a 'partially' processed pseudogene. Conclusion Based on the comparative genomic analysis of the alternatively spliced arthropod muscle myosin heavy chain genes we propose that the splicing process operates sequentially on the transcript. The process consists of the splicing of the mutually exclusive exons until one exon out of the cluster remains while retaining surrounding intronic sequence. In a second step splicing of introns takes place. A related mechanism could be responsible for

  11. Explaining preferences for home surroundings and locations

    DEFF Research Database (Denmark)

    Andersen, Hans Skifter

    2011-01-01

    This article is based on a survey carried out in Denmark that asked a random sample of the population about their preferences for home surroundings and locations. It shows that the characteristics of social surroundings are very important and can be divided into three independent dimensions......: avoiding social nuisances, preferring social homogeneity and living close to one’s social network and place of origin. The study shows that most people have many detailed preferences, whereas some have very few. This confirms an earlier theory that some people are very connected to certain places...... with given characteristics and thus do not have priorities regarding home surroundings and locations. For others, mostly young people and singles, home is just a place to sleep and relax, whereas life is lived elsewhere. For this group, there are only preferences for location and there are few specific...

  12. Geographical origin of Leucobryum boninense Sull. & Lesq. (Leucobryaceae, Musci) endemic to the Bonin Islands, Japan.

    Science.gov (United States)

    Oguri, Emiko; Yamaguchi, Tomio; Tsubota, Hiromi; Deguchi, Hironori; Murakami, Noriaki

    2013-04-01

    Leucobryum boninense is endemic to the Bonin Islands, Japan, and its related species are widely distributed in Asia and the Pacific. We aimed to clarify the phylogenetic relationships among Leucobryum species and infer the origin of L. boninense. We also describe the utility of the chloroplast trnK intron including matK for resolving the phylogenetic relationships among Leucobryum species, as phylogenetic analyses using trnK intron and/or matK have not been performed well in bryophytes to date. Fifty samples containing 15 species of Leucobryum from Asia and the Pacific were examined for six chloroplast DNA regions including rbcL, rps4, partial 5' trnK intron, matK, partial 3' trnK intron, and trnL-F intergenic spacer plus one nuclear DNA region including ITS. A molecular phylogenetic tree showed that L. boninense made a clade with L. scabrum from Japan, Taiwan and, Hong Kong; L. javense which is widely distributed in East and Southeast Asia, and L. pachyphyllum and L. seemannii restricted to the Hawaii Islands, as well as with L. scaberulum from the Ryukyus, Japan, Taiwan, and southeastern China. Leucobryum boninense from various islands of the Bonin Islands made a monophylic group that was closely related to L. scabrum and L. javense from Japan. Therefore, L. boninense may have evolved from L. scabrum from Japan, Taiwan, or Hong Kong, or L. javense from Japan. We also described the utility of trnK intron including matK. A percentage of the parsimony-informative characters in trnK intron sequence data (5.8%) was significantly higher than that from other chloroplast regions, rbcL (2.4%) and rps4 (3.2%) sequence data. Nucleotide sequence data of the trnK intron including matK are more informative than other chloroplast DNA regions for identifying the phylogenetic relationships among Leucobryum species.

  13. HIV behavioural surveillance among refugees and surrounding host ...

    African Journals Online (AJOL)

    We used a standardised behavioural surveillance survey (BSS), modified to be directly relevant to populations in conflict and post-conflict settings as well as to their surrounding host populations, to survey the populations of a refugee settlement in south-western Uganda and its surrounding area. Two-stage probability ...

  14. The evaluation of angiotensin-converting enzyme (ACE) gene I/D and IL-4 gene intron 3 VNTR polymorphisms in coronary artery disease.

    Science.gov (United States)

    Basol, Nursah; Celik, Atac; Karakus, Nevin; Ozturk, Sibel Demir; Ozsoy, Sibel Demir; Yigit, Serbulent

    2014-01-01

    Genetic polymorphism is a strong risk factor for coronary artery disease (CAD). In the present study, our aim was to evaluate angiotensin-converting enzyme (ACE) gene I/D polymorphism and interleukin-4 (IL-4) gene Intron 3 variable number of tandem repeat (VNTR) polymorphism in CAD. One hundred and twenty-four CAD patients and one hundred and twenty-three controls were enrolled. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) analyses. The risk associated with inheriting the combined genotypes for the two polymorphisms were evaluated and it was found that the individuals who were P2P2-homozygous at IL-4 gene intron 3 VNTR and DD-homozygous at ACE gene I/D have a higher risk of developing CAD. Although, there is no correlation between IL4 VNTR polymorphism and ACE gene polymorphism and CAD, there is a strong association between CAD and co-existence of IL-4 VNTR and ACE gene polymorphisms in the Turkish population. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  15. Whole Exome Sequencing for the differential diagnosis of primary adrenal insufficiency in children

    Directory of Open Access Journals (Sweden)

    Li F Chan

    2015-08-01

    Full Text Available Adrenal insufficiency is a rare, but potentially fatal medical condition. In children the cause is most commonly congenital and in recent years a growing number of causative gene mutations have been identified resulting in a myriad of syndromes that share adrenal insufficiency as one of the main characteristics. The evolution of adrenal insufficiency is dependent on the variant and the particular gene affected, meaning rapid and accurate diagnosis is imperative for effective treatment of the patient. Common practice is for candidate genes to be sequenced individually, which is a time consuming process and complicated by overlapping clinical phenotypes. However, with the availability, and increasing cost effectiveness of whole exome sequencing there is the potential for this to become a powerful diagnostic tool. Here we report the results of whole exome sequencing of 43 patients referred to us with a diagnosis of familial glucocorticoid deficiency (FGD who were mutation negative for MC2R, MRAP and STAR the most commonly mutated genes in FGD. WES provided a rapid genetic diagnosis in 17/43 sequenced patients, for the remaining 60% the gene defect may be within intronic/regulatory regions not covered by WES or may be in gene(s representing novel aetiologies. The diagnosis of isolated or familial glucocorticoid deficiency was only confirmed in 3 of the 17 patients, other genetic diagnoses were adrenal hypo- and hyperplasia, Triple A and autoimmune polyendocrinopathy syndrome type I, emphasizing both the difficulty of phenotypically distinguishing between disorders of PAI and the utility of WES as a tool to achieve this.

  16. Gene identification and protein classification in microbial metagenomic sequence data via incremental clustering

    Directory of Open Access Journals (Sweden)

    Li Weizhong

    2008-04-01

    Full Text Available Abstract Background The identification and study of proteins from metagenomic datasets can shed light on the roles and interactions of the source organisms in their communities. However, metagenomic datasets are characterized by the presence of organisms with varying GC composition, codon usage biases etc., and consequently gene identification is challenging. The vast amount of sequence data also requires faster protein family classification tools. Results We present a computational improvement to a sequence clustering approach that we developed previously to identify and classify protein coding genes in large microbial metagenomic datasets. The clustering approach can be used to identify protein coding genes in prokaryotes, viruses, and intron-less eukaryotes. The computational improvement is based on an incremental clustering method that does not require the expensive all-against-all compute that was required by the original approach, while still preserving the remote homology detection capabilities. We present evaluations of the clustering approach in protein-coding gene identification and classification, and also present the results of updating the protein clusters from our previous work with recent genomic and metagenomic sequences. The clustering results are available via CAMERA, (http://camera.calit2.net. Conclusion The clustering paradigm is shown to be a very useful tool in the analysis of microbial metagenomic data. The incremental clustering method is shown to be much faster than the original approach in identifying genes, grouping sequences into existing protein families, and also identifying novel families that have multiple members in a metagenomic dataset. These clusters provide a basis for further studies of protein families.

  17. An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10

    Directory of Open Access Journals (Sweden)

    Pavan William J

    2008-10-01

    Full Text Available Abstract Background A major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised. Results We have combined in vivo testing of DNA fragments in zebrafish and computational comparative genomics to identify the first regulatory regions of the zebrafish sox10 gene. Both approaches converged on the 3' end of the conserved 1st intron as being critical for spatial patterning of sox10 in the embryo. Importantly, we have defined a minimal region crucial for this function. We show that this region contains numerous binding sites for transcription factors known to be essential in early neural crest induction, including Tcf/Lef, Sox and FoxD3. We show that the identity and relative position of these binding sites are conserved between zebrafish and mammals. A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation. Furthermore, we show that β-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results. Conclusion We have thus identified two major sites of sox10 regulation in vertebrates and provided evidence supporting a role for at least three factors in driving sox10 expression in neural crest, otic epithelium and oligodendrocyte domains.

  18. Frequencies of VNTR and RFLP polymorphisms associated with factor VIII gene in Singapore

    Energy Technology Data Exchange (ETDEWEB)

    Fong, I.; Lai, P.S.; Ouah, T.C. [National Univ. of Singapore (Malaysia)] [and others

    1994-09-01

    The allelic frequency of any polymorphism within a population determines its usefulness for genetic counselling. This is important in populations of non-Caucasian origin as RFLPs may significantly differ among ethnic groups. We report a study of five intragenic polymorphisms in factor VIII gene carried out in Singapore. The three PCR-based RFLP markers studied were Intron 18/Bcl I, Intron 19/Hind III and Intron 22/Xba I. In an analysis of 148 unrelated normal X chromosomes, the allele frequencies were found to be A1 = 0.18, A2 = 0.82 (Bcl I RFLP), A1 = 0.80, A2 = 0.20 (Hind III RFLP) and A1 = 0.58, and A2 = 0.42 (Xba I RFLP). The heterozygosity rates of 74 females analyzed separately were 31%, 32% and 84.2%, respectively. Linkage disequilibrium was also observed to some degree between Bcl I and Hind III polymorphism in our population. We have also analyzed a sequence polymorphism in Intron 7 using hybridization with radioactive-labelled {sup 32}P allele-specific oligonucleotide probes. This polymorphism was not very polymorphic in our population with only 2% of 117 individuals analyzed being informative. However, the use of a hypervariable dinucleotide repeat sequence (VNTR) in Intron 13 showed that 25 of our of 27 (93%) females were heterozygous. Allele frequencies ranged from 1 to 55 %. We conclude that a viable strategy for molecular analysis of Hemophilia A families in our population should include the use of Intron 18/Bcl I and Intron 22/Xba I RFLP markers and the Intron 13 VNTR marker.

  19. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Science.gov (United States)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  20. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    International Nuclear Information System (INIS)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-01-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in λ phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (∼ 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia

  1. MR imaging of the temporomandibular joint. Part 2. Effect of flip angle on MR imaging with FLASH sequence

    International Nuclear Information System (INIS)

    Sakamoto, Maya; Sasano, Takashi; Higano, Shuichi; Takahashi, Shoki; Kurihara, Noriko

    1998-01-01

    In our previous study on MR imaging of the temporomandibular joint (TMJ), fast low angle shot (FLASH) showed the highest image contrast between disc and surrounding TMJ tissues compared with those of 4 other sequences (i,e., fast imaging with steady precession (FISP), conventional T1-weighted spin echo (SE) and fast spin echo (FSE, TR/TE/ETL: 1100/12/3, 3000/15/7)). Furthermore, FLASH also received a high score on visual evaluation including the position and contour of the disc, and the border between the disc and surrounding tissues. Therefore, we concluded that FLASH was the most suitable sequence for evaluating the TMJ disc. However, the image contrast and signal intensity on MR imaging with gradient echo pulse sequence are affected by flip angle. Consequently, in this report, to find the most suitable flip angle for MR scanning of the TMJ using a FLASH sequence (TR/TE: 450/11), ten TMJs of 5 volunteers were experimentally imaged with various flip angles from 10 degrees to 70 degrees at an interval of 10 degrees between 10 to 70. The image contrast and contrast-to-noise ratio (CNR) between the disc and surrounding tissues were compared. In addition, signal-to-noise ratio (SNR) of phantoms was also calculated using the same imaging parameters. Visual evaluation including position and contour of the disc, and the border between the disc and surrounding tissues, was also performed by 4 radiologists. As the flip angle increased, imaging contrast decreased while SNR increased. Images with flip angles between 30 and 60 degrees demonstrated high CNR. On visual evaluation, images using flip angles between 30 and 50 degrees received high scores. In conclusion, FLASH sequence with a flip angle between 30 and 50 degrees was considered most suitable for evaluating the TMJ disc based on the results of visual assessment and analysis of three major components of image diagnostic quality: image contrast, CNR and SNR. (author)

  2. Characterization of a Canine Tetranucleotide Microsatellite Marker Located in the First Intron of the Tumor Necrosis Factor Alpha Gene

    OpenAIRE

    WATANABE, Masashi; TANAKA, Kazuaki; TAKIZAWA, Tatsuya; SEGAWA, Kazuhito; NEO, Sakurako; TSUCHIYA, Ryo; MURATA, Michiko; MURAKAMI, Masaru; HISASUE, Masaharu

    2013-01-01

    ABSTRACT A polymorphic tetranucleotide (GAAT)n microsatellite in the first intron of the canine tumor necrosis factor alpha (TNFA) gene was characterized in this study; 139 dogs were analyzed: 22 Beagles, 26 Chihuahuas, 20 Miniature Dachshunds, 24 Miniature Poodles, 22 Pembroke Welsh Corgis and 25 Shiba Inus. We detected the presence of the 4 alleles (GAAT)5, (GAAT)6, (GAAT)7 and (GAAT)8, including 9 of the 10 expected genotypes. The expected heterozygosity (He) and the polymorphic informatio...

  3. Two complete chloroplast genome sequences of Cannabis sativa varieties.

    Science.gov (United States)

    Oh, Hyehyun; Seo, Boyoung; Lee, Seunghwan; Ahn, Dong-Ha; Jo, Euna; Park, Jin-Kyoung; Min, Gi-Sik

    2016-07-01

    In this study, we determined the complete chloroplast (cp) genomes from two varieties of Cannabis sativa. The genome sizes were 153,848 bp (the Korean non-drug variety, Cheungsam) and 153,854 bp (the African variety, Yoruba Nigeria). The genome structures were identical with 131 individual genes [86 protein-coding genes (PCGs), eight rRNA, and 37 tRNA genes]. Further, except for the presence of an intron in the rps3 genes of two C. sativa varieties, the cp genomes of C. sativa had conservative features similar to that of all known species in the order Rosales. To verify the position of C. sativa within the order Rosales, we conducted phylogenetic analysis by using concatenated sequences of all PCGs from 17 complete cp genomes. The resulting tree strongly supported monophyly of Rosales. Further, the family Cannabaceae, represented by C. sativa, showed close relationship with the family Moraceae. The phylogenetic relationship outlined in our study is well congruent with those previously shown for the order Rosales.

  4. Size Polymorphism in Alleles of the Myoglobin Gene from Biomphalaria Mollusks

    Directory of Open Access Journals (Sweden)

    Marcelo M. Santoro

    2010-10-01

    Full Text Available Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin and globin-like proteins are widely distributed in nature, being found even in prokaryotes and a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from Biomphalaria glabrata, B. straminea and B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR and analyzed by PCR-RFLP. Results showed that the size (number or nucleotides and the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2 and 3; this characterizes a homozygous/heterozygous profile and it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.

  5. Identification of a single‐copy gene encoding a Type I chlorophyll a/b‐binding polypeptide of photosystem I in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, Poul E; Kristensen, Michael; Hoff, Tine

    1992-01-01

    We have isolated and sequenced cDNA and genomic clones from Arabidopsis thaliana which specify a 241 residue protein with 84% sequence identity to a photosystem I Type I chlorophyll a/b-binding (CAB) protein from tomato. The open reading frame is interrupted by three introns which are found...... at equivalent positions as the corresponding introns in the tomato gene. Comparison to the amino acid sequence of other CAB proteins confirms that all CAB proteins share two regions of very high similarity. However, near the N-terminus and between the conserved regions this light-harvesting complex I (LHCI...

  6. Circumstances surrounding aneurysmal subarachnoid hemorrhage

    NARCIS (Netherlands)

    Schievink, W. I.; Karemaker, J. M.; Hageman, L. M.; van der Werf, D. J.

    1989-01-01

    The circumstances surrounding aneurysmal subarachnoid hemorrhage were investigated in a group of 500 consecutive patients admitted to a neurosurgical center. Subarachnoid hemorrhage occurred during stressful events in 42.8% of the patients, during nonstrenuous activities in 34.4%, and during rest or

  7. Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

    International Nuclear Information System (INIS)

    Hanafusa, Tadashi; Shinji, Toshiyuki; Shiraha, Hidenori; Nouso, Kazuhiro; Iwasaki, Yoshiaki; Yumoto, Eichiro; Ono, Toshiro; Koide, Norio

    2005-01-01

    Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells

  8. Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

    Science.gov (United States)

    Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

    2016-03-01

    Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

  9. Organellar maturases: A window into the evolution of the spliceosome.

    Science.gov (United States)

    Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

    2015-09-01

    During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover

  10. Association analysis between a VNTR intron 8 polymorphism of the dopamine transporter gene (SLC6A3 and obsessive- compulsive disorder in a Brazilian sample Análise de associação entre um polimorfismo VNTR no intron 8 do gene do transportador de dopamina (SLC6A3 e transtorno obsessivo-compulsivo em uma amostra brasileira

    Directory of Open Access Journals (Sweden)

    Karen Miguita

    2007-12-01

    Full Text Available Family, twin and segregation analysis have provided evidences that genetic factors are implicated in the susceptibility for obsessive-compulsive disorder (OCD. Several lines of research suggest that the dopaminergic system may be involved in the pathophysiology of OCD. Thus, the aim of the present study was to investigate a possible association between a polymorphism located in intron 8 of the dopamine transporter gene (SLC6A3 and OCD in a Brazilian sample composed by 208 patients and 865 healthy controls. No statistically differences were observed in allelic and genotype distributions between cases and controls. No association was also observed when the sample was divided according to specific phenotypic features such as gender, presence of tic disorders co-morbidity and age at onset of obsessive-compulsive symptoms (OCS. Our results suggest that the intron 8 VNTR of the SLC6A3 investigated in this study is not related to the susceptibility for OCD in our Brazilian sample.Estudos de família, gêmeos e de segregação têm demonstrado que fatores genéticos estão envolvidos na susceptibilidade para o desenvolvimento do transtorno obsessivo-compulsivo (TOC. Várias linhas de pesquisa sugerem que o sistema dopaminérgico possa estar envolvido na fisiopatologia do TOC. Assim, o objetivo do presente estudo foi investigar uma possível associação entre o polimorfismo localizado no intron 8 do gene do transportador da dopamina (SLC6A3 e o TOC em uma amostra brasileira composta por 208 pacientes e 865 controles sadios. Nenhuma diferença estatisticamente significante foi observada nas distribuições alélicas e genotípicas entre os grupos de pacientes e controles. Nenhuma associação também foi observada quando as amostras foram divididas de acordo com características fenotípicas específicas, tais como gênero, presença de co-morbidade com tiques e idade de início dos sintomas obsessivo-compulsivo (SOC. Nossos resultados sugerem que o VNTR

  11. Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1987-01-01

    The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

  12. Mason: a JavaScript web site widget for visualizing and comparing annotated features in nucleotide or protein sequences.

    Science.gov (United States)

    Jaschob, Daniel; Davis, Trisha N; Riffle, Michael

    2015-03-07

    Sequence feature annotations (e.g., protein domain boundaries, binding sites, and secondary structure predictions) are an essential part of biological research. Annotations are widely used by scientists during research and experimental design, and are frequently the result of biological studies. A generalized and simple means of disseminating and visualizing these data via the web would be of value to the research community. Mason is a web site widget designed to visualize and compare annotated features of one or more nucleotide or protein sequence. Annotated features may be of virtually any type, ranging from annotating transcription binding sites or exons and introns in DNA to secondary structure or domain boundaries in proteins. Mason is simple to use and easy to integrate into web sites. Mason has a highly dynamic and configurable interface supporting multiple sets of annotations per sequence, overlapping regions, customization of interface and user-driven events (e.g., clicks and text to appear for tooltips). It is written purely in JavaScript and SVG, requiring no 3(rd) party plugins or browser customization. Mason is a solution for dissemination of sequence annotation data on the web. It is highly flexible, customizable, simple to use, and is designed to be easily integrated into web sites. Mason is open source and freely available at https://github.com/yeastrc/mason.

  13. Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

    Science.gov (United States)

    Trujillano, Daniel; Perez, Belén; González, Justo; Tornador, Cristian; Navarrete, Rosa; Escaramis, Georgia; Ossowski, Stephan; Armengol, Lluís; Cornejo, Verónica; Desviat, Lourdes R; Ugarte, Magdalena; Estivill, Xavier

    2014-01-01

    Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughput-targeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth. PMID:23942198

  14. Evolutionary genomics of plant genes encoding N-terminal-TM-C2 domain proteins and the similar FAM62 genes and synaptotagmin genes of metazoans

    Directory of Open Access Journals (Sweden)

    Craxton Molly

    2007-07-01

    Full Text Available Abstract Background Synaptotagmin genes are found in animal genomes and are known to function in the nervous system. Genes with a similar domain architecture as well as sequence similarity to synaptotagmin C2 domains have also been found in plant genomes. The plant genes share an additional region of sequence similarity with a group of animal genes named FAM62. FAM62 genes also have a similar domain architecture. Little is known about the functions of the plant genes and animal FAM62 genes. Indeed, many members of the large and diverse Syt gene family await functional characterization. Understanding the evolutionary relationships among these genes will help to realize the full implications of functional studies and lead to improved genome annotation. Results I collected and compared plant Syt-like sequences from the primary nucleotide sequence databases at NCBI. The collection comprises six groups of plant genes conserved in embryophytes: NTMC2Type1 to NTMC2Type6. I collected and compared metazoan FAM62 sequences and identified some similar sequences from other eukaryotic lineages. I found evidence of RNA editing and alternative splicing. I compared the intron patterns of Syt genes. I also compared Rabphilin and Doc2 genes. Conclusion Genes encoding proteins with N-terminal-transmembrane-C2 domain architectures resembling synaptotagmins, are widespread in eukaryotes. A collection of these genes is presented here. The collection provides a resource for studies of intron evolution. I have classified the collection into homologous gene families according to distinctive patterns of sequence conservation and intron position. The evolutionary histories of these gene families are traceable through the appearance of family members in different eukaryotic lineages. Assuming an intron-rich eukaryotic ancestor, the conserved intron patterns distinctive of individual gene families, indicate independent origins of Syt, FAM62 and NTMC2 genes. Resemblances

  15. Solving nucleic acid structures by molecular replacement: examples from group II intron studies

    International Nuclear Information System (INIS)

    Marcia, Marco; Humphris-Narayanan, Elisabeth; Keating, Kevin S.; Somarowthu, Srinivas; Rajashankar, Kanagalaghatta; Pyle, Anna Marie

    2013-01-01

    Strategies for phasing nucleic acid structures by molecular replacement, using both experimental and de novo designed models, are discussed. Structured RNA molecules are key players in ensuring cellular viability. It is now emerging that, like proteins, the functions of many nucleic acids are dictated by their tertiary folds. At the same time, the number of known crystal structures of nucleic acids is also increasing rapidly. In this context, molecular replacement will become an increasingly useful technique for phasing nucleic acid crystallographic data in the near future. Here, strategies to select, create and refine molecular-replacement search models for nucleic acids are discussed. Using examples taken primarily from research on group II introns, it is shown that nucleic acids are amenable to different and potentially more flexible and sophisticated molecular-replacement searches than proteins. These observations specifically aim to encourage future crystallographic studies on the newly discovered repertoire of noncoding transcripts

  16. Polymorphisms in promoter sequences of MDM2, p53, and p16INK4a genes in normal Japanese individuals

    Directory of Open Access Journals (Sweden)

    Yasuhito Ohsaka

    2010-01-01

    Full Text Available Research has been conducted to identify sequence polymorphisms of gene promoter regions in patients and control subjects, including normal individuals, and to determine the influence of these polymorphisms on transcriptional regulation in cells that express wild-type or mutant p53. In this study we isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced the promoter regions of the MDM2, p53, and p16INK4a genes. We identified polymorphisms comprising 3 nucleotide substitutions at exon 1 and intron 1 regions of the MDM2 gene and 1 nucleotide insertion at a poly(C nucleotide position in the p53 gene. The Japanese individuals also exhibited p16INK4a polymorphisms at several positions, including position -191. Reporter gene analysis by using luciferase revealed that the polymorphisms of MDM2, p53, and p16INK4a differentially altered luciferase activities in several cell lines, including the Colo320DM, U251, and T98G cell lines expressing mutant p53. Our results indicate that the promoter sequences of these genes differ among normal Japanese individuals and that polymorphisms can alter gene transcription activity.

  17. Morphological character evolution of Amorphophallus (Araceae) based on a combined phylogenetic analysis of trnL, rbcL, and LEAFY second intron sequences

    NARCIS (Netherlands)

    Sedayu, A.; Eurlings, M.C.M.; Gravendeel, B.; Hetterscheid, W.L.A.

    2010-01-01

    Sequences of three different genes in 69 taxa of Amorphophallus were combined to reconstruct the molecular phylogeny of this species-rich Aroid genus. The data set was analyzed by three different methods, Maximum Parsimony, Maximum Likelihood and Bayesian analysis, producing slightly different tree

  18. Novel parvoviruses in reptiles and genome sequence of a lizard parvovirus shed light on Dependoparvovirus genus evolution.

    Science.gov (United States)

    Pénzes, Judit J; Pham, Hanh T; Benkö, Mária; Tijssen, Peter

    2015-09-01

    Here, we report the detection and partial genome characterization of two novel reptilian parvoviruses derived from a short-tailed pygmy chameleon (Rampholeon brevicaudatus) and a corn snake (Pantherophis guttatus) along with the complete genome analysis of the first lizard parvovirus, obtained from four bearded dragons (Pogona vitticeps). Both homology searches and phylogenetic tree reconstructions demonstrated that all are members of the genus Dependoparvovirus. Even though most dependoparvoviruses replicate efficiently only in co-infections with large DNA viruses, no such agents could be detected in one of the bearded dragon samples, hence the possibility of autonomous replication was explored. The alternative ORF encoding the full assembly activating protein (AAP), typical for the genus, could be obtained from reptilian parvoviruses for the first time, with a structure that appears to be more ancient than that of avian and mammalian parvoviruses. All three viruses were found to harbour short introns as previously observed for snake adeno-associated virus, shorter than that of any non-reptilian dependoparvovirus. According to the phylogenetic calculations based on full non-structural protein (Rep) and AAP sequences, the monophyletic cluster of reptilian parvoviruses seems to be the most basal out of all lineages of genus Dependoparvovirus. The suspected ability for autonomous replication, results of phylogenetic tree reconstruction, intron lengths and the structure of the AAP suggested that a single Squamata origin instead of the earlier assumed diapsid (common avian-reptilian) origin is more likely for the genus Dependoparvovirus of the family Parvoviridae.

  19. Allelic variation of the inducible costimulator (ICOS) gene: detection of polymorphisms, analysis of the promoter region, and extended haplotype estimation

    DEFF Research Database (Denmark)

    Andersen, A.D.H.; Lange, Marianne; Lillevang, S.T.

    2003-01-01

    The human chromosome region 2q33 including the three costimulatory molecules CD28, CTLA-4 and ICOS, has been subject to much attention due to its linkage to a number of autoimmune diseases. The search for the causal relationship of this linkage has revealed several polymorphisms, but no variations...... in the amino acid sequences except for one polymorphism in, the leader sequence of CTLA-4. In the present study, we examined the ICOS gene of an unrelated group of healthy donors from the Danish population. We were able to report 16 intronic SNP, one intronic G-insert and two repeat regions in intron 4......, consistent with the [T](n) and the [GT](n) regions reported in a Japanese study. Putative haplotypes for the established SNP and repeat polymorphisms have been estimated by computational analysis. Sequencing of similar to3500 by of the upstream region of ICOS revealed an additional eight SNP of which two...

  20. Separate introns gained within short and long soluble peridinin-chlorophyll a-protein genes during radiation of Symbiodinium (Dinophyceae) clade A and B lineages - PLoS One

    Science.gov (United States)

    Here we document introns in two Symbiodinium clades that were most likely gained following divergence of this genus from other peridinin-containing dinoflagellate lineages. Soluble peridinin-chlorophyll a-proteins (sPCP) occur in short and long forms in different species, and all...

  1. Cholinergic enhancement reduces orientation-specific surround suppression but not visual crowding

    Directory of Open Access Journals (Sweden)

    Anna A. Kosovicheva

    2012-09-01

    Full Text Available Acetylcholine (ACh reduces the spatial spread of excitatory fMRI responses in early visual cortex and the receptive field sizes of V1 neurons. We investigated the perceptual consequences of these physiological effects of ACh with surround suppression and crowding, two tasks that involve spatial interactions between visual field locations. Surround suppression refers to the reduction in perceived stimulus contrast by a high-contrast surround stimulus. For grating stimuli, surround suppression is selective for the relative orientations of the center and surround, suggesting that it results from inhibitory interactions in early visual cortex. Crowding refers to impaired identification of a peripheral stimulus in the presence of flankers and is thought to result from excessive integration of visual features. We increased synaptic ACh levels by administering the cholinesterase inhibitor donepezil to healthy human subjects in a placebo-controlled, double-blind design. In Exp. 1, we measured surround suppression of a central grating using a contrast discrimination task with three conditions: 1 surround grating with the same orientation as the center (parallel, 2 surround orthogonal to the center, or 3 no surround. Contrast discrimination thresholds were higher in the parallel than in the orthogonal condition, demonstrating orientation-specific surround suppression (OSSS. Cholinergic enhancement reduced thresholds only in the parallel condition, thereby reducing OSSS. In Exp. 2, subjects performed a crowding task in which they reported the identity of a peripheral letter flanked by letters on either side. We measured the critical spacing between the target and flanking letters that allowed reliable identification. Cholinergic enhancement had no effect on critical spacing. Our findings suggest that ACh reduces spatial interactions in tasks involving segmentation of visual field locations but that these effects may be limited to early visual cortical

  2. Genome-wide identification and functional prediction of nitrogen-responsive intergenic and intronic long non-coding RNAs in maize (Zea mays L.).

    Science.gov (United States)

    Lv, Yuanda; Liang, Zhikai; Ge, Min; Qi, Weicong; Zhang, Tifu; Lin, Feng; Peng, Zhaohua; Zhao, Han

    2016-05-11

    Nitrogen (N) is an essential and often limiting nutrient to plant growth and development. Previous studies have shown that the mRNA expressions of numerous genes are regulated by nitrogen supplies; however, little is known about the expressed non-coding elements, for example long non-coding RNAs (lncRNAs) that control the response of maize (Zea mays L.) to nitrogen. LncRNAs are a class of non-coding RNAs larger than 200 bp, which have emerged as key regulators in gene expression. In this study, we surveyed the intergenic/intronic lncRNAs in maize B73 leaves at the V7 stage under conditions of N-deficiency and N-sufficiency using ribosomal RNA depletion and ultra-deep total RNA sequencing approaches. By integration with mRNA expression profiles and physiological evaluations, 7245 lncRNAs and 637 nitrogen-responsive lncRNAs were identified that exhibited unique expression patterns. Co-expression network analysis showed that the nitrogen-responsive lncRNAs were enriched mainly in one of the three co-expressed modules. The genes in the enriched module are mainly involved in NADH dehydrogenase activity, oxidative phosphorylation and the nitrogen compounds metabolic process. We identified a large number of lncRNAs in maize and illustrated their potential regulatory roles in response to N stress. The results lay the foundation for further in-depth understanding of the molecular mechanisms of lncRNAs' role in response to nitrogen stresses.

  3. Evolution of the genus Aristolochia - Systematics, Molecular Evolution and Ecology

    OpenAIRE

    Wanke, Stefan

    2007-01-01

    Evolution of Piperales – matK gene and trnK intron sequence data reveal lineage specific resolution contrast. Piperales are one of the largest basal angiosperm orders with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 1,000 species), Peperomia (ca. 1,500-1,700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (excep...

  4. Elongation Factor-1α Accurately Reconstructs Relationships Amongst Psyllid Families (Hemiptera: Psylloidea), with Possible Diagnostic Implications.

    Science.gov (United States)

    Martoni, Francesco; Bulman, Simon R; Pitman, Andrew; Armstrong, Karen F

    2017-12-05

    The superfamily Psylloidea (Hemiptera: Sternorrhyncha) lacks a robust multigene phylogeny. This impedes our understanding of the evolution of this group of insects and, consequently, an accurate identification of individuals, of their plant host associations, and their roles as vectors of economically important plant pathogens. The conserved nuclear gene elongation factor-1 alpha (EF-1α) has been valuable as a higher-level phylogenetic marker in insects and it has also been widely used to investigate the evolution of intron/exon structure. To explore evolutionary relationships among Psylloidea, polymerase chain reaction amplification and nucleotide sequencing of a 250-bp EF-1α gene fragment was applied to psyllids belonging to five different families. Introns were detected in three individuals belonging to two families. The nine genera belonging to the family Aphalaridae all lacked introns, highlighting the possibility of using intron presence/absence as a diagnostic tool at a family level. When paired with cytochrome oxidase I gene sequences, the 250 bp EF-1α sequence appeared to be a very promising higher-level phylogenetic marker for psyllids. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Placental vascular responses are dependent on surrounding tissue

    DEFF Research Database (Denmark)

    Brøgger, Torbjørn Halle

    -depth understanding of the mechanism regulating blood flow and perfusion is necessary if we are to come up with new ideas for intervention and treatment. Method: From fresh born placentas stem villi arteries were carefully dissected. The artery branches were divided. The surrounding tissue was removed from one end...... and was left untouched in the other end. Then using wire myography they were investigated in terms of contractility and sensitivity to physiological relevant human-like agonists. Results: Sensitivity to PGF2α, Tx-analog, 5-HT and endothelin-1 was significantly lower in arteries with intact surrounding tissue...... compared to arteries stripped of the tissue. The maximal force development was also significantly lower in arteries with surrounding tissue, when they were depolarized high extracellular [K+] or stimulated with PGF2α or endotheline-1. Conclusion: The perivascular tissue significantly alters stem villi...

  6. Placental vascular responses are dependent on surrounding tissue

    DEFF Research Database (Denmark)

    Brøgger, Torbjørn Halle

    . Materials and methods. From fresh born placentas, stem villi arteries were carefully dissected. The artery branches were divided. The surrounding tissue was removed from one end and was left untouched in the other end.Then, using wire myography, they were investigated in terms of contractility...... and sensitivity to physiological relevant human-like agonists. Results. Sensitivity to PGF2α, Tx-analog, 5-HT and endothelin-1 was significantly lower in arteries with intact surrounding tissue compared to arteries stripped of the tissue. The maximal force development was also significantly lower in arteries...... with surrounding tissue when they were depolarized high extracellular [K+] or stimulated with PGF2α or endotheline-1. Conclusion. The perivascular tissue significantly alters stem villi arteries' sensitivity and force development in a suppressive way. This implicates a new aspect of blood flow regulation...

  7. The complete chloroplast genome sequence of the medicinal plant Andrographis paniculata.

    Science.gov (United States)

    Ding, Ping; Shao, Yanhua; Li, Qian; Gao, Junli; Zhang, Runjing; Lai, Xiaoping; Wang, Deqin; Zhang, Huiye

    2016-07-01

    The complete chloroplast genome of Andrographis paniculata, an important medicinal plant with great economic value, has been studied in this article. The genome size is 150,249 bp in length, with 38.3% GC content. A pair of inverted repeats (IRs, 25,300 bp) are separated by a large single copy region (LSC, 82,459 bp) and a small single-copy region (SSC, 17,190 bp). The chloroplast genome contains 114 unique genes, 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In these genes, 15 genes contained 1 intron and 3 genes comprised of 2 introns.

  8. A synchronous surround increases the motion strength gain of motion.

    Science.gov (United States)

    Linares, Daniel; Nishida, Shin'ya

    2013-11-12

    Coherent motion detection is greatly enhanced by the synchronous presentation of a static surround (Linares, Motoyoshi, & Nishida, 2012). To further understand this contextual enhancement, here we measured the sensitivity to discriminate motion strength for several pedestal strengths with and without a surround. We found that the surround improved discrimination of low and medium motion strengths, but did not improve or even impaired discrimination of high motion strengths. We used motion strength discriminability to estimate the perceptual response function assuming additive noise and found that the surround increased the motion strength gain, rather than the response gain. Given that eye and body movements continuously introduce transients in the retinal image, it is possible that this strength gain occurs in natural vision.

  9. Transcription Factor KLF5 Binds a Cyclin E1 Polymorphic Intronic Enhancer to Confer Increased Bladder Cancer Risk

    Science.gov (United States)

    Pattison, Jillian M.; Posternak, Valeriya; Cole, Michael D.

    2016-01-01

    It is well established that environmental toxins, such as exposure to arsenic, are risk factors in the development of urinary bladder cancer, yet recent genome-wide association studies (GWAS) provide compelling evidence that there is a strong genetic component associated with disease predisposition. A single nucleotide polymorphism (SNP), rs8102137, was identified on chromosome 19q12, residing 6 kb upstream of the important cell cycle regulator and proto-oncogene, Cyclin E1 (CCNE1). However, the functional role of this variant in bladder cancer predisposition has been unclear since it lies within a non-coding region of the genome. Here, it is demonstrated that bladder cancer cells heterozygous for this SNP exhibit biased allelic expression of CCNE1 with 1.5-fold more transcription occurring from the risk allele. Furthermore, using chromatin immunoprecipitation assays, a novel enhancer element was identified within the first intron of CCNE1 that binds Kruppel-like Factor 5 (KLF5), a known transcriptional activator in bladder cancer. Moreover, the data reveal that the presence of rs200996365, a SNP in high linkage disequilibrium with rs8102137 residing in the center of a KLF5 motif, alters KLF5 binding to this genomic region. Through luciferase assays and CRISPR-Cas9 genome editing, a novel polymorphic intronic regulatory element controlling CCNE1 transcription is characterized. These studies uncover how a cancer-associated polymorphism mechanistically contributes to an increased predisposition for bladder cancer development. Implications A polymorphic KLF5 binding site near the CCNE1 gene explains genetic risk identified through genome wide association studies. PMID:27514407

  10. Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation

    Directory of Open Access Journals (Sweden)

    Radulfus WN Slijkerman

    2016-01-01

    Full Text Available Usher syndrome (USH is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G was reported in 2012, leading to the insertion of a pseudoexon (PE40 into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

  11. DETECTING PRESENCE OF C/T POLYMORPHISM AT POSITION 34 SECOND INTRON OF THE MYOSTATIN GENE IN RABBITS

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    Agnieszka MARKOWSKA

    2011-01-01

    Full Text Available Myostatin gene is a negative regulator of skeletal muscles growth. It is responsible for normal development of skeletal muscles. The objective of the research was to detect variation of C/T at position 34 of the second intron of the MNST gene in rabbits. The research included 114 rabbits: 54 of them Polish Rabbits, and 60 of them White Flemish Giants, examined by means of the PCR-RFLP method using AluI restriction enzyme. We found allele C with a frequency of 0.6184 of the examined rabbit population, and allele T with a frequency of 0.3816 of the examined rabbits.

  12. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

    2012-04-25

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  13. Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

    International Nuclear Information System (INIS)

    Li, Long; Pintel, David J.

    2012-01-01

    Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

  14. [Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis].

    Science.gov (United States)

    Yu, Xiao-Dan; Jiang, Chao; Huang, Lu-Qi; Qin, Shuang-Shuang; Zeng, Xiang-Mei; Chen, Ping; Yuan, Yuan

    2013-08-01

    To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

  15. Insights into evolution in Andean Polystichum (Dryopteridaceae) from expanded understanding of the cytosolic phosphoglucose isomerase gene.

    Science.gov (United States)

    Lyons, Brendan M; McHenry, Monique A; Barrington, David S

    2017-07-01

    Cytosolic phosphoglucose isomerase (pgiC) is an enzyme essential to glycolysis found universally in eukaryotes, but broad understanding of variation in the gene coding for pgiC is lacking for ferns. We used a substantially expanded representation of the gene for Andean species of the fern genus Polystichum to characterize pgiC in ferns relative to angiosperms, insects, and an amoebozoan; assess the impact of selection versus neutral evolutionary processes on pgiC; and explore evolutionary relationships of selected Andean species. The dataset of complete sequences comprised nine accessions representing seven species and one hybrid from the Andes and Serra do Mar. The aligned sequences of the full data set comprised 3376 base pairs (70% of the entire gene) including 17 exons and 15 introns from two central areas of the gene. The exons are highly conserved relative to angiosperms and retain substantial homology to insect pgiC, but intron length and structure are unique to the ferns. Average intron size is similar to angiosperms; intron number and location in insects are unlike those of the plants we considered. The introns included an array of indels and, in intron 7, an extensive microsatellite array with potential utility in analyzing population-level histories. Bayesian and maximum-parsimony analysis of 129 variable nucleotides in the Andean polystichums revealed that 59 (1.7% of the 3376 total) were phylogenetically informative; most of these united sister accessions. The phylogenetic trees for the Andean polystichums were incongruent with previously published cpDNA trees for the same taxa, likely the result of rapid evolutionary change in the introns and contrasting stability in the exons. The exons code a total of seven amino-acid substitutions. Comparison of non-synonymous to synonymous substitutions did not suggest that the pgiC gene is under selection in the Andes. Variation in pgiC including two additional accessions represented by incomplete sequences

  16. Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.

    Science.gov (United States)

    Bui, Thi Thu Hien; Belli, Martina; Fassina, Lorenzo; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio

    2017-05-01

    Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species. © 2017 Wiley Periodicals, Inc.

  17. Deep intronic mutation and pseudo exon activation as a novel muscular hypertrophy modifier in cattle.

    Directory of Open Access Journals (Sweden)

    Claire Bouyer

    Full Text Available Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases.

  18. Ferritin gene organization: differences between plants and animals suggest possible kingdom-specific selective constraints.

    Science.gov (United States)

    Proudhon, D; Wei, J; Briat, J; Theil, E C

    1996-03-01

    Ferritin, a protein widespread in nature, concentrates iron approximately 10(11)-10(12)-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n = 7) is higher than in animals (n = 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may

  19. Biogeography of the Pistia clade (Araceae): based on chloroplast and mitochondrial DNA sequences and Bayesian divergence time inference.

    Science.gov (United States)

    Renner, Susanne S; Zhang, Li-Bing

    2004-06-01

    Pistia stratiotes (water lettuce) and Lemna (duckweeds) are the only free-floating aquatic Araceae. The geographic origin and phylogenetic placement of these unrelated aroids present long-standing problems because of their highly modified reproductive structures and wide geographical distributions. We sampled chloroplast (trnL-trnF and rpl20-rps12 spacers, trnL intron) and mitochondrial sequences (nad1 b/c intron) for all genera implicated as close relatives of Pistia by morphological, restriction site, and sequencing data, and present a hypothesis about its geographic origin based on the consensus of trees obtained from the combined data, using Bayesian, maximum likelihood, parsimony, and distance analyses. Of the 14 genera closest to Pistia, only Alocasia, Arisaema, and Typhonium are species-rich, and the latter two were studied previously, facilitating the choice of representatives that span the roots of these genera. Results indicate that Pistia and the Seychelles endemic Protarum sechellarum are the basalmost branches in a grade comprising the tribes Colocasieae (Ariopsis, Steudnera, Remusatia, Alocasia, Colocasia), Arisaemateae (Arisaema, Pinellia), and Areae (Arum, Biarum, Dracunculus, Eminium, Helicodiceros, Theriophonum, Typhonium). Unexpectedly, all Areae genera are embedded in Typhonium, which throws new light on the geographic history of Areae. A Bayesian analysis of divergence times that explores the effects of multiple fossil and geological calibration points indicates that the Pistia lineage is 90 to 76 million years (my) old. The oldest fossils of the Pistia clade, though not Pistia itself, are 45-my-old leaves from Germany; the closest outgroup, Peltandreae (comprising a few species in Florida, the Mediterranean, and Madagascar), is known from 60-my-old leaves from Europe, Kazakhstan, North Dakota, and Tennessee. Based on the geographic ranges of close relatives, Pistia likely originated in the Tethys region, with Protarum then surviving on the

  20. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.

    Science.gov (United States)

    van der Klift, Heleen M; Tops, Carli M J; Bik, Elsa C; Boogaard, Merel W; Borgstein, Anne-Marijke; Hansson, Kerstin B M; Ausems, Margreet G E M; Gomez Garcia, Encarna; Green, Andrew; Hes, Frederik J; Izatt, Louise; van Hest, Liselotte P; Alonso, Angel M; Vriends, Annette H J T; Wagner, Anja; van Zelst-Stams, Wendy A G; Vasen, Hans F A; Morreau, Hans; Devilee, Peter; Wijnen, Juul T

    2010-05-01

    Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, co-amplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3' end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2-specific PCR primers and MLPA probes, designed on PSVs, in the 3' duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. (c) 2010 Wiley-Liss, Inc.

  1. Geochronological correlation of the late Precambrian sequences on and around the stable zones of Equatorial Africa

    International Nuclear Information System (INIS)

    Cahen, L.

    1982-01-01

    Very few radiometric age determinations have a bearing on the late Precambrian sequences of the stable zones of Equatorial Africa. A geochronological correlation of these sequences must therefore rest upon a compilation of ages from these stable zones and the corresponding mobile zones. About 15 ages of varying precision more or less well tied up with the stratigraphical sequences, together with results of the study of stromatolite assemblages allow a provisional correlation to be made. One conclusion of this correlation is that most mixtites surrounding the Congo basin are in a 950 +- 50 Ma age bracket and are presumably to be correlated. (Auth.)

  2. Influence of Surrounding Colors in the Illuminant-Color Mode on Color Constancy

    Directory of Open Access Journals (Sweden)

    Kazuho Fukuda

    2011-05-01

    Full Text Available On color constancy, we showed that brighter surrounding colors had greater influence than dim colors (Uchikawa, Kitazawa, MacLeod, Fukuda, 2010 APCV. Increasing luminance of a stimulus causes the change in appearance from the surface-color to the illuminant-color mode. However it is unknown whether the visual system considers such color appearance mode of surrounding colors to achieve color constancy. We investigated the influence of surrounding colors that appeared illuminant on color constancy. The stimulus was composed of a central test stimulus and surrounding six colors: bright and dim red, green and blue. The observers adjusted the chromaticity of the test stimulus to be appeared as an achromatic surface. The luminance balance of three bright surrounding colors was equalized with that of the optimal colors in three illuminant conditions, then, the luminance of one of the three bright colors was varied in the range beyond the critical luminance of color appearance mode transition. The results showed that increasing luminance of a bright surrounding color shifted the observers' achromatic setting toward its chromaticity, but this effect diminished for the surrounding color in the illuminant-color mode. These results suggest that the visual system considers color appearance mode of surrounding colors to accomplish color constancy.

  3. P1-13: Color Induction from Surround Color under Interocular Suppression

    Directory of Open Access Journals (Sweden)

    Ichiro Kuriki

    2012-10-01

    Full Text Available The effect of surround colors on color appearance is known to subserve color constancy in humans, but how multiple mechanisms in the visual system are involved in this effect is controversial. We used an interocular-suppression technique to examine how the effect occurs at the level higher than the interaction of binocular information. A test color chip (1.7 × 1.7 deg visual angle was presented in a static surround either with continuous-flash suppression in the dominant eye (CFS condition to make the surround inperceptible or without the suppression (no-CFS condition. The surround stimulus was either a Mondrian or a uniform field of the same mean chromaticity. Stimuli were simulated OSA color chips under red, white (D65, or green illuminant color and were presented on a CRT display. Unique yellows were measured by asking the subjects to judge whether the test stimulus appeared reddish or greenish. Two sizes of the surround stimuli (widths of 1 deg and 4 deg were used. Results showed significant shifts in unique yellow even under the CFS conditions, except for the 1 deg uniform-surround condition. Under the no-CFS condition, the shifts showed remarkable difference between subjects, except for the 4 deg Mondrian-surround condition. Interestingly, trends of the shifts showed high consistency within each subject, across conditions. These results indicate that mechanisms at both higher and lower levels than the neuronal site of interocular suppression are involved, and that the color shifts follow each subject's strategy in the higher-order mechanisms when only insufficient clues are available in the surround to estimate illuminant color.

  4. Confronting, Confirming, and Dispelling Myths Surrounding ERP-in-the-Cloud

    DEFF Research Database (Denmark)

    Beaulieu, Tanya; C. Martin, Todd; Sarker, Saonee

    2015-01-01

    on the topic, there is substantial uncertainty surrounding the benefits and challenges of ERP cloud computing. Consequently, as often is the case with new technologies, popular myths surrounding the technology are used to make adoption and implementation decisions. As a first step toward providing an informed...... with stakeholders related to an ERP cloud-based solution. Our results dispel some of the myths, while supporting others, and highlight how ERP vendors work around the different types of challenges surrounding this technology. Our study also helps understand the benefits of ERP cloud computing, and informs about how...

  5. Plastome Sequencing of Ten Nonmodel Crop Species Uncovers a Large Insertion of Mitochondrial DNA in Cashew.

    Science.gov (United States)

    Rabah, Samar O; Lee, Chaehee; Hajrah, Nahid H; Makki, Rania M; Alharby, Hesham F; Alhebshi, Alawiah M; Sabir, Jamal S M; Jansen, Robert K; Ruhlman, Tracey A

    2017-11-01

    In plant evolution, intracellular gene transfer (IGT) is a prevalent, ongoing process. While nuclear and mitochondrial genomes are known to integrate foreign DNA via IGT and horizontal gene transfer (HGT), plastid genomes (plastomes) have resisted foreign DNA incorporation and only recently has IGT been uncovered in the plastomes of a few land plants. In this study, we completed plastome sequences for l0 crop species and describe a number of structural features including variation in gene and intron content, inversions, and expansion and contraction of the inverted repeat (IR). We identified a putative in cinnamon ( J. Presl) and other sequenced Lauraceae and an apparent functional transfer of to the nucleus of quinoa ( Willd.). In the orchard tree cashew ( L.), we report the insertion of an ∼6.7-kb fragment of mitochondrial DNA into the plastome IR. BLASTn analyses returned high identity hits to mitogenome sequences including an intact open reading frame. Using three plastome markers for five species of , we generated a phylogeny to investigate the distribution and timing of the insertion. Four species share the insertion, suggesting that this event occurred <20 million yr ago in a single clade in the genus. Our study extends the observation of mitochondrial to plastome IGT to include long-lived tree species. While previous studies have suggested possible mechanisms facilitating IGT to the plastome, more examples of this phenomenon, along with more complete mitogenome sequences, will be required before a common, or variable, mechanism can be elucidated. Copyright © 2017 Crop Science Society of America.

  6. Structure of the human hepatic triglyceride lipase gene

    International Nuclear Information System (INIS)

    Cai, Shengjian; Wong, D.M.; Chen, Sanhwan; Chan, L.

    1989-01-01

    The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residue 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains. The observations strongly support the common evolutionary origin of these two lipolytic enzymes

  7. The T -786C, G894T, and Intron 4 VNTR (4a/b) Polymorphisms of the Endothelial Nitric Oxide Synthase Gene in Prostate Cancer Cases.

    Science.gov (United States)

    Diler, S B; Öden, A

    2016-02-01

    In previously conducted some studies it has been revealed that nitric oxide (NO) and nitric oxide synthase (NOS) system play a significant role in carcinogenesis. Nitric oxide (NO) is regulated by endothelial nitric oxide synthase (eNOS) enzyme which is one of the isoenzymes of NO synthase (NOS). In this study we have tried to come to a conclusion about whether eNOS gene T -786C, G894T and Intron 4 VNTR (4a/b) polymorphisms might be considered as a risk factor causing prostate cancer (PCa) or not. A total of 200 subjects were included in this research. 84 patients with PCa (mean age 70.0 ± 6.4) and 116 healthy controls (mean age 69.9 ± 7.5) were recruited in this case-control study. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Maryland, USA), according to the manufacturer's guidelines. The T-786C, G894T and Intron 4 VNTR (4a/b) polymorphisms were amplified using polymerase chain reation (PCR), detected by restriction fragment length polymorphism (RFLP). For T -786C polymorphism CC genotype [odds ratio (OR): 0.34, 95% confidence interval (CI): 0.15-0.78, P = 0.009)] and allele frequency (OR: 0.631, CI: 0.421-0.946, P = 0.026) is significant for control. In patients with PCa eNOS G894T polymorphism, both GT (OR: 0.069, CI: 0.027-0.174; P = 0.0001) and TT (OR: 0.040, CI: 0.013-0.123; P = = 0.0001) genotype distribution, and also T allele frequency (OR: 0.237, CI: 0.155-0.362, P = 0.0001) were considered significant statistically. While genotype distribution for the other polymorphism eNOS, intron 4 VNTR (4a/b), is insignificant statistically, "a" allele frequency was found out to be significant (OR: 2.223, CI: 1.311-3.769, P = 0.003). In this study we indicated that genotype and allele frequencies of eNOS T -786C and G894T polymorphisms are statistically significant in patients with PCa. eNOS T -786C and G894T polymorphisms may be associated with PCa susceptibility in the Turkish population. In contrast, intron 4 VNTR (4a

  8. An integrative genomic approach reveals coordinated expression of intronic miR-335, miR-342, and miR-561 with deregulated host genes in multiple myeloma

    Directory of Open Access Journals (Sweden)

    Agnelli Luca

    2008-08-01

    Full Text Available Abstract Background The role of microRNAs (miRNAs in multiple myeloma (MM has yet to be fully elucidated. To identify miRNAs that are potentially deregulated in MM, we investigated those mapping within transcription units, based on evidence that intronic miRNAs are frequently coexpressed with their host genes. To this end, we monitored host transcript expression values in a panel of 20 human MM cell lines (HMCLs and focused on transcripts whose expression varied significantly across the dataset. Methods miRNA expression was quantified by Quantitative Real-Time PCR. Gene expression and genome profiling data were generated on Affymetrix oligonucleotide microarrays. Significant Analysis of Microarrays algorithm was used to investigate differentially expressed transcripts. Conventional statistics were used to test correlations for significance. Public libraries were queried to predict putative miRNA targets. Results We identified transcripts specific to six miRNA host genes (CCPG1, GULP1, EVL, TACSTD1, MEST, and TNIK whose average changes in expression varied at least 2-fold from the mean of the examined dataset. We evaluated the expression levels of the corresponding intronic miRNAs and identified a significant correlation between the expression levels of MEST, EVL, and GULP1 and those of the corresponding miRNAs miR-335, miR-342-3p, and miR-561, respectively. Genome-wide profiling of the 20 HMCLs indicated that the increased expression of the three host genes and their corresponding intronic miRNAs was not correlated with local copy number variations. Notably, miRNAs and their host genes were overexpressed in a fraction of primary tumors with respect to normal plasma cells; however, this finding was not correlated with known molecular myeloma groups. The predicted putative miRNA targets and the transcriptional profiles associated with the primary tumors suggest that MEST/miR-335 and EVL/miR-342-3p may play a role in plasma cell homing and

  9. Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders.

    Science.gov (United States)

    Codina-Solà, Marta; Rodríguez-Santiago, Benjamín; Homs, Aïda; Santoyo, Javier; Rigau, Maria; Aznar-Laín, Gemma; Del Campo, Miguel; Gener, Blanca; Gabau, Elisabeth; Botella, María Pilar; Gutiérrez-Arumí, Armand; Antiñolo, Guillermo; Pérez-Jurado, Luis Alberto; Cuscó, Ivon

    2015-01-01

    Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits. We performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants. We detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance. Integrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression.

  10. MRI of the coronary arteries: flip angle train optimization for 3D sequences

    International Nuclear Information System (INIS)

    Hietschold, V.; Kittner, T.; Abolmaali, N.

    2002-01-01

    Application of contrast agents in MRI of coronary arteries improves contrast-to-noise ratio (CNR), but widens the range of T 1 relaxation times of the tissues to be imaged. The flip angle train, generated for the measurement of all phase-encoding steps in the 3 rd spatial dimension of the navigator echo FLASH sequence used, is optimal only for one T 1 . Computer simulations show that it is not advisable to optimize the sequence on the basis of an extremely short T 1 relaxation time (such as in the case of contrast-enhanced vessels) because the imaging of the surrounding tissue would be negatively influenced. A sequence optimization to a T1 of approximately 200 ms seems to allow a CNR improvement of ≥ 50%. (orig.)

  11. A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

    Science.gov (United States)

    Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

    2013-11-06

    Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

  12. Complete Chloroplast Genome Sequences and Comparative Analysis of Chenopodium quinoa and C. album.

    Science.gov (United States)

    Hong, Su-Young; Cheon, Kyeong-Sik; Yoo, Ki-Oug; Lee, Hyun-Oh; Cho, Kwang-Soo; Suh, Jong-Taek; Kim, Su-Jeong; Nam, Jeong-Hwan; Sohn, Hwang-Bae; Kim, Yul-Ho

    2017-01-01

    The Chenopodium genus comprises ~150 species, including Chenopodium quinoa and Chenopodium album , two important crops with high nutritional value. To elucidate the phylogenetic relationship between the two species, the complete chloroplast (cp) genomes of these species were obtained by next generation sequencing. We performed comparative analysis of the sequences and, using InDel markers, inferred phylogeny and genetic diversity of the Chenopodium genus. The cp genome is 152,099 bp ( C. quinoa ) and 152,167 bp ( C. album ) long. In total, 119 genes (78 protein-coding, 37 tRNA, and 4 rRNA) were identified. We found 14 ( C. quinoa ) and 15 ( C. album ) tandem repeats (TRs); 14 TRs were present in both species and C. album and C. quinoa each had one species-specific TR. The trnI-GAU intron sequences contained one ( C. quinoa ) or two ( C. album ) copies of TRs (66 bp); the InDel marker was designed based on the copy number variation in TRs. Using the InDel markers, we detected this variation in the TR copy number in four species, Chenopodium hybridum, Chenopodium pumilio, Chenopodium ficifolium , and Chenopodium koraiense , but not in Chenopodium glaucum . A comparison of coding and non-coding regions between C. quinoa and C. album revealed divergent sites. Nucleotide diversity >0.025 was found in 17 regions-14 were located in the large single copy region (LSC), one in the inverted repeats, and two in the small single copy region (SSC). A phylogenetic analysis based on 59 protein-coding genes from 25 taxa resolved Chenopodioideae monophyletic and sister to Betoideae. The complete plastid genome sequences and molecular markers based on divergence hotspot regions in the two Chenopodium taxa will help to resolve the phylogenetic relationships of Chenopodium .

  13. MR imaging of cranial nerve lesions using six different high-resolution T1- and T2(*)-weighted 3D and 2D sequences

    Energy Technology Data Exchange (ETDEWEB)

    Seitz, J.; Held, P.; Strotzer, M.; Voelk, M.; Nitz, W.R.; Dorenbeck, U.; Feuerbach, S. [Univ. Hospital of Regensburg (Germany). Dept. of Diagnostic Radiology; Stamato, S. [Univ. of California, San Diego, CA (United States). Dept. of Radiology

    2002-07-01

    Purpose: To find a suitable high-resolution MR protocol for the visualization of lesions of all 12 cranial nerves. Material and Methods: Thirty-eight pathologically changed cranial nerves (17 patients) were studied with MR imaging at 1.5T using 3D T2*-weighted CISS, T1-weighted 3D MP-RAGE (without and with i.v. contrast medium), T2-weighted 3D TSE, T2-weighted 2D TSE and T1-weighted fat saturation 2D TSE sequences. Visibility of the 38 lesions of the 12 cranial nerves in each sequence was evaluated by consensus of two radiologists using an evaluation scale from 1 (excellently visible) to 4 (not visible). Results: The 3D CISS sequence provided the best resolution of the cranial nerves and their lesions when surrounded by CSF. In nerves which were not surrounded by CSF, the 2D T1-weighted contrast-enhanced fat suppression technique was the best sequence. Conclusions: A combination of 3D CISS, the 2D T1-weighted fat suppressed sequence and a 3D contrast-enhanced MP-RAGE proved to be the most useful sequence to visualize all lesions of the cranial nerves. For the determination of enhancement, an additional 3D MP-RAGE sequence without contrast medium is required. This sequence is also very sensitive for the detection of hemorrhage.

  14. MR imaging of cranial nerve lesions using six different high-resolution T1- and T2(*)-weighted 3D and 2D sequences

    International Nuclear Information System (INIS)

    Seitz, J.; Held, P.; Strotzer, M.; Voelk, M.; Nitz, W.R.; Dorenbeck, U.; Feuerbach, S.; Stamato, S.

    2002-01-01

    Purpose: To find a suitable high-resolution MR protocol for the visualization of lesions of all 12 cranial nerves. Material and Methods: Thirty-eight pathologically changed cranial nerves (17 patients) were studied with MR imaging at 1.5T using 3D T2*-weighted CISS, T1-weighted 3D MP-RAGE (without and with i.v. contrast medium), T2-weighted 3D TSE, T2-weighted 2D TSE and T1-weighted fat saturation 2D TSE sequences. Visibility of the 38 lesions of the 12 cranial nerves in each sequence was evaluated by consensus of two radiologists using an evaluation scale from 1 (excellently visible) to 4 (not visible). Results: The 3D CISS sequence provided the best resolution of the cranial nerves and their lesions when surrounded by CSF. In nerves which were not surrounded by CSF, the 2D T1-weighted contrast-enhanced fat suppression technique was the best sequence. Conclusions: A combination of 3D CISS, the 2D T1-weighted fat suppressed sequence and a 3D contrast-enhanced MP-RAGE proved to be the most useful sequence to visualize all lesions of the cranial nerves. For the determination of enhancement, an additional 3D MP-RAGE sequence without contrast medium is required. This sequence is also very sensitive for the detection of hemorrhage

  15. Ecological mechanisms linking protected areas to surrounding lands.

    Science.gov (United States)

    Hansen, Andrew J; DeFries, Ruth

    2007-06-01

    Land use is expanding and intensifying in the unprotected lands surrounding many of the world's protected areas. The influence of this land use change on ecological processes is poorly understood. The goal of this paper is to draw on ecological theory to provide a synthetic framework for understanding how land use change around protected areas may alter ecological processes and biodiversity within protected areas and to provide a basis for identifying scientifically based management alternatives. We first present a conceptual model of protected areas embedded within larger ecosystems that often include surrounding human land use. Drawing on case studies in this Invited Feature, we then explore a comprehensive set of ecological mechanisms by which land use on surrounding lands may influence ecological processes and biodiversity within reserves. These mechanisms involve changes in ecosystem size, with implications for minimum dynamic area, species-area effect, and trophic structure; altered flows of materials and disturbances into and out of reserves; effects on crucial habitats for seasonal and migration movements and population source/sink dynamics; and exposure to humans through hunting, poaching, exotics species, and disease. These ecological mechanisms provide a basis for assessing the vulnerability of protected areas to land use. They also suggest criteria for designing regional management to sustain protected areas in the context of surrounding human land use. These design criteria include maximizing the area of functional habitats, identifying and maintaining ecological process zones, maintaining key migration and source habitats, and managing human proximity and edge effects.

  16. Amino acid and nucleotide recurrence in aligned sequences: synonymous substitution patterns in association with global and local base compositions.

    Science.gov (United States)

    Nishizawa, M; Nishizawa, K

    2000-10-01

    The tendency for repetitiveness of nucleotides in DNA sequences has been reported for a variety of organisms. We show that the tendency for repetitive use of amino acids is widespread and is observed even for segments conserved between human and Drosophila melanogaster at the level of >50% amino acid identity. This indicates that repetitiveness influences not only the weakly constrained segments but also those sequence segments conserved among phyla. Not only glutamine (Q) but also many of the 20 amino acids show a comparable level of repetitiveness. Repetitiveness in bases at codon position 3 is stronger for human than for D.melanogaster, whereas local repetitiveness in intron sequences is similar between the two organisms. While genes for immune system-specific proteins, but not ancient human genes (i.e. human homologs of Escherichia coli genes), have repetitiveness at codon bases 1 and 2, repetitiveness at codon base 3 for these groups is similar, suggesting that the human genome has at least two mechanisms generating local repetitiveness. Neither amino acid nor nucleotide repetitiveness is observed beyond the exon boundary, denying the possibility that such repetitiveness could mainly stem from natural selection on mRNA or protein sequences. Analyses of mammalian sequence alignments show that while the 'between gene' GC content heterogeneity, which is linked to 'isochores', is a principal factor associated with the bias in substitution patterns in human, 'within gene' heterogeneity in nucleotide composition is also associated with such bias on a more local scale. The relationship amongst the various types of repetitiveness is discussed.

  17. A Comparison of the First Two Sequenced Chloroplast Genomes in Asteraceae: Lettuce and Sunflower

    Energy Technology Data Exchange (ETDEWEB)

    Timme, Ruth E.; Kuehl, Jennifer V.; Boore, Jeffrey L.; Jansen, Robert K.

    2006-01-20

    Asteraceae is the second largest family of plants, with over 20,000 species. For the past few decades, numerous phylogenetic studies have contributed to our understanding of the evolutionary relationships within this family, including comparisons of the fast evolving chloroplast gene, ndhF, rbcL, as well as non-coding DNA from the trnL intron plus the trnLtrnF intergenic spacer, matK, and, with lesser resolution, psbA-trnH. This culminated in a study by Panero and Funk in 2002 that used over 13,000 bp per taxon for the largest taxonomic revision of Asteraceae in over a hundred years. Still, some uncertainties remain, and it would be very useful to have more information on the relative rates of sequence evolution among various genes and on genome structure as a potential set of phylogenetic characters to help guide future phylogenetic structures. By way of contributing to this, we report the first two complete chloroplast genome sequences from members of the Asteraceae, those of Helianthus annuus and Lactuca sativa. These plants belong to two distantly related subfamilies, Asteroideae and Cichorioideae, respectively. In addition to these, there is only one other published chloroplast genome sequence for any plant within the larger group called Eusterids II, that of Panax ginseng (Araliaceae, 156,318 bps, AY582139). Early chloroplast genome mapping studies demonstrated that H. annuus and L. sativa share a 22 kb inversion relative to members of the subfamily Barnadesioideae. By comparison to outgroups, this inversion was shown to be derived, indicating that the Asteroideae and Cichorioideae are more closely related than either is to the Barnadesioideae. Later sequencing study found that taxa that share this 22 kb inversion also contain within this region a second, smaller, 3.3 kb inversion. These sequences also enable an analysis of patterns of shared repeats in the genomes at fine level and of RNA editing by comparison to available EST sequences. In addition, since

  18. The nature of surround-induced depolarizing responses in goldfish cones

    NARCIS (Netherlands)

    Kraaij, D. A.; Spekreijse, H.; Kamermans, M.

    2000-01-01

    Cones in the vertebrate retina project to horizontal and bipolar cells and the horizontal cells feedback negatively to cones. This organization forms the basis for the center/surround organization of the bipolar cells, a fundamental step in the visual signal processing. Although the surround

  19. Investigation of the readout electronics of DELPHI surround muon chamber

    International Nuclear Information System (INIS)

    Khovanskij, N.; Krumshtejn, Z.; Ol'shevskij, A.; Sadovskij, A.; Sedykh, Yu.; Molnar, J.; Sicho, P.; Tomsa, Z.

    1995-01-01

    The characteristics of the readout electronics of the DELPHI surround muon chambers with various AMPLEX chips (AMPLEX 16 and AMPLEX-SICAL) are presented. This electronics is studied in a cosmic rays test of the real surround muon chamber model. 4 refs., 6 figs., 1 tab

  20. Cloning, expression and characterisation of a novel gene encoding ...

    African Journals Online (AJOL)

    微软用户

    2012-01-12

    Jan 12, 2012 ... ... characterisation of a novel gene encoding a chemosensory protein from Bemisia ... The genomic DNA sequence comparisons revealed a 1490 bp intron ... have several conserved sequence motifs, including the. N-terminal ...

  1. FLAIR MR sequence in the diagnosis and follow-up of low-grade astrocytomas

    Directory of Open Access Journals (Sweden)

    Stošić-Opinćal Tatjana

    2005-01-01

    Full Text Available Aim. To evaluate the sensitivity of fluid-attenuated inversion recovery (FLAIR sequence in the diagnosis and follow-up of the patients with low-grade astrocytomas compared with T2-weighted (T2W sequence. Methods. Twenty-four patients with biopsy- confirmed low-grade astrocytoma (age range, 15-66 years underwent T1- weighted (T1W, T2W and FLAIR imaging with a superconducting unit 1.0 T. FLAIR images were qualitatively evaluated by comparison with T2W images by the three experienced neuroradiologists. To evaluate the diagnostic value of FLAIR, the neuroradiologists individually assessed the possibilities of the detection of lesions, as well as the possibilities of the differentiation of tumor from the surrounding edema on FLAIR vs. T2W images. Every examiner ranked FLAIR sequence vs. T2W in three degrees: worse, equal and better. Results. The comparison of FLAIR with T2W spin-echo (SE images with regard to the detection of the lesions showed that 82.8% of FLAIR studies were superior, 17.2% were of similar diagnostic value, and none was inferior to the T2W images. The comparison of images with regard to the differentiation of tumor boundaries vs. surrounding edema showed that 92.5% of FLAIR studies were superior, 7.5% were of similar diagnostic value, and none was inferior to the T2W images. Conclusion. Our results were similar to the previous studies' results concerning the advantages of FLAIR sequence in the diagnosis of low grade astrocytomas over T2W sequence. FLAIR was better at showing different tumor components, and at distinguishing CSF from the cystic component, and the postoperative cavity, compared with T2W images. Our conclusion was that FLAIR could be routinely used in the evaluation and follow-up of low-grade astrocytomas.

  2. Assembly and comparative analysis of complete mitochondrial genome sequence of an economic plant Salix suchowensis

    Directory of Open Access Journals (Sweden)

    Ning Ye

    2017-03-01

    Full Text Available Willow is a widely used dioecious woody plant of Salicaceae family in China. Due to their high biomass yields, willows are promising sources for bioenergy crops. In this study, we assembled the complete mitochondrial (mt genome sequence of S. suchowensis with the length of 644,437 bp using Roche-454 GS FLX Titanium sequencing technologies. Base composition of the S. suchowensis mt genome is A (27.43%, T (27.59%, C (22.34%, and G (22.64%, which shows a prevalent GC content with that of other angiosperms. This long circular mt genome encodes 58 unique genes (32 protein-coding genes, 23 tRNA genes and 3 rRNA genes, and 9 of the 32 protein-coding genes contain 17 introns. Through the phylogenetic analysis of 35 species based on 23 protein-coding genes, it is supported that Salix as a sister to Populus. With the detailed phylogenetic information and the identification of phylogenetic position, some ribosomal protein genes and succinate dehydrogenase genes are found usually lost during evolution. As a native shrub willow species, this worthwhile research of S. suchowensis mt genome will provide more desirable information for better understanding the genomic breeding and missing pieces of sex determination evolution in the future.

  3. SeqReporter: automating next-generation sequencing result interpretation and reporting workflow in a clinical laboratory.

    Science.gov (United States)

    Roy, Somak; Durso, Mary Beth; Wald, Abigail; Nikiforov, Yuri E; Nikiforova, Marina N

    2014-01-01

    A wide repertoire of bioinformatics applications exist for next-generation sequencing data analysis; however, certain requirements of the clinical molecular laboratory limit their use: i) comprehensive report generation, ii) compatibility with existing laboratory information systems and computer operating system, iii) knowledgebase development, iv) quality management, and v) data security. SeqReporter is a web-based application developed using ASP.NET framework version 4.0. The client-side was designed using HTML5, CSS3, and Javascript. The server-side processing (VB.NET) relied on interaction with a customized SQL server 2008 R2 database. Overall, 104 cases (1062 variant calls) were analyzed by SeqReporter. Each variant call was classified into one of five report levels: i) known clinical significance, ii) uncertain clinical significance, iii) pending pathologists' review, iv) synonymous and deep intronic, and v) platform and panel-specific sequence errors. SeqReporter correctly annotated and classified 99.9% (859 of 860) of sequence variants, including 68.7% synonymous single-nucleotide variants, 28.3% nonsynonymous single-nucleotide variants, 1.7% insertions, and 1.3% deletions. One variant of potential clinical significance was re-classified after pathologist review. Laboratory information system-compatible clinical reports were generated automatically. SeqReporter also facilitated quality management activities. SeqReporter is an example of a customized and well-designed informatics solution to optimize and automate the downstream analysis of clinical next-generation sequencing data. We propose it as a model that may envisage the development of a comprehensive clinical informatics solution. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. Religion's relationship with social boundaries surrounding gender ...

    African Journals Online (AJOL)

    Religion's relationship with social boundaries surrounding gender. ... is associated with segregation, marginalization and differentiation between men and women. ... are necessary in the society it should not be mistaken for gender inequality.

  5. Vasculature surrounding a nodule: A novel lung cancer biomarker.

    Science.gov (United States)

    Wang, Xiaohua; Leader, Joseph K; Wang, Renwei; Wilson, David; Herman, James; Yuan, Jian-Min; Pu, Jiantao

    2017-12-01

    To investigate whether the vessels surrounding a nodule depicted on non-contrast, low-dose computed tomography (LDCT) can discriminate benign and malignant screen detected nodules. We collected a dataset consisting of LDCT scans acquired on 100 subjects from the Pittsburgh Lung Screening study (PLuSS). Fifty subjects were diagnosed with lung cancer and 50 subjects had suspicious nodules later proven benign. For the lung cancer cases, the location of the malignant nodule in the LDCT scans was known; while for the benign cases, the largest nodule in the LDCT scan was used in the analysis. A computer algorithm was developed to identify surrounding vessels and quantify the number and volume of vessels that were connected or near the nodule. A nonparametric receiver operating characteristic (ROC) analysis was performed based on a single nodule per subject to assess the discriminability of the surrounding vessels to provide a lung cancer diagnosis. Odds ratio (OR) were computed to determine the probability of a nodule being lung cancer based on the vessel features. The areas under the ROC curves (AUCs) for vessel count and vessel volume were 0.722 (95% CI=0.616-0.811, plung cancer group 9.7 (±9.6) compared to the non-lung cancer group 4.0 (±4.3) CONCLUSION: Our preliminary results showed that malignant nodules are often surrounded by more vessels compared to benign nodules, suggesting that the surrounding vessel characteristics could serve as lung cancer biomarker for indeterminate nodules detected during LDCT lung cancer screening using only the information collected during the initial visit. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. SMRT Sequencing Revealed Mitogenome Characteristics and Mitogenome-Wide DNA Modification Pattern in Ophiocordyceps sinensis.

    Science.gov (United States)

    Kang, Xincong; Hu, Liqin; Shen, Pengyuan; Li, Rui; Liu, Dongbo

    2017-01-01

    Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis , with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3 , 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis , and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO 2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

  7. Deep transcriptome sequencing provides new insights into the structural and functional organization of the wheat genome.

    Science.gov (United States)

    Pingault, Lise; Choulet, Frédéric; Alberti, Adriana; Glover, Natasha; Wincker, Patrick; Feuillet, Catherine; Paux, Etienne

    2015-02-10

    Because of its size, allohexaploid nature, and high repeat content, the bread wheat genome is a good model to study the impact of the genome structure on gene organization, function, and regulation. However, because of the lack of a reference genome sequence, such studies have long been hampered and our knowledge of the wheat gene space is still limited. The access to the reference sequence of the wheat chromosome 3B provided us with an opportunity to study the wheat transcriptome and its relationships to genome and gene structure at a level that has never been reached before. By combining this sequence with RNA-seq data, we construct a fine transcriptome map of the chromosome 3B. More than 8,800 transcription sites are identified, that are distributed throughout the entire chromosome. Expression level, expression breadth, alternative splicing as well as several structural features of genes, including transcript length, number of exons, and cumulative intron length are investigated. Our analysis reveals a non-monotonic relationship between gene expression and structure and leads to the hypothesis that gene structure is determined by its function, whereas gene expression is subject to energetic cost. Moreover, we observe a recombination-based partitioning at the gene structure and function level. Our analysis provides new insights into the relationships between gene and genome structure and function. It reveals mechanisms conserved with other plant species as well as superimposed evolutionary forces that shaped the wheat gene space, likely participating in wheat adaptation.

  8. Childhood Suicide and Myths Surrounding It.

    Science.gov (United States)

    Greene, Dorothea B.

    1994-01-01

    Dispels five misconceptions surrounding the suicide of children: that children under the age of six do not commit suicide; that suicide in latency years is extremely rare; that psychodynamically and developmentally true depression is not possible in childhood; that child cannot understand finality of death; and that children are cognitively and…

  9. Genotyping Fanconi anemia patients from Serbia reveals three novel FANCD2 variants

    Directory of Open Access Journals (Sweden)

    Filipović-Tričković Jelena

    2017-01-01

    Full Text Available Fanconi anemia is rare inherited disease characterized by wide spectrum of congenital anomalies, progressive pancytopenia, and predisposition to hematological malignancies and solid tumors. Molecular genetic analysis of mutations in FANC genes is of a great importance for diagnosis confirmation, prenatal and carrier testing, as well as for prediction of chemotherapy outcome and disease complications. In this study we performed screening of frequently affected regions of FANCD2 gene for sequence variants in six unrelated FA-D2 patients in Serbia. This is the first molecular analysis of FANCD2 gene in Serbian FA-D2 patients. A total of 10 sequence variants were detected, one in homozygous, and nine in heterozygous state. Two variants were found within exons, and eight within introns, in deep intronic regions. In-silico analysis showed that among all detected variants one exon variant and three intron variants might have impact on splicing mechanism. Heterozygous variants found in intron 3, c.206-246delG; exon 26, c.2396 C>A and intron 28, c.2715+573 C>T were not previously reported. In-silico analysis revealed that among them, two (intron 3, c.206-246 delG and exon 26, c.2396 C>A could be novel disease-causing mutations. Many variants were found in more than one patient, including those unreported, indicating their possible ethnic association. Great number of variants in some patients suggests their non-random emergence in Fanconi anemia pathway. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173046

  10. Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer myostatin gene

    Directory of Open Access Journals (Sweden)

    Smith-Keune Carolyn

    2008-02-01

    Full Text Available Abstract Background Myostatin (MSTN is a member of the transforming growth factor-β superfamily that negatively regulates growth of skeletal muscle tissue. The gene encoding for the MSTN peptide is a consolidate candidate for the enhancement of productivity in terrestrial livestock. This gene potentially represents an important target for growth improvement of cultured finfish. Results Here we report molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer MSTN-1 gene. The barramundi MSTN-1 was encoded by three exons 379, 371 and 381 bp in length and translated into a 376-amino acid peptide. Intron 1 and 2 were 412 and 819 bp in length and presented typical GT...AG splicing sites. The upstream region contained cis-regulatory elements such as TATA-box and E-boxes. A first assessment of sequence variability suggested that higher mutation rates are found in the 5' flanking region with several SNP's present in this species. A putative micro RNA target site has also been observed in the 3'UTR (untranslated region and is highly conserved across teleost fish. The deduced amino acid sequence was conserved across vertebrates and exhibited characteristic conserved putative functional residues including a cleavage motif of proteolysis (RXXR, nine cysteines and two glycosilation sites. A qualitative analysis of the barramundi MSTN-1 expression pattern revealed that, in adult fish, transcripts are differentially expressed in various tissues other than skeletal muscles including gill, heart, kidney, intestine, liver, spleen, eye, gonad and brain. Conclusion Our findings provide valuable insights such as sequence variation and genomic information which will aid the further investigation of the barramundi MSTN-1 gene in association with growth. The finding for the first time in finfish MSTN of a miRNA target site in the 3'UTR provides an opportunity for the identification of regulatory mutations on the

  11. Making the most of RNA-seq: Pre-processing sequencing data with Opossum for reliable SNP variant detection [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Laura Oikkonen

    2017-01-01

    Full Text Available Identifying variants from RNA-seq (transcriptome sequencing data is a cost-effective and versatile alternative to whole-genome sequencing. However, current variant callers do not generally behave well with RNA-seq data due to reads encompassing intronic regions. We have developed a software programme called Opossum to address this problem. Opossum pre-processes RNA-seq reads prior to variant calling, and although it has been designed to work specifically with Platypus, it can be used equally well with other variant callers such as GATK HaplotypeCaller. In this work, we show that using Opossum in conjunction with either Platypus or GATK HaplotypeCaller maintains precision and improves the sensitivity for SNP detection compared to the GATK Best Practices pipeline. In addition, using it in combination with Platypus offers a substantial reduction in run times compared to the GATK pipeline so it is ideal when there are only limited time or computational resources available.

  12. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis

    Science.gov (United States)

    Diehn, Till A.; Pommerrenig, Benjamin; Bernhardt, Nadine; Hartmann, Anja; Bienert, Gerd P.

    2015-01-01

    Aquaporins (AQPs) are essential channel proteins that regulate plant water homeostasis and the uptake and distribution of uncharged solutes such as metalloids, urea, ammonia, and carbon dioxide. Despite their importance as crop plants, little is known about AQP gene and protein function in cabbage (Brassica oleracea) and other Brassica species. The recent releases of the genome sequences of B. oleracea and Brassica rapa allow comparative genomic studies in these species to investigate the evolution and features of Brassica genes and proteins. In this study, we identified all AQP genes in B. oleracea by a genome-wide survey. In total, 67 genes of four plant AQP subfamilies were identified. Their full-length gene sequences and locations on chromosomes and scaffolds were manually curated. The identification of six additional full-length AQP sequences in the B. rapa genome added to the recently published AQP protein family of this species. A phylogenetic analysis of AQPs of Arabidopsis thaliana, B. oleracea, B. rapa allowed us to follow AQP evolution in closely related species and to systematically classify and (re-) name these isoforms. Thirty-three groups of AQP-orthologous genes were identified between B. oleracea and Arabidopsis and their expression was analyzed in different organs. The two selectivity filters, gene structure and coding sequences were highly conserved within each AQP subfamily while sequence variations in some introns and untranslated regions were frequent. These data suggest a similar substrate selectivity and function of Brassica AQPs compared to Arabidopsis orthologs. The comparative analyses of all AQP subfamilies in three Brassicaceae species give initial insights into AQP evolution in these taxa. Based on the genome-wide AQP identification in B. oleracea and the sequence analysis and reprocessing of Brassica AQP information, our dataset provides a sequence resource for further investigations of the physiological and molecular functions of

  13. Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms.

    Science.gov (United States)

    Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

    2006-08-31

    Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats > or = 30 bp with a sequence identity > or = 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These

  14. Complete plastid genome sequence of Daucus carota: Implications for biotechnology and phylogeny of angiosperms

    Directory of Open Access Journals (Sweden)

    Ruhlman Tracey

    2006-08-01

    Full Text Available Abstract Background Carrot (Daucus carota is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. Results The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats ≥ 30 bp with a sequence identity ≥ 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP and maximum likelihood (ML were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. Conclusion The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap for the sister relationship of

  15. The complete chloroplast genome sequence of Taxus chinensis var. mairei (Taxaceae): loss of an inverted repeat region and comparative analysis with related species.

    Science.gov (United States)

    Zhang, Yanzhen; Ma, Ji; Yang, Bingxian; Li, Ruyi; Zhu, Wei; Sun, Lianli; Tian, Jingkui; Zhang, Lin

    2014-05-01

    Taxus chinensis var. mairei (Taxaceae) is a domestic variety of yew species in local China. This plant is one of the sources for paclitaxel, which is a promising antineoplastic chemotherapy drugs during the last decade. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of T. chinensis var. mairei. The T. chinensis var. mairei cp genome is 129,513 bp in length, with 113 single copy genes and two duplicated genes (trnI-CAU, trnQ-UUG). Among the 113 single copy genes, 9 are intron-containing. Compared to other land plant cp genomes, the T. chinensis var. mairei cp genome has lost one of the large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperm such as Cycas revoluta and Ginkgo biloba L. Compared to related species, the gene order of T. chinensis var. mairei has a large inversion of ~110kb including 91 genes (from rps18 to accD) with gene contents unarranged. Repeat analysis identified 48 direct and 2 inverted repeats 30 bp long or longer with a sequence identity greater than 90%. Repeated short segments were found in genes rps18, rps19 and clpP. Analysis also revealed 22 simple sequence repeat (SSR) loci and almost all are composed of A or T. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. RNA-Mediated Gene Duplication and Retroposons: Retrogenes, LINEs, SINEs, and Sequence Specificity

    Science.gov (United States)

    2013-01-01

    A substantial number of “retrogenes” that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 (LINE1, L1), has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes) and non-autonomous short interspersed elements (SINEs). The 3′-end sequences of various SINEs originated from a corresponding LINE. As the 3′-untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require “stringent” recognition of the 3′-end sequence of the RNA template. However, the 3′-ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of 3′-poly(A) repeats. Since the 3′-poly(A) repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A) repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A) repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution. PMID:23984183

  17. Complete Sequence and Analysis of the Mitochondrial Genome of Hemiselmis andersenii CCMP644 (Cryptophyceae

    Directory of Open Access Journals (Sweden)

    Bowman Sharen

    2008-05-01

    Full Text Available Abstract Background Cryptophytes are an enigmatic group of unicellular eukaryotes with plastids derived by secondary (i.e., eukaryote-eukaryote endosymbiosis. Cryptophytes are unusual in that they possess four genomes–a host cell-derived nuclear and mitochondrial genome and an endosymbiont-derived plastid and 'nucleomorph' genome. The evolutionary origins of the host and endosymbiont components of cryptophyte algae are at present poorly understood. Thus far, a single complete mitochondrial genome sequence has been determined for the cryptophyte Rhodomonas salina. Here, the second complete mitochondrial genome of the cryptophyte alga Hemiselmis andersenii CCMP644 is presented. Results The H. andersenii mtDNA is 60,553 bp in size and encodes 30 structural RNAs and 36 protein-coding genes, all located on the same strand. A prominent feature of the genome is the presence of a ~20 Kbp long intergenic region comprised of numerous tandem and dispersed repeat units of between 22–336 bp. Adjacent to these repeats are 27 copies of palindromic sequences predicted to form stable DNA stem-loop structures. One such stem-loop is located near a GC-rich and GC-poor region and may have a regulatory function in replication or transcription. The H. andersenii mtDNA shares a number of features in common with the genome of the cryptophyte Rhodomonas salina, including general architecture, gene content, and the presence of a large repeat region. However, the H. andersenii mtDNA is devoid of inverted repeats and introns, which are present in R. salina. Comparative analyses of the suite of tRNAs encoded in the two genomes reveal that the H. andersenii mtDNA has lost or converted its original trnK(uuu gene and possesses a trnS-derived 'trnK(uuu', which appears unable to produce a functional tRNA. Mitochondrial protein coding gene phylogenies strongly support a variety of previously established eukaryotic groups, but fail to resolve the relationships among higher

  18. Nanopore sequencing technology: a new route for the fast detection of unauthorized GMO.

    Science.gov (United States)

    Fraiture, Marie-Alice; Saltykova, Assia; Hoffman, Stefan; Winand, Raf; Deforce, Dieter; Vanneste, Kevin; De Keersmaecker, Sigrid C J; Roosens, Nancy H C

    2018-05-21

    In order to strengthen the current genetically modified organism (GMO) detection system for unauthorized GMO, we have recently developed a new workflow based on DNA walking to amplify unknown sequences surrounding a known DNA region. This DNA walking is performed on transgenic elements, commonly found in GMO, that were earlier detected by real-time PCR (qPCR) screening. Previously, we have demonstrated the ability of this approach to detect unauthorized GMO via the identification of unique transgene flanking regions and the unnatural associations of elements from the transgenic cassette. In the present study, we investigate the feasibility to integrate the described workflow with the MinION Next-Generation-Sequencing (NGS). The MinION sequencing platform can provide long read-lengths and deal with heterogenic DNA libraries, allowing for rapid and efficient delivery of sequences of interest. In addition, the ability of this NGS platform to characterize unauthorized and unknown GMO without any a priori knowledge has been assessed.

  19. MicroRNAs in Amoebozoa: deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs.

    Science.gov (United States)

    Avesson, Lotta; Reimegård, Johan; Wagner, E Gerhart H; Söderbom, Fredrik

    2012-10-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  20. Modifications of center-surround, spot detection and dot-pattern selective operators

    NARCIS (Netherlands)

    Petkov, Nicolai; Visser, Wicher T.

    2005-01-01

    This paper describes modifications of the models of center-surround and dot-pattern selective cells proposed previously. These modifications concern mainly the normalization of the difference of Gaussians (DoG) function used to model center-surround receptive fields, the normalization of

  1. Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.

    Directory of Open Access Journals (Sweden)

    Nicholas J Schurch

    Full Text Available The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3' untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3' polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1 gene and 3' UTR re-annotation (including extension of one 3' UTR by 5.9 kb; (2 disentangling of gene expression in complex regions; (3 clearer interpretation of small RNA expression and (4 identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

  2. Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars

    International Nuclear Information System (INIS)

    Baloch, M.J.; Jatoi, W.A.

    2012-01-01

    Physiological and yield traits such as stomatal conductance (mmol m-/sup 2/s/sup -1/), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 ( T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerab le

  3. Identification of new polymorphic regions and differentiation of cultivated olives (Olea europaea L.) through plastome sequence comparison

    Science.gov (United States)

    2010-01-01

    Background The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers. Results The complete plastid genome of the olive cultivar Frantoio was determined by direct sequence analysis using universal and novel PCR primers designed to amplify all overlapping regions. The chloroplast genome of the olive has an organisation and gene order that is conserved among numerous Angiosperm species and do not contain any of the inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses that have been found in the chloroplast genomes of the genera Jasminum and Menodora, from the same family as Olea. The annotated sequence was used to evaluate the content of coding genes, the extent, and distribution of repeated and long dispersed sequences and the nucleotide composition pattern. These analyses provided essential information for structural, functional and comparative genomic studies in olive plastids. Furthermore, the alignment of the olive plastome sequence to those of other varieties and species identified 30 new organellar polymorphisms within the cultivated olive. Conclusions In addition to identifying mutations that may play a functional role in modifying the metabolism and adaptation of olive cultivars, the new chloroplast markers represent a valuable tool to assess the level of olive intercultivar plastome variation for use in population genetic analysis, phylogenesis, cultivar characterisation and DNA food tracking. PMID:20868482

  4. Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes

    Science.gov (United States)

    López-Garriga, Juan; Cadilla, Carmen L.

    2016-01-01

    The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5’end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments. PMID:26824233

  5. Identification of eight novel SDHB, SDHC, SDHD germline variants in Danish pheochromocytoma/paraganglioma patients

    DEFF Research Database (Denmark)

    Bennedbæk, Marc; Rossing, Maria; Rasmussen, Åse K

    2016-01-01

    patients. METHODS: Mutational screening was performed by Sanger sequencing or next-generation sequencing. The frequencies of variants of unknown clinical significance, e.g. intronic, missense, and synonymous variants, were determined using the Exome Aggregation Consortium database, while the significance...

  6. Possession of ATM Sequence Variants as Predictor for Late Normal Tissue Responses in Breast Cancer Patients Treated With Radiotherapy

    International Nuclear Information System (INIS)

    Ho, Alice Y.; Fan, Grace; Atencio, David P.; Green, Sheryl; Formenti, Silvia C.; Haffty, Bruce G.; Iyengar, Preetha B.A.; Bernstein, Jonine L.; Stock, Richard G.; Cesaretti, Jamie A.; Rosenstein, Barry S.

    2007-01-01

    Purpose: The ATM gene product is a central component of cell cycle regulation and genomic surveillance. We hypothesized that DNA sequence alterations in ATM predict for adverse effects after external beam radiotherapy for early breast cancer. Methods and Materials: A total of 131 patients with a minimum of 2 years follow-up who had undergone breast-conserving surgery and adjuvant radiotherapy were screened for sequence alterations in ATM using DNA from blood lymphocytes. Genetic variants were identified using denaturing high performance liquid chromatography. The Radiation Therapy Oncology Group late morbidity scoring schemes for skin and subcutaneous tissues were applied to quantify the radiation-induced effects. Results: Of the 131 patients, 51 possessed ATM sequence alterations located within exons or in short intron regions flanking each exon that encompass putative splice site regions. Of these 51 patients, 21 (41%) exhibited a minimum of a Grade 2 late radiation response. In contrast, of the 80 patients without an ATM sequence variation, only 18 (23%) had radiation-induced adverse responses, for an odds ratio of 2.4 (95% confidence interval, 1.1-5.2). Fifteen patients were heterozygous for the G→A polymorphism at nucleotide 5557, which causes substitution of asparagine for aspartic acid at position 1853 of the ATM protein. Of these 15 patients, 8 (53%) exhibited a Grade 2-4 late response compared with 31 (27%) of the 116 patients without this alteration, for an odds ratio of 3.1 (95% confidence interval, 1.1-9.4). Conclusion: Sequence variants located in the ATM gene, in particular the 5557 G→A polymorphism, may predict for late adverse radiation responses in breast cancer patients

  7. Surrounding rock stress analysis of underground high level waste repository

    International Nuclear Information System (INIS)

    Liu Wengang; Wang Ju; Wang Guangdi

    2006-01-01

    During decay of nuclear waste, enormous energy was released, which results in temperature change of surrounding rock of depository. Thermal stress was produced because thermal expansion of rock was controlled. Internal structure of surrounding rock was damaged and strength of rock was weakened. So, variation of stress was a dynamic process with the variation of temperature. BeiShan region of Gansu province was determined to be the depository field in the future, it is essential to make research on granite in this region. In the process of experiment, basic physical parameters of granite were analyzed preliminary with MTS. Long range temperature and stress filed was simulated considering the damage effect of surrounding rock, and rules of temperature and stress was achieved. (authors)

  8. Making the most of RNA-seq: Pre-processing sequencing data with Opossum for reliable SNP variant detection [version 2; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Laura Oikkonen

    2017-03-01

    Full Text Available Identifying variants from RNA-seq (transcriptome sequencing data is a cost-effective and versatile complement to whole-exome (WES and whole-genome sequencing (WGS analysis. RNA-seq (transcriptome sequencing is primarily considered a method of gene expression analysis but it can also be used to detect DNA variants in expressed regions of the genome. However, current variant callers do not generally behave well with RNA-seq data due to reads encompassing intronic regions. We have developed a software programme called Opossum to address this problem. Opossum pre-processes RNA-seq reads prior to variant calling, and although it has been designed to work specifically with Platypus, it can be used equally well with other variant callers such as GATK HaplotypeCaller. In this work, we show that using Opossum in conjunction with either Platypus or GATK HaplotypeCaller maintains precision and improves the sensitivity for SNP detection compared to the GATK Best Practices pipeline. In addition, using it in combination with Platypus offers a substantial reduction in run times compared to the GATK pipeline so it is ideal when there are only limited time or computational resources available.

  9. The use of the hypervariable P8 region of trnL(UAA intron for identification of orchid species: Evidence from restriction site polymorphism analysis.

    Directory of Open Access Journals (Sweden)

    Rajkumar Kishor

    Full Text Available The P8 stem-loop region of the trnL intron, which is known to be hypervariable in size with multiple repeat motifs and created difficulties in alignment, is always excluded in phylogenetic as well as barcode analyses. This region was investigated for species discrimination in 98 taxa of orchids belonging to the tribe Vandeae using in silico mapping of restriction site polymorphism. The length of the P8 regions varied from 200 nucleotides in Aerides rosea to 669 nucleotides in Dendrophylax sallei. Forty two taxa had unique lengths, while as many as eight shared a common length of 521 nucleotides. Of the 35 restriction endonucleases producing digestions in the P8 regions, three, viz., AgsI, ApoI and TspDTI turned out to have recognition sites across all the 98 taxa being studied. When their restriction data were combined, 92 taxa could be discriminated leaving three taxon pairs. However, Acampe papillosa and Aeranthes arachnites despite having similar restriction sites differed in their P8 lengths. This is the first report on thorough investigation of the P8 region of trnL intron for search of species specific restriction sites and hence its use as a potential plant DNA barcode.

  10. Neutron spectrum in small iron pile surrounded by lead reflector

    International Nuclear Information System (INIS)

    Kimura, Itsuro; Hayashi, S.A.; Kobayashi, Katsuhei; Matsumura, Tetsuo; Nishihara, Hiroshi.

    1978-01-01

    In order to save the quantity of sample material, a possibility to assess group constants of a reactor material through measurement and analysis of neutron spectrum in a small sample pile surrounded by a reflector of heavy moderator, was investigated. As the sample and the reflector, we chose iron and lead, respectively. Although the time dispersion in moderation of neutrons was considerably prolonged by the lead reflector, this hardly interferes with the assessment of group constants. Theoretical calculation revealed that both the neutron flux spectrum and the sensitivity coefficient of group constants in an iron sphere, 35 cm in diameter surrounded by the lead reflector, 25 cm thick, were close to those of the bare iron sphere, 108 cm in diameter. The neutron spectra in a small iron pile surrounded by a lead reflector were experimentally obtained by the time-of-flight method with an electron linear accelerator and the result was compared with the predicted values. It could be confirmed that a small sample pile surrounded by a reflector, such as lead, was as useful as a much larger bulk pile for the assessment of group constants of a reactor material. (auth.)

  11. Smart Chips for Smart Surroundings -- 4S

    NARCIS (Netherlands)

    Schuler, Eberhard; König, Ralf; Becker, Jürgen; Rauwerda, G.K.; van de Burgwal, M.D.; Smit, Gerardus Johannes Maria; Cardoso, João M.P.; Hübner, Michael

    2011-01-01

    The overall mission of the 4S project (Smart Chips for Smart Surroundings) was to define and develop efficient flexible, reconfigurable core building blocks, including the supporting tools, for future Ambient System Devices. Reconfigurability offers the needed flexibility and adaptability, it

  12. A functional alternative splicing mutation in AIRE gene causes autoimmune polyendocrine syndrome type 1.

    Directory of Open Access Journals (Sweden)

    Junyu Zhang

    Full Text Available Autoimmune polyendocrine syndrome type 1 (APS-1 is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G>A (p.Gly155Ser in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G>A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203 containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1.

  13. Gene structure of the pregnancy-associated glycoprotein-like (PAG-L) in the Eurasian beaver (Castor fiber L.).

    Science.gov (United States)

    Lipka, Aleksandra; Majewska, Marta; Panasiewicz, Grzegorz; Bieniek-Kobuszewska, Martyna; Szafranska, Bozena

    2017-09-01

    The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1-9) and eight introns (A-H). The length of the CfPAG-L exons (59-200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288-1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3-72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.

  14. Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes.

    Science.gov (United States)

    Laopichienpong, Nararat; Muangmai, Narongrit; Chanhome, Lawan; Suntrarachun, Sunutcha; Twilprawat, Panupon; Peyachoknagul, Surin; Srikulnath, Kornsorn

    2017-03-01

    Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion–deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.

  15. Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

    Science.gov (United States)

    Godler, David E; Slater, Howard R; Bui, Quang M; Storey, Elsdon; Ono, Michele Y; Gehling, Freya; Inaba, Yoshimi; Francis, David; Hopper, John L; Kinsella, Glynda; Amor, David J; Hagerman, Randi J; Loesch, Danuta Z

    2012-03-01

    Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. Study participants included 74 control females (Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ 200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

  16. Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene.

    Directory of Open Access Journals (Sweden)

    Per Erixon

    Full Text Available BACKGROUND: Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare. METHODOLOGY/PRINCIPLE FINDINGS: We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family. Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs. CONCLUSIONS/SIGNIFICANCE: We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the

  17. Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae: Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer

    Directory of Open Access Journals (Sweden)

    Lemieux Claude

    2008-06-01

    Full Text Available Abstract Background To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales, Scenedesmus (Sphaeropleales, and Stigeoclonium (Chaetophorales revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade. Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales. Results Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns, and displays 99 different conserved genes and four long open reading frames (ORFs, three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members

  18. Comparison of C. elegans and C. briggsae genome sequences reveals extensive conservation of chromosome organization and synteny.

    Directory of Open Access Journals (Sweden)

    LaDeana W Hillier

    2007-07-01

    Full Text Available To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism-based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80-110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.

  19. Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient.

    Science.gov (United States)

    Greer, Kane; Mizzi, Kayla; Rice, Emily; Kuster, Lukas; Barrero, Roberto A; Bellgard, Matthew I; Lynch, Bryan J; Foley, Aileen Reghan; O Rathallaigh, Eoin; Wilton, Steve D; Fletcher, Sue

    2015-07-01

    We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

  20. Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

    Science.gov (United States)

    Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

    Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.