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Sample records for intronic cpg mediates

  1. 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.

  2. DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pathways

  3. Mendelian Randomization Analysis Identifies CpG Sites as Putative Mediators for Genetic Influences on Cardiovascular Disease Risk.

    Science.gov (United States)

    Richardson, Tom G; Zheng, Jie; Davey Smith, George; Timpson, Nicholas J; Gaunt, Tom R; Relton, Caroline L; Hemani, Gibran

    2017-10-05

    The extent to which genetic influences on cardiovascular disease risk are mediated by changes in DNA methylation levels has not been systematically explored. We developed an analytical framework that integrates genetic fine mapping and Mendelian randomization with epigenome-wide association studies to evaluate the causal relationships between methylation levels and 14 cardiovascular disease traits. We identified ten genetic loci known to influence proximal DNA methylation which were also associated with cardiovascular traits after multiple-testing correction. Bivariate fine mapping provided evidence that the individual variants responsible for the observed effects on cardiovascular traits at the ADCY3 and ADIPOQ loci were potentially mediated through changes in DNA methylation, although we highlight that we are unable to reliably separate causality from horizontal pleiotropy. Estimates of causal effects were replicated with results from large-scale consortia. Genetic variants and CpG sites identified in this study were enriched for histone mark peaks in relevant tissue types and gene promoter regions. Integrating our results with expression quantitative trait loci data, we provide evidence that variation at these regulatory regions is likely to also influence gene expression levels at these loci. Copyright © 2017 American Society of Human Genetics. All rights reserved.

  4. Plant siRNAs from introns mediate DNA methylation of host genes.

    Science.gov (United States)

    Chen, Dijun; Meng, Yijun; Yuan, Chunhui; Bai, Lin; Huang, Donglin; Lv, Shaolei; Wu, Ping; Chen, Ling-Ling; Chen, Ming

    2011-06-01

    Small RNAs (sRNAs), largely known as microRNAs (miRNAs) and short interfering RNAs (siRNAs), emerged as the critical components of genetic and epigenetic regulation in eukaryotic genomes. In animals, a sizable portion of miRNAs reside within the introns of protein-coding genes, designated as mirtron genes. Recently, high-throughput sequencing (HTS) revealed a huge amount of sRNAs that derived from introns in plants, such as the monocot rice (Oryza sativa). However, the biogenesis and the biological functions of this kind of sRNAs remain elusive. Here, we performed a genome-scale survey of intron-derived sRNAs in rice based on HTS data. Several introns were found to have great potential to form internal hairpin structures, and the short hairpins could generate miRNAs while the larger ones could produce siRNAs. Furthermore, 22 introns, termed "sirtrons," were identified from the rice protein-coding genes. The single-stranded sirtrons produced a diverse set of siRNAs from long hairpin structures. These sirtron-derived siRNAs are dominantly 21 nt, 22 nt, and 24 nt in length, whose production relied on DCL4, DCL2, and DCL3, respectively. We also observed a strong tendency for the sirtron-derived siRNAs to be coexpressed with their host genes. Finally, the 24-nt siRNAs incorporated with Argonaute 4 (AGO4) could direct DNA methylation on their host genes. In this regard, homeostatic self-regulation between intron-derived siRNAs and their host genes was proposed.

  5. Role of intron-mediated enhancement on accumulation of an Arabidopsis NB-LRR class R-protein that confers resistance to Cucumber mosaic virus.

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    Yukiyo Sato

    Full Text Available The accumulation of RCY1 protein, which is encoded by RESISTANCE TO CMV(Y (RCY1, a CC-NB-LRR class R-gene, is tightly correlated with the strength of the resistance to a yellow strain of Cucumber mosaic virus [CMV(Y] in Arabidopsis thaliana. In order to enhance resistance to CMV by overexpression of RCY1, A. thaliana was transformed with intron-less RCY1 cDNA construct under the control of strong CaMV35S promoter. Remarkably, a relative amount of RCY1 protein accumulation in the transformants was much lower than that in plants expressing genomic RCY1 under the control of its native promoter. To identify a regulatory element of RCY1 that could cause such differential levels of RCY1 accumulation, a series of RCY1 cDNA and genomic RCY1 constructs were transiently expressed in Nicotiana benthamiana leaves by the Agrobacterium-mediated infiltration method. Comparative analysis of the level of RCY1 accumulation in the leaf tissues transiently expressing each construct indicated that the intron located in the RCY1-coding region of genomic RCY1, but not the native RCY1 genomic promoter or the 5'-and 3'-untranslated regions of RCY1, was indispensable for high level RCY1 accumulation. The increased levels of RCY1 accelerated plant disease defense reactions. Interestingly, such intron-mediated enhancement of RCY1 accumulation depended neither on the abundance of the RCY1 transcript nor on the RCY1 specific-intron sequence. Taken together, intron-mediated RCY1 expression seems to play a key role in the expression of complete resistance to CMV(Y by maintaining RCY1 accumulation at high levels.

  6. Robust RNAi-mediated resistance to infection of seven potyvirids in soybean expressing an intron hairpin NIb RNA.

    Science.gov (United States)

    Yang, Xiangdong; Niu, Lu; Zhang, Wei; He, Hongli; Yang, Jing; Xing, Guojie; Guo, Dongquan; Du, Qian; Qian, Xueyan; Yao, Yao; Li, Qiyun; Dong, Yingshan

    2017-10-01

    Viral pathogens, such as soybean mosaic virus (SMV), are a major constraint in soybean production and often cause significant yield loss and quality deterioration. Engineering resistance by RNAi-mediated gene silencing is a powerful strategy for controlling viral diseases. In this study, a 248-bp inverted repeat of the replicase (nuclear inclusion b, NIb) gene was isolated from the SMV SC3 strain, driven by the leaf-specific rbcS2 promoter from Phaseolus vulgaris, and introduced into soybean. The transgenic lines had significantly lower average disease indices (ranging from 2.14 to 12.35) than did the non-transformed (NT) control plants in three consecutive generations, exhibiting a stable and significantly enhanced resistance to the SMV SC3 strain under field conditions. Furthermore, seed mottling did not occur in transgenic seeds, whereas the NT plants produced ~90% mottled seeds. Virus resistance spectrum screening showed that the greenhouse-grown transgenic lines exhibited robust resistance to five SMV strains (SC3, SC7, SC15, SC18, and a recombinant SMV), bean common mosaic virus, and watermelon mosaic virus. Nevertheless, no significantly enhanced resistance to bean pod mottle virus (BPMV, Comovirus) was observed in the transgenic lines relative to their NT counterparts. Consistent with the results of resistance evaluation, the accumulation of each potyvirid (but not of BPMV) was significantly inhibited in the transgenic plants relative to the NT controls as confirmed by quantitative real-time (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). These results demonstrate that robust RNAi-mediated resistance to multiple potyvirids in soybean was conferred by expressing an intron hairpin SMV NIb RNA.

  7. CH···O Hydrogen Bonds Mediate Highly Specific Recognition of Methylated CpG Sites by the Zinc Finger Protein Kaiso.

    Science.gov (United States)

    Nikolova, Evgenia N; Stanfield, Robyn L; Dyson, H Jane; Wright, Peter E

    2018-03-26

    Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein-DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression.

  8. Group I intron ribozymes

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2012-01-01

    Group I intron ribozymes constitute one of the main classes of ribozymes and have been a particularly important model in the discovery of key concepts in RNA biology as well as in the development of new methods. Compared to other ribozyme classes, group I intron ribozymes display considerable...... variation both in their structure and the reactions they catalyze. The best described pathway is the splicing pathway that results in a spliced out intron and ligated exons. This is paralleled by the circularization pathway that leads to full-length circular intron and un-ligated exons. In addition......, the intronic products of these pathways have the potential to integrate into targets and to form various types of circular RNA molecules. Thus, group I intron ribozymes and associated elements found within group I introns is a rich source of biological phenomena. This chapter provides a strategy and protocols...

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

    Directory of Open Access Journals (Sweden)

    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

  10. Ancient nature of alternative splicing and functions of introns

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Kemin; Salamov, Asaf; Kuo, Alan; Aerts, Andrea; Grigoriev, Igor

    2011-03-21

    Using four genomes: Chamydomonas reinhardtii, Agaricus bisporus, Aspergillus carbonarius, and Sporotricum thermophile with EST coverage of 2.9x, 8.9x, 29.5x, and 46.3x respectively, we identified 11 alternative splicing (AS) types that were dominated by intron retention (RI; biased toward short introns) and found 15, 35, 52, and 63percent AS of multiexon genes respectively. Genes with AS were more ancient, and number of AS correlated with number of exons, expression level, and maximum intron length of the gene. Introns with tendency to be retained had either stop codons or length of 3n+1 or 3n+2 presumably triggering nonsense-mediated mRNA decay (NMD), but introns retained in major isoforms (0.2-6percent of all introns) were biased toward 3n length and stop codon free. Stopless introns were biased toward phase 0, but 3n introns favored phase 1 that introduced more flexible and hydrophilic amino acids on both ends of introns which would be less disruptive to protein structure. We proposed a model in which minor RI intron could evolve into major RI that could facilitate intron loss through exonization.

  11. 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...

  12. Methylation-mediated deamination of 5-methylcytosine appears to give rise to mutations causing human inherited disease in CpNpG trinucleotides, as well as in CpG dinucleotides

    Directory of Open Access Journals (Sweden)

    Cooper David N

    2010-08-01

    Full Text Available Abstract The cytosine-guanine (CpG dinucleotide has long been known to be a hotspot for pathological mutation in the human genome. This hypermutability is related to its role as the major site of cytosine methylation with the attendant risk of spontaneous deamination of 5-methylcytosine (5mC to yield thymine. Cytosine methylation, however, also occurs in the context of CpNpG sites in the human genome, an unsurprising finding since the intrinsic symmetry of CpNpG renders it capable of supporting a semi-conservative model of replication of the methylation pattern. Recently, it has become clear that significant DNA methylation occurs in a CpHpG context (where H = A, C or T in a variety of human somatic tissues. If we assume that CpHpG methylation also occurs in the germline, and that 5mC deamination can occur within a CpHpG context, then we might surmise that methylated CpHpG sites could also constitute mutation hotspots causing human genetic disease. To test this postulate, 54,625 missense and nonsense mutations from 2,113 genes causing inherited disease were retrieved from the Human Gene Mutation Database http://www.hgmd.org. Some 18.2 per cent of these pathological lesions were found to be C → T and G → A transitions located in CpG dinucleotides (compatible with a model of methylation-mediated deamination of 5mC, an approximately ten-fold higher proportion than would have been expected by chance alone. The corresponding proportion for the CpHpG trinucleotide was 9.9 per cent, an approximately two-fold higher proportion than would have been expected by chance. We therefore estimate that ~5 per cent of missense/nonsense mutations causing human inherited disease may be attributable to methylation-mediated deamination of 5mC within a CpHpG context.

  13. Phylogenetic analyses suggest reverse splicing spread of group I introns in fungal ribosomal DNA

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    Simon Dawn M

    2005-11-01

    Full Text Available Abstract Background Group I introns have spread into over 90 different sites in nuclear ribosomal DNA (rDNA with greater than 1700 introns reported in these genes. These ribozymes generally spread through endonuclease-mediated intron homing. Another putative pathway is reverse splicing whereby a free group I intron inserts into a homologous or heterologous RNA through complementary base-pairing between the intron and exon RNA. Reverse-transcription of the RNA followed by general recombination results in intron spread. Here we used phylogenetics to test for reverse splicing spread in a taxonomically broadly sampled data set of fungal group I introns including 9 putatively ancient group I introns in the rDNA of the yeast-like symbiont Symbiotaphrina buchneri. Results Our analyses reveal a complex evolutionary history of the fungal introns with many cases of vertical inheritance (putatively for the 9 introns in S. buchneri and intron lateral transfer. There are several examples in which introns, many of which are still present in S. buchneri, may have spread through reverse splicing into heterologous rDNA sites. If the S. buchneri introns are ancient as we postulate, then group I intron loss was widespread in fungal rDNA evolution. Conclusion On the basis of these results, we suggest that the extensive distribution of fungal group I introns is at least partially explained by the reverse splicing movement of existing introns into ectopic rDNA sites.

  14. A cross-study analysis of prenatal exposures to environmental contaminants and the epigenome: support for stress-responsive transcription factor occupancy as a mediator of gene-specific CpG methylation patterning.

    Science.gov (United States)

    Martin, Elizabeth M; Fry, Rebecca C

    2016-01-01

    A biological mechanism by which exposure to environmental contaminants results in gene-specific CpG methylation patterning is currently unknown. We hypothesize that gene-specific CpG methylation is related to environmentally perturbed transcription factor occupancy. To test this hypothesis, a database of 396 genes with altered CpG methylation either in cord blood leukocytes or placental tissue was compiled from 14 studies representing assessments of six environmental contaminants. Subsequently, an in silico approach was used to identify transcription factor binding sites enriched among the genes with altered CpG methylation in relationship to the suite of environmental contaminants. For each study, the sequences of the promoter regions (representing -1000 to +500 bp from the transcription start site) of all genes with altered CpG methylation were analyzed for enrichment of transcription factor binding sites. Binding sites for a total of 56 unique transcription factors were identified to be enriched within the promoter regions of the genes. Binding sites for the Kidney-Enriched Krupple-like Factor 15, a known responder to endogenous stress, were enriched ( P support the transcription factor occupancy theory as a potential mechanism underlying environmentally-induced gene-specific CpG methylation.

  15. A cross-study analysis of prenatal exposures to environmental contaminants and the epigenome: support for stress-responsive transcription factor occupancy as a mediator of gene-specific CpG methylation patterning

    Science.gov (United States)

    Martin, Elizabeth M.; Fry, Rebecca C.

    2016-01-01

    Abstract A biological mechanism by which exposure to environmental contaminants results in gene-specific CpG methylation patterning is currently unknown. We hypothesize that gene-specific CpG methylation is related to environmentally perturbed transcription factor occupancy. To test this hypothesis, a database of 396 genes with altered CpG methylation either in cord blood leukocytes or placental tissue was compiled from 14 studies representing assessments of six environmental contaminants. Subsequently, an in silico approach was used to identify transcription factor binding sites enriched among the genes with altered CpG methylation in relationship to the suite of environmental contaminants. For each study, the sequences of the promoter regions (representing −1000 to +500 bp from the transcription start site) of all genes with altered CpG methylation were analyzed for enrichment of transcription factor binding sites. Binding sites for a total of 56 unique transcription factors were identified to be enriched within the promoter regions of the genes. Binding sites for the Kidney-Enriched Krupple-like Factor 15, a known responder to endogenous stress, were enriched ( P  contaminants. These data support the transcription factor occupancy theory as a potential mechanism underlying environmentally-induced gene-specific CpG methylation. PMID:27066266

  16. Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals

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    Keightley Peter D

    2008-09-01

    Full Text Available Abstract Background Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related. Results Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias. Conclusion Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of ad hoc methods of ancestral state assignment.

  17. Intronic variation at the

    NARCIS (Netherlands)

    Trimbos, K.B.; Kentie, R.; van der Velde, M.; Hooijmeijer, J.C.E.W.; Poley, C.; Musters, C.J.M.; de Snoo, G.R.; Piersma, T.

    2013-01-01

    Recently, Schroeder etal. (2010, Ibis 152: 368-377) suggested that intronic variation in the CHD1-Z gene of Black-tailed Godwits breeding in southwest Friesland, The Netherlands, correlated with fitness components. Here we re-examine this surprising result using an expanded dataset (2088 birds

  18. 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.

  19. Frequent Gain and Loss of Introns in Fungal Cytochrome b Genes

    Science.gov (United States)

    Yin, Liang-Fen; Hu, Meng-Jun; Wang, Fei; Kuang, Hanhui; Zhang, Yu; Schnabel, Guido; Li, Guo-Qing; Luo, Chao-Xi

    2012-01-01

    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. PMID:23145081

  20. Emergence and loss of spliceosomal twin introns

    OpenAIRE

    Flipphi, Michel; Ág, Norbert; Karaffa, Levente; Kavalecz, Napsugár; Cerqueira, Gustavo; Scazzocchio, Claudio; Fekete, Erzsébet

    2017-01-01

    Background In the primary transcript of nuclear genes, coding sequences—exons—usually alternate with non-coding sequences—introns. In the evolution of spliceosomal intron–exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns (“stwintrons”) are unconventional intervening sequences where a standard “internal” intron interrupts a canonical splicing motif of a second, “external” intron. The availability of genome sequences of ...

  1. YIDB: the Yeast Intron DataBase

    OpenAIRE

    Lopez, Pascal J.; Séraphin, Bertrand

    2000-01-01

    The Yeast Intron DataBase (YIDB) contains currently available information about all introns encoded in the nuclear and mitochondrial genomes of the yeast Saccharomyces cerevisiae. Introns are divided according to their mechanism of excision: group I and group II introns, pre-mRNA introns, tRNA introns and the HAC1 intron. Information about the host genome, the type of RNA in which they are inserted and their primary structure are provided together with references. For nuclear pre-mRNA introns...

  2. Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns.

    OpenAIRE

    Toor, Navtej; Zimmerly, Steven

    2002-01-01

    Group I and group II introns are unrelated classes of introns that each encode proteins that facilitate intron splicing and intron mobility. Here we describe a new subfamily of nine introns in fungi that are group II introns but encode LAGLIDADG ORFs typical of group I introns. The introns have fairly standard group IIB1 RNA structures and are inserted into three different sites in SSU and LSU rRNA genes. Therefore, introns should not be assumed to be group I introns based solely on the prese...

  3. Detained introns are a novel, widespread class of post-transcriptionally spliced introns.

    Science.gov (United States)

    Boutz, Paul L; Bhutkar, Arjun; Sharp, Phillip A

    2015-01-01

    Deep sequencing of embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within polyadenylated transcripts; we classified these as "detained" introns (DIs). We identified thousands of DIs, many of which are evolutionarily conserved, in human and mouse cell lines as well as the adult mouse liver. DIs can have half-lives of over an hour yet remain in the nucleus and are not subject to nonsense-mediated decay (NMD). Drug inhibition of Clk, a stress-responsive kinase, triggered rapid splicing changes for a specific subset of DIs; half showed increased splicing, and half showed increased intron detention, altering transcript pools of >300 genes. Srsf4, which undergoes a dramatic phosphorylation shift in response to Clk kinase inhibition, regulates the splicing of some DIs, particularly in genes encoding RNA processing and splicing factors. The splicing of some DIs-including those in Mdm4, a negative regulator of p53-was also altered following DNA damage. After 4 h of Clk inhibition, the expression of >400 genes changed significantly, and almost one-third of these are p53 transcriptional targets. These data suggest a widespread mechanism by which the rate of splicing of DIs contributes to the level of gene expression. © 2015 Boutz et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Intron V, not intron I of human thrombopoietin, improves expression in the milk of transgenic mice regulated by goat beta-casein promoter.

    Science.gov (United States)

    Li, Yan; Hao, Hu; Zhou, Mingqian; Zhou, Hongwei; Ye, Jianbin; Ning, Lijun; Ning, Yunshan

    2015-11-03

    Introns near 5' end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. Surprisingly, our previous experiments found that the vector containing the last intron (intron V) of human thromobopoietin (hTPO) expressed higher hTPO in cos-1 cell than the vector containing intron I regulated by cytomegalovirus promoter. Moreover, regulated by 1.0 kb rat whey acidic protein promoter, hTPO expression was higher in transgenic mice generated by intron V-TPOcDNA than in transgenic mice generated by TPOcDNA and TPOgDNA. However, it is unknown whether the enhancement of hTPO expression by intron I is decreased by uAUG7 at 5'-UTR of hTPO in vivo. Currently, we constructed vectors regulated by stronger 6.5 kb β-casein promoter, including pTPOGA (containing TPOcDNA), pTPOGB (containing TUR-TPOcDNA, TUR including exon1, intron I and non-coding exon2 of hTPO gene), pTPOGC (containing ΔTUR-TPOcDNA, nucleotides of TUR from uAUG7 to physiological AUG were deleted), pTPOGD (containing intron V-TPOcDNA) and pTPOGE (containing TPOgDNA), to evaluate the effect of intron I on hTPO expression and to further verify whether intron V enhances hTPO expression in the milk of transgenic mice. The results demonstrated that intron V, not intron I improved hTPO expression.

  5. DNA-methylation effect on cotranscriptional splicing is dependent on GC architecture of the exon-intron structure.

    Science.gov (United States)

    Gelfman, Sahar; Cohen, Noa; Yearim, Ahuvi; Ast, Gil

    2013-05-01

    DNA methylation is known to regulate transcription and was recently found to be involved in exon recognition via cotranscriptional splicing. We recently observed that exon-intron architectures can be grouped into two classes: one with higher GC content in exons compared to the flanking introns, and the other with similar GC content in exons and introns. The first group has higher nucleosome occupancy on exons than introns, whereas the second group exhibits weak nucleosome marking of exons, suggesting another type of epigenetic marker distinguishes exons from introns when GC content is similar. We find different and specific patterns of DNA methylation in each of the GC architectures; yet in both groups, DNA methylation clearly marks the exons. Exons of the leveled GC architecture exhibit a significantly stronger DNA methylation signal in relation to their flanking introns compared to exons of the differential GC architecture. This is accentuated by a reduction of the DNA methylation level in the intronic sequences in proximity to the splice sites and shows that different epigenetic modifications mark the location of exons already at the DNA level. Also, lower levels of methylated CpGs on alternative exons can successfully distinguish alternative exons from constitutive ones. Three positions at the splice sites show high CpG abundance and accompany elevated nucleosome occupancy in a leveled GC architecture. Overall, these results suggest that DNA methylation affects exon recognition and is influenced by the GC architecture of the exon and flanking introns.

  6. The Biology of Intron Gain and Loss

    DEFF Research Database (Denmark)

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

    2006-01-01

    eukaryote genomes during their evolution from an intron-poor ancestor. However, recent studies have shown that some eukaryotes lost many introns, whereas others accumulated and/or gained many introns. In this article, we discuss the growing evidence that these differences are subject to selection acting...... on introns depending on the biology of the organism and the gene involved....

  7. A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2016-01-01

    Introns, especially the first intron in the 5' untranslated region (5'UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

  8. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome fromtransposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG bindingdomain proteins are members of a class of proteins that bind tomethylated ...

  9. Tertiary architecture of the Oceanobacillus iheyensis group II intron

    Energy Technology Data Exchange (ETDEWEB)

    Toor, Navtej; Keating, Kevin S.; Fedorova, Olga; Rajashankar, Kanagalaghatta; Wang, Jimin; Pyle, Anna Marie (Yale); (Cornell)

    2010-05-03

    Group II introns are large ribozymes that act as self-splicing and retrotransposable RNA molecules. They are of great interest because of their potential evolutionary relationship to the eukaryotic spliceosome, their continued influence on the organization of many genomes in bacteria and eukaryotes, and their potential utility as tools for gene therapy and biotechnology. One of the most interesting features of group II introns is their relative lack of nucleobase conservation and covariation, which has long suggested that group II intron structures are stabilized by numerous unusual tertiary interactions and backbone-mediated contacts. Here, we provide a detailed description of the tertiary interaction networks within the Oceanobacillus iheyensis group IIC intron, for which a crystal structure was recently solved to 3.1 {angstrom} resolution. The structure can be described as a set of several intricately constructed tertiary interaction nodes, each of which contains a core of extended stacking networks and elaborate motifs. Many of these nodes are surrounded by a web of ribose zippers, which appear to further stabilize local structure. As predicted from biochemical and genetic studies, the group II intron provides a wealth of new information on strategies for RNA folding and tertiary structural organization.

  10. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2008-09-01

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

  11. Epigenetic Regulation of Intronic Transgenes in Arabidopsis.

    Science.gov (United States)

    Osabe, Kenji; Harukawa, Yoshiko; Miura, Saori; Saze, Hidetoshi

    2017-03-24

    Defense mechanisms of plant genomes can epigenetically inactivate repetitive sequences and exogenous transgenes. Loss of mutant phenotypes in intronic T-DNA insertion lines by interaction with another T-DNA locus, termed T-DNA suppression, has been observed in Arabidopsis thaliana, although the molecular basis of establishment and maintenance of T-DNA suppression is poorly understood. Here we show that maintenance of T-DNA suppression requires heterochromatinisation of T-DNA sequences and the nuclear proteins, INCREASED IN BONSAI METHYLATION 2 (IBM2) and ENHANCED DOWNY MILDEW 2 (EDM2), which prevent ectopic 3' end processing of mRNA in atypically long introns containing T-DNA sequences. Initiation of T-DNA suppression is mediated by the canonical RdDM pathway after hybridisation of two T-DNA strains, accompanied by DNA hypermethylation of T-DNA sequences in the F1 generation. Our results reveal the presence of a genome surveillance mechanism through genome hybridisation that masks repetitive DNAs intruding into transcription units.

  12. Origin and evolution of spliceosomal introns

    Directory of Open Access Journals (Sweden)

    Rogozin Igor B

    2012-04-01

    Full Text Available Abstract Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA. Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has

  13. Database for mobile group II introns

    OpenAIRE

    Dai, Lixin; Toor, Navtej; Olson, Robert; Keeping, Andrew; Zimmerly, Steven

    2003-01-01

    Group II introns are self-splicing RNAs and retroelements found in bacteria and lower eukaryotic organelles. During the past several years, they have been uncovered in surprising numbers in bacteria due to the genome sequencing projects; however, most of the newly sequenced introns are not correctly identified. We have initiated an ongoing web site database for mobile group II introns in order to provide correct information on the introns, particularly in bacteria. Information in the web site...

  14. AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

    OpenAIRE

    Keren, Ido; Bezawork-Geleta, Ayenachew; Kolton, Max; Maayan, Inbar; Belausov, Eduard; Levy, Maggie; Mett, Anahit; Gidoni, David; Shaya, Felix; Ostersetzer-Biran, Oren

    2009-01-01

    Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established...

  15. Emergence and loss of spliceosomal twin introns.

    Science.gov (United States)

    Flipphi, Michel; Ág, Norbert; Karaffa, Levente; Kavalecz, Napsugár; Cerqueira, Gustavo; Scazzocchio, Claudio; Fekete, Erzsébet

    2017-01-01

    In the primary transcript of nuclear genes, coding sequences-exons-usually alternate with non-coding sequences-introns. In the evolution of spliceosomal intron-exon structure, extant intron positions can be abandoned and new intron positions can be occupied. Spliceosomal twin introns ("stwintrons") are unconventional intervening sequences where a standard "internal" intron interrupts a canonical splicing motif of a second, "external" intron. The availability of genome sequences of more than a thousand species of fungi provides a unique opportunity to study spliceosomal intron evolution throughout a whole kingdom by means of molecular phylogenetics. A new stwintron was encountered in Aspergillus nidulans and Aspergillus niger . It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase ( lipS ). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders-in the transcript of the bifunctional biotin biosynthesis gene bioDA -occurs at the same position as a standard intron in many species of non-Dikarya. Nevertheless, also the bioDA stwintron has disappeared from certain lineages within the taxa where it occurs, i.e., Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes bioDA stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain. Molecular phylogeny of the peptide product was used to monitor

  16. Methylation of a CpG Island within the Uroplakin Ib Promoter: A Possible Mechanism for Loss of Uroplakin lb Expression in Bladder Carcinoma

    Directory of Open Access Journals (Sweden)

    Andrea E. Varga

    2004-03-01

    Full Text Available Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2′ -deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Spi/NFκB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

  17. 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.

  18. CpG island mapping by epigenome prediction.

    Directory of Open Access Journals (Sweden)

    Christoph Bock

    2007-06-01

    Full Text Available CpG islands were originally identified by epigenetic and functional properties, namely, absence of DNA methylation and frequent promoter association. However, this concept was quickly replaced by simple DNA sequence criteria, which allowed for genome-wide annotation of CpG islands in the absence of large-scale epigenetic datasets. Although widely used, the current CpG island criteria incur significant disadvantages: (1 reliance on arbitrary threshold parameters that bear little biological justification, (2 failure to account for widespread heterogeneity among CpG islands, and (3 apparent lack of specificity when applied to the human genome. This study is driven by the idea that a quantitative score of "CpG island strength" that incorporates epigenetic and functional aspects can help resolve these issues. We construct an epigenome prediction pipeline that links the DNA sequence of CpG islands to their epigenetic states, including DNA methylation, histone modifications, and chromatin accessibility. By training support vector machines on epigenetic data for CpG islands on human Chromosomes 21 and 22, we identify informative DNA attributes that correlate with open versus compact chromatin structures. These DNA attributes are used to predict the epigenetic states of all CpG islands genome-wide. Combining predictions for multiple epigenetic features, we estimate the inherent CpG island strength for each CpG island in the human genome, i.e., its inherent tendency to exhibit an open and transcriptionally competent chromatin structure. We extensively validate our results on independent datasets, showing that the CpG island strength predictions are applicable and informative across different tissues and cell types, and we derive improved maps of predicted "bona fide" CpG islands. The mapping of CpG islands by epigenome prediction is conceptually superior to identifying CpG islands by widely used sequence criteria since it links CpG island detection to

  19. Polycomb-like proteins link the PRC2 complex to CpG islands

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haojie; Liefke, Robert; Jiang, Junyi; Kurland, Jesse Vigoda; Tian, Wei; Deng, Pujuan; Zhang, Weidi; He, Qian; Patel, Dinshaw J.; Bulyk, Martha L.; Shi, Yang; Wang, Zhanxin

    2017-09-06

    The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression1,2 and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components3, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci4,5,6,7,8,9,10,11,12,13. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

  20. Milk: an epigenetic amplifier of FTO-mediated transcription? Implications for Western diseases.

    Science.gov (United States)

    Melnik, Bodo C

    2015-12-21

    Single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity-associated) gene are associated with enhanced FTO expression, increased body weight, obesity and type 2 diabetes mellitus (T2DM). The N (6) -methyladenosine (m(6)A) demethylase FTO plays a pivotal regulatory role for postnatal growth and energy expenditure. The purpose of this review is to provide translational evidence that links milk signaling with FTO-activated transcription of the milk recipient. FTO-dependent demethylation of m(6)A regulates mRNA splicing required for adipogenesis, increases the stability of mRNAs, and affects microRNA (miRNA) expression and miRNA biosynthesis. FTO senses branched-chain amino acids (BCAAs) and activates the nutrient sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), which plays a key role in translation. Milk provides abundant BCAAs and glutamine, critical components increasing FTO expression. CpG hypomethylation in the first intron of FTO has recently been associated with T2DM. CpG methylation is generally associated with gene silencing. In contrast, CpG demethylation generally increases transcription. DNA de novo methylation of CpG sites is facilitated by DNA methyltransferases (DNMT) 3A and 3B, whereas DNA maintenance methylation is controlled by DNMT1. MiRNA-29s target all DNMTs and thus reduce DNA CpG methylation. Cow´s milk provides substantial amounts of exosomal miRNA-29s that reach the systemic circulation and target mRNAs of the milk recipient. Via DNMT suppression, milk exosomal miRNA-29s may reduce the magnitude of FTO methylation, thereby epigenetically increasing FTO expression in the milk consumer. High lactation performance with increased milk yield has recently been associated with excessive miRNA-29 expression of dairy cow mammary epithelial cells (DCMECs). Notably, the galactopoietic hormone prolactin upregulates the transcription factor STAT3, which induces miRNA-29 expression. In a retrovirus-like manner

  1. Dualism of gene GC content and CpG pattern in regard to expression in the human genome: magnitude versus breadth.

    Science.gov (United States)

    Vinogradov, Alexander E

    2005-12-01

    In this article, I show that, in the human genome, the GC content in genes (but not the CpG island in the promoter) is related to the maximum level of gene expression among tissues, whereas the promoter CpG island and gene CpG level are more strongly related to the breadth of expression among tissues. The relevance of gene GC content to expression cannot be a consequence (i.e. a byproduct) of transcription because it does not correlate with expression in the germline. The variation of GC content and CpG level can determine the characteristics of gene expression in a synergistic interplay with transcription-factor-binding sites (mediated by chromatin condensation).

  2. The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate?

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-08-01

    Full Text Available Abstract Background Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes. Results I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron

  3. Structural Metals in the Group I Intron: A Ribozyme with a Multiple Metal Ion Core

    Energy Technology Data Exchange (ETDEWEB)

    Stahley,M.; Adams, P.; Wang, J.; Strobel, S.

    2007-01-01

    Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg2+ and K+ ions. Five of the metals bind within 12 Angstroms of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function.

  4. FGLamide Allatostatin genes in Arthropoda: introns early or late?

    Science.gov (United States)

    Martínez-Pérez, Francisco; Bendena, William G; Chang, Belinda S W; Tobe, Stephen S

    2009-07-01

    FGLamide allatostatins are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders and also show myomodulatory activity. The FGLamide allatostatin (AST) gene structure in Dictyoptera is intronless within the ORF, whereas in 9 species of Diptera, the FGLamide AST ORF has one intron. To investigate the evolutionary history of AST intron structure, (intron early versus intron late hypothesis), all available Arthropoda FGLamide AST gene sequences were examined from genome databases with reference to intron presence and position/phase. Three types of FGLamide AST ORF organization were found: intronless in I. scapularis and P. humanus corporis; one intron in D. pulex, A. pisum, A. mellifera and five Drosophila sp.; two introns in N. vitripennis, B. mori strains, A. aegypti, A. gambiae and C. quinquefasciatus. The literature suggests that for the majority of genes examined, most introns exist between codons (phase 0) which may reflect an ancient function of introns to separate protein modules. 60% of the FGLamide AST ORFs introns were between the first and second base within a codon (phase 1), 28% were between the second and third nucleotides within a codon (phase two) and 12% were phase 0. As would be required for correct intron splicing consensus sequence, 84% of introns were in codons starting with guanine. The positioning of introns was a maximum of 9 codons from a dibasic cleavage site. Our results suggest that the introns in the analyzed species support the intron late model.

  5. Liposomal SLA co-incorporated with PO CpG ODNs or PS CpG ODNs induce the same protection against the murine model of leishmaniasis.

    Science.gov (United States)

    Shargh, Vahid Heravi; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jaafari, Iman; Jalali, Seyed Amir; Abbasi, Azam; Badiee, Ali

    2012-06-06

    First generation Leishmania vaccines consisting of whole killed parasites with or without adjuvants have reached phase 3 trial and failed to show enough efficacy mainly due to the lack of an appropriate adjuvant. In this study, the nuclease-resistant phosphorothioate CpG oligodeoxynucleotides (PS CpG) or nuclease-sensitive phosphodiester CpG ODNs (PO CpG) were used as adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Due to the susceptibility of PO CpG to nuclease degradation, an efficient liposomal delivery system was developed to protect them from degradation. 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid was used because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODNs. To evaluate the role of liposomal formulation in protection rate and enhanced immune response, BALB/c mice were immunized subcutaneously with liposomal soluble Leishmania antigens (SLA) co-incorporated with PO CpG (Lip-SLA-PO CpG), Lip-SLA-PS CpG, SLA+PO CpG, SLA+PS CpG, SLA or buffer. As criteria for protection, footpad swelling at the site of challenge, parasite loads, the levels of IFN-γ and IL-4, and the IgG subtypes were evaluated. The groups of mice receiving Lip-SLA-PO CpG or Lip-SLA-PS CpG showed a high protection rate compared with the control groups. In addition, there was no significant difference in immune response generation between mice immunized with PS CpG and the group receiving PO CpG when incorporated into the liposomes. The results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Transcriptomic Insights into the Response of Placenta and Decidua Basalis to the CpG Oligodeoxynucleotide Stimulation in Non-Obese Diabetic Mice and Wild-Type Controls

    Directory of Open Access Journals (Sweden)

    Xiao-Rui Liu

    2016-08-01

    Full Text Available Intrauterine infection is one of the most frequent causes of miscarriage. CpG oligodeoxynucleotide (CpG ODN can mimic intrauterine infection. CpG ODN-induced embryo-resorption was observed consistently in the NK-cell deficient non-obese diabetic (NOD mice but not in the wild-type (WT mice. To elucidate the molecular mechanisms of differential pregnancy outcomes, differentially expressed genes (DEGs in the placenta and decidua basalis was revealed by RNA-Seq with CpG ODN or control ODN treatment. Common DEGs in the WT and NOD mice were enriched in antimicrobial/antibacterial humoral responses that may be activated as a primary response to bacterial infection. The susceptibility to CpG ODN-induced embryo-resorption in the NOD mice might mainly be attributed to M1 macrophage polarization and the immunodeficient status, such as the down-regulation in antigen processing and presentation, allograft rejection, and natural killer cell mediated cytotoxicity. In contrast, the WT mice with normal immune systems could activate multiple immune responses and be resistant to CpG ODN-induced embryo-resorption, such as M2 macrophage differentiation and activation regulated by complement component C1q and peroxisome proliferation-activated receptor (PPAR signaling pathways. Collectively, this study suggests that the immunodeficient status of NOD mice and the macrophage polarization regulated by C1q and PPAR signaling might be the basis for differential pregnancy outcomes between the NOD and WT mice.

  7. Functional intron+ and intron- rDNA in the same macronucleus of the ciliate Tetrahymena pigmentosa

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engberg, J

    1985-01-01

    alleles was followed in the total culture and in single cells during their vegetative segregation and it was observed that replication was non-preferential with respect to the two alleles. The diallelic clones were also used to demonstrate that intron-containing rDNA was transcribed and the transcript......Diallelic clones of Tetrahymena pigmentosa containing equal amounts of intron+ and intron- rDNA in the macronucleus were constructed. The macronucleus of the resulting strains divides amitotically during vegetative growth and the diallelic genotype is therefore unstable. The coexistence of the two...

  8. Intron size and genome size in plants.

    Science.gov (United States)

    J. Wendel; R. Cronn; I. Alvarez; B. Liu; R. Small; D. Senchina

    2002-01-01

    It has long been known that genomes vary over a remarkable range of sizes in both plants (Bennett, Cox, and Leitch 1997) and animals (Gregory 2001). It also has become evident that across the broad phylogenetic sweep, genome size may be correlated with intron size (Deutsch and Long 1999; Vinogradov 1999; McLysaght et al. 2000), suggesting that some component of genome...

  9. CPG Network Optimization for a Biomimetic Robotic Fish via PSO.

    Science.gov (United States)

    Yu, Junzhi; Wu, Zhengxing; Wang, Ming; Tan, Min

    2016-09-01

    In this brief, we investigate the parameter optimization issue of a central pattern generator (CPG) network governed forward and backward swimming for a fully untethered, multijoint biomimetic robotic fish. Considering that the CPG parameters are tightly linked to the propulsive performance of the robotic fish, we propose a method for determination of relatively optimized control parameters. Within the framework of evolutionary computation, we use a combination of dynamic model and particle swarm optimization (PSO) algorithm to seek the CPG characteristic parameters for an enhanced performance. The PSO-based optimization scheme is validated with extensive experiments conducted on the actual robotic fish. Noticeably, the optimized results are shown to be superior to previously reported forward and backward swimming speeds.

  10. For Group II Introns, More Heat Means More Mobility

    OpenAIRE

    Mohr, Georg; Ghanem, Eman; Lambowitz, Alan M.

    2010-01-01

    Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelements hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closel...

  11. 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.

  12. 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

    in which the intron terminal guanosine attacks the 5' splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron...

  13. 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.

  14. A high density of ancient spliceosomal introns in oxymonad excavates

    Directory of Open Access Journals (Sweden)

    Keeling Patrick J

    2006-04-01

    Full Text Available Abstract Background Certain eukaryotic genomes, such as those of the amitochondriate parasites Giardia and Trichomonas, have very low intron densities, so low that canonical spliceosomal introns have only recently been discovered through genome sequencing. These organisms were formerly thought to be ancient eukaryotes that diverged before introns originated, or at least became common. Now however, they are thought to be members of a supergroup known as excavates, whose members generally appear to have low densities of canonical introns. Here we have used environmental expressed sequence tag (EST sequencing to identify 17 genes from the uncultivable oxymonad Streblomastix strix, to survey intron densities in this most poorly studied excavate group. Results We find that Streblomastix genes contain an unexpectedly high intron density of about 1.1 introns per gene. Moreover, over 50% of these are at positions shared between a broad spectrum of eukaryotes, suggesting theyare very ancient introns, potentially present in the last common ancestor of eukaryotes. Conclusion The Streblomastix data show that the genome of the ancestor of excavates likely contained many introns and the subsequent evolution of introns has proceeded very differently in different excavate lineages: in Streblomastix there has been much stasis while in Trichomonas and Giardia most introns have been lost.

  15. Functional Analysis of Deep Intronic SNP rs13438494 in Intron 24 of PCLO Gene

    Science.gov (United States)

    Seo, Seunghee; Takayama, Kanako; Uno, Kyosuke; Ohi, Kazutaka; Hashimoto, Ryota; Nishizawa, Daisuke; Ikeda, Kazutaka; Ozaki, Norio; Nabeshima, Toshitaka; Miyamoto, Yoshiaki; Nitta, Atsumi

    2013-01-01

    The single nucleotide polymorphism (SNP) rs13438494 in intron 24 of PCLO was significantly associated with bipolar disorder in a meta-analysis of genome-wide association studies. In this study, we performed functional minigene analysis and bioinformatics prediction of splicing regulatory sequences to characterize the deep intronic SNP rs13438494. We constructed minigenes with A and C alleles containing exon 24, intron 24, and exon 25 of PCLO to assess the genetic effect of rs13438494 on splicing. We found that the C allele of rs13438494 reduces the splicing efficiency of the PCLO minigene. In addition, prediction analysis of enhancer/silencer motifs using the Human Splice Finder web tool indicated that rs13438494 induces the abrogation or creation of such binding sites. Our results indicate that rs13438494 alters splicing efficiency by creating or disrupting a splicing motif, which functions by binding of splicing regulatory proteins, and may ultimately result in bipolar disorder in affected people. PMID:24167553

  16. Intron retention as a component of regulated gene expression programs.

    Science.gov (United States)

    Jacob, Aishwarya G; Smith, Christopher W J

    2017-09-01

    Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as "noise". Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.

  17. CpG traffic lights are markers of regulatory regions in humans

    KAUST Repository

    Khamis, Abdullah M.

    2016-12-29

    DNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

  18. Patterns of intron gain and conservation in eukaryotic genes

    Directory of Open Access Journals (Sweden)

    Wolf Yuri I

    2007-10-01

    Full Text Available Abstract Background: The presence of introns in protein-coding genes is a universal feature of eukaryotic genome organization, and the genes of multicellular eukaryotes, typically, contain multiple introns, a substantial fraction of which share position in distant taxa, such as plants and animals. Depending on the methods and data sets used, researchers have reached opposite conclusions on the causes of the high fraction of shared introns in orthologous genes from distant eukaryotes. Some studies conclude that shared intron positions reflect, almost entirely, a remarkable evolutionary conservation, whereas others attribute it to parallel gain of introns. To resolve these contradictions, it is crucial to analyze the evolution of introns by using a model that minimally relies on arbitrary assumptions. Results: We developed a probabilistic model of evolution that allows for variability of intron gain and loss rates over branches of the phylogenetic tree, individual genes, and individual sites. Applying this model to an extended set of conserved eukaryotic genes, we find that parallel gain, on average, accounts for only ~8% of the shared intron positions. However, the distribution of parallel gains over the phylogenetic tree of eukaryotes is highly non-uniform. There are, practically, no parallel gains in closely related lineages, whereas for distant lineages, such as animals and plants, parallel gains appear to contribute up to 20% of the shared intron positions. In accord with these findings, we estimated that ancestral introns have a high probability to be retained in extant genomes, and conversely, that a substantial fraction of extant introns have retained their positions since the early stages of eukaryotic evolution. In addition, the density of sites that are available for intron insertion is estimated to be, approximately, one in seven basepairs. Conclusion: We obtained robust estimates of the contribution of parallel gain to the observed

  19. The evolution of an intron: Analysis of a long, deletion-prone intron in the human dystrophin gene

    Energy Technology Data Exchange (ETDEWEB)

    McNaughton, J.C.; Hughes, G.; Jones, W.A. [Univ. of Otago, Dunedin (New Zealand)] [and others

    1997-03-01

    The sequence of a 112-kb region of the human dystrophin (DMD/BMD) gene encompassing the deletion prone intron 7 (110 kb) and the much shorter intron 8 (1.1 kb) has been determined. Recognizable insertion sequences account for approximately 40% of intron 7. LINE-1 and THE-1/LTR sequences occur in intron 7 with significantly higher frequency than would be expected statistically while Alu sequences are underrepresented. Intron 7 also contains numerous mammalian-wide interspersed repeats, a diverse range of medium reiteration repeats of unknown origin, and a sequence derived from a mariner transposon. By contrast, the shorter intron 8 contains no detectable insertion sequences. Dating of the L1 and Alu sequences suggests that intron 7 has approximately doubled in size within the past 130 million years, and comparison with the corresponding intron from the pufferfish (Fugu rubripes) suggests that the intron has expanded some 44-fold over a period of 400 million years. The possible contribution of the insertion elements to the instability of intron 7 is discussed. 66 refs., 2 figs., 2 tabs.

  20. Collaborations between CpG sites in DNA methylation

    Science.gov (United States)

    Song, You; Ren, Honglei; Lei, Jinzhi

    2017-08-01

    DNA methylation patterns have profound impacts on genome stability, gene expression and development. The molecular base of DNA methylation patterns has long been focused at single CpG sites level. Here, we construct a kinetic model of DNA methylation with collaborations between CpG sites, from which a correlation function was established based on experimental data. The function consists of three parts that suggest three possible sources of the correlation: movement of enzymes along DNA, collaboration between DNA methylation and nucleosome modification, and global enzyme concentrations within a cell. Moreover, the collaboration strength between DNA methylation and nucleosome modification is universal for mouse early embryo cells. The obtained correlation function provides insightful understanding for the mechanisms of inheritance of DNA methylation patterns.

  1. CpG methylation controls reactivation of HIV from latency

    Czech Academy of Sciences Publication Activity Database

    Blažková, Jana; Trejbalová, Kateřina; Gondois-Rey, F.; Halfon, P.; Philibert, P.; Guiguen, A.; Verdin, E.; Olive, D.; Van Lint, C.; Hejnar, Jiří; Hirsch, I.

    2009-01-01

    Roč. 5, č. 8 (2009), e1000554-e1000554 E-ISSN 1553-7374 R&D Projects: GA ČR GA204/05/0939; GA ČR GP204/08/P616 Institutional research plan: CEZ:AV0Z50520514 Keywords : HIV-1 * proviral latency * CpG methylation * histone modifications * HAART * epigenetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.978, year: 2009

  2. Hypoxia-induced modulation of the respiratory CPG.

    Science.gov (United States)

    Bell, Harold J; Syed, Naweed I

    2009-01-01

    Despite recent advances in our understanding of the neural control of breathing, the precise cellular, synaptic, and molecular mechanisms underlying the generation and modulation of respiratory rhythm remain largely unknown. This lack of fundamental knowledge in the field of neural control of respiration is likely due to the complexity of the mammalian brain where synaptic connectivity between central respiratory neurons, motor neurons and their peripheral counterparts cannot be mapped reliably. We have therefore developed an invertebrate model system wherein the essential elements of the central pattern generator (CPG), the motor neurons and the peripheral chemosensory cells involved in respiratory control have been worked out both in vivo and in vitro. We discuss our recent identification of peripheral, hypoxia sensitive chemoreceptor elements in a sensory organ of the pulmonate freshwater pond snail Lymnaea stagnalis, which provide an excitatory drive to the respiratory CPG neuron RPeD1 via direct chemical synaptic connections. Further studies using this unique invertebrate model system may reveal highly conserved principles of CPG neuromodulation that will remain relevant to more complex mammalian systems.

  3. A Tth111I RFLP in intron 1 of the mouse Pgk-1 gene allows tracing of X chromosome inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Shanmugan, V.; Saha, B.K. [Emory Univ. School of Medicine, Atlanta, GA (United States)

    1994-09-01

    The X-linked immunodeficiency (xid) in CBA/N mice serves as a model for the X-linked agammaglobulinemia (XLA) syndrome in humans. Like the XLA carriers, the female mice heterozygous for xid (X{sup xid}/X{sup W}) are asymptomatic. The pattern of X chromosome inactivation in the F1 heterozygotes [CBA/N (X{sup xid}/X{sup xid}) X CAST/Ei (X{sup W}/Y)] was investigated by monitoring the methylation status of the two Pgk-1 alleles. Methylation of a CpG dinucleotide in the 5{prime} region of the Pgk-1 gene was previously shown to absolutely correlate with the inactivation of the corresponding X chromosome. In order to distinguish the two alleles, the proximal end of intron 1 of the Pgk-1 gene from CBA/N and CAST/Ei was sequenced. Several nucleotide polymorphisms, including a Tth111I RFLP, were detected in close proximity of the critical CpG dinucleotide. This allowed us to devise an assay based on PCR-amplification of a target DNA encompassing the CpG site as well as the Tth111I site. Results indicate that in circulating B lymphocytes of the female heterozygote only the X-chromosome carrying the normal allele is active (non-random inactivation of the X chromosome) whereas in non-B cells both the X chromosomes are active (random inactivation of the X chromosome). These results were further confirmed by direct measurement of transcription of the two alleles (X{sup xid} and X{sup W}).

  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. Intronic alternative splicing regulators identified by comparative genomics in nematodes.

    Directory of Open Access Journals (Sweden)

    Jennifer L Kabat

    2006-07-01

    Full Text Available Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (TGCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis

  6. CpG methylation controls reactivation of HIV from latency.

    Directory of Open Access Journals (Sweden)

    Jana Blazkova

    2009-08-01

    Full Text Available DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by CpG

  7. Identifying the mechanisms of intron gain: progress and trends

    Directory of Open Access Journals (Sweden)

    Yenerall Paul

    2012-09-01

    Full Text Available Abstract Continued improvements in Next-Generation DNA/RNA sequencing coupled with advances in gene annotation have provided researchers access to a plethora of annotated genomes. Subsequent analyses of orthologous gene structures have identified numerous intron gain and loss events that have occurred both recently and in the very distant past. This research has afforded exceptional insight into the temporal and lineage-specific rates of intron gain and loss among various species throughout evolution. Numerous studies have also attempted to identify the molecular mechanisms of intron gain and loss. However, even after considerable effort, very little is known about these processes. In particular, the mechanism(s of intron gain have proven exceptionally enigmatic and remain topics of considerable debate. Currently, there exists no definitive consensus as to what mechanism(s may generate introns. Because many introns are known to affect gene expression, it is necessary to understand the molecular process(es by which introns may be gained. Here we review the seven most commonly purported mechanisms of intron gain and, when possible, summarize molecular evidence for or against the occurrence of each of these mechanisms. Furthermore, we catalogue indirect evidence that supports the occurrence of each mechanism. Finally, because these proposed mechanisms fail to explain the mechanistic origin of many recently gained introns, we also look at trends that may aid researchers in identifying other potential mechanism(s of intron gain. Reviewers This article was reviewed by Eugene Koonin, Scott Roy (nominated by W. Ford Doolittle, and John Logsdon.

  8. Consolidated Recovered Materials Advisory Notice (RMAN) for the Comprehensive Procurement Guideline (CPG)

    Data.gov (United States)

    U.S. Environmental Protection Agency — EPA's Comprehensive Procurement Guideline (CPG) designates recycled content products that government agencies should buy. EPA publishes purchasing guidance and...

  9. CpG + CpNpG Analysis of Protein-Coding Sequences from Tomato

    DEFF Research Database (Denmark)

    Hobolth, Asger; Nielsen, Rasmus; Wang, Ying

    2006-01-01

    We develop codon-based models for simultaneously inferring the mutational effects of CpG and CpNpG methylation in coding regions. In a data set of 369 tomato genes, we show that there is very little effect of CpNpG methylation but a strong effect of CpG methylation affecting almost all genes. We...... further show that the CpNpG and CpG effects are largely uncorrelated. Our results suggest different roles of CpG and CpNpG methylation, with CpNpG methylation possibly playing a specialized role in defense against transposons and RNA viruses....

  10. Group I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts.

    OpenAIRE

    Ohta, E; Oda, K; Yamato, K; Nakamura, Y; Takemura, M; Nozato, N; Akashi, K; Ohyama, K; Michel, F

    1993-01-01

    The complete nucleotide sequence of the mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, contains thirty-two introns. Twenty-five of these introns possess the characteristic secondary structures and consensus sequences of group II introns. The remaining seven are group I introns, six of which happen to interrupt the gene coding for subunit 1 of cytochrome oxidase (cox1). Interestingly, the insertion sites of one group II and four group I introns in the cox1 gene coincide wit...

  11. Distribution of Conventional and Nonconventional Introns in Tubulin (α and β) Genes of Euglenids

    OpenAIRE

    Milanowski, Rafał; Karnkowska, Anna; Ishikawa, Takao; Zakryś, Bożena

    2013-01-01

    The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconvent...

  12. 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.

  13. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction....... From the perspective of mediatization research, the most important effect of the media stems from their embeddedness in culture and society....

  14. CpG islands or CpG clusters: how to identify functional GC-rich regions in a genome?

    Directory of Open Access Journals (Sweden)

    Han Leng

    2009-02-01

    Full Text Available Abstract Background CpG islands (CGIs, clusters of CpG dinucleotides in GC-rich regions, are often located in the 5' end of genes and considered gene markers. Hackenberg et al. (2006 recently developed a new algorithm, CpGcluster, which uses a completely different mathematical approach from previous traditional algorithms. Their evaluation suggests that CpGcluster provides a much more efficient approach to detecting functional clusters or islands of CpGs. Results We systematically compared CpGcluster with the traditional algorithm by Takai and Jones (2002. Our comparisons of (1 the number of islands versus the number of genes in a genome, (2 the distribution of islands in different genomic regions, (3 island length, (4 the distance between two neighboring islands, and (5 methylation status suggest that Takai and Jones' algorithm is overall more appropriate for identifying promoter-associated islands of CpGs in vertebrate genomes. Conclusion The generation of genome sequence and DNA methylation data is expected to accelerate greatly. The information in this study is important for its extensive utility in gene feature analysis and epigenomics including gene prediction and methylation chip design in different genomes.

  15. Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models

    Science.gov (United States)

    Jang, Julie K.; Khawli, Leslie A.; Canter, David C.; Hu, Peisheng; Zhu, Tian H.; Wu, Brian W.; Angell, Trevor E.; Li, Zhongjun; Epstein, Alan L.

    2016-01-01

    CpG oligodeoxynucleotides (CpG) potently activate the immune system by mimicking microbial DNA. Conjugation of CpG to chTNT-3, an antibody targeting the necrotic centers of tumors, enabled CpG to accumulate in tumors after systemic delivery, where it can activate the immune system in the presence of tumor antigens. CpG chemically conjugated to chTNT-3 (chTNT-3/CpG) were compared to free CpG in their ability to stimulate the immune system in vitro and reduce tumor burden in vivo. In subcutaneous Colon 26 adenocarcinoma and B16-F10 melanoma models in BALB/c and C57BL/6 mice, respectively, chTNT-3/CpG, free CpG, or several different control constructs were administered systemically. Intraperitoneal injections of chTNT-3/CpG delayed tumor growth and improved survival, and were comparable to intratumorally administered CpG. Compared to saline-treated mice, chTNT-3/CpG-treated mice had smaller average tumor volumes by as much as 72% in Colon 26-bearing mice and 79% in B16-bearing mice. Systemically delivered free CpG and CpG conjugated to an isotype control antibody did not reduce tumor burden or improve survival. In this study, chTNT-3/CpG retained immunostimulatory activity of the CpG moiety and enabled delivery to tumors. Because systemically administered CpG rapidly clear the body and do not accumulate into tumors, chTNT-3/CpG provide a solution to the limitations observed in preclinical and clinical trials. PMID:26960932

  16. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    Science.gov (United States)

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  17. The CpG island methylator phenotype: What's in a name?

    NARCIS (Netherlands)

    L.A.E. Hughes (Laura A.); V. Melotte (Veerle); J.D. Schrijver (Joachim De); M.P.M. de Maat (Moniek); V.T.H.B.M. Smit (Vincent); J.V.M.G. Bovée (Judith); P.J. French (Pim); P.A. van den Brandt (Piet); L. Schouten (Leo); T. Meyer (Thorsten); W. van Criekinge (Wim); N. Ahuja (Nita); J.G. Herman (James); M.P. Weijenberg (Matty); M. van Engeland (Manon)

    2013-01-01

    textabstractAlthough the CpG island methylator phenotype (CIMP) was first identified and has been most extensively studied in colorectal cancer, the term "CIMP" has been repeatedly used over the past decade to describe CpG island promoter methylation in other tumor types, including bladder, breast,

  18. Reproducibility of methylated CpG typing with the Illumina MiSeq

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Meyer, Olivia Strunge; Greby Schmidt, Suzanne

    2017-01-01

    DNA methylation patterns may be used for identification of body fluids and for age estimation of human individuals. We evaluated some of the challenges and pitfalls of studying methylated CpG sites. We compared the methylated CpG analysis of two different methods 1) massively parallel sequencing...

  19. Drosophila polytene chromosome bands formed by gene introns.

    Science.gov (United States)

    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.

  20. The counter regulatory response induced by CpG oligonucleotides prevents bleomycin induced pneumopathy

    Directory of Open Access Journals (Sweden)

    Kinjo Takeshi

    2012-06-01

    Full Text Available Abstract Bleomycin (BLM induces life-threatening pneumonitis and pulmonary fibrosis in 20% of patients, limiting its use as a chemotherapeutic agent. Oligonucleotides expressing immunostimulatory CpG motifs (CpG ODN stimulate cells that express Toll-like receptor 9 to initiate an inflammatory response. This short-lived inflammation is physiologically suppressed by a counter-regulatory process that peaks five days later. Using a murine model of BLM-induced lung injury, the effect of CpG ODN treatment on pulmonary inflammation, fibrosis and mortality was examined. Administering CpG ODN 5 days before BLM (so that the peak of the counter-regulatory process induced by CpG ODN coincided with BLM delivery resulted in a dose-dependent reduction in pulmonary toxicity (p 

  1. Genomewide analysis of intronic microRNAs in rice and Arabidopsis

    Indian Academy of Sciences (India)

    Here we report a comprehensive computational analysis to characterize intronic miRNAs in rice and Arabidopsis. RT-PCR analysis confirmed that the identified intronic miRNAs were derived from the real introns of host genes. Interestingly, 13 intronic miRNAs in rice and two in Arabidopsis were located within seven clusters ...

  2. Retrotransposition creates sloping shores: a graded influence of hypomethylated CpG islands on flanking CpG sites.

    Science.gov (United States)

    Grandi, Fiorella C; Rosser, James M; Newkirk, Simon J; Yin, Jun; Jiang, Xiaoling; Xing, Zhuo; Whitmore, Leanne; Bashir, Sanum; Ivics, Zoltán; Izsvák, Zsuzsanna; Ye, Ping; Yu, Y Eugene; An, Wenfeng

    2015-08-01

    Long interspersed elements (LINEs), through both self-mobilization and trans-mobilization of short interspersed elements and processed pseudogenes, have made an indelible impact on the structure and function of the human genome. One consequence is the creation of new CpG islands (CGIs). In fact, more than half of all CGIs in the genome are associated with repetitive DNA, three-quarters of which are derived from retrotransposons. However, little is known about the epigenetic impact of newly inserted CGIs. We utilized a transgenic LINE-1 mouse model and tracked DNA methylation dynamics of individual germline insertions during mouse development. The retrotransposed GFP marker sequence, a strong CGI, is hypomethylated in male germ cells but hypermethylated in somatic tissues, regardless of genomic location. The GFP marker is similarly methylated when delivered into the genome via the Sleeping Beauty DNA transposon, suggesting that the observed methylation pattern may be independent of the mode of insertion. Comparative analyses between insertion- and non-insertion-containing alleles further reveal a graded influence of the retrotransposed CGI on flanking CpG sites, a phenomenon that we described as "sloping shores." Computational analyses of human and mouse methylomic data at single-base resolution confirm that sloping shores are universal for hypomethylated CGIs in sperm and somatic tissues. Additionally, the slope of a hypomethylated CGI can be affected by closely positioned CGI neighbors. Finally, by tracing sloping shore dynamics through embryonic and germ cell reprogramming, we found evidence of bookmarking, a mechanism that likely determines which CGIs will be eventually hyper- or hypomethylated. © 2015 Grandi et al.; Published by Cold Spring Harbor Laboratory Press.

  3. CpG islands undermethylation in human genomic regions under selective pressure.

    Directory of Open Access Journals (Sweden)

    Sergio Cocozza

    Full Text Available DNA methylation at CpG islands (CGIs is one of the most intensively studied epigenetic mechanisms. It is fundamental for cellular differentiation and control of transcriptional potential. DNA methylation is involved also in several processes that are central to evolutionary biology, including phenotypic plasticity and evolvability. In this study, we explored the relationship between CpG islands methylation and signatures of selective pressure in Homo Sapiens, using a computational biology approach. By analyzing methylation data of 25 cell lines from the Encyclopedia of DNA Elements (ENCODE Consortium, we compared the DNA methylation of CpG islands in genomic regions under selective pressure with the methylation of CpG islands in the remaining part of the genome. To define genomic regions under selective pressure, we used three different methods, each oriented to provide distinct information about selective events. Independently of the method and of the cell type used, we found evidences of undermethylation of CGIs in human genomic regions under selective pressure. Additionally, by analyzing SNP frequency in CpG islands, we demonstrated that CpG islands in regions under selective pressure show lower genetic variation. Our findings suggest that the CpG islands in regions under selective pressure seem to be somehow more "protected" from methylation when compared with other regions of the genome.

  4. CpG oligodeoxynucleotide nanomedicines for the prophylaxis or treatment of cancers, infectious diseases, and allergies.

    Science.gov (United States)

    Hanagata, Nobutaka

    2017-01-01

    Unmethylated cytosine-guanine dinucleotide-containing oligodeoxynucleotides (CpG ODNs), which are synthetic agonists of Toll-like receptor 9 (TLR9), activate humoral and cellular immunity and are being developed as vaccine adjuvants to prevent or treat cancers, infectious diseases, and allergies. Free CpG ODNs have been used in many clinical trials implemented to verify their effects. However, recent research has reported that self-assembled CpG ODNs, protein/peptide-CpG ODN conjugates, and nanomaterial-CpG ODN complexes demonstrate higher adjuvant effects than free CpG ODNs, owing to their improved uptake efficiency into cells expressing TLR9. Moreover, protein/peptide-CpG ODN conjugates and nanomaterial-CpG ODN complexes are able to deliver CpG ODNs and antigens (or allergens) to the same types of cells, which enables a higher degree of prophylaxis or therapeutic effect. In this review, the author describes recent trends in the research and development of CpG ODN nanomedicines containing self-assembled CpG ODNs, protein/peptide-CpG ODN conjugates, and nanomaterial-CpG ODN complexes, focusing mainly on the results of preclinical and clinical studies.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tingting [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Chen, Man; Liu, Lian [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Cheng, Huaiyan [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Yan, You-E [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Feng, Ying-Hong, E-mail: yhfeng@usuhs.edu [Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland (United States); Wang, Hui, E-mail: wanghui19@whu.edu.cn [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2011-12-15

    Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: Black-Right-Pointing-Pointer Nicotine-induced StAR inhibition in two human adrenal cell models. Black-Right-Pointing-Pointer Nicotine-induced single CpG site methylation in StAR promoter. Black-Right-Pointing-Pointer Persistent StAR inhibition and single CpG methylation after nicotine termination

  7. Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

    International Nuclear Information System (INIS)

    Wang, Tingting; Chen, Man; Liu, Lian; Cheng, Huaiyan; Yan, You-E; Feng, Ying-Hong; Wang, Hui

    2011-01-01

    Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: ► Nicotine-induced StAR inhibition in two human adrenal cell models. ► Nicotine-induced single CpG site methylation in StAR promoter. ► Persistent StAR inhibition and single CpG methylation after nicotine termination. ► Single CpG methylation located at Pax6 binding motif regulates St

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. 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.

  12. Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites.

    Science.gov (United States)

    Lunghi, Matteo; Spano, Furio; Magini, Alessandro; Emiliani, Carla; Carruthers, Vern B; Di Cristina, Manlio

    2016-02-01

    Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.

  13. Reinforcement learning for a biped robot based on a CPG-actor-critic method.

    Science.gov (United States)

    Nakamura, Yutaka; Mori, Takeshi; Sato, Masa-aki; Ishii, Shin

    2007-08-01

    Animals' rhythmic movements, such as locomotion, are considered to be controlled by neural circuits called central pattern generators (CPGs), which generate oscillatory signals. Motivated by this biological mechanism, studies have been conducted on the rhythmic movements controlled by CPG. As an autonomous learning framework for a CPG controller, we propose in this article a reinforcement learning method we call the "CPG-actor-critic" method. This method introduces a new architecture to the actor, and its training is roughly based on a stochastic policy gradient algorithm presented recently. We apply this method to an automatic acquisition problem of control for a biped robot. Computer simulations show that training of the CPG can be successfully performed by our method, thus allowing the biped robot to not only walk stably but also adapt to environmental changes.

  14. CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors

    International Nuclear Information System (INIS)

    Mason, Kathryn A.; Neal, Robert; Hunter, Nancy; Ariga, Hisanori; Ang, Kian; Milas, Luka

    2006-01-01

    Background and purpose: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074-7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361-9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods: CpG ODN 1826 (100 μg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results: Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion: Overall results show that CpG ODN 1826 can markedly improve tumor response

  15. Mendelian inheritance of trimodal CpG methylation sites suggests distal cis-acting genetic effects.

    Science.gov (United States)

    Zaghlool, Shaza B; Al-Shafai, Mashael; Al Muftah, Wadha A; Kumar, Pankaj; Gieger, Christian; Waldenberger, Melanie; Falchi, Mario; Suhre, Karsten

    2016-01-01

    Environmentally influenced phenotypes, such as obesity and insulin resistance, can be transmitted over multiple generations. Epigenetic modifications, such as methylation of DNA cytosine-guanine (CpG) pairs, may be carriers of inherited information. At the population level, the methylation state of such "heritable" CpG sites is expected to follow a trimodal distribution, and their mode of inheritance should be Mendelian. Using the Illumina Infinium 450 K DNA methylation array, we determined DNA CpG-methylation in blood cells from a family cohort 123 individuals of Arab ethnicity, including 18 elementary father-mother-child trios, we asked whether Mendelian inheritance of CpG methylation is observed, and most importantly, whether it is independent of any genetic signals. Using 40× whole genome sequencing, we therefore excluded all CpG sites with possibly confounding genetic variants (SNP) within the binding regions of the Illumina probes. We identified a total of 955 CpG sites that displayed a trimodal distribution and confirmed trimodality in a study of 1805 unrelated Caucasians. Of 955 CpG sites, 99.9% observed a strict Mendelian pattern of inheritance and had no SNP within +/-110 nucleotides of the CpG site by design. However, in 97% of these cases a distal cis-acting SNP within a +/-1 Mbp window was found that explained the observed CpG distribution, excluding the hypothesis of epigenetic inheritance for these clear-cut trimodal sites. Using power analysis, we showed that in 46% of all cases, the closest CpG-associated SNP was located more than 1000 bp from the CpG site. Our findings suggest that CpG methylation is maintained over larger genomic distances. Furthermore, nearly half of the SNPs associated with these trimodal sites were also associated with the expression of nearby genes ( P  = 4.08 × 10 -6 ), implying a regulatory effect of these trimodal CpG sites.

  16. Subcellular RNA sequencing reveals broad presence of cytoplasmic intron-sequence retaining transcripts in mouse and rat neurons.

    Directory of Open Access Journals (Sweden)

    Mugdha Khaladkar

    Full Text Available Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs, hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-β mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts. The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the

  17. CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

    OpenAIRE

    Hsieh, C L; Lieber, M R

    1992-01-01

    The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100-fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromos...

  18. Prediction of CpG-island function: CpG clustering vs. sliding-window methods

    Directory of Open Access Journals (Sweden)

    Luque-Escamilla Pedro L

    2010-05-01

    Full Text Available Abstract Background Unmethylated stretches of CpG dinucleotides (CpG islands are an outstanding property of mammal genomes. Conventionally, these regions are detected by sliding window approaches using %G + C, CpG observed/expected ratio and length thresholds as main parameters. Recently, clustering methods directly detect clusters of CpG dinucleotides as a statistical property of the genome sequence. Results We compare sliding-window to clustering (i.e. CpGcluster predictions by applying new ways to detect putative functionality of CpG islands. Analyzing the co-localization with several genomic regions as a function of window size vs. statistical significance (p-value, CpGcluster shows a higher overlap with promoter regions and highly conserved elements, at the same time showing less overlap with Alu retrotransposons. The major difference in the prediction was found for short islands (CpG islets, often exclusively predicted by CpGcluster. Many of these islets seem to be functional, as they are unmethylated, highly conserved and/or located within the promoter region. Finally, we show that window-based islands can spuriously overlap several, differentially regulated promoters as well as different methylation domains, which might indicate a wrong merge of several CpG islands into a single, very long island. The shorter CpGcluster islands seem to be much more specific when concerning the overlap with alternative transcription start sites or the detection of homogenous methylation domains. Conclusions The main difference between sliding-window approaches and clustering methods is the length of the predicted islands. Short islands, often differentially methylated, are almost exclusively predicted by CpGcluster. This suggests that CpGcluster may be the algorithm of choice to explore the function of these short, but putatively functional CpG islands.

  19. Real-time Walking Pattern Generation for a Biped Robot with Hybrid CPG-ZMP Algorithm

    Directory of Open Access Journals (Sweden)

    Bin He

    2014-10-01

    Full Text Available Biped robots have better mobility than conventional wheeled robots. The bio-inspired method based on a central pattern generator (CPG can be used to control biped robot walking in a manner like human beings. However, to achieve stable locomotion, it is difficult to modulate the parameters for the neural networks to coordinate every degree of freedom of the walking robot. The zero moment point (ZMP method is very popular for the stability control of biped robot walking. However, the reference trajectories have low energy efficiency, lack naturalness and need significant offline calculation. This paper presents a new method for biped real-time walking generation using a hybrid CPG-ZMP control algorithm. The method can realize a stable walking pattern by combining the ZMP criterion with rhythmic motion control. The CPG component is designed to generate the desired motion for each robot joint, which is modulated by phase resetting according to foot contact information. By introducing the ZMP location, the activity of the CPG output signal is adjusted to coordinate the limbs’ motion and allow the robot to maintain balance during the process of locomotion. The numerical simulation results show that, compared with the CPG method, the new hybrid CPG-ZMP algorithm can enhance the robustness of the CPG parameters and improve the stability of the robot. In addition, the proposed algorithm is more energy efficient than the ZMP method. The results also demonstrate that the control system can generate an adaptive walking pattern through interactions between the robot, the CPG and the environment.

  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

    Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate...... the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have...

  1. A species-generalized probabilistic model-based definition of CpG islands.

    Science.gov (United States)

    Irizarry, Rafael A; Wu, Hao; Feinberg, Andrew P

    2009-01-01

    The DNA of most vertebrates is depleted in CpG dinucleotides, the target for DNA methylation. The remaining CpGs tend to cluster in regions referred to as CpG islands (CGI). CGI have been useful as marking functionally relevant epigenetic loci for genome studies. For example, CGI are enriched in the promoters of vertebrate genes and thought to play an important role in regulation. Currently, CGI are defined algorithmically as an observed-to-expected ratio (O/E) of CpG greater than 0.6, G+C content greater than 0.5, and usually but not necessarily greater than a certain length. Here we find that the current definition leaves out important CpG clusters associated with epigenetic marks, relevant to development and disease, and does not apply at all to nonvertabrate genomes. We propose an alternative Hidden Markov model-based approach that solves these problems. We fit our model to genomes from 30 species, and the results support a new epigenomic view toward the development of DNA methylation in species diversity and evolution. The O/E of CpG in islands and nonislands segregated closely phylogenetically and showed substantial loss in both groups in animals of greater complexity, while maintaining a nearly constant difference in CpG O/E between islands and nonisland compartments. Lists of CGI for some species are available at http://www.rafalab.org .

  2. Research on Walking Gait of Biped Robot Based on a Modified CPG Model

    Directory of Open Access Journals (Sweden)

    Qiang Lu

    2015-01-01

    Full Text Available The neurophysiological studies of animals locomotion have verified that the fundamental rhythmic movements of animals are generated by the central pattern generator (CPG. Many CPG models have been proposed by scientific researchers. In this paper, a modified CPG model whose output function is sin(x is presented. The paper proves that the modified model has stable periodic solution and characteristics of the rhythmic movement using the Lyapunov judgement theorem and the phase diagram. A modified locomotion model is established in which the credit-assignment cerebellar model articulation controller (CA-CMAC algorithm is used to realize the pattern mapping between the CPG output and the musculoskeletal system. And a seven-link biped robot is employed to simulate cyclic walking gait in order to test the validity of the locomotion model. The main findings include the following. (1 The modified CPG model can generate spontaneous oscillations which correspond to biological signals. (2 The analysis of the modified locomotion model reveals that the CA-CMAC algorithm can be used to realize the pattern mapping between the CPG output and the musculoskeletal system.

  3. Intron evolution in Neurospora: the role of mutational bias and selection.

    Science.gov (United States)

    Sun, Yu; Whittle, Carrie A; Corcoran, Pádraic; Johannesson, Hanna

    2015-01-01

    We used comparative and population genomics to study intron evolutionary dynamics in the fungal model genus Neurospora. For our investigation, we used well-annotated genomes of N. crassa, N. discreta, and N. tetrasperma, and 92 resequenced genomes of N. tetrasperma from natural populations. By analyzing the four well-annotated genomes, we identified 9495 intron sites in 7619 orthologous genes. Our data supports nonhomologous end joining (NHEJ) and tandem duplication as mechanisms for intron gains in the genus and the RT-mRNA process as a mechanism for intron loss. We found a moderate intron gain rate (5.78-6.89 × 10(-13) intron gains per nucleotide site per year) and a high intron loss rate (7.53-13.76 × 10(-10) intron losses per intron sites per year) as compared to other eukaryotes. The derived intron gains and losses are skewed to high frequencies, relative to neutral SNPs, in natural populations of N. tetrasperma, suggesting that selection is involved in maintaining a high intron turnover. Furthermore, our analyses of the association between intron population-level frequency and genomic features suggest that selection is involved in shaping a 5' intron position bias and a low intron GC content. However, intron sequence analyses suggest that the gained introns were not exposed to recent selective sweeps. Taken together, this work contributes to our understanding of the importance of mutational bias and selection in shaping the intron distribution in eukaryotic genomes. © 2015 Sun et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Limited MHC class I intron 2 repertoire variation in bonobos.

    Science.gov (United States)

    de Groot, Natasja G; Heijmans, Corrine M C; Helsen, Philippe; Otting, Nel; Pereboom, Zjef; Stevens, Jeroen M G; Bontrop, Ronald E

    2017-10-01

    Common chimpanzees (Pan troglodytes) experienced a selective sweep, probably caused by a SIV-like virus, which targeted their MHC class I repertoire. Based on MHC class I intron 2 data analyses, this selective sweep took place about 2-3 million years ago. As a consequence, common chimpanzees have a skewed MHC class I repertoire that is enriched for allotypes that are able to recognise conserved regions of the SIV proteome. The bonobo (Pan paniscus) shared an ancestor with common chimpanzees approximately 1.5 to 2 million years ago. To investigate whether the signature of this selective sweep is also detectable in bonobos, the MHC class I gene repertoire of two bonobo panels comprising in total 29 animals was investigated by Sanger sequencing. We identified 14 Papa-A, 20 Papa-B and 11 Papa-C alleles, of which eight, five and eight alleles, respectively, have not been reported previously. Within this pool of MHC class I variation, we recovered only 2 Papa-A, 3 Papa-B and 6 Papa-C intron 2 sequences. As compared to humans, bonobos appear to have an even more diminished MHC class I intron 2 lineage repertoire than common chimpanzees. This supports the notion that the selective sweep may have predated the speciation of common chimpanzees and bonobos. The further reduction of the MHC class I intron 2 lineage repertoire observed in bonobos as compared to the common chimpanzee may be explained by a founding effect or other subsequent selective processes.

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

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... [Ratanaphan A., Panomwan P., Canyuk B. and Maipang T. 2011 Identification of novel intronic BRCA1 variants of uncertain significance in a Thai hereditary ... DNA structures, possibly through a slipped strand mispairing mechanism during .... have been related to inherited human disease as in genetic.

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

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... with an asterisk. proband was transmitted to the proband's unaffected daugh- ter. In order to confirm these genetic variations, the exon– intron 7 boundary sequences of the remaining breast cancer patients were amplified, screened by SSCP and subsequently sequenced. Five oligonucleotide primer pairs ...

  7. 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...

  8. 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 ).

  9. CPG-based Locomotion Controller Design for a Boxfish-like Robot

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-06-01

    Full Text Available This paper focuses on a Central Pattern Generator (CPG-based locomotion controller design for a boxfish-like robot. The bio-inspired controller is aimed at flexible switching in multiple 3D swimming patterns and exact attitude control of yaw and roll such that the robot will swim more like a real boxfish. The CPG network comprises two layers, the lower layer is the network of coupled linear oscillators and the upper is the transition layer where the lower-dimensional locomotion stimuli are transformed into the higher-dimensional control parameters serving for all the oscillators. Based on such a two-layer framework, flexible switching between multiple three-dimensional swimming patterns, such as swimming forwards/backwards, turning left/right, swimming upwards/downwards and rolling clockwise/counter-clockwise, can be simply realized by inputting different stimuli. Moreover, the stability of the CPG network is strictly proved to guarantee the intrinsic stability of the swimming patterns. As to exact attitude control, based on this open-loop CPG network and the sensory feedback from the Inertial Measurement Unit (IMU, a closed-loop CPG controller is advanced for yaw and roll control of the robotic fish for the first time. This CPG-based online attitude control for a robotic fish will greatly facilitate high-level practical underwater applications. A series of relevant experiments with the robotic fish are conducted systematically to validate the effectiveness and stability of the open-loop and closed-loop CPG controllers.

  10. Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

    Directory of Open Access Journals (Sweden)

    Frese Marc-André

    2009-08-01

    Full Text Available Abstract Background Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes. Results Here we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing. Conclusion Multiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.

  11. Mitochondrial group I and group II introns in the sponge orders Agelasida and Axinellida

    OpenAIRE

    Huchon, Doroth?e; Szitenberg, Amir; Shefer, Sigal; Ilan, Micha; Feldstein, Tamar

    2015-01-01

    Background Self-splicing introns are present in the mitochondria of members of most eukaryotic lineages. They are divided into Group I and Group II introns, according to their secondary structure and splicing mechanism. Being rare in animals, self-splicing introns were only described in a few sponges, cnidarians, placozoans and one annelid species. In sponges, three types of mitochondrial Group I introns were previously described in two demosponge families (Tetillidae, and Aplysinellidae) and...

  12. How complex an intron may be? The example of the first intron of the CTP synthase gene of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Roberto Piergentili

    2013-02-01

    Full Text Available In eukaryotes, maturation of primary transcripts into mature messenger RNAs involves the elimination of parts of the gene called ‘introns’. The biological significance of introns is not yet completely understood. It has been demonstrated that introns may contain other genes, or regulatory sequences that may be involved in transcriptional control, or also being involved in alternative splicing mechanisms. However, these functions explain the role of only a small number of them, and it is very difficult to formulate any generalization. The CTP synthase gene of Drosophila melanogaster is characterized by the presence of a long first intron (approximately 7.2 kilobases whose role is currently unknown. In the present report we analyzed in silico the content of this intron, and found that it contains at least three interesting sub-sequences. Two of them are homologous to the CTP synthase itself and to a putative nucleotide pyrophosphatase, respectively. The third is a short stretch of DNA able to fold into a thermodynamically stable hairpin and showing homology with other 19 sequences from 21 genes inside the D. melanogaster genome. These findings suggest a complex yet very accurate way of controlling gene expression inside the fruit fly.

  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. Inheritance of the group I rDNA intron in Tetrahymena pigmentosa

    DEFF Research Database (Denmark)

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

    1992-01-01

    We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+ and intro...

  15. The impact of intragenic CpG content on gene expression.

    Science.gov (United States)

    Bauer, Asli Petra; Leikam, Doris; Krinner, Simone; Notka, Frank; Ludwig, Christine; Längst, Gernot; Wagner, Ralf

    2010-07-01

    The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1 alpha). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.

  16. Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

    Directory of Open Access Journals (Sweden)

    Turner Seán

    2007-09-01

    Full Text Available Abstract Background Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria. Results Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG, in large subunit (LSU rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of Synechococcus sp. C9, and the other two are in the LSU gene of Synechococcus lividus strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria. Conclusion We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same bacterial gene (multiple group I introns have been reported in at least one phage gene and one prophage gene. The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith–Wiedemann syndrome

    Science.gov (United States)

    Smilinich, Nancy J.; Day, Colleen D.; Fitzpatrick, Galina V.; Caldwell, Germaine M.; Lossie, Amy C.; Cooper, P. R.; Smallwood, Allan C.; Joyce, Johanna A.; Schofield, Paul N.; Reik, Wolf; Nicholls, Robert D.; Weksberg, Rosanna; Driscoll, D. J.; Maher, Eamonn R.; Shows, Thomas B.; Higgins, Michael J.

    1999-01-01

    Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith–Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription–PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains. PMID:10393948

  19. Physiological, anatomical and genetic identification of CPG neurons in the developing mammalian spinal cord

    DEFF Research Database (Denmark)

    Kiehn, Ole; Butt, Simon J.B.

    2003-01-01

    . These latter experiments have defined EphA4 as a molecular marker for mammalian excitatory hindlimb CPG neurons. We also review genetic approaches that can be applied to the mouse spinal cord. These include methods for identifying sub-populations of neurons by genetically encoded reporters, techniques to trace...... network connectivity with cell-specific genetically encoded tracers, and ways to selectively ablate or eliminate neuron populations from the CPG. We propose that by applying a multidisciplinary approach it will be possible to understand the network structure of the mammalian locomotor CPG......The basic motor patterns underlying rhythmic limb movements during locomotion are generated by neuronal networks located within the spinal cord. These networks are called Central Pattern Generators (CPGs). Isolated spinal cord preparations from newborn rats and mice have become increasingly...

  20. RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1.

    Science.gov (United States)

    Wang, Xingang; Duan, Cheng-Guo; Tang, Kai; Wang, Bangshing; Zhang, Huiming; Lei, Mingguang; Lu, Kun; Mangrauthia, Satendra K; Wang, Pengcheng; Zhu, Guohui; Zhao, Yang; Zhu, Jian-Kang

    2013-09-17

    DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3' distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.

  1. Both size and GC-content of minimal introns are selected in human populations.

    Directory of Open Access Journals (Sweden)

    Dapeng Wang

    Full Text Available BACKGROUND: We previously have studied the insertion and deletion polymorphism by sequencing no more than one hundred introns in a mixed human population and found that the minimal introns tended to maintain length at an optimal size. Here we analyzed re-sequenced 179 individual genomes (from African, European, and Asian populations from the data released by the 1000 Genome Project to study the size dynamics of minimal introns. PRINCIPAL FINDINGS: We not only confirmed that minimal introns in human populations are selected but also found two major effects in minimal intron evolution: (i Size-effect: minimal introns longer than an optimal size (87 nt tend to have a higher ratio of deletion to insertion than those that are shorter than the optimal size; (ii GC-effect: minimal introns with lower GC content tend to be more frequently deleted than those with higher GC content. The GC-effect results in a higher GC content in minimal introns than their flanking exons as opposed to larger introns (≥125 nt that always have a lower GC content than that of their flanking exons. We also observed that the two effects are distinguishable but not completely separable within and between populations. CONCLUSIONS: We validated the unique mutation dynamics of minimal introns in keeping their near-optimal size and GC content, and our observations suggest potentially important functions of human minimal introns in transcript processing and gene regulation.

  2. Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages

    Science.gov (United States)

    Shi, Yongyu; Felder, Mildred A.R.; Sondel, Paul M.; Rakhmilevich, Alexander L.

    2015-01-01

    Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy. PMID:25829245

  3. Universal PCR primers for ribosomal protein gene introns of fish

    Directory of Open Access Journals (Sweden)

    Seinen Chow

    2016-01-01

    Full Text Available Abstract Human ribosomal protein (RP gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC polymerase chain reaction (PCR. In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis, 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.

  4. Protective immunity against Megalocytivirus infection in rock bream (Oplegnathus fasciatus) following CpG ODN administration.

    Science.gov (United States)

    Jung, Myung-Hwa; Lee, Jehee; Ortega-Villaizan, M; Perez, Luis; Jung, Sung-Ju

    2017-06-27

    Rock bream iridovirus (RBIV) disease in rock bream (Oplegnathus fasciatus) remains an unsolved problem in Korea aquaculture farms. CpG ODNs are known as immunostimulant, can improve the innate immune system of fish providing resistance to diseases. In this study, we evaluated the potential of CpG ODNs to induce anti-viral status protecting rock bream from different RBIV infection conditions. We found that, when administered into rock bream, CpG ODN 1668 induces better antiviral immune responses compared to other 5 CpG ODNs (2216, 1826, 2133, 2395 and 1720). All CpG ODN 1668 administered fish (1/5µg) at 2days before infection (1.1×10 7 ) held at 26°C died even though mortality was delayed from 8days (1µg) and 4days (5µg). Similarly, CpG ODN 1668 administered (5µg) at 2days before infection (1.2×10 6 ) held at 23/20°C had 100% mortality; the mortality was delayed from 9days (23°C) and 11days (20°C). Moreover, when CpG ODN 1668 administered (1/5/10µg) at 2/4/7days before infection or virus concentration was decreased to 1.1×10 4 and held at 20°C had mortality rates of 20/60/30% (2days), 30/40/60% (4days) and 60/60/20% (7days), respectively, for the respective administration dose, through 100 dpi. To investigate the development of a protective immune response, survivors were re-infected with RBIV (1.1×10 7 ) at 100 and 400 dpi, respectively. While 100% of the previously unexposed fish died, 100% of the previously infected fish survived. The high survival rate of fish following re-challenge with RBIV indicates that protective immunity was established in the surviving rock bream. Our results showed the possibility of developing preventive measures against RBIV using CpG ODN 1668 by reducing RBIV replication speed (i.e. water temperature of 20°C and infection dose of 1.1×10 4 ). Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Diversity of sponge mitochondrial introns revealed by cox 1 sequences of Tetillidae

    Directory of Open Access Journals (Sweden)

    Rot Chagai

    2010-09-01

    Full Text Available Abstract Background Animal mitochondrial introns are rare. In sponges and cnidarians they have been found in the cox 1 gene of some spirophorid and homosclerophorid sponges, as well as in the cox 1 and nad 5 genes of some Hexacorallia. Their sporadic distribution has raised a debate as to whether these mobile elements have been vertically or horizontally transmitted among their hosts. The first sponge found to possess a mitochondrial intron was a spirophorid sponge from the Tetillidae family. To better understand the mode of transmission of mitochondrial introns in sponges, we studied cox 1 intron distribution among representatives of this family. Results Seventeen tetillid cox 1 sequences were examined. Among these sequences only six were found to possess group I introns. Remarkably, three different forms of introns were found, named introns 714, 723 and 870 based on their different positions in the cox 1 alignment. These introns had distinct secondary structures and encoded LAGLIDADG ORFs belonging to three different lineages. Interestingly, sponges harboring the same intron form did not always form monophyletic groups, suggesting that their introns might have been transferred horizontally. To evaluate whether the introns were vertically or horizontally transmitted in sponges and cnidarians we used a host parasite approach. We tested for co-speciation between introns 723 (the introns with the highest number of sponge representatives and their nesting cox 1 sequences. Reciprocal AU tests indicated that the intron and cox 1 tree are significantly different, while a likelihood ratio test was not significant. A global test of co-phylogeny had significant results; however, when cnidarian sequences were analyzed separately the results were not significant. Conclusions The co-speciation analyses thus suggest that a vertical transmission of introns in the ancestor of sponges and cnidarians, followed by numerous independent losses, cannot solely

  6. Transdermal baicalin delivery using diethylene glycol monoethyl ether-mediated cubic phase gel.

    Science.gov (United States)

    Zhang, Yongtai; Zhang, Kai; Guo, Teng; Li, Yuan; Zhu, Chunyun; Feng, Nianping

    2015-02-01

    This study investigated the transdermal permeability of baicalin, a hydrophobic and readily hydrolyzed drug, delivered by glyceryl monooleate (GMO)-based cubic phase gel (CPG) mediated with Transcotol(®) P (TP, diethylene glycol monoethyl ether). A range of CPGs was produced by varying GMO, water, and TP levels. Examination of their physicochemical properties revealed that the optically isotropic CPG showed higher viscosity than lamellar phase gels (LPG), and the baicalin cargo increased CPG viscosity. The GMO:TP ratio and water content also altered viscosity. CPG-mediated delivery increased baicalin's skin permeation, with 76.65- to 200.24-fold higher (p<0.05) transdermal flux than that of a Carbopol(®)-based hydrogel (HDG), and 6.72- to 17.55-fold (p<0.05) higher than that of LPG, with the same water content. Rat in vivo microdialysis showed that CPG produced sustained baicalin release, with superior pharmacokinetic parameters to those of HDG. Furthermore, cutaneous drug absorption was more efficient on rat abdominal skin, compared to that in the chest or scapular region. Effective fusion between the CPG lipid matrix and the stratum corneum may explain this enhancement of transdermal permeation. CPG containing TP therefore, achieved excellent transdermal drug delivery and good baicalin stability, indicating that this system represents a promising transdermal delivery vehicle. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Single nucleotide polymorphisms in intron 1 and intron 2 of Larimichthys crocea growth hormone gene are correlated with growth traits

    Science.gov (United States)

    Ni, Jing; You, Feng; Xu, Jianhe; Xu, Dongdong; Wen, Aiyun; Wu, Zhihao; Xu, Yongli; Zhang, Peijun

    2012-03-01

    The growth hormone gene ( GH) affects animal growth and is a potential target for genetic studies of variation related to growth traits. In this study, we analyzed single nucleotide polymorphisms (SNPs) in GH intron regions and their associations with growth traits in large yellow croaker, Larimichthys crocea, from Zhejiang and Fujian stocks. The results of PCR-single strand conformation polymorphism showed two haplotypes of intron 1, named AA and AB genotypes, in Zhejiang stock. AB exhibited an SNP at position 196 (G→A) that was negatively correlated with body height and positively correlated with standard length/body height ( P≤0.05). Two different genotypes, CC and CD, were identified in intron 2 in Fujian stock, with CD showing an SNP at position 692 (T→C). The CD genotype had a significantly positive correlation with both weight and total length ( P≤0.01). These basic data highlight the potential for using GH as a genetic marker of fish growth in marker assisted selection.

  8. Epigenetic silencing of the 3p22 tumor suppressor DLEC1 by promoter CpG methylation in non-Hodgkin and Hodgkin lymphomas

    Directory of Open Access Journals (Sweden)

    Wang Zhaohui

    2012-10-01

    Full Text Available Abstract Background Inactivaion of tumor suppressor genes (TSGs by promoter CpG methylation frequently occurs in tumorigenesis, even in the early stages, contributing to the initiation and progression of human cancers. Deleted in lung and esophageal cancer 1 (DLEC1, located at the 3p22-21.3 TSG cluster, has been identified frequently silenced by promoter CpG methylation in multiple carcinomas, however, no study has been performed for lymphomas yet. Methods We examined the expression of DLEC1 by semi-quantitative reverse transcription (RT-PCR, and evaluated the promoter methylation of DLEC1 by methylation-specific PCR (MSP and bisulfite genomic sequencing (BGS in common lymphoma cell lines and tumors. Results Here we report that DLEC1 is readily expressed in normal lymphoid tissues including lymph nodes and PBMCs, but reduced or silenced in 70% (16/23 of non-Hodgkin and Hodgkin lymphoma cell lines, including 2/6 diffuse large B-cell (DLBCL, 1/2 peripheral T cell lymphomas, 5/5 Burkitt, 6/7 Hodgkin and 2/3 nasal killer (NK/T-cell lymphoma cell lines. Promoter CpG methylation was frequently detected in 80% (20/25 of lymphoma cell lines and correlated with DLEC1 downregulation/silencing. Pharmacologic demethylation reversed DLEC1 expression in lymphoma cell lines along with concomitant promoter demethylation. DLEC1 methylation was also frequently detected in 32 out of 58 (55% different types of lymphoma tissues, but not in normal lymph nodes. Furthermore, DLEC1 was specifically methylated in the sera of 3/13 (23% Hodgkin lymphoma patients. Conclusions Thus, methylation-mediated silencing of DLEC1 plays an important role in multiple lymphomagenesis, and may serve as a non-invasive tumor marker for lymphoma diagnosis.

  9. 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.

  10. Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba.

    Science.gov (United States)

    Gast, R J; Fuerst, P A; Byers, T J

    1994-01-01

    The discovery of group I introns in small subunit nuclear rDNA (nsrDNA) is becoming more common as the effort to generate phylogenies based upon nsrDNA sequences grows. In this paper we describe the discovery of the first two group I introns in the nsrDNA from the genus Acanthamoeba. The introns are in different locations in the genes, and have no significant primary sequence similarity to each other. They are identified as group I introns by the conserved P, Q, R and S sequences (1), and the ability to fit the sequences to a consensus secondary structure model for the group I introns (1, 2). Both introns are absent from the mature srRNA. A BLAST search (3) of nucleic acid sequences present in GenBank and EMBL revealed that the A. griffini intron was most similar to the nsrDNA group I intron of the green alga Dunaliella parva. A similar search found that the A. lenticulata intron was not similar to any of the other reported group I introns. Images PMID:8127708

  11. Structural and functional characterization of ribosomal protein gene introns in sponges.

    Science.gov (United States)

    Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.

  12. Isolation and characterization of functional tripartite group II introns using a Tn5-based genetic screen.

    Directory of Open Access Journals (Sweden)

    Christine Ritlop

    Full Text Available BACKGROUND: Group II introns are RNA enzymes that splice themselves from pre-mRNA transcripts. Most bacterial group II introns harbour an open reading frame (ORF, coding for a protein with reverse transcriptase, maturase and occasionally DNA binding and endonuclease activities. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA target sites. From an evolutionary perspective, group II introns are hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs--U1, U2, U4, U5 and U6 that are important functional elements of the spliceosome machinery. The ability of some group II introns fragmented in two or three pieces to assemble and undergo splicing in trans supports the theory that spliceosomal snRNAs evolved from portions of group II introns. METHODOLOGY/PRINCIPAL FINDINGS: We used a transposon-based genetic screen to explore the ability of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis to be fragmented into three pieces in vivo. Trans-splicing tripartite variants of Ll.LtrB were selected using a highly efficient and sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support tripartite trans-splicing, showing that this intron is remarkably tolerant to fragmentation. CONCLUSIONS/SIGNIFICANCE: This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo. The selected introns represent the first evidence of functional tripartite group II introns in bacteria and provide experimental support for the proposed evolutionary relationship between group II introns and snRNAs.

  13. Methylated CpG site count of dapper homolog 1 (DACT1) promoter prediction the poor survival of gastric cancer.

    Science.gov (United States)

    Deng, Jingyu; Liang, Han; Zhang, Rupeng; Ying, Guoguang; Xie, Xingmin; Yu, Jun; Fan, Daiming; Hao, Xishan

    2014-01-01

    To elucidate the clinical significance of the methylated status of CpG sites of dapper homolog 1 (DACT1) promoter in the survival prediction in gastric cancer (GC). The large scale GC patients (n=459) were analyzed for the quantitatively methylated status of CpG sites of DACT1 DNA promoter with the bisulphite sequencing PCR (BSP). With gene sequencing analysis, the methylated statuses of 12 cytosine-phosphate-guanine (CpG) sites in DACT1 promoter were detected to supply detailed information for the precisely prognostic prediction. Associations between molecular, clinicopathologic, and survival data were analyzed. With the MSP detection, different methylated levels of DACT1 promoter were identified in the 25 GC tissues, while none of 25 normal gastric mucosal tissues were found to be methylated. DACT1 promoter methylation was found in 28.32% in 459 patients. GC patients with 4 or more methylated CpG sites of DACT1 promoter was significantly associated with the poorer survival (P=0.19). The methylated statuses of CpG -515, CpG -435, and CpG -430 sites were also identified to provide the elaborate survival discrimination for 459 GC patients, respectively (P=0.049, =0.006, and =0.037). In addition, we demonstrated that the methylated CpG site count had smallest AIC and BIC values than other three methylated status of CpG sites for prediction the survival of 459 GC patients. The methylated CpG site count of DACT1 promoter had the significant applicability for clinical evaluation the prognosis of GC.

  14. A survey on CPG-inspired control models and system implementation.

    Science.gov (United States)

    Yu, Junzhi; Tan, Min; Chen, Jian; Zhang, Jianwei

    2014-03-01

    This paper surveys the developments of the last 20 years in the field of central pattern generator (CPG) inspired locomotion control, with particular emphasis on the fast emerging robotics-related applications. Functioning as a biological neural network, CPGs can be considered as a group of coupled neurons that generate rhythmic signals without sensory feedback; however, sensory feedback is needed to shape the CPG signals. The basic idea in engineering endeavors is to replicate this intrinsic, computationally efficient, distributed control mechanism for multiple articulated joints, or multi-DOF control cases. In terms of various abstraction levels, existing CPG control models and their extensions are reviewed with a focus on the relative advantages and disadvantages of the models, including ease of design and implementation. The main issues arising from design, optimization, and implementation of the CPG-based control as well as possible alternatives are further discussed, with an attempt to shed more light on locomotion control-oriented theories and applications. The design challenges and trends associated with the further advancement of this area are also summarized.

  15. High CpG island methylation of p16 gene and loss of p16 protein ...

    Indian Academy of Sciences (India)

    SI-JU GAO

    proposed for cell therapy based on interventions in heart fail- ure, and HFC senescence is associated with upregulation of p16 expression (Golubnitschaja et al. 2003; Ball and Levine. 2005). Although, the involvement of CpG island methylation in suppression of p16 gene expression has been discussed in cancer settings ...

  16. High CpG island methylation of p16 gene and loss of p16 protein ...

    Indian Academy of Sciences (India)

    ... in p16 promoters in ToF patients was negatively correlated with p16 protein and gene expression (both P < 0.05). Our study reports that high CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of ToF, which may have significant therapeutic applications ...

  17. CpG oligodeoxynucleotides as TLR9 agonists: therapeutic applications in cancer.

    Science.gov (United States)

    Murad, Yanal M; Clay, Timothy M

    2009-01-01

    Toll-like receptors (TLRs) are part of the innate immune system, and they belong to the pattern recognition receptors (PRR) family. The PRR family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are not generated by the host and are restricted and essential to micro-organisms. TLR9, which recognizes unmethylated CpG (cytosine guanosine dinucleotide), is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells and B cells, and results in potent T helper-1 (T(h)1)-type immune responses and antitumor responses in mouse tumor models and in patients. Several pharmaceutical companies, such as Pfizer, Idera, and Dynavax, are developing CpG oligodeoxynucleotides (ODNs) for the treatment of cancer, along with other conditions, such as infections and allergy. CpG ODNs have shown promising results as vaccine adjuvants and in combination with cancer immunotherapy. Several TLR9 agonists are being developed and have entered clinical trials to evaluate their safety and efficacy for the treatment of several hematopoietic and solid tumors. In this review, we discuss the use of CpG ODNs in several phase I and II clinical trials for the treatment of NHL, renal cell carcinoma, melanoma, and non-small cell lung cancer, either alone or in combination with other agents.

  18. On the role of sensory feedbacks in Rowat-Selverston CPG to improve robot legged locomotion

    Directory of Open Access Journals (Sweden)

    Elmira eAmrollah

    2010-12-01

    Full Text Available This paper presents the use of Rowat and Selverston-type of CPG to control locomotion. It focuses on the role of afferent exteroceptive and proprioceptive signals in the dynamic phase synchronization in CPG legged robots. The sensori-motor neural network architecture is evaluated to control a two-joint planar robot leg that slips on a rail. Then, the closed loop between the CPG and the mechanical system allows to study the modulation of rhythmic patterns and the effect of the sensing loop via sensory neurons during the locomotion task. Firstly simulations show that the proposed architecture easily allows to modulate rhythmic patterns of the leg, and therefore the velocity of the robot. Secondly, simulations show that sensori-feedbacks from foot/ground contact of the leg make the hip velocity smoother and larger. The results show that the Rowat-Selverston-type CPG with sensory feedbacks is an effective choice for building adaptive Neural Central Pattern Generators for legged robots.

  19. 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.

  20. Evidence for the late origin of introns in chloroplast genes from an evolutionary analysis of the genus Euglena.

    Science.gov (United States)

    Thompson, M D; Copertino, D W; Thompson, E; Favreau, M R; Hallick, R B

    1995-01-01

    The origin of present day introns is a subject of spirited debate. Any intron evolution theory must account for not only nuclear spliceosomal introns but also their antecedents. The evolution of group II introns is fundamental to this debate, since group II introns are the proposed progenitors of nuclear spliceosomal introns and are found in ancient genes from modern organisms. We have studied the evolution of chloroplast introns and twintrons (introns within introns) in the genus Euglena. Our hypothesis is that Euglena chloroplast introns arose late in the evolution of this lineage and that twintrons were formed by the insertion of one or more introns into existing introns. In the present study we find that 22 out of 26 introns surveyed in six different photosynthesis-related genes from the plastid DNA of Euglena gracilis are not present in one or more basally branching Euglena spp. These results are supportive of a late origin for Euglena chloroplast group II introns. The psbT gene in Euglena viridis, a basally branching Euglena species, contains a single intron in the identical position to a psbT twintron from E.gracilis, a derived species. The E.viridis intron, when compared with 99 other Euglena group II introns, is most similar to the external intron of the E.gracilis psbT twintron. Based on these data, the addition of introns to the ancestral psbT intron in the common ancester of E.viridis and E.gracilis gave rise to the psbT twintron in E.gracilis. Images PMID:8532514

  1. Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: Introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes

    OpenAIRE

    Landthaler, Markus; Shub, David A.

    1999-01-01

    Analysis of RNA that can be labeled with GTP indicates the existence of group I introns in genes of at least three transcriptional classes in the genome of Staphylococcus aureus bacteriophage Twort. A single ORF of 142 amino acids (Orf142) is interrupted by three self-splicing group I introns, providing the first example of a phage gene with multiple intron insertions. Twort Orf142 is encoded in a message that is abundant 15–20 min after infection and is highly similar to a late gene product ...

  2. Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium.

    Science.gov (United States)

    Wang, Hsiao-Lin V; Dinwiddie, Brandon L; Lee, Herman; Chekanova, Julia A

    2015-02-01

    Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3' UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr-siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. © 2015 Wang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. Diversity, mobility, and structural and functional evolution of group II introns carrying an unusual 3' extension

    OpenAIRE

    Tourasse, Nicolas J; Stabell, Fredrik B; Kolstø, Anne-Brit

    2011-01-01

    Background Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes) that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the Bacillu...

  6. Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-beta knock-out mice

    DEFF Research Database (Denmark)

    Matheu, Victor; Treschow, Alexandra; Teige, Ingrid

    2005-01-01

    by therapy with CpG-ODN is only slightly and partially dependent on IFN-beta, while IFN-beta is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination....... of CpG-ODN is not known. OBJECTIVE: Here, we aimed to elucidate the role of IFN-beta in the anti-allergic effect of CpG motifs. METHODS: We assessed the immune response in OVA-primed/OVA-challenged IFN-beta knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment......BACKGROUND: CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-beta in the immunomodulatory effects...

  7. Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla species (Orchidaceae).

    Science.gov (United States)

    Besse, Pascale; Da Silva, Denis; Bory, Séverine; Noirot, Michel; Grisoni, Michel

    2009-04-01

    Variation in intron length in caffeic acid O-methyltransferase (COMT) in Vanilla was studied and demonstrated that COMT genes in Vanilla are organized with four exons and three introns. At least two to four different versions (either allelic or paralogous) of the COMT multigenic family in the genus Vanilla (in terms of intron sizes) were detected. The three introns were differentially variable, with intron-1 being the most length-polymorphic. Patterns of variations were in accordance with known phylogenetic relationships in the genus obtained with neutral markers. In particular, the genus displayed a strong Old World versus New World differentiation with American fragrant species being characterized by a specific 99bp intron-1 size-variant and a unique 226bp intron-3 variant. Conversely, leafless species of the genus displayed unexpected variations in intron lengths. Due to their role in primary (lignin) and secondary (phenolics, e.g., vanillin, alkaloids) metabolisms, COMT genes might not be neutral markers, and represent candidate functional markers for resistance, aromatic or medicinal properties of Vanilla species. Investigating the orthologous/paralogous status of the different genes revealed (in terms of intron size) will allow the evolution of the COMT genes to be studied. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

  8. An overabundance of phase 0 introns immediately after the start codon in eukaryotic genes

    Directory of Open Access Journals (Sweden)

    Wernersson Rasmus

    2006-10-01

    Full Text Available Abstract Background A knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. Despite this, there has been relatively little focus on the distribution of intron positions in genes. Results In proteins with signal peptides, there is an overabundance of phase 1 introns around the region of the signal peptide cleavage site. This has been described before. But in proteins without signal peptides, a novel phenomenon is observed: There is a sharp peak of phase 0 intron positions immediately following the start codon, i.e. between codons 1 and 2. This effect is seen in a wide range of eukaryotes: Vertebrates, arthropods, fungi, and flowering plants. Proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. In addition, there is a peak of phase 0 introns at position 5 in Drosophila genes with signal peptides, predominantly representing cuticle proteins. Conclusion There is an overabundance of phase 0 introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. We give a detailed description of these start codon introns and the proteins that contain them.

  9. An overabundance of phase 0 introns immediately after the start codon in eukaryotic genes.

    Science.gov (United States)

    Nielsen, Henrik; Wernersson, Rasmus

    2006-10-11

    A knowledge of the positions of introns in eukaryotic genes is important for understanding the evolution of introns. Despite this, there has been relatively little focus on the distribution of intron positions in genes. In proteins with signal peptides, there is an overabundance of phase 1 introns around the region of the signal peptide cleavage site. This has been described before. But in proteins without signal peptides, a novel phenomenon is observed: There is a sharp peak of phase 0 intron positions immediately following the start codon, i.e. between codons 1 and 2. This effect is seen in a wide range of eukaryotes: Vertebrates, arthropods, fungi, and flowering plants. Proteins carrying this start codon intron are found to comprise a special class of relatively short, lysine-rich and conserved proteins with an overrepresentation of ribosomal proteins. In addition, there is a peak of phase 0 introns at position 5 in Drosophila genes with signal peptides, predominantly representing cuticle proteins. There is an overabundance of phase 0 introns immediately after the start codon in eukaryotic genes, which has been described before only for human ribosomal proteins. We give a detailed description of these start codon introns and the proteins that contain them.

  10. The Chloroplast Genome of Euglena mutabilis-Cluster Arrangement, Intron Analysis, and Intrageneric Trends.

    Science.gov (United States)

    Dabbagh, Nadja; Preisfeld, Angelika

    2017-01-01

    A comparative analysis of the chloroplast genome of Euglena mutabilis underlined a high diversity in the evolution of plastids in euglenids. Gene clusters in more derived Euglenales increased in complexity with only a few, but remarkable changes in the genus Euglena. Euglena mutabilis differed from other Euglena species in a mirror-inverted arrangement of 12 from 15 identified clusters, making it very likely that the emergence at the base of the genus Euglena, which has been considered a long branch artifact, is truly a probable position. This was corroborated by many similarities in gene arrangement and orientation with Strombomonas and Monomorphina, rendering the genome organization of E. mutabilis in certain clusters as plesiomorphic feature. By RNA analysis exact exon-intron boundaries and the type of the 77 introns identified were mostly determined unambiguously. A detailed intron study of psbC pointed at two important issues: First, the number of introns varied even between species, and no trend from few to many introns could be observed. Second, mat1 was localized in Eutreptiales exclusively in intron 1, and mat2 was not identified. With the emergence of Euglenaceae in most species, a new intron containing mat2 inserted in front of the previous intron 1 and thereby became intron 2 with mat1. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  11. A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

    OpenAIRE

    Hebbar, S K; Belcher, S M; Perlman, P S

    1992-01-01

    Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or...

  12. CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

    Science.gov (United States)

    Stefansson, Olafur Andri; Hermanowicz, Stefan; van der Horst, Jasper; Hilmarsdottir, Holmfridur; Staszczak, Zuzanna; Jonasson, Jon Gunnlaugur; Tryggvadottir, Laufey; Gudjonsson, Thorkell; Sigurdsson, Stefan

    2017-07-05

    DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20

  13. Group-II intron splicing factors in higher-plants mitochondria

    Directory of Open Access Journals (Sweden)

    Gregory G. Brown

    2014-02-01

    Full Text Available Group-II introns are large catalytic RNAs (ribozymes which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group-II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has retained in the mtDNA of angiosperms: matR encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4. Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4, which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory

  14. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.

    Science.gov (United States)

    Pan, Xichun; Li, Bin; Kuang, Mei; Liu, Xin; Cen, Yanyan; Qin, Rongxin; Ding, Guofu; Zheng, Jiang; Zhou, Hong

    2016-02-22

    Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  15. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Xichun Pan

    2016-02-01

    Full Text Available Toll-like receptor (TLR 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN. Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS- and lipopeptide (PAM3CSK4-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  16. Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

    Science.gov (United States)

    Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

    2004-09-01

    Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

  17. 7-Deaza-2'-deoxyguanosine allows PCR and sequencing reactions from CpG islands.

    Science.gov (United States)

    Jung, A; Ruckert, S; Frank, P; Brabletz, T; Kirchner, T

    2002-02-01

    CpG islands are GC rich sequences that are found in the promoters of many genes in higher eukaryotes. They contain a high frequency of CG dinucleotides, which are substrates for DNA methylases. Methylation leads to transcriptional silencing of promoters. Owing to their high GC content CpG islands exhibit strong base-base interactions, which lead to superstructures and consequently to regions with higher melting temperatures. Therefore, Taq polymerases (especially sequenases) fall off their templates, causing premature termination of the polymerase chain reaction (PCR) or sequencing reactions. The results from such reactions are thus insufficient for further analysis. Therefore, we have evaluated the use of 7-deaza-2'-deoxyguanosine for PCR amplification of the human p16(INK4A) promoter and sequencing of HUMARA exon 1 PCR products. Our results show that the addition of 7-deaza-2'-deoxyguanosine significantly improves results, particularly when small amounts of poor quality DNA are available as starting material.

  18. 7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands

    Science.gov (United States)

    Jung, A; Ruckert, S; Frank, P; Brabletz, T; Kirchner, T

    2002-01-01

    CpG islands are GC rich sequences that are found in the promoters of many genes in higher eukaryotes. They contain a high frequency of CG dinucleotides, which are substrates for DNA methylases. Methylation leads to transcriptional silencing of promoters. Owing to their high GC content CpG islands exhibit strong base–base interactions, which lead to superstructures and consequently to regions with higher melting temperatures. Therefore, Taq polymerases (especially sequenases) fall off their templates, causing premature termination of the polymerase chain reaction (PCR) or sequencing reactions. The results from such reactions are thus insufficient for further analysis. Therefore, we have evaluated the use of 7-deaza-2`-deoxyguanosine for PCR amplification of the human p16INK4A promoter and sequencing of HUMARA exon 1 PCR products. Our results show that the addition of 7-deaza-2`-deoxyguanosine significantly improves results, particularly when small amounts of poor quality DNA are available as starting material. PMID:11836448

  19. Genome-wide DNA Methylation Profiling of CpG Islands in Hypospadias

    Science.gov (United States)

    Choudhry, Shweta; Deshpande, Archana; Qiao, Liang; Beckman, Kenneth; Sen, Saunak; Baskin, Laurence S.

    2013-01-01

    Purpose Hypospadias is one of the most frequent genital malformations in the male newborn, and results from abnormal penile and urethral development. The etiology of hypospadias remains largely unknown despite intensive investigations. Fetal androgens have a crucial role in genital differentiation. Recent studies have suggested that molecular mechanisms that underlie the effects of androgens on the fetus may involve disruption of epigenetic programming of gene expression during development. We assessed whether epigenetic modification of DNA methylation is associated with hypospadias in a case-control study of 12 hypospadias and 8 control subjects. Materials and Methods Genome-wide DNA methylation profiling was performed on the study subjects using the Illumina Infinium® HumanMethylation450 Bead-Chip, which enables the direct investigation of methylation status of more than 485,000 individual CpG sites throughout the genome. The methylation level at each CpG site was compared between cases and controls using the t test and logistic regression. Results We identified 14 CpG sites that were associated with hypospadias with p hypospadias using a unique and novel epigenetic approach. Our findings suggest DNA methylation patterns are useful in identifying new genes such as SCARB1 and MYBPH that may be involved in the etiology of hypospadias. PMID:22906644

  20. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration.

    Science.gov (United States)

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn't affect Egf mRNA expression during the re-organization phase.

  1. A novel CpG island set identifies tissue-specific methylation at developmental gene loci.

    Directory of Open Access Journals (Sweden)

    Robert Illingworth

    2008-01-01

    Full Text Available CpG islands (CGIs are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%-8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

  2. Electrophysiological representation of scratching CpG activity in the cerebellum.

    Science.gov (United States)

    Martínez-Silva, Lourdes; Manjarrez, Elias; Gutiérrez-Ospina, Gabriel; Quevedo, Jorge N

    2014-01-01

    We analyzed the electrical activity of neuronal populations in the cerebellum and the lumbar spinal cord during fictive scratching in adult decerebrate cats before and after selective sections of the Spino-Reticulo Cerebellar Pathway (SRCP) and the Ventral-Spino Cerebellar Tract (VSCT). During fictive scratching, we found a conspicuous sinusoidal electrical activity, called Sinusoidal Cerebellar Potentials (SCPs), in the cerebellar vermis, which exhibited smaller amplitude in the paravermal and hemisphere cortices. There was also a significant spino-cerebellar coherence between these SCPs and the lumbar sinusoidal cord dorsum potentials (SCDPs). However, during spontaneous activity such spino-cerebellar coherence between spontaneous potentials recorded in the same regions decreased. We found that the section of the SRCP and the VSCT did not abolish the amplitude of the SCPs, suggesting that there are additional pathways conveying information from the spinal CPG to the cerebellum. This is the first evidence that the sinusoidal activity associated to the spinal CPG circuitry for scratching has a broad representation in the cerebellum beyond the sensory representation from hindlimbs previously described. Furthermore, the SCPs represent the global electrical activity of the spinal CPG for scratching in the cerebellar cortex.

  3. Smooth transition for CPG-based body shape control of a snake-like robot

    International Nuclear Information System (INIS)

    Nor, Norzalilah Mohamad; Ma, Shugen

    2014-01-01

    This paper presents a locomotion control based on central pattern generator (CPG) of a snake-like robot. The main point addressed in this paper is a method that produces a smooth transition of the body shape of a snake-like robot. Body shape transition is important for snake-like robot locomotion to adapt to different space widths and also for obstacle avoidance. By manipulating the phase difference of the CPG outputs instantly, it will results in a sharp point or discontinuity which lead to an unstable movement of the snake-like robot. To tackle the problem, we propose a way of controlling the body shape: by incorporating activation function in the phase oscillator CPG model. The simplicity of the method promises an easy implementation and simple control. Simulation results and torque analysis confirm the effectiveness of the proposed control method and thus, can be used as a locomotion control in various potential applications of a snake-like robot. (paper)

  4. Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats

    Directory of Open Access Journals (Sweden)

    Guadalupe S. López-Álvarez

    2017-01-01

    Full Text Available The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4 using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.

  5. Dynamic Modelling of a CPG-Controlled Amphibious Biomimetic Swimming Robot

    Directory of Open Access Journals (Sweden)

    Rui Ding

    2013-04-01

    Full Text Available This paper focuses on the modelling and control problems of a self-propelled, multimodal amphibious robot. Inspired by the undulatory body motions of fish and dolphins, the amphibious robot propels itself underwater by oscillations of several modular fish-like propelling units coupled with a pair of pectoral fins capable of non-continuous 360 degree rotation. In order to mimic fish-like undulating propulsion, a control architecture based on Central Pattern Generator (CPG is applied to the amphibious robot for robust swimming gaits, including forward and backward swimming and turning, etc. With the simplification of the robot as a multi-link serial mechanism, a Lagrangian function is employed to establish the hydrodynamic model for steady swimming. The CPG motion control law is then imported into the Lagrangian-based dynamic model, where an associated system of kinematics and dynamics is formed to solve real-time movements and, further, to guide the exploration of the CPG parameters and steady locomotion gaits. Finally, comparative results between the simulations and experiments are provided to show the effectiveness of the built control models.

  6. Genomewide analysis of intronic microRNAs in rice and Arabidopsis

    Indian Academy of Sciences (India)

    Keywords. rice; Arabidopsis; intronic miRNA; host gene; bioinformatics; function. Abstract. MicroRNAs (miRNAs) are potent regulators of gene transcription and posttranscriptional processes. The majority of miRNAs are localized within intronic regions of protein-coding genes (host genes) and have diverse functions in ...

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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...

  10. Increased expression of prion protein gene is accompanied by demethylation of CpG sites in a mouse embryonal carcinoma cell line, P19C6

    Science.gov (United States)

    DALAI, Wuyun; MATSUO, Eiko; TAKEYAMA, Natsumi; KAWANO, Junichi; SAEKI, Keiichi

    2017-01-01

    Elucidation of the processes regulating the prion protein gene (Prnp) is an important key to understanding the development of prion disorders. In this study, we explored the involvement of DNA methylation in Prnp transcriptional regulation during neuronal differentiation of embryonic carcinoma P19C6 cells. When P19C6 cells were differentiated into neuronal cells, the expression of Prnp was markedly increased, while CpG methylation was significantly demethylated at the nucleotide region between −599 and −238 from the transcription start site. In addition, when P19C6 cells were applied in a DNA methyltransferase inhibitor, RG108, Prnp transcripts were also significantly increased in relation to the decreased methylation statuses. These findings helped to elucidate the DNA methylation-mediated regulation of Prnp expression during neuronal differentiation. PMID:28132962

  11. Polyethyleneimine-functionalized boron nitride nanospheres as efficient carriers for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Zhang HJ

    2015-08-01

    Full Text Available Huijie Zhang,1 Shini Feng,1 Ting Yan,1 Chunyi Zhi,2 Xiao-Dong Gao,1 Nobutaka Hanagata3,41The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, People’s Republic of China; 2Department of Physics and Materials Science, City University of Hong Kong, Kowlong, Hong Kong SAR, People’s Republic of China; 3Biomaterials Unit, International Center for Materials Nanoarchitectonics, National Institute for Materials Science, Ibaraki, Japan; 4Nanotechnology Innovation Station, National Institute for Materials Science, Ibaraki, JapanAbstract: CpG oligodeoxynucleotides (ODNs stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyethyleneimine (PEI-functionalized boron nitride nanospheres (BNNS. PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS–PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS–PEI complexes with concentrations up to 100 µg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS–PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS–PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS–PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon

  12. Intron gain by tandem genomic duplication: a novel case in a potato gene encoding RNA-dependent RNA polymerase

    Directory of Open Access Journals (Sweden)

    Ming-Yue Ma

    2016-07-01

    Full Text Available The origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. However, the mechanisms underlying intron gain remain unclear because there are few proven cases of recently gained introns. In an RNA-dependent RNA polymerase (RdRp gene, we found that a tandem duplication occurred after the divergence of potato and its wild relatives among other Solanum plants. The duplicated sequence crosses the intron-exon boundary of the first intron and the second exon. A new intron was detected at this duplicated region, and it includes a small previously exonic segment of the upstream copy of the duplicated sequence and the intronic segment of the downstream copy of the duplicated sequence. The donor site of this new intron was directly obtained from the small previously exonic segment. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers, and the GC contents differed between the intron and exon. In the widely cited model of intron gain by tandem genomic duplication, the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. Our results illustrate that the tandem duplication model of intron gain should be diverse in terms of obtaining the proper splicing signals.

  13. Insertion of a self-splicing intron into the mtDNA of atriploblastic animal

    Energy Technology Data Exchange (ETDEWEB)

    Valles, Y.; Halanych, K.; Boore, J.L.

    2006-04-14

    Nephtys longosetosa is a carnivorous polychaete worm that lives in the intertidal and subtidal zones with worldwide distribution (pleijel&rouse2001). Its mitochondrial genome has the characteristics typical of most metazoans: 37 genes; circular molecule; almost no intergenic sequence; and no significant gene rearrangements when compared to other annelid mtDNAs (booremoritz19981995). Ubiquitous features as small intergenic regions and lack of introns suggested that metazoan mtDNAs are under strong selective pressures to reduce their genome size allowing for faster replication requirements (booremoritz19981995Lynch2005). Yet, in 1996 two type I introns were found in the mtDNA of the basal metazoan Metridium senile (FigureX). Breaking a long-standing rule (absence of introns in metazoan mtDNA), this finding was later supported by the further presence of group I introns in other cnidarians. Interestingly, only the class Anthozoa within cnidarians seems to harbor such introns. Although several hundreds of triploblastic metazoan mtDNAs have been sequenced, this study is the first evidence of mitochondrial introns in triploblastic metazoans. The cox1 gene of N. longosetosa has an intron of almost 2 kbs in length. This finding represents as well the first instance of a group II intron (anthozoans harbor group I introns) in all metazoan lineages. Opposite trends are observed within plants, fungi and protist mtDNAs, where introns (both group I and II) and other non-coding sequences are widespread. Plant, fungal and protist mtDNA structure and organization differ enormously from that of metazoan mtDNA. Both, plant and fungal mtDNA are dynamic molecules that undergo high rates of recombination, contain long intergenic spacer regions and harbor both group I and group II introns. However, as metazoans they have a conserved gene content. Protists, on the other hand have a striking variation of gene content and introns that account for the genome size variation. In contrast to

  14. CpG Oligodeoxynucleotides Induce Differential Cytokine and Chemokine Gene Expression Profiles in Dapulian and Landrace Pigs.

    Science.gov (United States)

    Hu, Jiaqing; Yang, Dandan; Wang, Hui; Li, Chuanhao; Zeng, Yongqing; Chen, Wei

    2016-01-01

    Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) mimic the immunostimulatory activity of microbial DNA by interacting with Toll-like receptor 9 (TLR9) to activate both the innate and adaptive immune responses in different species. However, few studies have been published to compare the effects of CpG ODN on different pig breeds. Therefore, in this study, whole blood gene expression profiles of DPL and Landrace pigs treated with CpG ODN were studied using RNA-seq technology. Five Hundred differentially expressed genes (DEGs) were identified between the two breeds. DPL pigs had significantly higher number of immune-relevant DEGs than the Landrace pigs after CpG ODN treatment. Pathway analysis showed that cytokine-cytokine receptor interaction and chemokine signaling pathway were the major enriched pathways of the immune-relevant DEGs. Further in vitro experiments showed that PBMCs of the DPL pigs had significantly higher levels of TLR9 mRNA than those of the Landrace pigs, both before and after CpG ODN stimulation. Cytokine and chemokine induction in the PBMCs of both breeds were also measured after CpG ODN stimulation. Our data showed that mRNA levels of cytokines (IFNα, IL8, IL12 p40) and chemokines (CXCL9, CXCL13) were significantly higher in the PBMCs of the DPL pigs than those of the Landrace pigs. Taken together, our data provide new information regarding the pig breed difference in response to CpG ODN stimulation and that higher levels of TLR9 mRNA in DPL pigs may be a major contributor for disease resistance.

  15. GaussianCpG: a Gaussian model for detection of CpG island in human genome sequences.

    Science.gov (United States)

    Yu, Ning; Guo, Xuan; Zelikovsky, Alexander; Pan, Yi

    2017-05-24

    As crucial markers in identifying biological elements and processes in mammalian genomes, CpG islands (CGI) play important roles in DNA methylation, gene regulation, epigenetic inheritance, gene mutation, chromosome inactivation and nuclesome retention. The generally accepted criteria of CGI rely on: (a) %G+C content is ≥ 50%, (b) the ratio of the observed CpG content and the expected CpG content is ≥ 0.6, and (c) the general length of CGI is greater than 200 nucleotides. Most existing computational methods for the prediction of CpG island are programmed on these rules. However, many experimentally verified CpG islands deviate from these artificial criteria. Experiments indicate that in many cases %G+C is human genome. We analyze the energy distribution over genomic primary structure for each CpG site and adopt the parameters from statistics of Human genome. The evaluation results show that the new model can predict CpG islands efficiently by balancing both sensitivity and specificity over known human CGI data sets. Compared with other models, GaussianCpG can achieve better performance in CGI detection. Our Gaussian model aims to simplify the complex interaction between nucleotides. The model is computed not by the linear statistical method but by the Gaussian energy distribution and accumulation. The parameters of Gaussian function are not arbitrarily designated but deliberately chosen by optimizing the biological statistics. By using the pseudopotential analysis on CpG islands, the novel model is validated on both the real and artificial data sets.

  16. The Effect of TLR9 Agonist CpG Oligodeoxynucleotides on the Intestinal Immune Response of Cobia (Rachycentron canadum

    Directory of Open Access Journals (Sweden)

    Omkar Byadgi

    2014-01-01

    Full Text Available Cytosine-guanine oligodeoxynucleotide (CpG ODN motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9 and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1β. Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds and B (30 folds isoforms at 10 dpi (CpG ODN 1668 and MyD88 (21 folds at 6 dpv (CpG ODN 2395. Subsequently, IL-1β increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.

  17. Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    Science.gov (United States)

    Nichol Edamura, Kerrie; Leonard, Michelle R.; Pearson, Christopher E.

    2005-01-01

    Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation. PMID:15625623

  18. Resequencing PNMT in European hypertensive and normotensive individuals: no common susceptibilily variants for hypertension and purifying selection on intron 1

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    Viigimaa Margus

    2007-07-01

    Full Text Available Abstract Background Human linkage and animal QTL studies have indicated the contribution of genes on Chr17 into blood pressure regulation. One candidate gene is PNMT, coding for phenylethanolamine-N-methyltransferase, catalyzing the synthesis of epinephrine from norepinephrine. Methods Fine-scale variation of PNMT was screened by resequencing hypertensive (n = 50 and normotensive (n = 50 individuals from two European populations (Estonians and Czechs. The resulting polymorphism data were analyzed by statistical genetics methods using Genepop 3.4, PHASE 2.1 and DnaSP 4.0 software programs. In silico prediction of transcription factor binding sites for intron 1 was performed with MatInspector 2.2 software. Results PNMT was characterized by minimum variation and excess of rare SNPs in both normo- and hypertensive individuals. None of the SNPs showed significant differences in allelic frequencies among population samples, as well as between screened hypertensives and normotensives. In the joint case-control analysis of the Estonian and the Czech samples, hypertension patients had a significant excess of heterozygotes for two promoter region polymorphisms (SNP-184; SNP-390. The identified variation pattern of PNMT reflects the effect of purifying selection consistent with an important role of PNMT-synthesized epinephrine in the regulation of cardiovascular and metabolic functions, and as a CNS neurotransmitter. A striking feature is the lack of intronic variation. In silico analysis of PNMT intron 1 confirmed the presence of a human-specific putative Glucocorticoid Responsive Element (GRE, inserted by Alu-mediated transfer. Further analysis of intron 1 supported the possible existence of a full Glucocorticoid Responsive Unit (GRU predicted to consist of multiple gene regulatory elements known to cooperate with GRE in driving transcription. The role of these elements in regulating PNMT expression patterns and thus determining the dynamics of the

  19. 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.

  20. Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses

    Science.gov (United States)

    Da Lage, Jean-Luc; Maczkowiak, Frédérique; Cariou, Marie-Louise

    2011-01-01

    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. PMID:21611157

  1. Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses

    OpenAIRE

    Da Lage, Jean-Luc; Maczkowiak, Frédérique; Cariou, Marie-Louise

    2011-01-01

    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, al...

  2. Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Senejani Alireza G

    2009-12-01

    Full Text Available Abstract Background Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life. Results To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites. Conclusions These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult

  3. Diversity, mobility, and structural and functional evolution of group II introns carrying an unusual 3' extension

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    Tourasse Nicolas J

    2011-12-01

    Full Text Available Abstract Background Group II introns are widespread genetic elements endowed with a dual functionality. They are catalytic RNAs (ribozymes that are able of self-splicing and they are also mobile retroelements that can invade genomic DNA. The group II intron RNA secondary structure is typically made up of six domains. However, a number of unusual group II introns carrying a unique extension of 53-56 nucleotides at the 3' end have been identified previously in bacteria of the Bacillus cereus group. Methods In the present study, we conducted combined sequence comparisons and phylogenetic analyses of introns, host gene, plasmid and chromosome of host strains in order to gain insights into mobility, dispersal, and evolution of the unusual introns and their extension. We also performed in vitro mutational and kinetic experiments to investigate possible functional features related to the extension. Results We report the identification of novel copies of group II introns carrying a 3' extension including the first two copies in bacteria not belonging to the B. cereus group, Bacillus pseudofirmus OF4 and Bacillus sp. 2_A_57_CT2, an uncharacterized species phylogenetically close to B. firmus. Interestingly, the B. pseudofirmus intron has a longer extension of 70 bases. From sequence comparisons and phylogenetic analyses, several possible separate events of mobility involving the atypical introns could be identified, including both retrohoming and retrotransposition events. In addition, identical extensions were found in introns that otherwise exhibit little sequence conservation in the rest of their structures, with the exception of the conserved and catalytically critical domains V and VI, suggesting either separate acquisition of the extra segment by different group II introns or a strong selection pressure acting on the extension. Furthermore, we show by in vitro splicing experiments that the 3' extension affects the splicing properties differently in

  4. Antagonistic factors control the unproductive splicing of SC35 terminal intron.

    Science.gov (United States)

    Dreumont, Natacha; Hardy, Sara; Behm-Ansmant, Isabelle; Kister, Liliane; Branlant, Christiane; Stévenin, James; Bourgeois, Cyril F

    2010-03-01

    Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3' untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3' splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.

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

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    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.

  6. 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.

  7. Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating.

    Science.gov (United States)

    Lian, Tiantian; Yang, Tao; Sun, Junde; Guo, Suping; Yang, Huaijun; Dong, Caihong

    2014-08-01

    Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns.

  8. 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.

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

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    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. Comparison of mitochondrial genomes provides insights into intron dynamics and evolution in the caterpillar fungus Cordyceps militaris.

    Science.gov (United States)

    Zhang, Yongjie; Zhang, Shu; Zhang, Guozhen; Liu, Xingzhong; Wang, Chengshu; Xu, Jianping

    2015-04-01

    Intra-specific comparison of mitochondrial genomes can help elucidate the evolution of a species, however it has not been performed for hypocrealean fungi that form diverse symbiotic associations with other organisms. In this study, comparative analyses of three completely sequenced mitochondrial genomes of a hypocrealean fungus, Cordyceps militaris, the type species of Cordyceps genus, revealed that the introns were the main contributors to mitochondrial genome size variations among strains. Mitochondrial genes in C. militaris have been invaded by group I introns in at least eight positions. PCR assays of various C. militaris isolates showed abundant variations of intron presence/absence among strains at seven of the eight intronic loci. Although the ancestral intron pattern was inferred to contain all eight introns, loss and/or gain events occurred for seven of the eight introns. These introns invaded the C. militaris mitochondrial genome probably by horizontal transfer from other fungi, and intron insertions into intronless genes in C. militaris were accompanied by co-conversions of upstream exon sequences especially for those introns targeting protein-coding genes. We also detected phylogenetic congruence between the intron and exon trees at each individual locus, consistent with the ancestral mitochondria of C. militaris as having all eight introns. This study helps to explain the evolution of C. militaris mitochondrial genomes and will facilitate population genetic studies of this medicinally important fungus. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    Science.gov (United States)

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  12. Origin and evolution of chloroplast group I introns in lichen algae.

    Science.gov (United States)

    Del Hoyo, Alicia; Álvarez, Raquel; Gasulla, Francisco; Casano, Leonardo Mario; Del Campo, Eva María

    2018-02-01

    The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free-living Trebouxiophyceae compared to free-living non-Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non-Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free-living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free-living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms. © 2017 Phycological Society of America.

  13. Did group II intron proliferation in an endosymbiont-bearing archaeon create eukaryotes?

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    Poole Anthony M

    2006-12-01

    Full Text Available Abstract Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the

  14. The Role of Liposomal CpG ODN on the Course of L. major Infection in BALB/C Mice

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    H Hejazi

    2010-02-01

    Full Text Available "nBackground: Historically, leishmanization is the most effective protective measure against Cutaneous Leishmaniasis (CL, CL lesion induced by leishmanization sometimes takes a long time to heal. Ma­nipulation of leishmanization inoculums needed to induce a mild and acceptable CL lesion. The aim of this study was to explore if liposomal form of CpG ODN (Cytosin phosphate Guanin Oligodeoxynu­cleotides mixed with Leishmania major   would induce a milder lesion size in Balb/c mice."nMethods: This study was performed in Biotechnology Research Center, Mashhad, and Center for Re­search and Training in Skin Diseases and Leprosy, Tehran, Iran during 2008-2009.  mice were subcutaneously (SC inoculated with L. major mixed with liposomal form of CpG ODN, or L. major plus free CpG ODN, or L. major mixed with empty liposomes or L. major in PBS. The lesion onset and the size of lesion were recorded; the death rate was also monitored. "nResult: Footpad thickness was significantly (P<0.01 smaller, death rate was also significantly (P<0.05 lower in the mice received L. major mixed with liposomal CpG ODN or free CpG ODN than control groups received L. major in PBS or L. major plus liposomes, also mice which received L. ma­jor mixed with CpG ODN in soluble form showed a significantly (P < 0.001 smaller lesion size than control groups."nConclusion: CpG ODN seems to be an appropriate immunopotentiator mixed with Leishmania stabi­late in leishmanization.

  15. Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin.

    Science.gov (United States)

    Bina, Minou; Wyss, Phillip; Novorolsky, Elise; Zulkelfi, Noorfatin; Xue, Jing; Price, Randi; Fay, Matthew; Gutmann, Zach; Fogler, Brian; Wang, Daidong

    2013-12-28

    Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven "bookmarked" regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the memory of highly active genes

  16. In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron

    DEFF Research Database (Denmark)

    Vader, A; Nielsen, Henrik; Johansen, S

    1999-01-01

    as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron......The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF......) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well...

  17. 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.

  18. Anxiety Associated Increased CpG Methylation in the Promoter of Asb1: A Translational Approach Evidenced by Epidemiological and Clinical Studies and a Murine Model.

    Science.gov (United States)

    Emeny, Rebecca T; Baumert, Jens; Zannas, Anthony S; Kunze, Sonja; Wahl, Simone; Iurato, Stella; Arloth, Janine; Erhardt, Angelika; Balsevich, Georgia; Schmidt, Mathias V; Weber, Peter; Kretschmer, Anja; Pfeiffer, Liliane; Kruse, Johannes; Strauch, Konstantin; Roden, Michael; Herder, Christian; Koenig, Wolfgang; Gieger, Christian; Waldenberger, Melanie; Peters, Annette; Binder, Elisabeth B; Ladwig, Karl-Heinz

    2018-01-01

    Epigenetic regulation in anxiety is suggested, but evidence from large studies is needed. We conducted an epigenome-wide association study (EWAS) on anxiety in a population-based cohort and validated our finding in a clinical cohort as well as a murine model. In the KORA cohort, participants (n=1522, age 32-72 years) were administered the Generalized Anxiety Disorder (GAD-7) instrument, whole blood DNA methylation was measured (Illumina 450K BeadChip), and circulating levels of hs-CRP and IL-18 were assessed in the association between anxiety and methylation. DNA methylation was measured using the same instrument in a study of patients with anxiety disorders recruited at the Max Planck Institute of Psychiatry (MPIP, 131 non-medicated cases and 169 controls). To expand our mechanistic understanding, these findings were reverse translated in a mouse model of acute social defeat stress. In the KORA study, participants were classified according to mild, moderate, or severe levels of anxiety (29.4%/6.0%/1.5%, respectively). Severe anxiety was associated with 48.5% increased methylation at a single CpG site (cg12701571) located in the promoter of the gene encoding Asb1 (β-coefficient=0.56 standard error (SE)=0.10, p (Bonferroni)=0.005), a protein hypothetically involved in regulation of cytokine signaling. An interaction between IL-18 and severe anxiety with methylation of this CpG cite showed a tendency towards significance in the total population (p=0.083) and a significant interaction among women (p=0.014). Methylation of the same CpG was positively associated with Panic and Agoraphobia scale (PAS) scores (β=0.005, SE=0.002, p=0.021, n=131) among cases in the MPIP study. In a murine model of acute social defeat stress, Asb1 gene expression was significantly upregulated in a tissue-specific manner (p=0.006), which correlated with upregulation of the neuroimmunomodulating cytokine interleukin 1 beta. Our findings suggest epigenetic regulation of the stress

  19. Analysis of CpG SNPs in 34 genes: association test with suicide attempt in schizophrenia.

    Science.gov (United States)

    Bani-Fatemi, Ali; Gonçalves, Vanessa F; Zai, Clement; de Souza, Renan; Le Foll, Bernard; Kennedy, James L; Wong, Albert H; De Luca, Vincenzo

    2013-07-01

    Suicide is the act of intentionally causing one's own death. The lifetime suicide risk in schizophrenia is 4.9% and 20% to 50% of patients with SCZ will attempt suicide during their life. The other risk factors for suicidal behavior in schizophrenia include prior history of suicide attempts, active psychosis, depression and substance abuse. To date, there are no robust genetic or epigenetic predictors of suicide or suicide attempt in this specific population. We collected detailed clinical information and DNA samples from 241 schizophrenia patients and performed the genetic analyses in suicide attempters and non-attempters, among these patients. Using the structured research interview, we determined the presence of suicide attempt lifetime and then we tested 384 DNA variants in candidate genes supposed to be involved in the neurobiology of schizophrenia. We applied a novel mapping analysis using a specific bioinformatic tool that analyzed only the polymorphic CpG sites in our SNP panel. This analysis looked at the presence or absence of methylation sites affected by the SNP allele. The SNPs in the candidate genes were studied under a different perspective considering their direct contribution to the availability of methylation sites within the gene of interest. The level of potential methylation was compared using a linear model in attempters and non-attempters. Among the 384 SNPs selected from the Illumina Bead Chip only the rs2661319 in the RGS4 gene was significantly associated with suicide attempt (p = 0.002). There were 119 CpG SNPs in the aforementioned panel. The gene-wise potential methylation level of RGS4 was 55% in the attempters and 65% in the non-attempters with a p-value of 0.005. The total level of potential metylation in the overall panel (119 SNPs combined) was not associated with suicide attempt. However, when considering the potential methylation at chromosome 1, we found that suicide attempt (p = 0.036) was associated with lower methylation. The

  20. Glutamatergic mechanisms for speed control and network operation in the rodent locomotor CPG

    DEFF Research Database (Denmark)

    Talpalar, Adolfo E.; Kiehn, Ole

    2010-01-01

    in mammals have produced conflicting results regarding the necessity and role of the different ionotropic glutamate receptors (GluRs) in the CPG function. Here, we use electrophysiological and pharmacological techniques in the in vitro neonatal mouse lumbar spinal cord to investigate the role of a broad...... mechanisms acting at various network levels. AMPA and kainate receptors are necessary for generating the highest locomotor frequencies. For coordination, NMDARs are more important than non-NMDARs for conveying the rhythmic signal from the network to the motor neurons during long-lasting and steady locomotor...

  1. Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection

    Science.gov (United States)

    Frazier, Courtney L.; San Filippo, Joseph; Lambowitz, Alan M.; Mills, David A.

    2003-01-01

    Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. PMID:12571038

  2. Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium

    Directory of Open Access Journals (Sweden)

    Marie-Mathilde Perrineau

    2015-06-01

    Full Text Available Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

  3. DNA-methyltransferase1 (DNMT1) binding to CpG rich GABAergic and BDNF promoters is increased in the brain of schizophrenia and bipolar disorder patients.

    Science.gov (United States)

    Dong, E; Ruzicka, W B; Grayson, D R; Guidotti, A

    2015-09-01

    The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity. Copyright © 2014. Published by Elsevier B.V.

  4. CpG preconditioning regulates miRNA expression that modulates genomic reprogramming associated with neuroprotection against ischemic injury

    Science.gov (United States)

    Vartanian, Keri B; Mitchell, Hugh D; Stevens, Susan L; Conrad, Valerie K; McDermott, Jason E; Stenzel-Poore, Mary P

    2015-01-01

    Cytosine-phosphate-guanine (CpG) preconditioning reprograms the genomic response to stroke to protect the brain against ischemic injury. The mechanisms underlying genomic reprogramming are incompletely understood. MicroRNAs (miRNAs) regulate gene expression; however, their role in modulating gene responses produced by CpG preconditioning is unknown. We evaluated brain miRNA expression in response to CpG preconditioning before and after stroke using microarray. Importantly, we have data from previous gene microarrays under the same conditions, which allowed integration of miRNA and gene expression data to specifically identify regulated miRNA gene targets. CpG preconditioning did not significantly alter miRNA expression before stroke, indicating that miRNA regulation is not critical for the initiation of preconditioning-induced neuroprotection. However, after stroke, differentially regulated miRNAs between CpG- and saline-treated animals associated with the upregulation of several neuroprotective genes, implicating these miRNAs in genomic reprogramming that increases neuroprotection. Statistical analysis revealed that the miRNA targets were enriched in the gene population regulated in the setting of stroke, implying that miRNAs likely orchestrate this gene expression. These data suggest that miRNAs regulate endogenous responses to stroke and that manipulation of these miRNAs may have the potential to acutely activate novel neuroprotective processes that reduce damage. PMID:25388675

  5. CpG adjuvant enhances the mucosal immunogenicity and efficacy of a Treponema pallidum DNA vaccine in rabbits.

    Science.gov (United States)

    Zhao, Feijun; Liu, Shuangquan; Zhang, Xiaohong; Yu, Jian; Zeng, Tiebing; Gu, Weiming; Cao, Xunyu; Chen, Xi; Wu, Yimou

    2013-04-01

    The protective response against Treponema pallidum (Tp) infection of a DNA vaccine enhanced by an adjuvant CpG ODN was investigated. The mucosal adjuvant CpG ODN enhanced the production of higher levels of anti-TpGpd antibodies induced by pcD/Gpd-IL-2 in rabbits. It also resulted in higher levels of secretion of IL-2 and IFN-γ, and facilitated T cell proliferation and differentiation (p0.05). Furthermore, CpG ODN stimulated the production of mucosa-specific anti-sIgA antibodies and resulted in the lowest Tp-positive rate (6.7%) for Tp-infection of skin lesions and the lowest rates (8.3%) of ulceration lesions, thus achieving better protective effects. New Zealand rabbits were immunized with the eukaryotic vector encoding recombinant pcD/Gpd-IL-2 using intramuscular multi-injection or together with mucosal enhancement via a nasal route. The effect of the mucosal adjuvant CpG ODN was examined. The CpG ODN adjuvant significantly enhances the humoral and cellular immune effects of the immunization by pcD/Gpd-IL-2 with mucosal enhancement via nasal route. It also stimulates strong mucosal immune effects, thus initiating more efficient immune-protective effects.

  6. Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation.

    Science.gov (United States)

    Kamalakaran, Sitharthan; Kendall, Jude; Zhao, Xiaoyue; Tang, Chunlao; Khan, Sohail; Ravi, Kandasamy; Auletta, Theresa; Riggs, Michael; Wang, Yun; Helland, Aslaug; Naume, Bjørn; Dimitrova, Nevenka; Børresen-Dale, Anne-Lise; Hicks, Jim; Lucito, Robert

    2009-07-01

    Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25,000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.

  7. Towards autonomous locomotion: CPG-based control of smooth 3D slithering gait transition of a snake-like robot.

    Science.gov (United States)

    Bing, Zhenshan; Cheng, Long; Chen, Guang; Röhrbein, Florian; Huang, Kai; Knoll, Alois

    2017-04-04

    Snake-like robots with 3D locomotion ability have significant advantages of adaptive travelling in diverse complex terrain over traditional legged or wheeled mobile robots. Despite numerous developed gaits, these snake-like robots suffer from unsmooth gait transitions by changing the locomotion speed, direction, and body shape, which would potentially cause undesired movement and abnormal torque. Hence, there exists a knowledge gap for snake-like robots to achieve autonomous locomotion. To address this problem, this paper presents the smooth slithering gait transition control based on a lightweight central pattern generator (CPG) model for snake-like robots. First, based on the convergence behavior of the gradient system, a lightweight CPG model with fast computing time was designed and compared with other widely adopted CPG models. Then, by reshaping the body into a more stable geometry, the slithering gait was modified, and studied based on the proposed CPG model, including the gait transition of locomotion speed, moving direction, and body shape. In contrast to sinusoid-based method, extensive simulations and prototype experiments finally demonstrated that smooth slithering gait transition can be effectively achieved using the proposed CPG-based control method without generating undesired locomotion and abnormal torque.

  8. Comparative analysis of information contents relevant to recognition of introns in many species

    Directory of Open Access Journals (Sweden)

    Gotoh Osamu

    2011-01-01

    Full Text Available Abstract Background The basic process of RNA splicing is conserved among eukaryotic species. Three signals (5' and 3' splice sites and branch site are commonly used to directly conduct splicing, while other features are also related to the recognition of an intron. Although there is experimental evidence pointing to the significant species specificities in the features of intron recognition, a quantitative evaluation of the divergence of these features among a wide variety of eukaryotes has yet to be conducted. Results To better understand the splicing process from the viewpoints of evolution and information theory, we collected introns from 61 diverse species of eukaryotes and analyzed the properties of the nucleotide sequences relevant to splicing. We found that trees individually constructed from the five features (the three signals, intron length, and nucleotide composition within an intron roughly reflect the phylogenetic relationships among the species but sometimes extensively deviate from the species classification. The degree of topological deviation of each feature tree from the reference trees indicates the lowest discordance for the 5' splicing signal, followed by that for the 3' splicing signal, and a considerably greater discordance for the other three features. We also estimated the relative contributions of the five features to short intron recognition in each species. Again, moderate correlation was observed between the similarities in pattern of short intron recognition and the genealogical relationships among the species. When mammalian introns were categorized into three subtypes according to their terminal dinucleotide sequences, each subtype segregated into a nearly monophyletic group, regardless of the host species, with respect to the 5' and 3' splicing signals. It was also found that GC-AG introns are extraordinarily abundant in some species with high genomic G + C contents, and that the U12-type spliceosome might make a

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

    Directory of Open Access Journals (Sweden)

    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

  10. 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)....

  11. Unusual group II introns in bacteria of the Bacillus cereus group.

    Science.gov (United States)

    Tourasse, Nicolas J; Stabell, Fredrik B; Reiter, Lillian; Kolstø, Anne-Brit

    2005-08-01

    A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the alpha subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

  12. Clinical significance of intronic variants in BRAF inhibitor resistant melanomas with altered BRAF transcript splicing

    OpenAIRE

    Pupo, Gulietta M.; Boyd, Suzanah C.; Fung, Carina; Carlino, Matteo S.; Menzies, Alexander M.; Pedersen, Bernadette; Johansson, Peter; Hayward, Nicholas K.; Kefford, Richard F.; Scolyer, Richard A.; Long, Georgina V.; Rizos, Helen

    2017-01-01

    Alternate BRAF splicing is the most common mechanism of acquired resistance to BRAF inhibitor treatment in melanoma. Recently, alternate BRAF exon 4?8 splicing was shown to involve an intronic mutation, located 51 nucleotides upstream of BRAF exon 9 within a predicted splicing branch point. This intronic mutation was identified in a single cell line but has not been examined in vivo. Herein we demonstrate that in three melanomas biopsied from patients with acquired resistance to BRAF inhibito...

  13. Strand-specific CpG hemimethylation, a novel epigenetic modification functional for genomic imprinting.

    Science.gov (United States)

    Patiño-Parrado, Iris; Gómez-Jiménez, Álvaro; López-Sánchez, Noelia; Frade, José M

    2017-09-06

    Imprinted genes are regulated by allele-specific differentially DNA-methylated regions (DMRs). Epigenetic methylation of the CpGs constituting these DMRs is established in the germline, resulting in a 5-methylcytosine-specific pattern that is tightly maintained in somatic tissues. Here, we show a novel epigenetic mark, characterized by strand-specific hemimethylation of contiguous CpG sites affecting the germline DMR of the murine Peg3, but not Snrpn, imprinted domain. This modification is enriched in tetraploid cortical neurons, a cell type where evidence for a small proportion of formylmethylated CpG sites within the Peg3-controlling DMR is also provided. Single nucleotide polymorphism (SNP)-based transcriptional analysis indicated that these epigenetic modifications participate in the maintainance of the monoallelic expression pattern of the Peg3 imprinted gene. Our results unexpectedly demonstrate that the methylation pattern observed in DMRs controlling defined imprinting regions can be modified in somatic cells, resulting in a novel epigenetic modification that gives rise to strand-specific hemimethylated domains functional for genomic imprinting. We anticipate the existence of a novel molecular mechanism regulating the transition from fully methylated CpGs to strand-specific hemimethylated CpGs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Frequency Modulation and Spatiotemporal Stability of the sCPG in Preterm Infants with RDS

    Directory of Open Access Journals (Sweden)

    Steven M. Barlow

    2012-01-01

    Full Text Available The nonnutritive suck (NNS is an observable and accessible motor behavior which is often used to make inference about brain development and pre-feeding skill in preterm and term infants. The purpose of this study was to model NNS burst compression pressure dynamics in the frequency and time domain among two groups of preterm infants, including those with respiratory distress syndrome (RDS, N=15 and 17 healthy controls. Digitized samples of NNS compression pressure waveforms recorded at a 1-week interval were collected 15 minutes prior to a scheduled feed. Regression analysis and ANOVA revealed that healthy preterm infants produced longer NNS bursts and the mean burst initiation cycle frequencies were higher when compared to the RDS group. Moreover, the initial 5 cycles of the NNS burst manifest a frequency modulated (FM segment which is a significant feature of the suck central pattern generator (sCPG, and differentially expressed in healthy and RDS infants. The NNS burst structure revealed significantly lower spatiotemporal index values for control versus RDS preterm infants during FM, and provides additional information on the microstructure of the sCPG which may be used to gauge the developmental status and progression of oromotor control systems among these fragile infants.

  15. FPGA implementation of a configurable neuromorphic CPG-based locomotion controller.

    Science.gov (United States)

    Barron-Zambrano, Jose Hugo; Torres-Huitzil, Cesar

    2013-09-01

    Neuromorphic engineering is a discipline devoted to the design and development of computational hardware that mimics the characteristics and capabilities of neuro-biological systems. In recent years, neuromorphic hardware systems have been implemented using a hybrid approach incorporating digital hardware so as to provide flexibility and scalability at the cost of power efficiency and some biological realism. This paper proposes an FPGA-based neuromorphic-like embedded system on a chip to generate locomotion patterns of periodic rhythmic movements inspired by Central Pattern Generators (CPGs). The proposed implementation follows a top-down approach where modularity and hierarchy are two desirable features. The locomotion controller is based on CPG models to produce rhythmic locomotion patterns or gaits for legged robots such as quadrupeds and hexapods. The architecture is configurable and scalable for robots with either different morphologies or different degrees of freedom (DOFs). Experiments performed on a real robot are presented and discussed. The obtained results demonstrate that the CPG-based controller provides the necessary flexibility to generate different rhythmic patterns at run-time suitable for adaptable locomotion. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

    Science.gov (United States)

    Myers, Christopher A; Kuhla, Birte; Cusack, Stephen; Lambowitz, Alan M

    2002-03-05

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

  17. 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.

  18. 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.

  19. Group II introns break new boundaries: presence in a bilaterian's genome.

    Directory of Open Access Journals (Sweden)

    Yvonne Vallès

    Full Text Available Group II introns are ribozymes, removing themselves from their primary transcripts, as well as mobile genetic elements, transposing via an RNA intermediate, and are thought to be the ancestors of spliceosomal introns. Although common in bacteria and most eukaryotic organelles, they have never been reported in any bilaterian animal genome, organellar or nuclear. Here we report the first group II intron found in the mitochondrial genome of a bilaterian worm. This location is especially surprising, since animal mitochondrial genomes are generally distinct from those of plants, fungi, and protists by being small and compact, and so are viewed as being highly streamlined, perhaps as a result of strong selective pressures for fast replication while establishing germ plasm during early development. This intron is found in the mtDNA of an annelid worm, (an undescribed species of Nephtys, where the complete sequence revealed a 1819 bp group II intron inside the cox1 gene. We infer that this intron is the result of a recent horizontal gene transfer event from a viral or bacterial vector into the mitochondrial genome of Nephtys sp. Our findings hold implications for understanding mechanisms, constraints, and selective pressures that account for patterns of animal mitochondrial genome evolution.

  20. Protecting exons from deleterious R-loops: a potential advantage of having introns

    Directory of Open Access Journals (Sweden)

    Niu Deng-Ke

    2007-04-01

    Full Text Available Abstract Background Accumulating evidence indicates that the nascent RNA can invade and pair with one strand of DNA, forming an R-loop structure that threatens the stability of the genome. In addition, the cost and benefit of introns are still in debate. Results At least three factors are likely required for the R-loop formation: 1 sequence complementarity between the nascent RNA and the target DNA, 2 spatial juxtaposition between the nascent RNA and the template DNA, and 3 accessibility of the template DNA and the nascent RNA. The removal of introns from pre-mRNA reduces the complementarity between RNA and the template DNA and avoids the spatial juxtaposition between the nascent RNA and the template DNA. In addition, the secondary structures of group I and group II introns may act as spatial obstacles for the formation of R-loops between nearby exons and the genomic DNA. Conclusion Organisms may benefit from introns by avoiding deleterious R-loops. The potential contribution of this benefit in driving intron evolution is discussed. I propose that additional RNA polymerases may inhibit R-loop formation between preceding nascent RNA and the template DNA. This idea leads to a testable prediction: intermittently transcribed genes and genes with frequently prolonged transcription should have higher intron density. Reviewers This article was reviewed by Dr. Eugene V. Koonin, Dr. Alexei Fedorov (nominated by Dr. Laura F Landweber, and Dr. Scott W. Roy (nominated by Dr. Arcady Mushegian.

  1. Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice.

    Science.gov (United States)

    Yoshida, Hiroyuki; Nishikawa, Makiya; Kiyota, Tsuyoshi; Toyota, Hiroyasu; Takakura, Yoshinobu

    2011-07-15

    Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Prophylactic application of CpG oligonucleotides augments the early host response and confers protection in acute melioidosis.

    Directory of Open Access Journals (Sweden)

    Barbara M Judy

    Full Text Available Prophylactic administration of CpG oligodeoxynucleotides (CpG ODNs is known to confer protection against lethal sepsis caused by Burkholderia pseudomallei in the mouse model. The mechanisms whereby CpG regulates the innate immune response to provide protection against B. pseudomallei, however, are poorly characterized. In the present study, we demonstrate that intranasal treatment of mice with Class C CpG, results in recruitment of inflammatory monocytes and neutrophils to the lung at 48 h post-treatment. Mice infected with B. pseudomallei 48 h post-CpG treatment had reduced organ bacterial load and significantly altered cytokine and chemokine profiles concomitant with protection as compared to control animals. CpG administration reduced the robust production of chemokines and pro-inflammatory cytokines in blood, lung and spleen, observed following infection of non-treated animals. Death of control animals coincided with the time of peak cytokine production (day 1-3, while a moderate; sustained cytokine production in CpG-treated animals was associated with survival. In general, CpG treatment resulted in diminished expression of cytokines and chemokines post-infection, though IL-12p40 was released in larger quantities in CpG treated animals. In contrast to CpG-treated animals, the lungs of infected control animals were infiltrated with leukocytes, especially neutrophils, and large numbers of necrotic lesions were observed in lung sections. Therapeutic treatment of B. pseudomallei-infected animals with CpG at 24 h post-infection did not impact survival compared to control animals. In summary, protection of CpG-treated animals was associated with recruitment of inflammatory monocytes and neutrophils into the lungs prior to infection. These responses correspond with early control of bacterial growth, a dampened inflammatory cytokine/chemokine response, reduced lung pathology, and greatly increased survival. In contrast, a delay in recruitment of

  3. Protection of Balb/c mice against infection with FMDV by immunostimulation with CpG oligonucleotides

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Frimann, Tine; Barfoed, Annette Malene

    2006-01-01

    Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are potent stimulators of the innate immune system, and are capable of aborting several infections in a non-specific manner. We here report studies of the capacity of such ODN to protect mice against infection with foot and mouth...... serotypes, when compared to no treatment or treatment with a control ODN. The effect was observed when ODN was administered simultaneously with, or up to 12 h after, infection with FMDV, and lasted for 14 days post treatment. The potential application of CpG ODN for control of FMDV during an outbreak...

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

    Directory of Open Access Journals (Sweden)

    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.

  5. The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns.

    Directory of Open Access Journals (Sweden)

    Cyril Férandon

    Full Text Available In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a "Homing Endonuclease Gene" (heg encoding a DNA endonuclease acting in transfer and site-specific integration ("homing" and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.

  6. Self-splicing of a group IIC intron: 5? exon recognition and alternative 5? splicing events implicate the stem?loop motif of a transcriptional terminator

    OpenAIRE

    Toor, Navtej; Robart, Aaron R.; Christianson, Joshua; Zimmerly, Steven

    2006-01-01

    Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interesting...

  7. Dual activity of phosphorothioate CpG oligodeoxynucleotides on HIV: reactivation of latent provirus and inhibition of productive infection in human T cells.

    Science.gov (United States)

    Scheller, Carsten; Ullrich, Anett; Lamla, Stefan; Dittmer, Ulf; Rethwilm, Axel; Koutsilieri, Eleni

    2006-12-01

    CpG oligodeoxynucleotides (CpG ODNs) bind to toll-like receptor-9 (TLR-9) and activate immune cells with antigen-presenting activity, including B cells and dendritic cells. Here we show that treatment of the latently human immunodeficiency virus (HIV)-infected T cell line ACH-2 with the CpG ODNs 2006 or 2040 triggers activation of viral gene expression, demonstrating that CpG-signaling activity can also be found in T cells. The CpG ODNs g12AAC and g12GTC had no effect on virus reactivation. In contrast to the stimulating effects on viral gene expression in latently infected cells, CpG ODNs potently suppressed HIV replication in productively infected MT4 T cells or PBLs. Inhibition of virus replication was not related to the CpG motif but similarly occurred with non-CpG phosphorothioate (PTO)-ODNs. Thus, virus inhibition was likely caused by the PTO backbone of the CpG ODNs, probably by interfering with events prior to integration of the viral cDNA into the host genome. The ability of CpG PTO-ODNs to trigger reactivation of latent HIV in combination with their antiviral activity on productive infection makes this substance class an interesting candidate for further test to asses their potential as supplements in HIV therapy.

  8. GRK5 intronic (CAn polymorphisms associated with type 2 diabetes in Chinese Hainan Island.

    Directory of Open Access Journals (Sweden)

    Zhenfang Xia

    Full Text Available A genome-wide association study had showed G-protein-coupled receptor kinase 5 (GRK5 rs10886471 was related to the risk of type 2 diabetes mellitus (T2DM through upregulated GRK5 mRNA expression. Rs10886471 is located in the intron region of GRK5. However, the mechanism by which intronic SNP affects gene expression remains unclear, whether the effect on gene expression depends on the intronic short tandem repeat (STR (CAn splicing regulator or not. Here we investigated the STR (CAn polymorphism in rs10886471 and further discussed its role in the T2DM risk of Chinese Hainan Island individuals. A total of 1164 subjects were recruited and classified into a normal fasting glucose (NFG group, an impaired fasting glucose (IFG group, an impaired glucose tolerance (IGT group, and a T2DM group. STR (CAn polymorphisms were detected through polymerase chain reaction and sequencing. Five intronic (CAn alleles, (CA15 to (CA19, were identified in GRK5 rs10886471. Only the (CA16 allele was significantly associated with increased prediabetes and T2DM risk [odds ratio (OR>1, P<0.05]. Conversely, multiple alleles without any (CA16 protected against prediabetes and T2DM (0intronic SNP causes GRK5 overexpression the subsequent risk of T2DM may be due to the rs10886471 intronic STR (CAn splicing enhancer. Further studies should focus on verifying these finding using a large sample size and analyzing the splicing mechanism of intronic (CAn in rs10886471.

  9. Multiple recent horizontal transfers of the cox1 intron in Solanaceae and extended co-conversion of flanking exons.

    Science.gov (United States)

    Sanchez-Puerta, Maria V; Abbona, Cinthia C; Zhuo, Shi; Tepe, Eric J; Bohs, Lynn; Olmstead, Richard G; Palmer, Jeffrey D

    2011-09-27

    The most frequent case of horizontal transfer in plants involves a group I intron in the mitochondrial gene cox1, which has been acquired via some 80 separate plant-to-plant transfer events among 833 diverse angiosperms examined. This homing intron encodes an endonuclease thought to promote the intron's promiscuous behavior. A promising experimental approach to study endonuclease activity and intron transmission involves somatic cell hybridization, which in plants leads to mitochondrial fusion and genome recombination. However, the cox1 intron has not yet been found in the ideal group for plant somatic genetics - the Solanaceae. We therefore undertook an extensive survey of this family to find members with the intron and to learn more about the evolutionary history of this exceptionally mobile genetic element. Although 409 of the 426 species of Solanaceae examined lack the cox1 intron, it is uniformly present in three phylogenetically disjunct clades. Despite strong overall incongruence of cox1 intron phylogeny with angiosperm phylogeny, two of these clades possess nearly identical intron sequences and are monophyletic in intron phylogeny. These two clades, and possibly the third also, contain a co-conversion tract (CCT) downstream of the intron that is extended relative to all previously recognized CCTs in angiosperm cox1. Re-examination of all published cox1 genes uncovered additional cases of extended co-conversion and identified a rare case of putative intron loss, accompanied by full retention of the CCT. We infer that the cox1 intron was separately and recently acquired by at least three different lineages of Solanaceae. The striking identity of the intron and CCT from two of these lineages suggests that one of these three intron captures may have occurred by a within-family transfer event. This is consistent with previous evidence that horizontal transfer in plants is biased towards phylogenetically local events. The discovery of extended co

  10. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with {sup 99m}Tc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    Energy Technology Data Exchange (ETDEWEB)

    Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J. [Cancer Research Targeting and Imaging Group, Royal Free Campus of Royal Free and University College Medical School, NW3 2PF, London (United Kingdom); Mather, S.J. [Cancer Research Dept. Nuclear Medicine, St Bartholomew' s Hospital, EC1A 7BE, London (United Kingdom); Waibel, R. [Paul Scherrer Institute, Center for Radiopharmaceutical Science, Villigen PSI (Switzerland)

    2004-08-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was {sup 99m}Tc-carbonyl [{sup 99m}Tc(H{sub 2}O){sub 3}(CO){sub 3}]{sup +} (abbreviated to TcCO) mediated labelling of {sup 99m}Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  11. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    International Nuclear Information System (INIS)

    Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J.; Mather, S.J.; Waibel, R.

    2004-01-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99m Tc-carbonyl [ 99m Tc(H 2 O) 3 (CO) 3 ] + (abbreviated to TcCO) mediated labelling of 99m Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  12. Long-range autocorrelations of CpG islands in the human genome.

    Directory of Open Access Journals (Sweden)

    Benjamin Koester

    Full Text Available In this paper, we use a statistical estimator developed in astrophysics to study the distribution and organization of features of the human genome. Using the human reference sequence we quantify the global distribution of CpG islands (CGI in each chromosome and demonstrate that the organization of the CGI across a chromosome is non-random, exhibits surprisingly long range correlations (10 Mb and varies significantly among chromosomes. These correlations of CGI summarize functional properties of the genome that are not captured when considering variation in any particular separate (and local feature. The demonstration of the proposed methods to quantify the organization of CGI in the human genome forms the basis of future studies. The most illuminating of these will assess the potential impact on phenotypic variation of inter-individual variation in the organization of the functional features of the genome within and among chromosomes, and among individuals for particular chromosomes.

  13. Gene Silencing Triggers Polycomb Repressive Complex 2 Recruitment to CpG Islands Genome Wide

    DEFF Research Database (Denmark)

    Riising, Eva Madi; Vacher-Comet, Itys; Leblanc, Benjamin Olivier

    2014-01-01

    Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show that knoc......Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show......-wide ectopic PRC2 recruitment to endogenous PcG target genes found in other tissues. PRC2 binding analysis shows that it is restricted to nucleosome-free CpG islands (CGIs) of untranscribed genes. Our results show that it is the transcriptional state that governs PRC2 binding, and we propose that it binds...

  14. CpG Type A Induction of an Early Protective Environment in Experimental Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    James Crooks

    2017-01-01

    Full Text Available Experimental autoimmune encephalomyelitis (EAE is an inflammatory, demyelinating disease of the CNS that mimics human multiple sclerosis (MS, and it is thought to be driven by Th1 and Th17 myelin-reactive cells. Although adaptive immunity is clearly pivotal in the pathogenesis of EAE, with an essential role of CD4+ T cells, little is known of early, innate responses in this experimental setting. CpG-rich oligodeoxynucleotides (ODNs, typically found in microbial genomes, are potent activators of TLR9 in plasmacytoid dendritic cells (pDCs. In this study, we compared the effects of two types of CpG, namely, type A and type B, on EAE. We found that treatment with CpG type A ODN (CpG-A, known to induce high amounts of IFN-α in pDCs, significantly reduced disease severity in EAE, relative to controls (12.63±1.86 versus 23.49±1.46, resp.; p=0.001. Treatment also delayed onset of neurological deficits and reduced spinal cord demyelination, while increasing the percentage of splenic regulatory (Foxp3+ CD4+ T cells. CpG-A likewise reduced the levels of IL-17 and IFN-γ in the CNS. Mechanistic insight into those events showed that CpG-A promoted a regulatory phenotype in pDCs. Moreover, adoptive transfer of pDCs isolated from CpG-A-treated mice inhibited CNS inflammation and induced disease remission in acute-phase EAE. Our data thus identify a link between TLR9 activation by specific ligands and the induction of tolerance via innate immunity mechanisms.

  15. CpG site DNA methylation patterns reveal a novel regulatory element in the mouse prion protein gene

    Science.gov (United States)

    DALAI, Wuyun; MATSUO, Eiko; TAKEYAMA, Natsumi; KAWANO, Junichi; SAEKI, Keiichi

    2016-01-01

    The cellular isoform of the prion protein (PrPC) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prnp) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at −218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at −599 to −238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at −576 was negatively correlated with the PrP mRNA level (Pearson’s r = −0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner. PMID:27666463

  16. Inhibitory effects of unmethylated CpG oligodeoxynucleotides on MHC class I-deficient and -proficient HPV16-associated tumours

    Czech Academy of Sciences Publication Activity Database

    Reiniš, Milan; Šímová, Jana; Bubeník, Jan

    2006-01-01

    Roč. 118, č. 7 (2006), s. 1836-1842 ISSN 0020-7136 R&D Projects: GA ČR(CZ) GA301/04/0492 Institutional research plan: CEZ:AV0Z50520514 Keywords : HPV16 * immunotherapy * CpG oligodeoxynucleotides Subject RIV: EC - Immunology Impact factor: 4.693, year: 2006

  17. Methylation of CpG sites in RNF180 DNA promoter prediction poor survival of gastric cancer.

    Science.gov (United States)

    Deng, Jingyu; Liang, Han; Ying, Guoguang; Zhang, Rupeng; Wang, Baogui; Yu, Jun; Fan, Daiming; Hao, Xishan

    2014-05-30

    RNF 180, a novel tumor suppressor, has been implicated in the carcinogenesis and progress of gastric cancer. At present study, we found that lower expression of RNA180 was specific in gastric cancer tissues, and the inconsistently methylated levels of RNF180 promoter were identified in the gastric cancer tissues. Importantly, we demonstrated that the methylated CpG site count and four hypermethylated CpG sites (-116, -80, +97, and +102) were significantly associated with the survival of 400 gastric cancer patients, respectively. With multivariate survival analyses, we demonstrated that both the methylation of combined CpG (-116, -80, +97, and +102) sites and N stage were the independent indictor of prognosis of gastric cancer patients. Eventually, the methylation of combined CpG (-116, -80, +97, and +102) sites was identified to have smaller AIC value than N stage by mean of AIC calculation with the Cox proportional hazards model. These findings indicated that the quantitative detection of RNF180 promoter methylation had the intensive feasibility for evaluation the prognosis of gastric cancer patients in clinic.

  18. Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

    Energy Technology Data Exchange (ETDEWEB)

    Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

  19. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

    DEFF Research Database (Denmark)

    Tariq, M.; Saze, H.; Probst, A.

    2003-01-01

    In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from...

  20. Protection of Balb/c mice against infection with FMDV by immunostimulation with CpG oligonucleotides

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Frimann, Tine; Barfoed, Annette Malene

    2006-01-01

    Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are potent stimulators of the innate immune system, and are capable of aborting several infections in a non-specific manner. We here report studies of the capacity of such ODN to protect mice against infection with foot and mouth dise...

  1. Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort

    OpenAIRE

    Landthaler, Markus; Begley, Ulrike; Lau, Nelson C.; Shub, David A.

    2002-01-01

    We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome. Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE). Reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from Staphylococcus a...

  2. Short-term sequence evolution and vertical inheritance of the Naegleria twin-ribozyme group I intron

    Directory of Open Access Journals (Sweden)

    De Jonckheere Johan F

    2006-05-01

    Full Text Available Abstract Background Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516 has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2 and a homing endonuclease gene (NaHEG. NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. Results To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, contains hallmarks of a degenerated NaHEG. Phylogenetic analyses performed on separate data sets representing NaGIR1, NaGIR2, NaHEG, and ITS1-5.8S-ITS2 ribosomal DNA are consistent with an overall vertical inheritance pattern of the intron within the Naegleria genus. Conclusion The Nae.S516 twin-ribozyme intron was gained early in the Naegleria evolution with subsequent vertical inheritance. The intron was lost in the majority of isolates (70%, leaving a widespread but scattered distribution pattern. Why the apparent asexual Naegleria amoebae harbors active intron homing endonucleases, dependent on sexual reproduction for its function, remains a puzzle.

  3. Evolution of small putative group I introns in the SSU rRNA gene locus of Phialophora species

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    Harris Lorena B

    2011-07-01

    Full Text Available Abstract Background Group I introns (specifically subgroup IC1 are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt in length, several small putative (or degenerate group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the PaSSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme. Findings Here, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola. Conclusions The small putative group I introns in Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses.

  4. Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus).

    Science.gov (United States)

    Shao, Chaopeng; Xiong, Wen; Wang, Wei

    2004-01-01

    The DVa(Hus) was previously investigated through cDNA analysis, which revealed an RHD-CE(5)-D hybrid allele. However, the 5' and 3' breakpoints remain unknown. In this article, gene recombinations between the RHD and RHCE alleles were investigated by a combination approach of a sequence-specific primer PCR (PCR-SSP) and an RHD full-length coding region sequencing method on two Chinese subjects with weak D phenotypes. The hybrid Rhesus box of each individual was also investigated through an established PCR-based method. As a result, two partial D phenotypes, DVa(Hus) and DVI type III, were identified, each carrying one hybrid RHD-CE-D allele. The two samples were also serotyped with Rh phontypes of DccEe and DCcee, respectively. Other sequencing analyses of the DVaHus sample showed that the sequence of intron 4 is identical with RHD, whereas the whole sequence of exon 5 and intron 5 is identical with RHCE except for seven polymorphisms in the intron 5. We may concluded that in the case of this Chinese DVa(Hus), the whole exon 5 and complete intron 5 of a total segment of 1801 nucleotides were replaced by RHCE suggesting that the breakpoints of the replaced region are the 5' end of the exon 5 and the 3' end of the intron 5.

  5. Introns regulate gene expression in Cryptococcus neoformans in a Pab2p dependent pathway.

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    Carolin Goebels

    Full Text Available Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A polymerase, respectively, has no effect on intronless allele expression.

  6. Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway

    Science.gov (United States)

    Goebels, Carolin; Thonn, Aline; Gonzalez-Hilarion, Sara; Rolland, Olga; Moyrand, Frederique; Beilharz, Traude H.; Janbon, Guilhem

    2013-01-01

    Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression. PMID:23966870

  7. PCR primers for an aldolase-B intron in acanthopterygian fishes

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    Jones William J

    2001-11-01

    Full Text Available Abstract Background Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. Results Aldolase intron 'G' was amplified from four fish species using previously described primer sets that target several loci indiscriminately. Phylogenetic analyses were used to group these fragments and other full-length aldolase proteins from teleost fishes into orthologous clades and a primer set was designed to target specifically an intron within the aldolase-B locus in acanthopterygian fishes. DNA amplifications were tried in a variety of acanthopterygian fishes and amplification products, identifiable as aldolase-B intron 'G', were observed in all atherinomorph and percomorph taxa examined. Sequence variation within this locus was found within and among several species examined. Conclusions Using 'universal' primer sets coupled with phylogenetic analyses it was possible to develop a genetic assay to target a specific locus in a variety of fish taxa. Sequence variation was observed within and among species suggesting that this targeted assay might facilitate interspecific and intraspecific comparisons.

  8. Long-term evolution of the S788 fungal nuclear small subunit rRNA group I introns

    OpenAIRE

    HAUGEN, PEIK; RUNGE, HENRY JOSEPH; BHATTACHARYA, DEBASHISH

    2004-01-01

    More than 1000 group I introns have been identified in fungal rDNA. Little is known, however, of the splicing and secondary structure evolution of these ribozymes. Here, we use a combination of comparative and biochemical methods to address the evolution and splicing of a vertically inherited group I intron found at position 788 in the fungal small subunit (S) rRNA. The ancestral state of the S788 intron contains a highly conserved core and an extended P5 domain typical of IC1 introns. In con...

  9. Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

    Science.gov (United States)

    Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

    2017-03-09

    It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

  10. tRNALeu intron (UAA) of Ficus carica L.: genetic diversity and evolutionary patterns.

    Science.gov (United States)

    Baraket, G; Abdelkrim, A B; Salhi-Hannachi, A

    2015-04-22

    Cytoplasmic chloroplast DNA was explored to establish genetic relationships among Ficus carica cultivars and elucidate the molecular evolution of the species. The results suggest the occurrence of haplotype and nucleotide diversity. Conserved group I intron sequence motifs were detected and showed a common secondary structure, despite the presence of some mutations on their sequences. The neighbor-joining dendrogram showed a continuous diversity that characterizes local resources. The maximum parsimony tree, with an RI index of 0.507, indicated minimal homoplasy within the data set. Furthermore, our results demonstrate that the trnL intron is the seat of numerous substitutions. Herein, new insight on the mechanism involved in the evolution of the trnL intron in the fig is presented. From the study, it appears that there is an explicit rejection of the null hypothesis in F. carica. A scenario of positive selection and recent expansion of F. carica genotypes across Tunisia seems to be retained.

  11. Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani.

    Science.gov (United States)

    McNeil, Bonnie A; Zimmerly, Steven

    2014-06-01

    Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5' splice site located 8 nt upstream of the usual 5' GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1-EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5' splice site is shown to be affected by structures in addition to IBS1-EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3' exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression. © 2014 McNeil and Zimmerly; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Effective suppression of dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain.

    Science.gov (United States)

    Carter, James R; Keith, James H; Fraser, Tresa S; Dawson, James L; Kucharski, Cheryl A; Horne, Kate M; Higgs, Stephen; Fraser, Malcolm J

    2014-06-13

    Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS

  13. Regulation of Retention of FosB Intron 4 by PTB

    OpenAIRE

    Marinescu, Victor; Loomis, Patricia A.; Ehmann, Svetlana; Beales, Mitchell; Potashkin, Judith A.

    2007-01-01

    One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed DeltaFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3' end of intron 4 (I4...

  14. Evidence for transitional stages in the evolution of euglenid group II introns and twintrons in the Monomorphina aenigmatica plastid genome.

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    Jean-François Pombert

    Full Text Available Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome (74,746 bp, 70.6% A+T, 87 genes contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate.

  15. An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

    Science.gov (United States)

    Sirand-Pugnet, P; Durosay, P; Brody, E; Marie, J

    1995-09-11

    Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.

  16. CpG DNA facilitate the inactivated transmissible gastroenteritis virus in enhancing the local and systemic immune response of pigs via oral administration.

    Science.gov (United States)

    Lin, Jian; Tu, Chongzhi; Mou, Chunxiao; Chen, Xiaojuan; Yang, Qian

    2016-04-01

    Transmissible gastroenteritis virus (TGEV) replicates in the small intestine and induces enteritis and watery diarrhea. Establishment of local immunity in the intestine would thus prevent TGEV transmission. CpG DNA has been reported as a promising mucosal adjuvant in some animals. The effects of oral immunization of CpG DNA together with inactivated TGEV (ITGEV) were investigated in this study. Pigs (6 weeks old) were orally immunized with ITGEV plus CpG DNA. The TGEV-specific IgA level in the intestinal tract and the TGEV-specific IgG level in serum significantly increased following immunization with ITGEV plus CpG DNA (P ≤ 0.05). Moreover, populations of IgA-secreting cells, CD3+ T lymphocytes and intraepithelial lymphocytes (IELs), in the intestine increased significantly after immunization with ITGEV plus CpG DNA (P ≤ 0.05). Furthermore, the expression of IL-6, IL-12 and interferon-γ (IFN-γ) in ligated intestine segments increased significantly after injection with ITGEV plus CpG DNA (P ≤ 0.05). Taken together, these data suggest that oral immunization of ITGEV plus CpG DNA elicits a local immune response. Further studies are required to determine whether this immunity provides protection against TGEV in pigs. Copyright © 2016. Published by Elsevier B.V.

  17. 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

  18. Methylation of the BIN1 gene promoter CpG island associated with breast and prostate cancer

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    Khomyakova Anastasiya

    2007-01-01

    Full Text Available Abstract Background Loss of BIN1 tumor suppressor expression is abundant in human cancer and its frequency exceeds that of genetic alterations, suggesting the role of epigenetic regulators (DNA methylation. BIN1 re-expression in the DU145 prostate cancer cell line after 5-aza-2'-deoxycytidine treatment was recently reported but no methylation of the BIN1 promoter CpG island was found in DU145. Methods Methylation-sensitive arbitrarily-primed PCR was used to detect genomic loci abnormally methylated in breast cancer. BIN1 CpG island fragment was identified among the differentially methylated loci as a result of direct sequencing of the methylation-sensitive arbitrarily-primed PCR product and subsequent BLAST alliance. BIN1 CpG island cancer related methylation in breast and prostate cancers was confirmed by bisulphite sequencing and its methylation frequency was evaluated by methylation sensitive PCR. Loss of heterozygosity analysis of the BIN1 region was performed with two introgenic and one closely adjacent extragenic microsatellite markers.BIN1 expression was evaluated by real-time RT-PCR. Results We have identified a 3'-part of BIN1 promoter CpG island among the genomic loci abnormally methylated in breast cancer. The fragment proved to be methylated in 18/99 (18% and 4/46 (9% breast and prostate tumors, correspondingly, as well as in MCF7 and T47D breast cancer cell lines, but was never methylated in normal tissues and lymphocytes as well as in DU145 and LNCaP prostate cancer cell lines. The 5'-part of the CpG island revealed no methylation in all samples tested. BIN1 expression losses were detected in MCF7 and T47D cells and were characteristic of primary breast tumors (10/13; 77%, while loss of heterozygosity was a rare event in tissue samples (2/22 informative cases; 9% and was ruled out for MCF7. Conclusion BIN1 promoter CpG island is composed of two parts differing drastically in the methylation patterns in cancer. This appears to be a

  19. 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.

  20. Comprehensive phylogenetic analysis of bacterial group II intron-encoded ORFs lacking the DNA endonuclease domain reveals new varieties.

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    Nicolás Toro

    Full Text Available Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP ORF within the ribozyme domain IV (DIV. This ORF encodes an N-terminal reverse transcriptase (RT domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D region followed by a DNA endonuclease (En domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns. On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

  1. Design and Experimental Evolution of trans-Splicing Group I Intron Ribozymes

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    Ulrich F. Müller

    2017-01-01

    Full Text Available Group I intron ribozymes occur naturally as cis-splicing ribozymes, in the form of introns that do not require the spliceosome for their removal. Instead, they catalyze two consecutive trans-phosphorylation reactions to remove themselves from a primary transcript, and join the two flanking exons. Designed, trans-splicing variants of these ribozymes replace the 3′-portion of a substrate with the ribozyme’s 3′-exon, replace the 5′-portion with the ribozyme’s 5′-exon, or insert/remove an internal sequence of the substrate. Two of these designs have been evolved experimentally in cells, leading to variants of group I intron ribozymes that splice more efficiently, recruit a cellular protein to modify the substrate’s gene expression, or elucidate evolutionary pathways of ribozymes in cells. Some of the artificial, trans-splicing ribozymes are promising as tools in therapy, and as model systems for RNA evolution in cells. This review provides an overview of the different types of trans-splicing group I intron ribozymes that have been generated, and the experimental evolution systems that have been used to improve them.

  2. 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.

  3. A method for construction, cloning and expression of intron-less gene from unannotated genomic DNA.

    Science.gov (United States)

    Agrawal, Vineet; Gupta, Bharti; Banerjee, Uttam Chand; Roy, Nilanjan

    2008-11-01

    The present century has witnessed an unprecedented rise in genome sequences owing to various genome-sequencing programs. However, the same has not been replicated with cDNA or expressed sequence tags (ESTs). Hence, prediction of protein coding sequence of genes from this enormous collection of genomic sequences presents a significant challenge. While robust high throughput methods of cloning and expression could be used to meet protein requirements, lack of intron information creates a bottleneck. Computational programs designed for recognizing intron-exon boundaries for a particular organism or group of organisms have their own limitations. Keeping this in view, we describe here a method for construction of intron-less gene from genomic DNA in the absence of cDNA/EST information and organism-specific gene prediction program. The method outlined is a sequential application of bioinformatics to predict correct intron-exon boundaries and splicing by overlap extension PCR for spliced gene synthesis. The gene construct so obtained can then be cloned for protein expression. The method is simple and can be used for any eukaryotic gene expression.

  4. Allelic polymorphism in introns 1 and 2 of the HLA-DQA1 gene.

    Science.gov (United States)

    Voorter, C E M; de Groot, N G; Meertens, C M H; Bontrop, R E; van den Berg-Loonen, E M

    2005-01-01

    Human leukocyte antigen (HLA) class II antigens are highly polymorphic membrane glycoproteins, encoded by the A and B genes of DR, DQ, and DP. The polymorphism is mainly located in exon 2, with the exception of DQA1. Of the 27 DQA1 alleles presently known, 18 cannot be identified on the basis of exon 2 alone, but need additional information from the other exons. DQA1 has been reported to be the most ancient class II gene. For evolutionary comparison and to assess the degree of polymorphism outside the exons, the sequences of introns 1 and 2 were determined from 30 different cell lines, encompassing 15 different DQA1 alleles. The sequences revealed major nucleotide differences between the different lineages, whereas within each lineage few differences were present. Phylogenetic analysis of intron and exon sequences confirmed this lineage specificity. Altogether, the present data indicate that the HLA-DQA1 lineages represent ancient entities. The observed variation of the introns in alleles with identical exon sequences implicates conservative selection of the exons within a given lineage. Intron sequences may provide the means to set up an accurate typing system.

  5. 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...

  6. When a mid-intronic variation of DMD gene creates an ESE site.

    Science.gov (United States)

    Trabelsi, Madiha; Beugnet, Caroline; Deburgrave, Nathalie; Commere, Virgine; Orhant, Lucie; Leturcq, France; Chelly, Jamel

    2014-12-01

    Duchenne and Becker muscular dystrophy are X-linked allelic disorders caused by mutations in the DMD gene. The majority (65%) of these mutations are intragenic deletions/duplications that often lead to frameshift errors. Among the remaining ones, we find the mid-intronic mutations that usually create cryptic exons by activating potential splice sites. In this report, we identified, in a Becker muscular dystrophy patient, a mid-intronic variation that creates two ESE sites in intron 26 of DMD gene resulting in the insertion of a new cryptic exon in mRNA. Despite the out of frame character of this mutation, we observed the production of a reduced amount of full-size dystrophin which could be explained by the alternation between normal and altered splicing of dystrophin mRNA in this patient. To our knowledge, this is the first case report describing this novel pathogenic mechanism of mid-intronic variations of DMD gene. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. 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...

  8. Genomewide analysis of intronic microRNAs in rice and Arabidopsis

    Indian Academy of Sciences (India)

    mented with 3% sucrose and stratified at 4. ◦. C for two days in the dark prior to germination. .... RT-PCR results and the deep sequencing analysis together strongly suggest that most of the identified intronic miRNAs ...... Published on the Web: 13 December 2012. 324. Journal of Genetics, Vol. 91, No. 3, December 2012.

  9. An Orchestrated Intron Retention Program in Meiosis Controls Timely Usage of Transcripts during Germ Cell Differentiation.

    Science.gov (United States)

    Naro, Chiara; Jolly, Ariane; Di Persio, Sara; Bielli, Pamela; Setterblad, Niclas; Alberdi, Antonio J; Vicini, Elena; Geremia, Raffaele; De la Grange, Pierre; Sette, Claudio

    2017-04-10

    Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Correlating CpG islands, motifs, and sequence variants in human chromosome 21

    Directory of Open Access Journals (Sweden)

    Cercone Nick

    2011-07-01

    Full Text Available Abstract Background CpG islands are important regions in DNA. They usually appear at the 5’ end of genes containing GC-rich dinucleotides. When DNA methylation occurs, gene regulation is affected and it sometimes leads to carcinogenesis. We propose a new detection program using a hidden-markov model alongside the Viterbi algorithm. Methods Our solution provides a graphical user interface not seen in many of the other CGI detection programs and we unify the detection and analysis under one program to allow researchers to scan a genetic sequence, detect the significant CGIs, and analyze the sequence once the scan is complete for any noteworthy findings. Results Using human chromosome 21, we show that our algorithm finds a significant number of CGIs. Running an analysis on a dataset of promoters discovered that the characteristics of methylated and unmethylated CGIs are significantly different. Finally, we detected significantly different motifs between methylated and unmethylated CGI promoters using MEME and MAST. Conclusions Developing this new tool for the community using powerful algorithms has shown that combining analysis with CGI detection will improve the continued research within the field of epigenetics.

  11. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm†

    Science.gov (United States)

    Navarro-Costa, Paulo; Nogueira, Paulo; Carvalho, Marta; Leal, Fernanda; Cordeiro, Isabel; Calhaz-Jorge, Carlos; Gonçalves, João; Plancha, Carlos E.

    2010-01-01

    BACKGROUND Successful gametogenesis requires the establishment of an appropriate epigenetic state in developing germ cells. Nevertheless, an association between abnormal spermatogenesis and epigenetic disturbances in germline-specific genes remains to be demonstrated. METHODS In this study, the DNA methylation pattern of the promoter CpG island (CGI) of two germline regulator genes—DAZL and DAZ, was characterized by bisulphite genomic sequencing in quality-fractioned ejaculated sperm populations from normozoospermic (NZ) and oligoasthenoteratozoospermic (OAT) men. RESULTS OAT patients display increased methylation defects in the DAZL promoter CGI when compared with NZ controls. Such differences are recorded when analyzing sperm fractions enriched either in normal or defective germ cells (P< 0.001 in both cases). Significant differences in DNA methylation profiles are also observable when comparing the qualitatively distinct germ cell fractions inside the NZ and OAT groups (P= 0.003 and P= 0.007, respectively). Contrastingly, the unmethylation pattern of the DAZ promoter CGI remains correctly established in all experimental groups. CONCLUSIONS An association between disrupted DNA methylation of a key spermatogenesis gene and abnormal human sperm is described here for the first time. These results suggest that incorrect epigenetic marks in germline genes may be correlated with male gametogenic defects. PMID:20685756

  12. Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms.

    Science.gov (United States)

    Löhne, Cornelia; Borsch, Thomas

    2005-02-01

    Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences

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

    Directory of Open Access Journals (Sweden)

    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

  14. In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases.

    Directory of Open Access Journals (Sweden)

    Julia Arand

    2012-06-01

    Full Text Available The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites. The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM to calculate the relative contribution of DNA methyltransferases (Dnmts for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.

  15. Genome-wide CpG island methylation analysis implicates novel genes in the pathogenesis of renal cell carcinoma

    OpenAIRE

    Ricketts, Christopher J.; Morris, Mark R.; Gentle, Dean; Brown, Michael; Wake, Naomi; Woodward, Emma R.; Clarke, Noel; Latif, Farida; Maher, Eamonn R.

    2012-01-01

    In order to identify novel candidate tumor suppressor genes (TSGs) implicated in renal cell carcinoma (RCC), we performed genome-wide methylation profiling of RCC using the HumanMethylation27 BeadChips to assess methylation at >14,000 genes. Two hundred and twenty hypermethylated probes representing 205 loci/genes were identified in genomic CpG islands. A subset of TSGs investigated in detail exhibited frequent tumor methylation, promoter methylation associated transcriptional silencing an...

  16. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal

    2010-07-21

    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  17. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal

    2010-01-01

    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  18. Applicability of the methylated CpG sites of paired box 5 (PAX5) promoter for prediction the prognosis of gastric cancer.

    Science.gov (United States)

    Deng, Jingyu; Liang, Han; Zhang, Rupeng; Dong, Qiuping; Hou, Yachao; Yu, Jun; Fan, Daiming; Hao, Xishan

    2014-09-15

    Paired box gene 5 (PAX5), a member of the paired box gene family, is involved in control of organ development and tissue differentiation. In previous study, PAX5 promoter methylation was found in gastric cancer (GC) cells and tissues. At present study, we found that the inconsistently methylated levels of PAX5 promoter were identified in the different GC tissues. The methylated CpG site count and the methylated statuses of four CpG sites (-236, -183, -162, and -152) were significantly associated with the survival of 460 GC patients, respectively. Ultimately, the methylated CpG -236 was the optimal prognostic predictor of patients identified by using the Cox regression with AIC value calculation. These findings indicated that the methylated CpG -236 of PAX5 promoter has the potential applicability for clinical evaluation the prognosis of GC.

  19. PRIMEGENS-v2: genome-wide primer design for analyzing DNA methylation patterns of CpG islands.

    Science.gov (United States)

    Srivastava, Gyan P; Guo, Juyuan; Shi, Huidong; Xu, Dong

    2008-09-01

    DNA methylation plays important roles in biological processes and human diseases, especially cancers. High-throughput bisulfite genomic sequencing based on new generation of sequencers, such as the 454-sequencing system provides an efficient method for analyzing DNA methylation patterns. The successful implementation of this approach depends on the use of primer design software capable of performing genome-wide scan for optimal primers from in silico bisulfite-treated genome sequences. We have developed a method, which fulfills this requirement and conduct primer design for sequences including regions of given promoter CpG islands. The developed method has been implemented using the C and JAVA programming languages. The primer design results were tested in the PCR experiments of 96 selected human DNA sequences containing CpG islands in the promoter regions. The results indicate that this method is efficient and reliable for designing sequence-specific primers. The sequence-specific primer design for DNA meth-ylated sequences including CpG islands has been integrated into the second version of PRIMEGENS as one of the primer design features. The software is freely available for academic use at http://digbio.missouri.edu/primegens/.

  20. CpG ODN and ISCOMATRIX Adjuvant: A Synergistic Adjuvant Combination Inducing Strong T-Cell IFN-γ Responses

    Directory of Open Access Journals (Sweden)

    Michael J. McCluskie

    2013-01-01

    Full Text Available For the induction of robust humoral and cellular immune responses, a strong rationale exists to use vaccine-adjuvant combinations possessing both immune modulatory and enhanced delivery capabilities. Herein, we evaluated the combination of 2 different adjuvants, a TLR9 agonist, composed of synthetic oligodeoxynucleotides (ODN containing immunostimulatory CpG motifs (CpG, and ISCOMATRIX adjuvant (ISCOMATRIX, composed of saponin, phospholipid, and cholesterol, which possesses both immunostimulatory and delivery properties. While both individual adjuvants have been shown effective in numerous preclinical and clinical studies, it is likely that for optimal adjuvant activity a combined adjuvant approach will be necessary. Herein, using three different antigens, namely, hepatitis B surface antigen (HBsAg, ovalbumin (OVA, and influenza A haemagglutinin antigen (HA, we show in mice that some adjuvant effects of CpG and ISCOMATRIX are further enhanced if they are used in combination. In particular, with all three antigens, IFN-γ levels were greatly increased with the CpG/ISCOMATRIX combination. The ability of the CpG/ISCOMATRIX combination to induce antitumor responses when administered with OVA following administration to mice of a highly metastatic OVA-secreting tumor cell line (B16-OVA melanoma was also demonstrated. Thus the CpG/ISCOMATRIX combination may prove to be a valuable tool in the development of novel or improved vaccines.

  1. A Panel of CpG Methylation Sites Distinguishes Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Kevin Huang

    2014-01-01

    Full Text Available Whether human induced pluripotent stem cells (hiPSCs are epigenetically identical to human embryonic stem cells (hESCs has been debated in the stem cell field. In this study, we analyzed DNA methylation patterns in a large number of hiPSCs (n = 114 and hESCs (n = 155, and identified a panel of 82 CpG methylation sites that can distinguish hiPSCs from hESCs with high accuracy. We show that 12 out of the 82 CpG sites were subject to hypermethylation in part by DNMT3B. Notably, DNMT3B contributes directly to aberrant hypermethylation and silencing of the signature gene, TCERG1L. Overall, we conclude that DNMT3B is involved in a wave of de novo methylation during reprogramming, a portion of which contributes to the unique hiPSC methylation signature. These 82 CpG methylation sites may be useful as biomarkers to distinguish between hiPSCs and hESCs.

  2. Dengue-1 envelope protein domain III along with PELC and CpG oligodeoxynucleotides synergistically enhances immune responses.

    Directory of Open Access Journals (Sweden)

    Chen-Yi Chiang

    Full Text Available The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.

  3. Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis.

    Science.gov (United States)

    Heravi Shargh, Vahid; Jaafari, Mahmoud Reza; Khamesipour, Ali; Jalali, Seyed Amir; Firouzmand, Hengameh; Abbasi, Azam; Badiee, Ali

    2012-07-01

    Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.

  4. A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray

    Directory of Open Access Journals (Sweden)

    Qiao Yingjuan

    2008-01-01

    Full Text Available Abstract Background DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. Results We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray, provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. Conclusion This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

  5. CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

    Science.gov (United States)

    Sepulveda, Antonia R.; Jones, Dan; Ogino, Shuji; Samowitz, Wade; Gulley, Margaret L.; Edwards, Robin; Levenson, Victor; Pratt, Victoria M.; Yang, Bin; Nafa, Khedoudja; Yan, Liying; Vitazka, Patrick

    2009-01-01

    Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics. PMID:19541921

  6. CpG island clusters and pro-epigenetic selection for CpGs in protein-coding exons of HOX and other transcription factors

    OpenAIRE

    Branciamore, Sergio; Chen, Zhao-Xia; Riggs, Arthur D.; Rodin, Sergei N.

    2010-01-01

    CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and ∼5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synony...

  7. CpG island clusters and pro-epigenetic selection for CpGs in protein-coding exons of HOX and other transcription factors.

    Science.gov (United States)

    Branciamore, Sergio; Chen, Zhao-Xia; Riggs, Arthur D; Rodin, Sergei N

    2010-08-31

    CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and approximately 5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synonymous codons with the potential for CpG. That is, we studied "silent" changes that do not affect protein products but could damage epigenetic marking. We find that DNA-binding transcription factors and other developmentally relevant genes show, only in methylated genomes, a bimodal distribution of CpG usage. Several genetic code-based tests indicate, again for methylated genomes only, that the frequency of silent CpGs in Hox genes is much greater than expectation. Also informative are NCG-GNN and NCC-GNN codon doublets, for which an unusually high rate of G to C and C to G transversions was observed at the third (silent) position of the first codon. Together these results are interpreted as evidence for strong "pro-epigenetic" selection acting to preserve CpG sites in coding regions of many genes controlling development. We also report that DNA-binding transcription factors and developmentally important genes are dramatically overrepresented in or near clusters of three or more CpG islands, suggesting a possible relationship between evolutionary preservation of CpG dinucleotides in both coding regions and CpG islands.

  8. Phase 1 trial of AMA1-C1/Alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria.

    Directory of Open Access Journals (Sweden)

    Gregory E D Mullen

    2008-08-01

    Full Text Available Apical Membrane Antigen 1 (AMA1, a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909.A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15, 80 microg of AMA1-C1/Alhydrogel (n = 30, or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30.Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG were detected by enzyme-linked immunosorbent assay (ELISA, and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition.The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing.ClinicalTrials.gov NCT00344539.

  9. 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.

  10. The rates of G:C[yields]T:A and G:C[yields]C:G transversions at CpG dinucleotides in the human factor IX gene

    Energy Technology Data Exchange (ETDEWEB)

    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))

    1994-05-01

    The authors have identified eight independent transversions at CpG in 290 consecutive families with hemophilia B. These eight transversions account for 16.3% of all independent transversions in the sample, yet the expected frequency of CpG transversions at random in the factor IX gene is only 2.6% (P<0.1). The aggregate data suggest that the two types of CpG transversions (G:C[yields]T:A and G:C[yields]C:G) possess similar mutation rates (24.8 [times] 10[sup [minus]10] and 20.6 [times] 10[sup [minus]10], respectively), which are about fivefold greater than the comparable rates for transversions at non-CpG dinucleotides. The enhancement of transversions at CpG suggest that the model by which mutations occur at CpG may need to be reevaluated. The relationship, if any, between deamination of 5-methyl cytosine and enhancement of transversions at CpG remains to be defined. 28 refs., 2 tabs.

  11. [Interaction between polycyclic aromatic hydrocarbons andp16,FHITgene CpG island methylation in patients with cervical intraepithelial neoplasias].

    Science.gov (United States)

    Wang, L; Liu, X Z; Ren, Z Y; Ding, L; Nan, J; Liu, C L; Song, Z C; Feng, M J; Yang, Q; Wang, J T

    2017-08-10

    Objective: To explore the effect of polycyclic aromatic hydrocarbons (PAHs) and p16, FHIT gene CpG island methylation, as well as their interaction in cervical intraepithelial neoplasias. Methods: Objects of this study were from a cohort of cervical lesions study in Yangqu county of Shanxi province. All the patients were diagnosed pathologically, that including 83 patients with high-grade cervical intraepithelial neoplasia (CINⅡ/Ⅲ), 86 patients with low-grade cervical intraepithelial neoplasia (CINⅠ) and another 91 women under normal cervical (NC) condition. 1-hydroxy pyrene in the urine was detected by high performance liquid chromatography (HPLC) while CpG island methylation status of tumor suppressor gene p16 and FHIT were measured by methylation-specifc polymerase chain reaction (MSP). Data were analyzed with Kruskal-Wallis H test, chi-square test and trend of chi-square test. Logistic regression models were used to estimate the odds ratio ( OR ) and corresponding 95% confidence intervals (95% CI ) between influencing factors and the cervical disease by using the SPSS statistical software (version 20.0). The interaction under study was evaluated by using the generalized multifactor dimensionality reduction (GMDR) model. Results: Level of 1-hydroxy pyrene ( H =50.743, P neoplasia. The CpG island methylation rates of p16, FHIT in CINⅠand CINⅡ/Ⅲ group were higher than that in NC group, and gradually increasing along with the severity of cervical intraepithelial neoplasia (trend χ (2)=9.75, P =0.002; trend χ (2)=10.39, P =0.001). Results from the GMDR model showed that interaction existed among the high exposure of 1-hydroxy pyrene and the CpG island methylation of p16, FHIT in CINⅠ and CINⅡ/Ⅲ group. Conclusion: Under the high exposure of 1-hydroxy pyrene and the CpG island methylation of p16, FHIT appeared to have increased the risk of cervical intraepithelial neoplasia and causing synergistic effect in cervical intraepithelial neoplasia.

  12. 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

  13. The role of the CpG island methylator phenotype on survival outcome in colon cancer.

    Science.gov (United States)

    Kang, Ki Joo; Min, Byung Hoon; Ryu, Kyung Ju; Kim, Kyoung Mee; Chang, Dong Kyung; Kim, Jae J; Rhee, Jong Chul; Kim, Young Ho

    2015-03-01

    CpG island methylator phenotype (CIMP)- high colorectal cancers (CRCs) have distinct clinicopathologi-cal features from their CIMP-low/negative CRC counterparts. However, controversy exists regarding the prognosis of CRC according to the CIMP status. Therefore, this study examined the prognosis of Korean patients with colon cancer according to the CIMP status. Among a previous cohort pop-ulation with CRC, a total of 154 patients with colon cancer who had available tissue for DNA extraction were included in the study. CIMP-high was defined as ≥3/5 methylated mark-ers using the five-marker panel (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1). CIMP-high and CIMP-low/neg-ative cancers were observed in 27 patients (17.5%) and 127 patients (82.5%), respectively. Multivariate analysis adjust-ing for age, gender, tumor location, tumor stage and CIMP and microsatellite instability (MSI) statuses indicated that CIMP-high colon cancers were associated with a significant increase in colon cancer-specific mortality (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.20 to 8.69; p=0.02). In microsatellite stable cancers, CIMP-high cancer had a poor survival outcome compared to CIMP-low/negative cancer (HR, 2.91; 95% CI, 1.02 to 8.27; p=0.04). Re-gardless of the MSI status, CIMP-high cancers had poor sur-vival outcomes in Korean patients. (Gut Liver, 2015;9202-207).

  14. Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex

    Science.gov (United States)

    Nyikó, Tünde; Kerényi, Farkas; Szabadkai, Levente; Benkovics, Anna H.; Major, Péter; Sonkoly, Boglárka; Mérai, Zsuzsanna; Barta, Endre; Niemiec, Emilia; Kufel, Joanna; Silhavy, Dániel

    2013-01-01

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3′untranslated region (UTR). In yeasts, unusually long 3′UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3′UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3′UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD. PMID:23666629

  15. 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

    The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors...... a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein...

  16. 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.

  17. Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus.

    Science.gov (United States)

    Yanagihara, Toyoshi; Sanematsu, Fumiyuki; Sato, Tetsuya; Uruno, Takehito; Duan, Xuefeng; Tomino, Takahiro; Harada, Yosuke; Watanabe, Mayuki; Wang, Yuqing; Tanaka, Yoshihiko; Nakanishi, Yoichi; Suyama, Mikita; Yoshinori, Fukui

    2015-11-04

    The thymus has spatially distinct microenvironments, the cortex and the medulla, where the developing T-cells are selected to mature or die through the interaction with thymic stromal cells. To establish the immunological self in the thymus, medullary thymic epithelial cells (mTECs) express diverse sets of tissue-specific self-antigens (TSAs). This ectopic expression of TSAs largely depends on the transcriptional regulator Aire, yet the mechanism controlling Aire expression itself remains unknown. Here, we show that Jmjd6, a dioxygenase that catalyses lysyl hydroxylation of splicing regulatory proteins, is critical for Aire expression. Although Jmjd6 deficiency does not affect abundance of Aire transcript, the intron 2 of Aire gene is not effectively spliced out in the absence of Jmjd6, resulting in marked reduction of mature Aire protein in mTECs and spontaneous development of multi-organ autoimmunity in mice. These results highlight the importance of intronic regulation in controlling Aire protein expression.

  18. 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.

  19. 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.

  20. Antagonistic factors control the unproductive splicing of SC35 terminal intron

    OpenAIRE

    Dreumont, Natacha; Hardy, Sara; Behm-Ansmant, Isabelle; Kister, Liliane; Branlant, Christiane; St?venin, James; Bourgeois, Cyril F.

    2009-01-01

    Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3? untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements respon...

  1. Intronic TP53 Germline Sequence Variants Modify the Risk in German Breast/Ovarian Cancer Families

    Directory of Open Access Journals (Sweden)

    Liu Xuan

    2004-07-01

    Full Text Available Abstract To establish the contribution of TP53 germline mutations to familial breast/ovarian cancer in Germany we screened the complete coding region of the TP53 gene in a series of German breast/ovarian cancer families negative for mutations in the BRCA1 and BRCA2 genes. Two different intronic TP53 sequence variants were identified in 6/48 (12.5% breast/ovarian cancer families. A novel A to T nucleotide change at position 17708 in intron 10 segregating with the disease was detected in three breast cancer families (6.2%. One 17708 A>T-associated breast tumour showed loss of the wild-type allele. This variant was also found in 5/112 (4.5% healthy controls indicating that it is a polymorphism. A second sequence variant changing a G to C at position 13964 in intron 6 not segregating with the disease was found in two breast cancer families and one breast-ovarian cancer family (6.2%. This variant has previously been shown to occur at an elevated frequency in hereditary breast cancer patients from North America and to be of functional importance leading to inhibition of apoptosis and prolongation of cell survival after DNA-damage. Screening of 185 consecutive unselected German breast cancer patients revealed the 13964 G>C variant in four patients (2.2%. Immunohistochemical analysis of the TP53 protein showed negative immunoreactivity in normal and tumour tissues of one 17708 A>T and six 13964 G>C carriers. TP53 overexpression was detected in the tumour tissue of one sporadic breast cancer patient carrying the 13964 G>C variant. Our results show that intronic changes of the TP53 gene may act as or be associated with risk modifiers in familial breast cancer.

  2. Intronic non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees.

    Science.gov (United States)

    Cingolani, Pablo; Cao, Xiaoyi; Khetani, Radhika S; Chen, Chieh-Chun; Coon, Melissa; Sammak, Alya'a; Bollig-Fischer, Aliccia; Land, Susan; Huang, Yun; Hudson, Matthew E; Garfinkel, Mark D; Zhong, Sheng; Robinson, Gene E; Ruden, Douglas M

    2013-09-30

    Previous whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing. We confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites. Cytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns.

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

    OpenAIRE

    Chalamcharla Venkata R; Edgell David R; Belfort Marlene

    2011-01-01

    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 bacter...

  4. 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

    The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5' end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors...... a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein...... 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...

  5. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

  6. 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.

  7. 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.

  8. Characterization of cryptic splicing in germline PTEN intronic variants in Cowden syndrome.

    Science.gov (United States)

    Chen, Hannah Jinlian; Romigh, Todd; Sesock, Kaitlin; Eng, Charis

    2017-10-01

    Germline mutations in the tumor-suppressor gene PTEN predispose to subsets of Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and autism. Evidence-based classification of PTEN variants as either deleterious or benign is urgently needed for accurate molecular diagnosis and gene-informed genetic counseling. We studied 34 different germline PTEN intronic variants from 61 CS patients, characterized their PTEN mRNA processing, and analyzed PTEN expression and downstream readouts of P-AKT and P-ERK1/2. While we found that many mutations near splice junctions result in exon skipping, we also identified the presence of cryptic splicing that resulted in premature termination or a shift in isoform usage. PTEN protein expression is significantly lower in the group with splicing changes while P-AKT, but not P-ERK1/2, is significantly increased. Our observations of these PTEN intronic variants should contribute to the determination of pathogenicity of PTEN intronic variants and aid in genetic counseling. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

  9. Three Group-I introns in 18S rDNA of Endosymbiotic Algae of Paramecium bursaria from Japan

    Science.gov (United States)

    Hoshina, Ryo; Kamako, Shin-ichiro; Imamura, Nobutaka

    2004-08-01

    In the nuclear encoded small subunit ribosomal DNA (18S rDNA) of symbiotic alga of Paramecium bursaria (F36 collected in Japan) possesses three intron-like insertions (Hoshina et al., unpubl. data, 2003). The present study confirmed these exact lengths and insertion sites by reverse transcription-PCR. Two of them were inserted at Escherichia coli 16S rRNA genic position 943 and 1512 that are frequent intron insertion positions, but another insertion position (nearly 1370) was the first finding. Their secondary structures suggested they belong to Group-I intron; one belongs to subgroup IE, others belong to subgroup IC1. Similarity search indicated these introns are ancestral ones.

  10. 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.

  11. Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3

    Directory of Open Access Journals (Sweden)

    Kandul Nikolai P

    2009-10-01

    Full Text Available Abstract Background Alternative splicing (AS of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs in the Drosophila RNA-binding Bruno-3 (Bru-3 gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described. Results Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates. Conclusion We found that large introns can promote AS via exon-skipping and exon turnover during

  12. High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing.

    Science.gov (United States)

    Sepulveda, Jorge L; Gutierrez-Pajares, Jorge L; Luna, Aesis; Yao, Yuan; Tobias, John W; Thomas, Steven; Woo, Yanghee; Giorgi, Federico; Komissarova, Elena V; Califano, Andrea; Wang, Timothy C; Sepulveda, Antonia R

    2016-02-01

    Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.

  13. Diet and lifestyle factor associations with CpG island methylator phenotype and BRAF mutations in colon cancer.

    Science.gov (United States)

    Slattery, Martha L; Curtin, Karen; Sweeney, Carol; Levin, Theodore R; Potter, John; Wolff, Roger K; Albertsen, Hans; Samowitz, Wade S

    2007-02-01

    It has been proposed that dietary factors such as folate, alcohol and methionine may be associated with colon cancer because of their involvement in DNA methylation processes. Data from a large population-based case-control study of incident colon cancer were used to evaluate whether intake of dietary, obesity, physical activity and nonsteroidal antiinflammatory drugs are associated with a CpG island methylator phenotype (CIMP). The BRAF V600E mutation and 5 CpG island markers (MINT1, MINT2, MINT31, p16 and hMLH1) were assessed in 1154 cases of colon cancer. We hypothesized that dietary factors involved in DNA methylation, cruciferous vegetables and use of aspirin/NSAIDs would be associated with CIMP-high tumors. Dietary folate, vitamins B(6) and B(12), methionine and alcohol were not associated with increased likelihood of colon tumors with the CIMP-high (2 or more markers methylated) phenotype. Dietary fiber, physical activity and aspirin and other nonsteroidal antiinflammatory drugs were inversely associated with both CIMP-low and CIMP-high tumors. Our results also suggested non-CIMP pathways as well. Obese individuals were at 2-fold increased risk of having a CIMP-low tumor. Alcohol was associated with an increased risk of tumors that were MSI+ and CIMP-low. In the presence of smoking 20 or more cigarettes per day, use of NSAIDs did not protect against a BRAF mutation. Our data suggest multiple pathways to colon cancer. They do not support a unique role for dietary folate, alcohol, vitamins B(6) and B(12) and methionine in a CpG island methylator phenotype.

  14. Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Farrar William L

    2010-10-01

    Full Text Available Abstract Background Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation. Results Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1. Conclusions Using this

  15. hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

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    Snijders Peter JF

    2010-06-01

    Full Text Available Abstract Background Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells. Methods Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG and quantitative methylation specific PCR (qMSP analysis of two regions flanking the hTERT core promoter. Results We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity. By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls. Conclusions Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA

  16. 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...

  17. Analysis of Geologic Parameters on the Performance of CO2-Plume Geothermal (CPG) Systems in a Multi-Layered Reservoirs

    Science.gov (United States)

    Garapati, N.; Randolph, J.; Saar, M. O.

    2013-12-01

    CO2-Plume Geothermal (CPG) involves injection of CO2 as a working fluid to extract heat from naturally high permeable sedimentary basins. The injected CO2 forms a large subsurface CO2 plume that absorbs heat from the geothermal reservoir and eventually buoyantly rises to the surface. The heat density of sedimentary basins is typically relatively low.However, this drawback is likely counteracted by the large accessible volume of natural reservoirs compared to artificial, hydrofractured, and thus small-scale, reservoirs. Furthermore, supercritical CO2has a large mobility (inverse kinematic viscosity) and expansibility compared to water resulting in the formation of a strong thermosiphon which eliminates the need for parasitic pumping power requirements and significantly increasing electricity production efficiency. Simultaneously, the life span of the geothermal power plant can be increased by operating the CPG system such that it depletes the geothermal reservoir heat slowly. Because the produced CO2 is reinjected into the ground with the main CO2 sequestration stream coming from a CO2 emitter, all of the CO2 is ultimately geologically sequestered resulting in a CO2 sequestering geothermal power plant with a negative carbon footprint. Conventional geothermal process requires pumping of huge amount of water for the propagation of the fractures in the reservoir, but CPG process eliminates this requirement and conserves water resources. Here, we present results for performance of a CPG system as a function of various geologic properties of multilayered systemsincludingpermeability anisotropy, rock thermal conductivity, geothermal gradient, reservoir depth and initial native brine salinity as well as spacing between the injection and production wells. The model consists of a 50 m thick, radially symmetric grid with a semi-analytic heat exchange and no fluid flow at the top and bottom boundaries and no fluid and heat flow at the lateral boundaries. We design Plackett

  18. Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium

    DEFF Research Database (Denmark)

    Pedersen, G; Andresen, Lars; Matthiessen, M W

    2005-01-01

    Recognition of repeat CpG motifs, which are common in bacterial, but not in mammalian, DNA, through Toll-like receptor (TLR)9 is an integral part of the innate immune system. As the role of TLR9 in the human gut is unknown, we determined the spectrum of TLR9 expression in normal and inflamed colon...... in vitro despite spontaneous TLR9 gene expression. This suggests that the human epithelium is able to avoid inappropriate immune responses to luminal bacterial products through modulation of the TLR9 pathway....

  19. 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.

  20. Prevention and Treatment of Cutaneous Leishmaniasis in Primates by Using Synthetic Type D/A Oligodeoxynucleotides Expressing CpG Motifs

    Science.gov (United States)

    Flynn, Barbara; Wang, Vivian; Sacks, David L.; Seder, Robert A; Verthelyi, Daniela

    2005-01-01

    Oligodeoxynucleotides (ODN) containing CpG motifs mimic microbial DNA and are recognized by toll-like receptor 9 on immune cells. The resulting response limits the early spread of infectious organisms and promotes the development of adaptive immunity. In this regard, CpG ODN show promise as immunoprotective agents and as vaccine adjuvants. Previous studies of nonhuman primates showed that administration of CpG ODN type D (also known as type A) at the site of infection 3 days before and after a challenge with Leishmania major enhanced host resistance and reduced the lesion's severity. In this study, we show that systemic administration of D/A ODN limits the size of lesions following an intradermal infection with L. major. Importantly, the reduced morbidity was not associated with a reduction in long-term immunity, as such treated macaques were still protected following a secondary challenge. Finally, administration of D/A ODN to macaques that had established cutaneous lesions reduced the severity of the lesions, suggesting a potential role for CpG ODN in L. major treatment. Together, these findings support the development of clinical studies to assess the use of CpG ODN types D/A as immunoprotective and therapeutic agents. PMID:16041009

  1. Proinflammatory Stimulation of Toll-Like Receptor 9 with High Dose CpG ODN 1826 Impairs Endothelial Regeneration and Promotes Atherosclerosis in Mice.

    Directory of Open Access Journals (Sweden)

    Alexander O Krogmann

    Full Text Available Toll-like receptors (TLR of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice.TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/- mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects.Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology.

  2. 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

  3. Organization of the Mammalian Locomotor CPG: Review of Computational Model and Circuit Architectures Based on Genetically Identified Spinal Interneurons

    Science.gov (United States)

    Dougherty, Kimberly J.; Shevtsova, Natalia A.

    2015-01-01

    Abstract The organization of neural circuits that form the locomotor central pattern generator (CPG) and provide flexor–extensor and left–right coordination of neuronal activity remains largely unknown. However, significant progress has been made in the molecular/genetic identification of several types of spinal interneurons, including V0 (V0D and V0V subtypes), V1, V2a, V2b, V3, and Shox2, among others. The possible functional roles of these interneurons can be suggested from changes in the locomotor pattern generated in mutant mice lacking particular neuron types. Computational modeling of spinal circuits may complement these studies by bringing together data from different experimental studies and proposing the possible connectivity of these interneurons that may define rhythm generation, flexor–extensor interactions on each side of the cord, and commissural interactions between left and right circuits. This review focuses on the analysis of potential architectures of spinal circuits that can reproduce recent results and suggest common explanations for a series of experimental data on genetically identified spinal interneurons, including the consequences of their genetic ablation, and provides important insights into the organization of the spinal CPG and neural control of locomotion. PMID:26478909

  4. Polymorphism in folate- and methionine-metabolizing enzyme and aberrant CpG island hypermethylation in uterine cervical cancer.

    Science.gov (United States)

    Kang, Sokbom; Kim, Jae Weon; Kang, Gyeong Hoon; Park, Noh Hyun; Song, Yong Sang; Kang, Soon Beom; Lee, Hyo Pyo

    2005-01-01

    This study was conducted to explore the association between the CpG island hypermethylation of tumor-associated genes and the polymorphisms of methyl group metabolizing enzymes in uterine cervical cancer. We analyzed CpG island hypermethylation in 15 genes (APC, CDH1, COX2, DAPK, FHIT, GSTP1, HLTF1, hMLH1, MGMT, p14, p16, RASSF1A, RUNX3, THBS1, and TIMP3) and its association with the methylene-tetrahydrofolate reductase (MTHFR) C677T and A1298C and the methionine synthase (MS) A2756G polymorphisms in 82 Korean women with uterine cervical cancer. All uterine cervical cancer samples had at least one gene methylated. The average number of methylated genes was lower in patients with the heterozygous genotype of MTHFR and MS than in those with the common homozygous genotype, although this difference was not significant. The MTHFR 677 CT genotype was significantly associated with the decreased promoter hypermethylation of O(6)-methylguanine DNA methyltransferase (MGMT) (OR = 0.22, 95% confidence interval (CI) 0.07-0.70, P = 0.011). However, the MTHFR C677T and A1298C and the MS A2756G polymorphisms were not associated with an increased risk of uterine cervical cancer. These findings suggest that there is a possible interaction between epigenetic and genetic factors in uterine cervical cancer.

  5. Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer.

    Science.gov (United States)

    Diede, Scott J; Yao, Zizhen; Keyes, C Chip; Tyler, Ashlee E; Dey, Joyoti; Hackett, Christopher S; Elsaesser, Katrina; Kemp, Christopher J; Neiman, Paul E; Weiss, William A; Olson, James M; Tapscott, Stephen J

    2013-12-01

    Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.

  6. Biophysical Attributes of CpG Presentation Control TLR9 Signaling to Differentially Polarize Systemic Immune Responses

    Directory of Open Access Journals (Sweden)

    Jardin A. Leleux

    2017-01-01

    Full Text Available It is currently unknown whether and how mammalian pathogen recognition receptors (PRRs respond to biophysical patterns of pathogen-associated molecular danger signals. Using synthetic pathogen-like particles (PLPs that mimic physical properties of bacteria or large viruses, we have discovered that the quality and quantity of Toll-like receptor 9 (TLR9 signaling by CpG in mouse dendritic cells (mDCs are uniquely dependent on biophysical attributes; specifically, the surface density of CpG and size of the presenting PLP. These physical patterns control DC programming by regulating the kinetics and magnitude of MyD88-IRAK4 signaling, NF-κB-driven responses, and STAT3 phosphorylation, which, in turn, controls differential T cell responses and in vivo immune polarization, especially T helper 1 (Th1 versus T helper 2 (Th2 antibody responses. Our findings suggest that innate immune cells can sense and respond not only to molecular but also pathogen-associated physical patterns (PAPPs, broadening the tools for modulating immunity and helping to better understand innate response mechanisms to pathogens and develop improved vaccines.

  7. A hybrid CPG-ZMP control system for stable walking of a simulated flexible spine humanoid robot.

    Science.gov (United States)

    Or, Jimmy

    2010-04-01

    Biped humanoid robots have gained much popularity in recent years. These robots are mainly controlled by two major control methods, the biologically-inspired approach based on Central Pattern Generator (CPG) and the engineering-oriented approach based on Zero Moment Point (ZMP). Given that flexibility in the body torso is required in some human activities, we believe that it is beneficial for the next generation of humanoid robots to have a flexible spine as humans do. In order to cope with the increased complexity in controlling this type of robot, a new kind of control system is necessary. Currently, there is no controller that allows a flexible spine humanoid robot to maintain stability in real-time while walking with dynamic spine motions. This paper presents a new hybrid CPG-ZMP control system for the walking of a realistically simulated flexible spine humanoid robot. Experimental results showed that using our control method, the robot is able to adapt its spine motions in real-time to allow stable walking. Our control system could be used for the control of the next generation humanoid robots. Copyright 2009 Elsevier Ltd. All rights reserved.

  8. An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands.

    Science.gov (United States)

    Dai, Zunyan; Weichenhan, Dieter; Wu, Yue-Zhong; Hall, Julia L; Rush, Laura J; Smith, Laura T; Raval, Aparna; Yu, Li; Kroll, Daniela; Muehlisch, Joerg; Frühwald, Michael C; de Jong, Pieter; Catanese, Joe; Davuluri, Ramana V; Smiraglia, Dominic J; Plass, Christoph

    2002-10-01

    Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.

  9. Vaccination with Toxoplasma lysate antigen and CpG oligodeoxynucleotides: comparison of immune responses in intranasal versus intramuscular administrations.

    Science.gov (United States)

    EL-Malky, Mohamed A; Al-Harthi, Saeed A; Mohamed, Raafat T; EL Bali, Mohamed A; Saudy, Niveen S

    2014-06-01

    Toxoplasma gondii (T. gondii) is one of the most successful intracellular protozoan parasites on earth and highly prevalent in most warm-blooded vertebrates. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance in disease control. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T-helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against intracellular pathogen. In this study, we compare the immune responses of Toxoplasma susceptible C57BL/6 mice following intranasal and intramuscular vaccination with Toxoplasma lysate antigen (TLA) with or without CpG ODN as adjuvant. Immunized and control non-immunized mice were challenged with 85 cyst of the moderately virulent Beverley strain of T. gondii. Intranasal vaccination gave significantly a higher protection compared to other groups as indicated by prolonged survival and significantly reduced brain cyst burden (P intramuscular vaccination enhanced humoral immunity towards a type Th1 pattern characterized by a significant increase of specific IgG and Ig2a. Our results suggest that intranasal administration of CpG/TLA would provide a stable, pronounced, and effective vaccine against toxoplasmosis through stimulation of Th1 cellular immunity and mucosal IgA.

  10. Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak.

    Science.gov (United States)

    Hsu, William; Mercado, Augustus T; Hsiao, George; Yeh, Jui-Ming; Chen, Chung-Yung

    2017-05-15

    Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R 2 =0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. 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

  12. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  13. Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics.

    Science.gov (United States)

    Singh, Vandana; Kumari, Bhavini; Maity, Banibrata; Seth, Debabrata; Das, Prolay

    2014-01-01

    Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Methylation of the estrogen receptor CpG island distinguishes spontaneous and plutonium-induced tumors from nitrosamine-induced lung tumors

    Energy Technology Data Exchange (ETDEWEB)

    Belinsky, S.A.; Baylin, S.B.; Issa, J.J. [Johns Hopkins Univ., Baltimore, MD (United States)

    1995-12-01

    CpG islands located in the promoter region of genes constitute one mechanism for regulating transcription. These islands are normally free of methylation, regardless of the expression state of the gene. Hypermethylation of CpG islands, the addition of a methyl group to the internal cytosine within CpG dinucleotides, can cause silencing of a gene. Hypermethylation has been detected as an early event at specific chromosome loci during the development of colon cancer and represents one mechanism used by neoplatic cells to inactivate tumor suppressor genes. Recent studies have demonstrated this mechanism in inactivation of the VHL tumor suppressor gene in 19% of sporadic renal tumors and the p16 {sup INK4a} tumor suppressor gene in 30% of non-small cell lung cancers. A recent report indicates that the estrogen receptor gene could also be inactivated through methylation. In addition, estrogen receptor CpG island methylation arises as a direct function of age in normal colonic mucosa and is present in virtually all colonic tumors. In cultured colon cancer cells, methylation-associated loss of expression of the estrogen receptor gene results in deregulated growth, suggesting a role for the estrogen receptor in colon cancer development. These results provide further evidence that gene silencing through methylation could be a predominant epigenetic mechanism underlying the development of many different types of cancer. The purpose of the current investigation was to determine whether estrogen receptor CpG island methylation is involved in the development of lung cancer. The frequency for methylation of the estrogen receptor CpG island in rodent lung tumors is summarized.

  15. Methylation of the estrogen receptor CpG island distinguishes spontaneous and plutonium-induced tumors from nitrosamine-induced lung tumors

    International Nuclear Information System (INIS)

    Belinsky, S.A.; Baylin, S.B.; Issa, J.J.

    1995-01-01

    CpG islands located in the promoter region of genes constitute one mechanism for regulating transcription. These islands are normally free of methylation, regardless of the expression state of the gene. Hypermethylation of CpG islands, the addition of a methyl group to the internal cytosine within CpG dinucleotides, can cause silencing of a gene. Hypermethylation has been detected as an early event at specific chromosome loci during the development of colon cancer and represents one mechanism used by neoplatic cells to inactivate tumor suppressor genes. Recent studies have demonstrated this mechanism in inactivation of the VHL tumor suppressor gene in 19% of sporadic renal tumors and the p16 INK4a tumor suppressor gene in 30% of non-small cell lung cancers. A recent report indicates that the estrogen receptor gene could also be inactivated through methylation. In addition, estrogen receptor CpG island methylation arises as a direct function of age in normal colonic mucosa and is present in virtually all colonic tumors. In cultured colon cancer cells, methylation-associated loss of expression of the estrogen receptor gene results in deregulated growth, suggesting a role for the estrogen receptor in colon cancer development. These results provide further evidence that gene silencing through methylation could be a predominant epigenetic mechanism underlying the development of many different types of cancer. The purpose of the current investigation was to determine whether estrogen receptor CpG island methylation is involved in the development of lung cancer. The frequency for methylation of the estrogen receptor CpG island in rodent lung tumors is summarized

  16. Deep intronic GBE1 mutation in manifesting heterozygous patients with adult polyglucosan body disease.

    Science.gov (United States)

    Akman, H Orhan; Kakhlon, Or; Coku, Jorida; Peverelli, Lorenzo; Rosenmann, Hanna; Rozenstein-Tsalkovich, Lea; Turnbull, Julie; Meiner, Vardiella; Chama, Liat; Lerer, Israela; Shpitzen, Shoshi; Leitersdorf, Eran; Paradas, Carmen; Wallace, Mary; Schiffmann, Raphael; DiMauro, Salvatore; Lossos, Alexander; Minassian, Berge A

    2015-04-01

    We describe a deep intronic mutation in adult polyglucosan body disease. Similar mechanisms can also explain manifesting heterozygous cases in other inborn metabolic diseases. To explain the genetic change consistently associated with manifesting heterozygous patients with adult polyglucosan body disease. This retrospective study took place from November 8, 2012, to November 7, 2014. We studied 35 typical patients with adult polyglucosan body disease, of whom 16 were heterozygous for the well-known c.986A>C mutation in the glycogen branching enzyme gene (GBE1) but harbored no other known mutation in 16 exons. All 16 manifesting heterozygous patients had lower glycogen branching activity compared with homozygous patients, which showed inactivation of the apparently normal allele. We studied the messenger ribonucleic acid (mRNA) structure and the genetic change due to the elusive second mutation. When we reverse transcribed and sequenced the mRNA of GBE1, we found that all manifesting heterozygous patients had the c.986A>C mutant mRNA and complete lack of mRNA encoded by the second allele. We identified a deep intronic mutation in this allele, GBE1-IVS15+5289_5297delGTGTGGTGGinsTGTTTTTTACATGACAGGT, which acts as a gene trap, creating an ectopic last exon. The mRNA transcript from this allele missed the exon 16 and 3'UTR and encoded abnormal GBE causing further decrease of enzyme activity from 18% to 8%. We identified the deep intronic mutation, which acts as a gene trap. This second-most common adult polyglucosan body disease mutation explains another founder effect in all Ashkenazi-Jewish cases.

  17. Strong signature of natural selection within an FHIT intron implicated in prostate cancer risk.

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    Yan Ding

    Full Text Available 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 chi(2 = 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 chi(2 = 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 (pi = 0.0072, Tajima's D = 3.31, 14 SNPs and the Japanese (pi = 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.

  18. The relationship between polymorphisms in intron 8 of vitamin D receptor gene and occult HBV infection

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    Rezvani ME

    2009-08-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 Background: Occult hepatitis B infection (OBI is a form of hepatitis in which despite absence of detectable HBsAg, HBV-DNA is present in peripheral blood of patients. The responsible mechanisms for progression of OBI yet to be clarified, but some investigators believed that the genetics and immunological parameters are different in resistant individuals and patients. Vitamin D3 and its receptor interaction could be involved in anti-viral immune response. The aim of this study was to investigate the association between polymorphisms in intron 8 of VDR with OBI."n"n Methods: In this experimental study, the plasma samples of 3700 blood donors were collected and tested for HBsAg and anti-HBs by ELISA. The HBsAg negative and anti-HBc positive samples were selected and screened for HBV-DNA using PCR. HBV-DNA positive samples were assigned as OBI cases and PCR-RFLP was performed to examine the polymorphisms in intron 8 of VDR genes."n"n Results: Results of current study indicated that 352 (9.5% of 3700 blood samples were HBsAg- and anti-HBc+. HBV-DNA was detected in 57/352 (16.1% of HBsAg- and anti-HBc+ samples. Our results showed that no significant difference was observed in Apa-1 polymorphisms of intron 8 of VDR and OBI patients."n"n Conclusion: Our results demonstrated that there are not any association between Apa-1 detected alleles and OBI, hence, it can be concluded that these alleles are not associated with OBI and other researchers should evaluate relation between other polymorphisms of VDR with OBI.

  19. Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

    Science.gov (United States)

    Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

    2016-06-01

    Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  20. Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

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    Du Yantao

    2012-06-01

    Full Text Available Abstract Background Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC-related methylation of miR CpG islands and its relationship with miRNA expression. Methods Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. Results Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210 gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P  Conclusions In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC

  1. Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

    International Nuclear Information System (INIS)

    Du, Yantao; Liu, Zhaojun; Gu, Liankun; Zhou, Jing; Zhu, Bu-dong; Ji, Jiafu; Deng, Dajun

    2012-01-01

    Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may

  2. The Tgif2 gene contains a retained intron within the coding sequence

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    Wotton David

    2006-01-01

    Full Text Available Abstract Background TGIF and TGIF2 are homeodomain proteins, which act as TGFβ specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFβ activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron. Results By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25–50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFβ dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3. Conclusion These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic

  3. 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

  4. 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

  5. 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.

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

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    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.

  7. 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.

  8. 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.

  9. 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

  10. Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

    International Nuclear Information System (INIS)

    Wasserkort, Reinhold; Kalmar, Alexandra; Valcz, Gabor; Spisak, Sandor; Krispin, Manuel; Toth, Kinga; Tulassay, Zsolt; Sledziewski, Andrew Z; Molnar, Bela

    2013-01-01

    The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to

  11. Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample.

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    Katsuhiko Nosho

    Full Text Available The CpG island methylator phenotype (CIMP is a distinct phenotype associated with microsatellite instability (MSI and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. METHOLODOLOGY/PRINCIPAL FINDINGS: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16, CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight. In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1, CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers, multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1 activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also

  12. AtMBD6, a methyl CpG binding domain protein, maintains gene ...

    Indian Academy of Sciences (India)

    ... targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved inRNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 alsointeracts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM ...

  13. An intronic ABCA3 mutation that is responsible for respiratory disease.

    Science.gov (United States)

    Agrawal, Amit; Hamvas, Aaron; Cole, F Sessions; Wambach, Jennifer A; Wegner, Daniel; Coghill, Carl; Harrison, Keith; Nogee, Lawrence M

    2012-06-01

    Member A3 of the ATP-binding cassette family of transporters (ABCA3) is essential for surfactant metabolism. Nonsense, missense, frameshift, and splice-site mutations in the ABCA3 gene (ABCA3) have been reported as causes of neonatal respiratory failure (NRF) and interstitial lung disease. We tested the hypothesis that mutations in noncoding regions of ABCA3 may cause lung disease. ABCA3-specific cDNA was generated and sequenced from frozen lung tissue from a child with fatal lung disease with only one identified ABCA3 mutation. ABCA3 was sequenced from genomic DNA prepared from blood samples obtained from the proband, parents, and other children with NRF. ABCA3 cDNA from the proband contained sequences derived from intron 25 that would be predicted to alter the structure and function of the ABCA3 protein. Genomic DNA sequencing revealed a heterozygous C>T transition in intron 25 trans to the known mutation, creating a new donor splice site. Seven additional infants with an ABCA3-deficient phenotype and inconclusive genetic findings had this same variant, which was not found in 2,132 control chromosomes. These findings support that this variant is a disease-causing mutation that may account for additional cases of ABCA3 deficiency with negative genetic studies.

  14. 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. © 2015 Qin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Repetition priming of motor activity mediated by a central pattern generator: the importance of extrinsic vs. intrinsic program initiators

    Science.gov (United States)

    Siniscalchi, Michael J.; Jing, Jian; Weiss, Klaudiusz R.

    2016-01-01

    Repetition priming is characterized by increased performance as a behavior is repeated. Although this phenomenon is ubiquitous, mediating mechanisms are poorly understood. We address this issue in a model system, the feeding network of Aplysia. This network generates both ingestive and egestive motor programs. Previous data suggest a chemical coding model: ingestive and egestive inputs to the feeding central pattern generator (CPG) release different modulators, which act via different second messengers to prime motor activity in different ways. The ingestive input to the CPG (neuron CBI-2) releases the peptides feeding circuit activating peptide and cerebral peptide 2, which produce an ingestive pattern of activity. The egestive input to the CPG (the esophageal nerve) releases the peptide small cardioactive peptide. This model is based on research that focused on a single aspect of motor control (radula opening). Here we ask whether repetition priming is observed if activity is triggered with a neuron within the core CPG itself and demonstrate that it is not. Moreover, previous studies demonstrated that effects of modulatory neurotransmitters that induce repetition priming persist. This suggests that it should be possible to “prime” motor programs triggered from within the CPG by first stimulating extrinsic modulatory inputs. We demonstrate that programs triggered after ingestive input activation are ingestive and programs triggered after egestive input activation are egestive. We ask where this priming occurs and demonstrate modifications within the CPG itself. This arrangement is likely to have important consequences for “task” switching, i.e., the cessation of one type of motor activity and the initiation of another. PMID:27466134

  16. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells.

    Science.gov (United States)

    Takahashi, Yuta; Wu, Jun; Suzuki, Keiichiro; Martinez-Redondo, Paloma; Li, Mo; Liao, Hsin-Kai; Wu, Min-Zu; Hernández-Benítez, Reyna; Hishida, Tomoaki; Shokhirev, Maxim Nikolaievich; Esteban, Concepcion Rodriguez; Sancho-Martinez, Ignacio; Belmonte, Juan Carlos Izpisua

    2017-05-05

    CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease. Copyright © 2017, American Association for the Advancement of Science.

  17. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells

    KAUST Repository

    Takahashi, Yuta

    2017-05-05

    CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease.

  18. Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant

    Directory of Open Access Journals (Sweden)

    Sarah Reeman

    2017-12-01

    Full Text Available Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

  19. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups

    DEFF Research Database (Denmark)

    Sogin, M L; Ingold, A; Karlok, M

    1986-01-01

    Previous work has demonstrated the presence of a self-splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups....... This phylogeny was consistent with the groupings suggested by comparisons of other biochemical characters including cytoskeletal proteins, isozyme analyses, and restriction maps of complete rRNA transcription units. The homology groupings that were based upon the intron sequence data do not agree....... In an attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree...

  20. 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.

  1. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    Science.gov (United States)

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2012-10-01

    Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Metronomic Doses of Temozolomide Enhance the Efficacy of Carbon Nanotube CpG Immunotherapy in an Invasive Glioma Model.

    Directory of Open Access Journals (Sweden)

    Mao Ouyang

    Full Text Available Even when treated with aggressive current therapies, most patients with glioblastoma survive less than two years. Rapid tumor growth, an invasive nature, and the blood-brain barrier, which limits the penetration of large molecules into the brain, all contribute to the poor tumor response associated with conventional therapies. Immunotherapy has emerged as a therapeutic approach that may overcome these challenges. We recently reported that single-walled carbon nanotubes (SWCNTs can be used to dramatically increase the immunotherapeutic efficacy of CpG oligonucleotides in a mouse model of glioma. Following implantation in the mouse brain, the tumor cell line used in these previous studies (GL261 tends to form a spherical tumor with limited invasion into healthy brain. In order to evaluate SWCNT/CpG therapy under more clinically-relevant conditions, here we report the treatment of a more invasive mouse glioma model (K-Luc that better recapitulates human disease. In addition, a CpG sequence previously tested in humans was used to formulate the SWCNT/CpG which was combined with temozolomide, the standard of care chemotherapy for glioblastoma patients. We found that, following two intracranial administrations, SWCNT/CpG is well-tolerated and improves the survival of mice bearing invasive gliomas. Interestingly, the efficacy of SWCNT/CpG was enhanced when combined with temozolomide. This enhanced anti-tumor efficacy was correlated to an increase of tumor-specific cytotoxic activity in splenocytes. These results reinforce the emerging understanding that immunotherapy can be enhanced by combining it with chemotherapy and support the continued development of SWCNT/CpG.

  3. CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression

    Czech Academy of Sciences Publication Activity Database

    Hejnar, Jiří; Hájková, P.; Plachý, Jiří; Elleder, Daniel; Stepanets, Volodymyr; Svoboda, Jan

    2001-01-01

    Roč. 98, č. 2 (2001), s. 565-569 ISSN 0027-8424 R&D Projects: GA ČR GA312/97/P082; GA ČR GA312/98/0825 Keywords : CpG island * provirus silencing * DNA methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.890, year: 2001

  4. Signal management in pharmacovigilance and human risk assessment of CpG 7909, integrating embryo-fetal and post-natal developmental toxicity studies in rats and rabbits.

    Science.gov (United States)

    Delannois, Frédérique; Planty, Camille; Giordano, Giulia; Destexhe, Eric; Stanislaus, Dinesh; Da Silva, Fernanda Tavares; Stegmann, Jens-Ulrich; Thacker, Karen; Reynaud, Lucie; Garçon, Nathalie; Segal, Lawrence

    2018-01-01

    The potential reproductive and developmental toxicity of the synthetic oligodeoxynucleotide (ODN) CpG 7909, a component of GSK's AS15 immunostimulant, was examined in rat and rabbit studies following intermittent intramuscular injections. Previous studies using subcutaneous and intraperitoneal injections in mice, rats and rabbits revealed that CpG ODNs induced developmental effects. To analyze the safety signal, GSK conducted additional animal studies using the intended clinical route of administration. CpG 7909 injections were administered intramuscularly to rats or rabbits 28 and 14days before pairing, on 4 or 5 occasions during gestation, and on lactation day 7. The No Observed Adverse Effect Level for female fertility, embryo-fetal and pre- and post-natal development was 4.2mg/kg in both species, approximately 500-fold higher than the anticipated human dose. In conclusion, the anticipated risk to humans is considered low for sporadic intramuscular exposure to CpG 7909. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. A stable nanoparticulate DDA/MMG formulation acts synergistically with CpG ODN 1826 to enhance the CD4(+) T-cell response

    DEFF Research Database (Denmark)

    Karlsen, Kasper; Korsholm, Karen Smith; Mortensen, Rasmus

    2014-01-01

    AIM: To combine the dimethyldioctadecyl ammonium/monomycoloyl glycerol (DDA/MMG) liposomal vaccine adjuvant with the Toll-like receptor (TLR) ligands poly(I:C) (TLR3), flagellin (TLR5) or CpG oligodeoxynucleotide 1826 (TLR9) and investigate their physicochemical properties as well as their CD4(+)...

  6. Effect of neonatal sublingual vaccination with native or denatured ovalbumin and adjuvant CpG or cholera toxin on systemic and mucosal immunity in mice.

    Science.gov (United States)

    Huang, C-F; Wu, T-C; Chu, Y-H; Hwang, K-S; Wang, C-C; Peng, H-J

    2008-11-01

    Sublingual immunotherapy has been applied for allergic diseases, but whether sublingual immunization in neonates can prevent sensitization has not been studied. In this study, we evaluate the effect of neonatal sublingual vaccination with native or denatured allergens alone or plus adjuvant on allergy prevention. Newborn BALB/c mice were sublingually vaccinated daily for the first 3 days with native or denatured ovalbumin (OVA) only, or combined adjuvant CpG or cholera toxin (CT). They were sensitized with OVA adsorbed onto alum 7 weeks after the last vaccination. Specific secretory IgA antibody responses were readily induced by neonatal vaccination with antigen plus CpG or CT, but not with antigen alone. Whereas vaccination with denatured OVA plus CpG markedly enhanced T helper 1 (Th1) responses and inhibited IgE production, vaccination with denatured OVA plus CT increased cervical lymph node cell production of interleukin-4 (IL-4), IL-5, IL-6, and serum IgG1 responses. These data demonstrate that neonatal sublingual vaccination with denatured OVA and CpG not only preferentially induces systemic Th1 responses and mucosal immunity, but also simultaneously abrogates IgE production. Neonatal sublingual vaccines may play a role for the strategy of allergy prevention.

  7. 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.

  8. A palindromic CpG-containing phosphodiester oligodeoxynucleotide as a mucosal adjuvant stimulates plasmacytoid dendritic cell-mediated T(H1 immunity.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Maeyama

    Full Text Available BACKGROUND: CpG oligodeoxynucleotides (ODNs, resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC-mediated T(H1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. METHODS: T(H1 and T(H2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT through nasal vaccination. RESULTS: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of T(H1, but not T(H2-type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. CONCLUSIONS: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated T(H1 immunity.

  9. CpG ODN 1668 induce innate and adaptive immune responses in rock bream (Oplegnathus fasciatus) against rock bream iridovirus (RBIV) infection.

    Science.gov (United States)

    Jung, Myung-Hwa; Jung, Sung-Ju

    2017-10-01

    Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream in Korea. CpG ODN 1668 showed promise as immunoprotective agents against RBIV infection in rock bream. In this study, we assessed innate/adaptive-related gene expression patterns in RBIV-infected rock bream with and without CpG ODN 1668 administration to determine important immune defense related factors that may affect fish survival. In the CpG ODN 1668+virus-injected group, virus copies were more than 7.4- to 790591-fold lower than in the virus-injected group at 4 d (8.79 × 10 4 and 6.58 × 10 5 /μl, respectively), 7 d (5.30 × 10 2 and 2.29 × 10 7 /μl, respectively) and 10 dpi (7.79 × 10 1 and 6.16 × 10 7 /μl, respectively). Furthermore, in the CpG ODN 1668+virus-injected group, significantly higher levels of MyD88 (6 h, 1 d, 4 d and 7 dpi), IL1β (1 d, 2 d and 7 dpi) and perforin/granzyme (1 dpi) expression were observed, whereas these genes were not significantly expressed in the virus-injected group at that time points. Mx, ISG15 and PKR were significantly highly expressed at 4 d and 7 dpi and reduced when low viral loads at 10 dpi in the CpG ODN 1668+virus-injected group. Conversely, in the virus-injected group, Mx, ISG15 and PKR expression were significantly higher than the control group until 10 dpi. However, MHC class I, CD8, Fas, Fas ligand and caspases (3, 8 and 9) expression levels showed no statistically significant differences between virus- and CpG ODN 1668+virus-injected group. In summary, CpG ODN 1668 administration in fish induces innate immune response or cell death pathway, which could be a major contributing factor to effective fish control over viral transcription on 4 d to 10 dpi. Expression of MyD88, IL1β, perforin and granzyme-related immune gene response is critical factor for inhibition of RBIV replication. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Methyl-CpG binding protein MBD2 is implicated in methylation-mediated suppression of miR-373 in hilar cholangiocarcinoma.

    Science.gov (United States)

    Chen, Yongjun; Gao, Wei; Luo, Jian; Tian, Rui; Sun, Huawen; Zou, Shengquan

    2011-02-01

    Aberrant expression of miRNAs is associated with particular cancers showing tissue- and clinical-feature-specificity patterns. Some miRNA genes harboring or being embedded in CpG islands undergo methylation mediated silencing. MBP, methyl CpG binding protein, suppresses transcription through binding to methylated CpG dinucleotides. Expression of miR-373 has been reported to be suppressed in malignant bile duct cell lines. Bioinformatic prediction reveals that the transcription start site (TSS) of miR-373 is implanted in a 402 bp canonical CpG island containing 26 CpG dinucleotides. In this study, we aim to determine the epigenetic regulation of miR-373 gene in hilar cholangiocarcinoma. Taqman microRNA assay shows that down-regulation of miR-373 is closely associated with poor cell differentiation, advanced clinical stage and shorter overall and disease-free survival in hilar cholangiocarcinomas. Methylation analysis shows that the promoter-associated CpG island is hypermethylated which is consistent with the inhibition of miR-373. Chromatin immunoprecipitation (ChIP) assay indicates that down-regulation of miR-373 results from the selective recruitment of MBD2 to methylated CpG islands. In contrast, MBD2 knock-down by use of a specific siRNA promoted the expression of miR-373. Reactivation of miR-373 by pharmacologic induction of 5-aza-CdR and trichostatin A (TSA) led to decreased enrichment of MBD2 at CpG island regions. Enhanced expression of exogenous MBD2 in stable QBC939 cells which stably express pGL4-m373-prom induces strengthened recruitment of MBD2. Our findings suggest that miR-373 is a methylation-mediated gene and the implication of MBD2 in methylation-mediated suppression of miR-373 plays an important role in tumourigenesis and development in hilar cholangiocarcinoma.

  11. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups

    DEFF Research Database (Denmark)

    Sogin, M L; Ingold, A; Karlok, M

    1986-01-01

    Previous work has demonstrated the presence of a self-splicing intron in the large subunit ribosomal RNA coding region in some strains of the ciliate protozoan Tetrahymena. Sequence comparisons of the intron regions from six Tetrahymena species showed these to fall into three homology groups....... In an attempt to evaluate the evolutionary origins of the intervening sequences, we have now determined complete small subunit ribosomal RNA gene sequences from 13 species of Tetrahymena and the absolute number of nucleotide differences between the sequences was used to construct a phylogenetic tree...

  12. The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Travis B White

    Full Text Available Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3' end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ. Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and

  13. Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma.

    Science.gov (United States)

    Hallberg, Andrea R; Vorrink, Sabine U; Hudachek, Danielle R; Cramer-Morales, Kimberly; Milhem, Mohammed M; Cornell, Robert A; Domann, Frederick E

    2014-12-01

    Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.

  14. Distribution of CpG Motifs in Upstream Gene Domains in a Reef Coral and Sea Anemone: Implications for Epigenetics in Cnidarians.

    Directory of Open Access Journals (Sweden)

    Adam G Marsh

    Full Text Available Coral reefs are under assault from stressors including global warming, ocean acidification, and urbanization. Knowing how these factors impact the future fate of reefs requires delineating stress responses across ecological, organismal and cellular scales. Recent advances in coral reef biology have integrated molecular processes with ecological fitness and have identified putative suites of temperature acclimation genes in a Scleractinian coral Acropora hyacinthus. We wondered what unique characteristics of these genes determined their coordinate expression in response to temperature acclimation, and whether or not other corals and cnidarians would likewise possess these features. Here, we focus on cytosine methylation as an epigenetic DNA modification that is responsive to environmental stressors. We identify common conserved patterns of cytosine-guanosine dinucleotide (CpG motif frequencies in upstream promoter domains of different functional gene groups in two cnidarian genomes: a coral (Acropora digitifera and an anemone (Nematostella vectensis. Our analyses show that CpG motif frequencies are prominent in the promoter domains of functional genes associated with environmental adaptation, particularly those identified in A. hyacinthus. Densities of CpG sites in upstream promoter domains near the transcriptional start site (TSS are 1.38x higher than genomic background levels upstream of -2000 bp from the TSS. The increase in CpG usage suggests selection to allow for DNA methylation events to occur more frequently within 1 kb of the TSS. In addition, observed shifts in CpG densities among functional groups of genes suggests a potential role for epigenetic DNA methylation within promoter domains to impact functional gene expression responses in A. digitifera and N. vectensis. Identifying promoter epigenetic sequence motifs among genes within specific functional groups establishes an approach to describe integrated cellular responses to

  15. Neonatal sublingual vaccination with Salmonella proteins and adjuvant cholera toxin or CpG oligodeoxynucleotides induces mucosal and systemic immunity in mice.

    Science.gov (United States)

    Huang, Ching-Feng; Wang, Chih-Chien; Wu, Tzee-Chung; Wu, Keh-Gong; Lee, Chin-Cheng; Peng, Ho-Jen

    2008-03-01

    Salmonella enteritidis is one of the most common enteric pathogens that cause acute gastroenteritis. A vaccine that can induce systemic and mucosal immune responses by a simple, noninvasive pathway and provide protection against this mucosal pathogen is needed. Newborn BALB/c mice were sublingually vaccinated daily for the first 3 days with sonicated Salmonella proteins (SSP) only, or SSP combined with adjuvant CpG or cholera toxin (CT). A booster vaccination was given 7 weeks after the last treatment. Serum and saliva antibody responses, cytokine profiles of spleen cells, survival rate, and intestinal morphology after live S enteritidis challenge were investigated. Saliva-specific secretory IgA (SIgA) antibody responses were markedly enhanced by neonatal sublingual vaccination with SSP together with adjuvant CpG or CT. Whereas vaccination with SSP and CpG enhanced spleen cell interferon-gamma production and serum-specific IgG2a antibody responses, vaccination with SSP and CT increased spleen cell interleukin (IL)-4, IL-5, IL-6, and interferon-gamma production and serum-specific IgG1 and IgG2a antibody responses. Vaccination with SSP and CpG or CT protected against intestinal necrosis and was associated with a higher survival rate after oral challenge with live S enteritidis. The vaccinated mice with higher specific IgG and saliva-specific secretory IgA antibody levels had a better survival rate. Neonatal sublingual vaccination with adjuvant CpG or CT can induce both mucosal and systemic immunity and may play a crucial role in protection against enteric pathogens.

  16. In silico enhanced restriction enzyme based methylation analysis of the human glioblastoma genome using Agilent 244K CpG Island microarrays

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    Anh Tran

    2010-01-01

    Full Text Available Genome wide methylation profiling of gliomas is likely to provide important clues to improving treatment outcomes. Restriction enzyme based approaches have been widely utilized for methylation profiling of cancer genomes and will continue to have importance in combination with higher density microarrays. With the availability of the human genome sequence and microarray probe sequences, these approaches can be readily characterized and optimized via in silico modeling. We adapted the previously described HpaII/MspI based Methylation Sensitive Restriction Enzyme (MSRE assay for use with two-color Agilent 244K CpG island microarrays. In this assay, fragmented genomic DNA is digested in separate reactions with isoschizomeric HpaII (methylation-sensitive and MspI (methylation-insensitive restriction enzymes. Using in silico hybridization, we found that genomic fragmentation with BfaI was superior to MseI, providing a maximum effective coverage of 22,362 CpG islands in the human genome. In addition, we confirmed the presence of an internal control group of fragments lacking HpaII/MspI sites which enable separation of methylated and unmethylated fragments. We used this method on genomic DNA isolated from normal brain, U87MG cells, and a glioblastoma patient tumor sample and confirmed selected differentially methylated CpG islands using bisulfite sequencing. Along with additional validation points, we performed a receiver operating characteristics (ROC analysis to determine the optimal threshold (p ≤ 0.001. Based on this threshold, we identified ~2400 CpG islands common to all three samples and 145 CpG islands unique to glioblastoma. These data provide more general guidance to individuals seeking to maximize effective coverage using restriction enzyme based methylation profiling approaches.

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

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    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. An intronic microRNA links Rb/E2F and EGFR signaling.

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    Mary Truscott

    2014-07-01

    Full Text Available The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.

  19. VEZF1 elements mediate protection from DNA methylation.

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    Jacqueline Dickson

    2010-01-01

    Full Text Available There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

  20. HLA-DQA1 introns 2 and 3 sequencing: DQA1 sequencing-based typing and characterization of a highly polymorphic microsatellite at intron 3 of DQA1*0505.

    Science.gov (United States)

    Balas, Antonio; Aviles, Maria J; Alonso-Nieto, Manuela; Zarapuz, Loreto; Blanco, Lydia; García-Sánchez, Felix; Vicario, Jose L

    2005-08-01

    DQA1 class II gene encodes the alpha-chain of the human leukocyte antigen (HLA)-DQ heterodimer. Sequencing-based typing (SBT) for HLA genes is the most powerful methodology described. However, most of the SBT procedures reported for HLA class II genes are not able to define complete exon 2 region. For that purpose, we have characterized introns 2 and 3 from most DQA1 alleles to design amplification procedures that were able to obtain complete exon 2 and 3 sequences from DQA1 genes. This coding information allowed us to reduce the number of ambiguities for DQA1 typing. DQA1 intron 2 and 3 characterization demonstrated the presence of two polymorphisms for alleles with the same exons 2 and 3 sequence from DQA1*05 group. Different samples including the DQA1*050101 alleles showed a single nucleotide polymorphism at position 53 of intron 2 (G53T). Additional haplotypic analysis showed the possible association of T53 allele with the Ax-Cw5-B18-DR17-DQ2 extended haplotype. On the other hand, DQA1*0505 sequencing from different control samples noticed the existence of a microsatellite (TTTC/AAAG)n located at position 126 of intron 3. Fragment length analysis demonstrated a high polymorphism for this short tandem repeat system (0505STR), defining alleles that ranged from 8 to 20 repetitions in our population.

  1. Effect of introns and AT-rich sequences on expression of the bacterial hygromycin B resistance gene in the basidiomycete Schizophyllum commune

    NARCIS (Netherlands)

    Scholtmeijer, K; Wosten, HAB; Springer, J; Wessels, JGH

    Previously, it was shown that introns are required for efficient mRNA accumulation in Schizophyllum commune and that the presence of AT-rich sequences in the coding region of genes can result in truncation of transcripts in this homobasidiomycete. Here we show that intron-dependent mRNA accumulation

  2. An Intron 7 Polymorphism in APP Affects the Age of Onset of Dementia in Down Syndrome

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    Emma L. Jones

    2011-01-01

    Full Text Available People with Down syndrome (DS develop Alzheimer's disease (AD with an early age of onset. A tetranucleotide repeat, attt5−8, in intron 7 of the amyloid precursor protein has been associated with the age of onset of AD in DS in a preliminary study. The authors examine the impact of this polymorphism in a larger cohort of individuals with DS. Adults with DS were genotyped for attt5−8 and APOE. The results were analysed with respect to the age of onset of dementia. The presence of three copies of the six-repeat allele resulted in onset of dementia seven years earlier than in the presence of other genotypes. Further study is essential to elucidate the mechanism by which this polymorphism functions, with an exciting opportunity to identify novel treatment targets relevant for people with DS and AD.

  3. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites...... and other features found in the adjacent exons and introns. By restricting the training of neural network algorithms to 'pure' UTRs (not extending partially into protein coding regions), we for the first time investigate the predictive power of the splicing signal proper, in contrast to conventional splice...... in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-.3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also...

  4. [The chromosomal genes for black widow spider neurotoxins do not contain introns].

    Science.gov (United States)

    Danilevich, V N; Grishin, E V

    2000-12-01

    The overlapping fragments of the chromosomal DNA from black widow spider Latrodectus mactans carrying genes for high-molecular-mass protein neurotoxins, alpha- and delta-latroinsectotoxins (alpha-LIT and delta-LIT) and alpha-latrotoxin (alpha-LTX), were PCR-amplified and cloned. Restriction analysis of the PCR products showed that the distribution and sizes of the restriction fragments coincided with those deduced from the earlier sequencing of cDNAs of the corresponding genes. It thus followed that the alpha-LIT and delta-LIT genes are intronless. Along with our data on the structure of the alpha-latrocrustotoxin (alpha-LCT), this implies that the lack of introns is a common feature of the black widow spider genes encoding high molecular mass neurotoxins.

  5. Deep intronic mutation and pseudo exon activation as a novel muscular hypertrophy modifier in cattle.

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    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.

  6. Evolution of Fungal U3 snoRNAs: Structural Variation and Introns

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    Sebastian Canzler

    2017-01-01

    Full Text Available The U3 small nucleolar RNA (snoRNA is an essential player in the initial steps of ribosomal RNA biogenesis which is ubiquitously present in Eukarya. It is exceptional among the small nucleolar RNAs in its size, the presence of multiple conserved sequence boxes, a highly conserved secondary structure core, its biogenesis as an independent gene transcribed by polymerase III, and its involvement in pre-rRNA cleavage rather than chemical modification. Fungal U3 snoRNAs share many features with their sisters from other eukaryotic kingdoms but differ from them in particular in their 5’ regions, which in fungi has a distinctive consensus structure and often harbours introns. Here we report on a comprehensive homology search and detailed analysis of the evolution of sequence and secondary structure features covering the entire kingdom Fungi.

  7. An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10

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    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.

  8. Recessive inheritance of population-specific intronic LINE-1 insertion causes a rotor syndrome phenotype.

    Science.gov (United States)

    Kagawa, Tatehiro; Oka, Akira; Kobayashi, Yoshinao; Hiasa, Yoichi; Kitamura, Tsuneo; Sakugawa, Hiroshi; Adachi, Yukihiko; Anzai, Kazuya; Tsuruya, Kota; Arase, Yoshitaka; Hirose, Shunji; Shiraishi, Koichi; Shiina, Takashi; Sato, Tadayuki; Wang, Ting; Tanaka, Masayuki; Hayashi, Hideki; Kawabe, Noboru; Robinson, Peter N; Zemojtel, Tomasz; Mine, Tetsuya

    2015-03-01

    Sequences of long-interspersed elements (LINE-1, L1) make up ∼17% of the human genome. De novo insertions of retrotransposition-active L1s can result in genetic diseases. It has been recently shown that the homozygous inactivation of two adjacent genes SLCO1B1 and SLCO1B3 encoding organic anion transporting polypeptides OATP1B1 and OATP1B3 causes a benign recessive disease presenting with conjugated hyperbilirubinemia, Rotor syndrome. Here, we examined SLCO1B1 and SLCO1B3 genes in six Japanese diagnosed with Rotor syndrome on the basis of laboratory data and laparoscopy. All six Japanese patients were homozygous for the c.1738C>T nonsense mutation in SLCO1B1 and homozygous for the insertion of a ∼6.1-kbp L1 retrotransposon in intron 5 of SLCO1B3, which altogether make up a Japanese-specific haplotype. RNA analysis revealed that the L1 insertion induced deleterious splicing resulting in SLCO1B3 transcripts lacking exon 5 or exons 5-7 and containing premature stop codons. The expression of OATP1B1 and OATP1B3 proteins was not detected in liver tissues. This is the first documented case of a population-specific polymorphic intronic L1 transposon insertion contributing to molecular etiology of recessive genetic disease. Since L1 activity in human genomes is currently seen as a major source of individual genetic variation, further investigations are warranted to determine whether this phenomenon results in other autosomal-recessive diseases. © 2014 WILEY PERIODICALS, INC.

  9. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

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    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  10. Precise mapping of 17 deletion breakpoints within the central hotspot deletion region (introns 50 and 51) of the DMD gene.

    Science.gov (United States)

    Esposito, Gabriella; Tremolaterra, Maria Roberta; Marsocci, Evelina; Tandurella, Igor Cm; Fioretti, Tiziana; Savarese, Maria; Carsana, Antonella

    2017-12-01

    Exon deletions in the human DMD gene, which encodes the dystrophin protein, are the molecular defect in 50-70% of cases of Duchenne/Becker muscular dystrophies. Deletions are preferentially clustered in the 5' (exons 2-20) and the central (exons 45-53) region of DMD, likely because local DNA structure predisposes to specific breakage or recombination events. Notably, innovative therapeutic strategies may rescue dystrophin function by homology-based specific targeting of sequences within the central DMD hot spot deletion region. To further study molecular mechanisms that generate such frequent genome variations and to identify residual intronic sequences, we sequenced 17 deletion breakpoints within introns 50 and 51 of DMD and analyzed the surrounding genomic architecture. There was no breakpoint clustering within the introns nor extensive homology between sequences adjacent to each junction. However, at or near the breakpoint, we found microhomology, short tandem repeats, interspersed repeat elements and short sequence stretches that predispose to DNA deletion or bending. Identification of such structural elements contributes to elucidate general mechanisms generating deletion within the DMD gene. Moreover, precise mapping of deletion breakpoints and localization of repeated elements are of interest, because residual intronic sequences may be targeted by therapeutic strategies based on genome editing correction.

  11. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

    Science.gov (United States)

    Mohr, Sabine; Ghanem, Eman; Smith, Whitney; Sheeter, Dennis; Qin, Yidan; King, Olga; Polioudakis, Damon; Iyer, Vishwanath R.; Hunicke-Smith, Scott; Swamy, Sajani; Kuersten, Scott; Lambowitz, Alan M.

    2013-01-01

    Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility (“retrohoming”) by a process that requires reverse transcription of a highly structured, 2–2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications. PMID:23697550

  12. Phase 1 testing of detoxified LPS/group B meningococcal outer membrane protein vaccine with and without synthetic CPG 7909 adjuvant for the prevention and treatment of sepsis.

    Science.gov (United States)

    Cross, Alan S; Greenberg, Nancy; Billington, Melissa; Zhang, Lei; DeFilippi, Christopher; May, Ryan C; Bajwa, Kanwaldeep K

    2015-11-27

    Gram-negative bacteria (GNB) are a leading cause of nosocomial infection and sepsis. Increasing multi-antibiotic resistance has left clinicians with fewer therapeutic options. Antibodies to GNB lipopolysaccharide (LPS, or endotoxin) have reduced morbidity and mortality as a result of infection and are not subject to the resistance mechanisms deployed by bacteria against antibiotics. In this phase 1 study, we administered a vaccine that elicits antibodies against a highly conserved portion of LPS with and without a CpG oligodeoxynucleotide (ODN) TLR9 agonist as adjuvant. A vaccine composed of the detoxified LPS (dLPS) from E. coli O111:B4 (J5 mutant) non-covalently complexed to group B meningococcal outer membrane protein (OMP). Twenty healthy adult subjects received three doses at 0, 29 and 59 days of antigen (10 μg dLPS) with or without CPG 7909 (250 or 500 μg). Subjects were evaluated for local and systemic adverse effects and laboratory findings. Anti-J5 LPS IgG and IgM antibody levels were measured by electrochemiluminesence. Due to premature study termination, not all subjects received all three doses. All vaccine formulations were well-tolerated with no local or systemic events of greater than moderate severity. The vaccine alone group achieved a ≥ 4-fold "responder" response in IgG and IgM antibody in only one of 6 subjects. In contrast, the vaccine plus CPG 7909 groups appeared to have earlier and more sustained (to 180 days) responses, greater mean-fold increases, and a higher proportion of "responders" achieving ≥ 4-fold increases over baseline. Although the study was halted before all enrolled subjects received all three doses, the J5dLPS/OMP vaccine, with or without CpG adjuvant, was safe and well-tolerated. The inclusion of CpG increased the number of subjects with a ≥ 4-fold antibody response, evident even after the second of three planned doses. A vaccine comprising J5dLPS/OMP antigen with CpG adjuvant merits further investigation. Clinical

  13. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

    International Nuclear Information System (INIS)

    Xiang, Shengyan; Liu, Zhaojun; Zhang, Baozhen; Zhou, Jing; Zhu, Bu-Dong; Ji, Jiafu; Deng, Dajun

    2010-01-01

    Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively

  14. Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

    Science.gov (United States)

    Waldsich, C; Semrad, K; Schroeder, R

    1998-12-01

    The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

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

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    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.

  16. Emulsified nanoparticles containing inactivated influenza virus and CpG oligodeoxynucleotides critically influences the host immune responses in mice.

    Directory of Open Access Journals (Sweden)

    Ming-Hsi Huang

    2010-08-01

    Full Text Available Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1 formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity.After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span(R85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 microg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2.Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a

  17. Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

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    Hashizume Toko

    2011-05-01

    Full Text Available Abstract Background Group 1 introns (ribozymes are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. Results Intron-F's (inserted at L798 position were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921 at 12% and intron-H's (inserted at L2563 at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR. Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. Conclusion The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

  18. Lenalidomide modulates gene expression in human ABC-DLBCL cells by regulating IKAROS interaction with an intronic control region of SPIB.

    Science.gov (United States)

    Solomon, Lauren A; Batista, Carolina R; DeKoter, Rodney P

    2017-12-01

    Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is associated with a poor prognosis compared with other DLBCL types and therefore represents a top priority for developing novel therapies. Lenalidomide, an immunomodulatory drug in trials for treatment of ABC-DLBCL, targets the transcription factor IKAROS for degradation by the cereblon E3 ubiquitin ligase complex. In this study, we investigated whether the gene encoding the transcription factor SPI-B is a target of IKAROS. Using cultured ABC-DLBCL cell lines, we found that high levels of SPI-B expression conferred resistance to lenalidomide. Lenalidomide treatment of ABC-DLBCL cells resulted in downregulation of SPIB at the level of transcription. SPIB was regulated directly by IKAROS through a binding site located in the first intron of the gene. Inhibition of IKAROS binding using CRISPR/Cas9-mediated transcriptional repression downregulated endogenous SPIB transcription. Finally, ectopic expression of IKAROS protected SPIB from downregulation. These results show that the mechanism of action of lenalidomide in ABC-DLBCL cells involves downregulation of SPIB transcription by cereblon-induced degradation of IKAROS. These results have implications for the design of synthetic lethal therapy for the treatment of ABC-DLBCL. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  19. CpGislandEVO: A Database and Genome Browser for Comparative Evolutionary Genomics of CpG Islands

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    Guillermo Barturen

    2013-01-01

    Full Text Available Hypomethylated, CpG-rich DNA segments (CpG islands, CGIs are epigenome markers involved in key biological processes. Aberrant methylation is implicated in the appearance of several disorders as cancer, immunodeficiency, or centromere instability. Furthermore, methylation differences at promoter regions between human and chimpanzee strongly associate with genes involved in neurological/psychological disorders and cancers. Therefore, the evolutionary comparative analyses of CGIs can provide insights on the functional role of these epigenome markers in both health and disease. Given the lack of specific tools, we developed CpGislandEVO. Briefly, we first compile a database of statistically significant CGIs for the best assembled mammalian genome sequences available to date. Second, by means of a coupled browser front-end, we focus on the CGIs overlapping orthologous genes extracted from OrthoDB, thus ensuring the comparison between CGIs located on truly homologous genome segments. This allows comparing the main compositional features between homologous CGIs. Finally, to facilitate nucleotide comparisons, we lifted genome coordinates between assemblies from different species, which enables the analysis of sequence divergence by direct count of nucleotide substitutions and indels occurring between homologous CGIs. The resulting CpGislandEVO database, linking together CGIs and single-cytosine DNA methylation data from several mammalian species, is freely available at our website.

  20. Class A CpG Oligonucleotide Priming Rescues Mice from Septic Shock via Activation of Platelet-Activating Factor Acetylhydrolase

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    Yoshinari Yamamoto

    2017-08-01

    Full Text Available Sepsis is a life-threatening, overwhelming immune response to infection with high morbidity and mortality. Inflammatory response and blood clotting are caused by sepsis, which induces serious organ damage and death from shock. As a mechanism of pathogenesis, platelet-activating factor (PAF induces excessive inflammatory responses and blood clotting. In this study, we demonstrate that a Class A CpG oligodeoxynucleotide (CpG-A1585 strongly induced PAF acetylhydrolase, which generates lyso-PAF. CpG-A1585 rescued mice from acute lethal shock and decreased fibrin deposition, a hallmark of PAF-induced disseminated intravascular coagulation. Furthermore, CpG-A1585 improved endotoxin shock induced by lipopolysaccharide, which comprises the cell wall of Gram-negative bacteria and inhibits inflammatory responses induced by cytokines such as interleukin-6 and tumor necrosis factor-α. These results suggest that CpG-A1585 is a potential therapeutic target to prevent sepsis-related induction of PAF.

  1. CpGislandEVO: A Database and Genome Browser for Comparative Evolutionary Genomics of CpG Islands

    Science.gov (United States)

    Barturen, Guillermo; Dios, Francisco; Hamberg, E. J. Maarten; Oliver, José L.

    2013-01-01

    Hypomethylated, CpG-rich DNA segments (CpG islands, CGIs) are epigenome markers involved in key biological processes. Aberrant methylation is implicated in the appearance of several disorders as cancer, immunodeficiency, or centromere instability. Furthermore, methylation differences at promoter regions between human and chimpanzee strongly associate with genes involved in neurological/psychological disorders and cancers. Therefore, the evolutionary comparative analyses of CGIs can provide insights on the functional role of these epigenome markers in both health and disease. Given the lack of specific tools, we developed CpGislandEVO. Briefly, we first compile a database of statistically significant CGIs for the best assembled mammalian genome sequences available to date. Second, by means of a coupled browser front-end, we focus on the CGIs overlapping orthologous genes extracted from OrthoDB, thus ensuring the comparison between CGIs located on truly homologous genome segments. This allows comparing the main compositional features between homologous CGIs. Finally, to facilitate nucleotide comparisons, we lifted genome coordinates between assemblies from different species, which enables the analysis of sequence divergence by direct count of nucleotide substitutions and indels occurring between homologous CGIs. The resulting CpGislandEVO database, linking together CGIs and single-cytosine DNA methylation data from several mammalian species, is freely available at our website. PMID:24205506

  2. Detection of Turner syndrome using X-chromosome inactivation specific differentially methylated CpG sites: A pilot study.

    Science.gov (United States)

    Zhang, Qiang; Guo, Xiaohong; Tian, Tian; Wang, Teng; Li, Qiaoli; Wang, Lei; Liu, Yun; Xing, Qinghe; He, Lin; Zhao, Xinzhi

    2017-05-01

    Early diagnosis of Turner syndrome (TS) may improve preventive measures and treatment. X-chromosome inactivation specific differentially methylated CpG sites (XIDMSs) that are high methylated in inactive X chromosomes (Xi) and unmethylated in active X chromosomes (Xa) may be potential makers for TS detection. The candidate XIDMSs were screened from 9 male and 12 female DNA samples with normal karyotypes using the Illumina 450k array and validated by bisulfite sequencing PCR and pyrosequencing assay. X chromosome dosage was calculated according to the methylation level of multiple XIDMSs. Overall, 108 candidate XIDMSs were screened by the 450k array. Validations indicated that XIDMSs gathered and formed the X-chromosome inactivation specific differentially methylated regions (XIDMRs). Using 3 XIDMRs at SAT1, UXT and UTP14A loci, 36 TS, 22 normal female and 6 male samples were analyzed. Methylation levels of the 20 XIDMSs in the XIDMRs could distinguish between TS and normal female DNA samples, the X chromosome dosage was consistent with karyotyping data. Analyzing samples of 2 triple X syndrome and 3 Klinefelter syndrome patients suggested that this method could be used to detect X chromosome aneuploids other than TS. XIDMSs are widely spread along the X chromosome and might be effective markers for detection of TS and other X chromosome aneuploids. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Virus-like attachment sites and plastic CpG islands:landmarks of diversity in plant Del retrotransposons.

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    Guilherme M Q Cruz

    Full Text Available Full-length Del elements from ten angiosperm genomes, 5 monocot and 5 dicot, were retrieved and putative attachment (att sites were identified. In the 2432 Del elements, two types of U5 att sites and a single conserved type of U3 att site were identified. Retroviral att sites confer specificity to the integration process, different att sites types therefore implies lineage specificity. While some features are common to all Del elements, CpG island patterns within the LTRs were particular to lineage specific clusters. All eudicot copies grouped into one single clade while the monocots harbour a more diverse collection of elements. Furthermore, full-length Del elements and truncated copies were unevenly distributed amongst chromosomes. Elements of Del lineage are organized in plants into three clusters and each cluster is composed of elements with distinct LTR features. Our results suggest that the Del lineage efficiently amplified in the monocots and that one branch is probably a newly emerging sub-lineage. Finally, sequences in all groups are under purifying selection. These results show the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.

  4. Interaction between Methylation and CpG Single-Nucleotide Polymorphisms in the HTR2A Gene: Association Analysis with Suicide Attempt in Schizophrenia.

    Science.gov (United States)

    Bani-Fatemi, Ali; Howe, Aaron S; Matmari, Michelle; Koga, Arthur; Zai, Clement; Strauss, John; De Luca, Vincenzo

    2016-01-01

    Dysfunctional mechanisms in the serotonergic system have been implicated in suicidal behavior among patients with schizophrenia. However, previous association analyses of major serotonin genes have provided inconsistent findings regarding their role in suicidal behavior. The goal of the current study was to identify single-nucleotide polymorphisms (SNP) within HTR2A that directly affect CpG methylation sites in schizophrenic patients with suicidal behavior. Furthermore, direct methylation analysis was performed using genomic DNA from peripheral leukocytes employing bisulfite pyrosequencing to assess the contributions of six CpG sites in HTR2A exon I in 67 schizophrenia patients assessed for lifetime suicide attempt. Potential methylation in 25 CpG SNPs across the entire HTR2A gene was analyzed considering their direct contribution to methylation. When we compared direct methylation between attempters and nonattempters, we found that only the polymorphic T102C (rs6313) was significantly different between the two groups (p = 0.02). Furthermore, in the potential methylation analysis, we found a nominal association with suicide attempt for six of the 25 SNPs analyzed, i.e. rs2770293 (p = 0.045), rs6313 (p = 0.033), rs17068986 (p = 0.029), rs4942578 (p = 0.024), rs1728872 (p = 0.014), and rs9534511 (p = 0.003). The results of this investigation provide preliminary evidence that the combined analysis of CpG SNPs and methylation may be useful for investigating the genetic and epigenetic factors involved in suicidal behavior. © 2016 S. Karger AG, Basel.

  5. The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing

    Czech Academy of Sciences Publication Activity Database

    Šenigl, Filip; Plachý, Jiří; Hejnar, Jiří

    2008-01-01

    Roč. 82, č. 16 (2008), s. 7818-7827 ISSN 0022-538X R&D Projects: GA ČR GA204/05/0939; GA ČR GA523/07/1171 Institutional research plan: CEZ:AV0Z50520514 Keywords : anti-methylation protection * retroviral vector * CpG island Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.308, year: 2008

  6. B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

    Science.gov (United States)

    Hagn, Magdalena; Blackwell, Sue E.; Beyer, Thamara; Ebel, Verena; Fabricius, Dorit; Lindner, Stefanie; Stilgenbauer, Stefan; Simmet, Thomas; Tam, Constantine; Neeson, Paul; Trapani, Joseph A.; Schrezenmeier, Hubert; Weiner, George J.

    2014-01-01

    CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL. PMID:24497611

  7. Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

    Science.gov (United States)

    Mur, Pilar; Rodríguez de Lope, Ángel; Díaz-Crespo, Francisco Javier; Hernández-Iglesias, Teresa; Ribalta, Teresa; Fiaño, Concepción; García, Juan Fernando; Rey, Juan Antonio; Mollejo, Manuela; Meléndez, Bárbara

    2015-05-01

    Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

  8. MECHANISM AND REGULATION OF NONSENSE-MEDIATED MRNA DECAY (NMD, AN ESSENTIAL QUALITY CONTROL SYSTEM OF PLANTS

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    D. Silhavy

    2008-09-01

    Full Text Available In eukaryotic cell, various quality control mechanisms have evolved to ensure that only perfect mRNAs could be translated. Nonsense-mediated mRNA decay (NMD is a quality control system that identifies and eliminates mRNAs containing premature termination codons, thereby preventing the accumulation of potentially harmful truncated proteins. While NMD is well-characterized in yeast, in invertebrates and in mammals, plant NMD is poorly understood. In yeast and in invertebrates unusually long 3'untranslated regions (3'UTRs render an mRNA subject to NMD, while in mammals' 3'UTR located introns trigger NMD. UPF1, 2 and 3 are the key trans-acting NMD factors in yeast as well as in animals. However, in mammals, the core components of the Exon Junction Complex (Mago, Y14, eIF4A3 and MLN51 are also required for NMD. It was proposed that long 3’UTR-induced NMD is the ancient type and that it was changed to a more complex intron-based NMD in mammals. To better understand the evolution of eukaryotic NMD systems, we have studied the NMD machinery of plants, as plants are outgroup relative to fungi and animals. We have elaborated various transient assays to analyze plant NMD. Using these assays we defined the cis elements of plant NMD and characterized several trans-acting plant NMD factors. We demonstrated that two plant NMD pathways co-exist, one pathway, as yeast or invertebrate NMD systems, eliminates mRNAs with long 3'UTRs, while a distinct pathway, like mammalian NMD, degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2, and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. We also provide evidence that the molecular mechanism of long 3'UTR-based plant NMD resembles yeast NMD, while the intron-based NMD is similar to mammalian NMD. Moreover we have found that the SMG-7 component of plant NMD is targeted by NMD suggesting that plant NMD is autoregulated. We propose that in

  9. Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers.

    Science.gov (United States)

    Krizek, Beth A

    2015-10-12

    The AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers. Transgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5' sequence and 919 bp of 3' sequence (AIL6:cAIL6-3') fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5' and 3' sequence (AIL6:gAIL6-3') can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5' sequence and 919 bp of 3' sequence (AIL6m:gAIL6-3') complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3' and AIL6m:gAIL6-3' lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation. The results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.

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

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    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.

  11. Conformation effects of CpG methylation on single-stranded DNA oligonucleotides: analysis of the opioid peptide dynorphin-coding sequences.

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    Malik Mumtaz Taqi

    Full Text Available Single-stranded DNA (ssDNA is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE, circular dichroism (CD spectroscopy and nuclear magnetic resonance (NMR spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4°C, and some also at 37°C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA.

  12. Immortalization of T-Cells Is Accompanied by Gradual Changes in CpG Methylation Resulting in a Profile Resembling a Subset of T-Cell Leukemias

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    Sofie Degerman

    2014-07-01

    Full Text Available We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs, islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.

  13. A STAT6 Intronic Single-Nucleotide Polymorphism is Associated with Clinical Malaria in Ghanaian Children

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    Daniel Amoako-Sakyi

    2016-01-01

    Full Text Available Malaria pathogenesis may be influenced by IgE responses and cytokine cross-regulation. Several mutations in the IL-4/STAT6 signaling pathway can alter cytokine cross-regulation and IgE responses during a Plasmodium falciparum malarial infection. This study investigated the relationship between a STAT6 intronic single-nucleotide polymorphism (rs3024974, total IgE, cytokines, and malaria severity in 238 Ghanaian children aged between 0.5 and 13 years. Total IgE and cytokine levels were measured by ELISA, while genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (RFLP. Compared with healthy controls, heterozygosity protected against clinical malaria: uncomplicated malaria (odds ratios [OR] = 0.13, P < 0.001, severe malarial anemia (OR = 0.18, P < 0.001, and cerebral malaria (OR = 0.39, P = 0.022. Levels of total IgE significantly differed among malaria phenotypes (P = 0.044 and rs3024974 genotypes (P = 0.037. Neither cytokine levels nor IL-6/IL-10 ratios were associated with malaria phenotypes or rs3024974 genotypes. This study suggests a role for rs3024974 in malaria pathogenesis and offers further insights into an IL-4/STAT6 pathway mutation in malaria pathogenesis.

  14. Phylogenetic inferences in Avena based on analysis of FL intron2 sequences.

    Science.gov (United States)

    Peng, Yuan-Ying; Wei, Yu-Ming; Baum, Bernard R; Yan, Ze-Hong; Lan, Xiu-Jin; Dai, Shou-Fen; Zheng, You-Liang

    2010-09-01

    The development and application of molecular methods in oats has been relatively slow compared with other crops. Results from the previous analyses have left many questions concerning species evolutionary relationships unanswered, especially regarding the origins of the B and D genomes, which are only known to be present in polyploid oat species. To investigate the species and genome relationships in genus Avena, among 13 diploid (A and C genomes), we used the second intron of the nuclear gene FLORICAULA/LEAFY (FL int2) in seven tetraploid (AB and AC genomes), and five hexaploid (ACD genome) species. The Avena FL int2 is rather long, and high levels of variation in length and sequence composition were found. Evidence for more than one copy of the FL int2 sequence was obtained for both the A and C genome groups, and the degree of divergence of the A genome copies was greater than that observed within the C genome sequences. Phylogenetic analysis of the FL int2 sequences resulted in topologies that contained four major groups; these groups reemphasize the major genomic divergence between the A and C genomes, and the close relationship among the A, B, and D genomes. However, the D genome in hexaploids more likely originated from a C genome diploid rather than the generally believed A genome, and the C genome diploid A. clauda may have played an important role in the origination of both the C and D genome in polyploids.

  15. DNA methylation in an intron of the IBM1 histone demethylase gene stabilizes chromatin modification patterns.

    Science.gov (United States)

    Rigal, Mélanie; Kevei, Zoltán; Pélissier, Thierry; Mathieu, Olivier

    2012-06-29

    The stability of epigenetic patterns is critical for genome integrity and gene expression. This highly coordinated process involves interrelated positive and negative regulators that impact distinct epigenetic marks, including DNA methylation and dimethylation at histone H3 lysine 9 (H3K9me2). In Arabidopsis, mutations in the DNA methyltransferase MET1, which maintains CG methylation, result in aberrant patterns of other epigenetic marks, including ectopic non-CG methylation and the relocation of H3K9me2 from heterochromatin into gene-rich chromosome regions. Here, we show that the expression of the H3K9 demethylase IBM1 (increase in BONSAI methylation 1) requires DNA methylation. Surprisingly, the regulatory methylated region is contained in an unusually large intron that is conserved in IBM1 orthologues. The re-establishment of IBM1 expression in met1 mutants restored the wild-type H3K9me2 nuclear patterns, non-CG DNA methylation and transcriptional patterns at selected loci, which included DNA demethylase genes. These results provide a mechanistic explanation for long-standing puzzling observations in met1 mutants and reveal yet another layer of control in the interplay between DNA methylation and histone modification, which stabilizes DNA methylation patterns at genes.

  16. Deep intronic variants introduce DMD pseudoexon in patient with muscular dystrophy.

    Science.gov (United States)

    Zaum, Ann-Kathrin; Stüve, Burkhard; Gehrig, Andrea; Kölbel, Heike; Schara, Ulrike; Kress, Wolfram; Rost, Simone

    2017-07-01

    Dystrophinopathies are X-linked muscle diseases caused by mutations in the large DMD gene. The most common mutations are detected by standard diagnostic techniques. However, some patients remain without detectable mutation, most likely due to changes in the non-coding sequence. We report on a boy with complete absence of dystrophin in muscle biopsy but no causative mutation according to standard diagnostics. To search for deep intronic variations (DIV) in the DMD gene we isolated mRNA from muscle tissue and amplified overlapping cDNA fragments using RT-PCR. One cDNA product revealed an augmented fragment size showing an insertion of 77 bp between the exons 7 and 8 by sequencing. We sequenced the flanking sequences of gDNA and found two hemizygous single nucleotide variants (c.650-39575 A>C and c.650-39498 A>G) surrounding the inserted fragment. Both variants create cryptic splice sites which initiate the formation of a pseudoexon that produces a frameshift in the DMD gene. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Identification of Six Introns in a Partial Sequence of Echinococcus granulosus Paramyosin Gene.

    Science.gov (United States)

    Esmaelizad, Majid; Ramezan, Atefeh; Razmaraii, Nasser; Mirjalili, Ali

    2015-03-01

    Paramyosin is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses. In this study, we attempted to characterize the noncoding sequence of the paramyosin gene. Genomic DNA was isolated from G1 Iranian hydatid cysts. A DNA fragment of 3200 bp in length was amplified from the paramyosin gene. The polymerase chain reaction (PCR) product was cloned to the pTZ57T vector and sequenced by M13 primers and then compared with unique cDNA coding sequences of E. granulosus (Z21787) and Taenia solium (AY034087). Six introns I (107 bp), II (75 bp), III (47 bp), IV (921 bp), V (19 bp), and VI (456 bp) were identified in the partial sequence of the paramyosin gene. Some nucleotide changes were observed in three exons I, IV, and VI. This data could help scientists in better understanding the possible alternative splicing and designing a real-time PCR technique for the evaluation of the transcription levels of paramyosin in the stages of the Echinococcus sp. life cycle.

  18. Becker muscular dystrophy due to an intronic splicing mutation inducing a dual dystrophin transcript.

    Science.gov (United States)

    Todeschini, Alice; Gualandi, Francesca; Trabanelli, Cecilia; Armaroli, Annarita; Ravani, Anna; Fanin, Marina; Rota, Silvia; Bello, Luca; Ferlini, Alessandra; Pegoraro, Elena; Padovani, Alessandro; Filosto, Massimiliano

    2016-10-01

    We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and "BRCA-Like" Status, in Both Blood and Tumour DNA.

    Directory of Open Access Journals (Sweden)

    Sarah L Daniels

    Full Text Available Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007. The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively. Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively. This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies.

  20. Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant.

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    Fan Wu

    Full Text Available In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2. Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene's structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol (7dC, but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.

  1. Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis.

    Science.gov (United States)

    Romagnolo, Donato F; Papoutsis, Andreas J; Laukaitis, Christina; Selmin, Ornella I

    2015-12-29

    Only 5-10% of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95% of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. We evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague-Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)α-negative sporadic UACC-3199 and ERα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist α-naphthoflavone (αNF) modulated BRCA-1 and ERα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples. Mammary tumors induced with DMBA had reduced BRCA-1 and ERα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with αNF rescued BRCA-1 and ERα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, αNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERα. TNBC exhibited increased basal AhR and BRCA-1

  2. 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

  3. Deep intronic mis-splicing mutation in JAK3 gene underlies T-B+NK- severe combined immunodeficiency phenotype.

    Science.gov (United States)

    Stepensky, Polina; Keller, Baerbel; Shamriz, Oded; NaserEddin, Adeeb; Rumman, Nisreen; Weintraub, Michael; Warnatz, Klaus; Elpeleg, Orly; Barak, Yaacov

    2016-02-01

    Severe combined immune deficiency (SCID) is a group of genetically heterogeneous diseases caused by an early block in T cell differentiation and present with life threatening infections, often within the first year of life. Janus kinase (JAK)3 gene mutations have been found to cause autosomal recessive T-B+ SCID phenotype. In this study we describe three patients with a novel deep intronic mis-splicing mutation in JAK3 as a cause of T-B+NK- SCID highlighting the need for careful evaluation of intronic regulatory elements of known genes associated with clearly defined clinical phenotypes. We present the cases and discuss the current literature. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Gene encoding the human. beta. -hexosaminidase. beta. chain: Extensive homology of intron placement in the. alpha. - and. beta. -chain genes

    Energy Technology Data Exchange (ETDEWEB)

    Proia, R.L. (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD (USA))

    1988-03-01

    Lysosomal {beta}-hexosaminidase is composed of two structurally similar chains, {alpha} and {beta}, that are the products of different genes. Mutations in either gene causing {beta}-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the {alpha} and {beta} chains, the {beta}-chain gene was isolated, and its organization was characterized. The {beta}-chain coding region is divided into 14 exons distributed over {approx}40 kilobases of DNA. Comparison with the {alpha}-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions. This extensive sharing of intron placement demonstrates that the {alpha} and {beta} chains evolved by way of the duplication of a common ancestor.

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

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    Cayla M Johnson

    Full Text Available 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. Genomic organization of the mouse src gene. Sequencing of src introns revealed a new chromosome 2 microsatellite marker

    Czech Academy of Sciences Publication Activity Database

    Fučík, Vladimír; Beran, Jaroslav; Černý, Zbyněk; Mácha, J.; Jonák, Jiří

    2002-01-01

    Roč. 48, č. 1 (2002), s. 34-39 ISSN 0015-5500 R&D Projects: GA ČR GV312/96/K205; GA AV ČR IPP2052002 Institutional research plan: CEZ:AV0Z5052915 Keywords : Mus musculus, c-src introns, microsatellite Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.615, year: 2002

  7. Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera : Culicidae)

    OpenAIRE

    Lardeux, Frédéric; Aliaga, Claudia; Tejerina, Rosenka; Ursic-Bedoya, Raul

    2012-01-01

    International audience; Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged ...

  8. 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.

  9. 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.

  10. Single dose CpG immunization protects against a heterosubtypic challenge and generates antigen specific memory T cells

    Directory of Open Access Journals (Sweden)

    Alexander eVogel

    2015-06-01

    Full Text Available Despite extensive research, influenza A virus (IAV remains a major cause of morbidity, mortality, and healthcare expenditure. Emerging pandemics from highly pathogenic IAV strains such as H5N1 and pandemic H1N1 highlight the need for universal, cross-protective vaccines. Current vaccine formulations generate strain-specific neutralizing antibodies primarily against the outer coat proteins hemagglutinin and neuraminidase. In contrast to these highly mutable proteins, internal proteins of IAV are more conserved and are a favorable target for developing vaccines that induce strong T cell responses in addition to humoral immunity. Here, we found that intranasal administration with a single dose of CpG and inactivated x31 (H3N2 reduced viral titers and partially protected mice from a heterosubtypic challenge with a lethal dose of PR8 (H1N1. Early after immunization, vaccinated mice showed increased innate immune activation with high levels of MHCII and CD86 expression on dendritic cells in both the draining lymph nodes and lungs. Three days after immunization, CD4 and CD8 cells in the lung upregulated CD69, suggesting that activated lymphocytes are present at the site of vaccine administration. The ensuing effector Th1 responses were capable of producing multiple cytokines and were present at least 30 days after immunization. Furthermore, functional memory responses were observed, as antigen specific IFN-γ+ and GrB+ cells were detected early after lethal infection. Together, this work provides evidence for using pattern recognition receptor agonists as a mucosal vaccine platform for inducing robust T cell responses capable of protecting against heterologous IAV challenges.

  11. Rare k-mer DNA: Identification of sequence motifs and prediction of CpG island and promoter.

    Science.gov (United States)

    Mohamed Hashim, Ezzeddin Kamil; Abdullah, Rosni

    2015-12-21

    Empirical analysis on k-mer DNA has been proven as an effective tool in finding unique patterns in DNA sequences which can lead to the discovery of potential sequence motifs. In an extensive study of empirical k-mer DNA on hundreds of organisms, the researchers found unique multi-modal k-mer spectra occur in the genomes of organisms from the tetrapod clade only which includes all mammals. The multi-modality is caused by the formation of the two lowest modes where k-mers under them are referred as the rare k-mers. The suppression of the two lowest modes (or the rare k-mers) can be attributed to the CG dinucleotide inclusions in them. Apart from that, the rare k-mers are selectively distributed in certain genomic features of CpG Island (CGI), promoter, 5' UTR, and exon. We correlated the rare k-mers with hundreds of annotated features using several bioinformatic tools, performed further intrinsic rare k-mer analyses within the correlated features, and modeled the elucidated rare k-mer clustering feature into a classifier to predict the correlated CGI and promoter features. Our correlation results show that rare k-mers are highly associated with several annotated features of CGI, promoter, 5' UTR, and open chromatin regions. Our intrinsic results show that rare k-mers have several unique topological, compositional, and clustering properties in CGI and promoter features. Finally, the performances of our RWC (rare-word clustering) method in predicting the CGI and promoter features are ranked among the top three, in eight of the CGI and promoter evaluations, among eight of the benchmarked datasets. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Transitions at CpG dinucleotides, geographic clustering of TP53 mutations and food availability patterns in colorectal cancer.

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    Fabio Verginelli

    Full Text Available BACKGROUND: Colorectal cancer is mainly attributed to diet, but the role exerted by foods remains unclear because involved factors are extremely complex. Geography substantially impacts on foods. Correlations between international variation in colorectal cancer-associated mutation patterns and food availabilities could highlight the influence of foods on colorectal mutagenesis. METHODOLOGY: To test such hypothesis, we applied techniques based on hierarchical clustering, feature extraction and selection, and statistical pattern recognition to the analysis of 2,572 colorectal cancer-associated TP53 mutations from 12 countries/geographic areas. For food availabilities, we relied on data extracted from the Food Balance Sheets of the Food and Agriculture Organization of the United Nations. Dendrograms for mutation sites, mutation types and food patterns were constructed through Ward's hierarchical clustering algorithm and their stability was assessed evaluating silhouette values. Feature selection used entropy-based measures for similarity between clusterings, combined with principal component analysis by exhaustive and heuristic approaches. CONCLUSION/SIGNIFICANCE: Mutations clustered in two major geographic groups, one including only Western countries, the other Asia and parts of Europe. This was determined by variation in the frequency of transitions at CpGs, the most common mutation type. Higher frequencies of transitions at CpGs in the cluster that included only Western countries mainly reflected higher frequencies of mutations at CpG codons 175, 248 and 273, the three major TP53 hotspots. Pearson's correlation scores, computed between the principal components of the datamatrices for mutation types, food availability and mutation sites, demonstrated statistically significant correlations between transitions at CpGs and both mutation sites and availabilities of meat, milk, sweeteners and animal fats, the energy-dense foods at the basis of

  13. Epigenetic silencing of BTB and CNC homology 2 and concerted promoter CpG methylation in gastric cancer.

    Science.gov (United States)

    Haam, Keeok; Kim, Hee-Jin; Lee, Kyung-Tae; Kim, Jeong-Hwan; Kim, Mirang; Kim, Seon-Young; Noh, Seung-Moo; Song, Kyu-Sang; Kim, Yong Sung

    2014-09-01

    BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription factor with a prominent role in B-cell development. Genetic polymorphisms within a single locus encoding BACH2 are associated with various autoimmune diseases and allergies. In this study, restriction landmark genomic scanning revealed methylation at a NotI site in a CpG island covering the BACH2 promoter in gastric cancer cell lines and primary gastric tumors. Increased methylation of the BACH2 promoter was observed in 52% (43/83) of primary gastric tumors, and BACH2 hypermethylation was significantly associated with decreased gene expression. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin. A restored BACH2 expression in BACH2-silenced gastric cancer cell lines, and knockdown of BACH2 using short hairpin RNA (i.e. RNA interference) increased cell proliferation in gastric cancer cells. Clinicopathologic data showed that decreased BACH2 expression occurred significantly more frequently in intestinal-type (27/44, 61%) compared with diffuse-type (13/50, 26%) gastric cancers (P<0.001). Furthermore, BACH2 promoter methylation paralleled that of previously identified targets, such as LRRC3B, LIMS2, PRKD1 and POPDC3, in a given set of gastric tumors. We propose that concerted methylation in many promoters plays a role in accelerating gastric tumor formation and that methylated promoter loci may be targets for therapeutic treatment, such as the recently introduced technique of epigenetic editing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. IGFBP3 Promoter Methylation in Colorectal Cancer: Relationship with Microsatellite Instability, CpG Island Methylator Phenotype, p53

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    Takako Kawasaki

    2007-12-01

    Full Text Available Insulin-like growth factor binding protein 3 (IGFBP3, which is induced by wild-type p53, regulates IGF and interacts with the TGF-β pathway. IGFBP3 promoter methylation may occur in colorectal cancer with or without the CpG island methylator phenotype (CIMP, which is associated with microsatellite instability (MSI and TGFBR2 mutation. We examined the relationship between IGFBP3 methylation, p53 expression, CIMP and MSI in 902 population-based colorectal cancers. Utilizing real-time PCR (MethyLight, we quantified promoter methylation in IGFBP3 and eight other CIMP-high-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1. IGFBP3 methylation was far more frequent in non-MSI-high CIMP-high tumors (85% = 35/41 than in MSI-high CIMPhigh (49% = 44/90, P < .0001, MSI-high non-CIMP-high (17% = 6/36, P < .0001, non-MSI-high non-CIMP-high tumors (22% = 152/680, P < .0001. Among CIMPhigh tumors, the inverse relationship between MSI and IGFBP3 methylation persisted in p53-negative tumors (P < .0001, but not in p53-positive tumors. IGFBP3 methylation was associated inversely with TGFBR2 mutation in MSI-high non-CIMP-high tumors (P = .02. In conclusion, IGFBP3 methylation is inversely associated with MSI in CIMP-high colorectal cancers, this relationship is limited to p53-negative tumors. Our data suggest complex relationship between global genomic/epigenomic phenomena (such as MSI/ CIMP, single molecular events (e.g., IGFBP3 methylation, TP53 mutation, TGFBR2 mutation, the related pathways.

  15. Aging of blood can be tracked by DNA methylation changes at just three CpG sites.

    Science.gov (United States)

    Weidner, Carola Ingrid; Lin, Qiong; Koch, Carmen Maike; Eisele, Lewin; Beier, Fabian; Ziegler, Patrick; Bauerschlag, Dirk Olaf; Jöckel, Karl-Heinz; Erbel, Raimund; Mühleisen, Thomas Walter; Zenke, Martin; Brümmendorf, Tim Henrik; Wagner, Wolfgang

    2014-02-03

    Human aging is associated with DNA methylation changes at specific sites in the genome. These epigenetic modifications may be used to track donor age for forensic analysis or to estimate biological age. We perform a comprehensive analysis of methylation profiles to narrow down 102 age-related CpG sites in blood. We demonstrate that most of these age-associated methylation changes are reversed in induced pluripotent stem cells (iPSCs). Methylation levels at three age-related CpGs--located in the genes ITGA2B, ASPA and PDE4C--were subsequently analyzed by bisulfite pyrosequencing of 151 blood samples. This epigenetic aging signature facilitates age predictions with a mean absolute deviation from chronological age of less than 5 years. This precision is higher than age predictions based on telomere length. Variation of age predictions correlates moderately with clinical and lifestyle parameters supporting the notion that age-associated methylation changes are associated more with biological age than with chronological age. Furthermore, patients with acquired aplastic anemia or dyskeratosis congenita--two diseases associated with progressive bone marrow failure and severe telomere attrition--are predicted to be prematurely aged. Our epigenetic aging signature provides a simple biomarker to estimate the state of aging in blood. Age-associated DNA methylation changes are counteracted in iPSCs. On the other hand, over-estimation of chronological age in bone marrow failure syndromes is indicative for exhaustion of the hematopoietic cell pool. Thus, epigenetic changes upon aging seem to reflect biological aging of blood.

  16. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

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    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  17. [Whole exon 5 and intron 5 replaced by RHD/CE in partial D phenotype DVa (Hus)].

    Science.gov (United States)

    Zhou, Yi-Yan; Xiong, Wen; Shao, Chao-Peng

    2005-02-01

    The study was purposed to analyze DNA and allele structure of the partial D phenotypes D(Va) and D(VI) of the Rhesus blood group in Chinese. Through polymerase chain reaction (PCR) and direct genomic DNA sequencing, the RHD gene was detected in three weak D individuals identified serologically. The results showed that among the three weak D individuals, one was identified as partial D phenotype D(Va) (Hus) type and genotyped DccEe; another two were testified as D(VI) III type and genotyped DCcee. Moreover, the breakpoints of the replaced region by RHCE in D(Va) (Hus) were 5' end of the exon 5 and 3' end of the intron 5, and there were 7 novel polymorphisms in intron 5: 23-25(GCA)2, 98G>A, 168-169insG, 205-206insT, 494-495insA, 1256-1257insC, 1347G>T. In conclusion the whole exon 5 and intron 5 are replaced by RHCE in D(Va) (Hus) detected in Chinese.

  18. 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.

  19. Why Selection Might Be Stronger When Populations Are Small: Intron Size and Density Predict within and between-Species Usage of Exonic Splice Associated cis-Motifs

    Science.gov (United States)

    Wu, XianMing; Hurst, Laurence D.

    2015-01-01

    The nearly neutral theory predicts that small effective population size provides the conditions for weakened selection. This is postulated to explain why our genome is more “bloated” than that of, for example, yeast, ours having large introns and large intergene spacer. If a bloated genome is also an error prone genome might it, however, be the case that selection for error-mitigating properties is stronger in our genome? We examine this notion using splicing as an exemplar, not least because large introns can predispose to noisy splicing. We thus ask whether, owing to genomic decay, selection for splice error-control mechanisms is stronger, not weaker, in species with large introns and small populations. In humans much information defining splice sites is in cis-exonic motifs, most notably exonic splice enhancers (ESEs). These act as splice-error control elements. Here then we ask whether within and between-species intron size is a predictor of the commonality of exonic cis-splicing motifs. We show that, as predicted, the proportion of synonymous sites that are ESE-associated and under selection in humans is weakly positively correlated with the size of the flanking intron. In a phylogenetically controlled framework, we observe, also as expected, that mean intron size is both predicted by Ne.μ and is a good predictor of cis-motif usage across species, this usage coevolving with splice site definition. Unexpectedly, however, across taxa intron density is a better predictor of cis-motif usage than intron size. We propose that selection for splice-related motifs is driven by a need to avoid decoy splice sites that will be more common in genes with many and large introns. That intron number and density predict ESE usage within human genes is consistent with this, as is the finding of intragenic heterogeneity in ESE density. As intronic content and splice site usage across species is also well predicted by Ne.μ, the result also suggests an unusual circumstance in

  20. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly

    OpenAIRE

    Haseloff, Jim; Siemering, Kirby R.; Prasher, Douglas C.; Hodge, Sarah

    1997-01-01

    The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains seq...

  1. [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.

  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. RNA-Seq Analysis of Differential Splice Junction Usage and Intron Retentions by DEXSeq.

    Directory of Open Access Journals (Sweden)

    Yafang Li

    Full Text Available Alternative splicing is an important biological process in the generation of multiple functional transcripts from the same genomic sequences. Differential analysis of splice junctions (SJs and intron retentions (IRs is helpful in the detection of alternative splicing events. In this study, we conducted differential analysis of SJs and IRs by use of DEXSeq, a Bioconductor package originally designed for differential exon usage analysis in RNA-seq data analysis. We set up an analysis pipeline including mapping of RNA-seq reads, the preparation of count tables of SJs and IRs as the input files, and the differential analysis in DEXSeq. We analyzed the public RNA-seq datasets generated from RNAi experiments on Drosophila melanogaster S2-DRSC cells to deplete RNA-binding proteins (GSE18508. The analysis confirmed previous findings on the alternative splicing of the trol and Ant2 (sesB genes in the CG8144 (ps-depletion experiment and identified some new alternative splicing events in other RNAi experiments. We also identified IRs that were confirmed in our SJ analysis. The proposed method used in our study can output the genomic coordinates of differentially used SJs and thus enable sequence motif search. Sequence motif search and gene function annotation analysis helped us infer the underlying mechanism in alternative splicing events. To further evaluate this method, we also applied the method to public RNA-seq data from human breast cancer (GSE45419 and the plant Arabidopsis (SRP008262. In conclusion, our study showed that DEXSeq can be adapted to differential analysis of SJs and IRs, which will facilitate the identification of alternative splicing events and provide insights into the molecular mechanisms of transcription processes and disease development.

  4. Characterization of the mouse cyclin D3 gene: Exon/intron organization and promoter activity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhengyu; Zhang, Ying; Ravid, K. [Boston Univ. School of Medicine, Boston, MA (United States)] [and others

    1996-07-01

    The three D-type cyclins have been shown to be differentially expressed in a number of cell types, suggesting that they play distinct roles in cell cycle regulation in particular cell lineages. We have determined the complete nucleotide sequence (-1681 to +6582) of the mouse cyclin D3 gene, which encodes a G1 phase cyclin. The gene consists of five exons and four introns, varying in length from 422 to 2472 bp. Primer extension analysis revealed one major transcription initiation site at the position 107 bp 5{prime} upstream of the translation start. The promoter region lacks both canonical {open_quotes}TATA{close_quotes} and {open_quotes}CAAT{close_quotes} boxes. It contains, however, multiple transcription factor recognition by GATA, NF-{kappa}B, ATF, E2F, and TRE/AP1 transcription factors, E box binding myogenic factors, and the IL-6 induced-transcription factor, APRF. Promoter activity of the 1681-bp fragment upstream of the transcription initiation site was confirmed by linking it to a reporter gene and subjecting it to transient expression experiments in various cell types. Promoter activity was high in cell lines that expressed high levels of endogenous D3 mRNA, as indicated by Northern blot analysis, and was significantly reduced when the promoter was truncated to -122 bp. The characterization of the mouse cyclin D3 gene and insight into its promoter region will allow further studies defining the molecular events regulating the expression of this cyclin in proliferating and quiescent cells. 60 refs., 4 figs., 1 tab.

  5. Effect of sublingual administration with a native or denatured protein allergen and adjuvant CpG oligodeoxynucleotides or cholera toxin on systemic T(H)2 immune responses and mucosal immunity in mice.

    Science.gov (United States)

    Huang, Ching-Feng; Wang, Chih-Chien; Wu, Tzee-Chung; Chu, Chia-Hsiang; Peng, Ho-Jen

    2007-11-01

    Sublingual immunotherapy has been recently used for allergic diseases, but its mechanisms are still unclear. To examine the effect of sublingual administration of a native or denatured allergen alone or plus adjuvant on systemic T(H)2 responses and mucosal immunity in mice. Naive or sensitized BALB/c mice were sublingually vaccinated biweekly for 3 weeks with ovalbumin (OVA) or urea-denatured OVA (CM-OVA) only or plus adjuvant CpG oligodeoxynucleotides (CpG) or cholera toxin (CT). Two weeks later, their specific serum IgG, IgG1, IgG2a, IgE, and saliva secretory IgA (SIgA) antibody responses and the cytokine profiles of spleen and cervical lymph node cells were investigated. Specific SIgA antibody responses were induced by vaccination with CM-OVA plus CpG or CT. Whereas vaccination with CM-OVA and CpG enhanced T(H)1 responses but inhibited IgE production, vaccination with CT and CM-OVA or OVA increased cervical lymph node cell production of interleukin (IL) 4, IL-5, and IL-6 and serum IgG1 antibody responses. In previously sensitized mice, sublingual vaccination with OVA or CM-OVA plus CT or CpG stimulated mucosal SIgA antibody responses, but did not enhance ongoing IgE antibody responses. Sublingual vaccination with OVA or CM-OVA plus adjuvant CT or CpG all can induce systemic and mucosal immunity, but CM-OVA plus CpG had the best prophylactic and therapeutic effects on IgE antibody production. It is likely that sublingual vaccines may have a role for the prophylaxis and immunotherapy of allergic reactions.

  6. Human Vav1 expression in hematopoietic and cancer cell lines is regulated by c-Myb and by CpG methylation.

    Directory of Open Access Journals (Sweden)

    Lena Ilan

    Full Text Available Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA. Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5' regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

  7. Efficient in vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naive prostate cancer patients.

    Science.gov (United States)

    Karbach, Julia; Neumann, Antje; Atmaca, Akin; Wahle, Claudia; Brand, Kathrin; von Boehmer, Lotta; Knuth, Alexander; Bender, Armin; Ritter, Gerd; Old, Lloyd J; Jäger, Elke

    2011-02-15

    NY-ESO-1, one of the most immunogenic tumor antigens, is expressed in 15% to 25% of metastatic prostate cancers. The immunological and clinical effects of vaccination with recombinant NY-ESO-1 protein combined with CpG as adjuvant were evaluated. In a phase I clinical study, patients with advanced prostate cancer were vaccinated with recombinant NY-ESO-1 protein (100 μg) mixed with CpG 7909 (2.5 mg) every 3 weeks intradermally for 4 doses. Objectives of the study were the safety of the vaccine and changes of specific humoral and cellular immunological responses to NY-ESO-1 in relation to detectable NY-ESO-1 expression in the individual tumor. All 12 baseline sero-negative patients developed high-titer NY-ESO-1 antibody responses. B-cell epitope mapping identified NY-ESO-1 p91-110 to be recognized most frequently by vaccine-induced antibodies. Two patients developed significant antibody titers against the adjuvant CpG. NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were induced in 9 patients (69%). Five of these 9 patients did not express NY-ESO-1 in the autologous tumor. Postvaccine CD8+ T-cell clones recognized and lyzed HLA-matched tumor cell lines in an antigen-specific manner. Our data provide clear evidence for the capacity of NY-ESO-1 protein/CpG vaccine to induce integrated antigen-specific immune responses in vivo and to efficiently prime CD8+ T-cell responses in NY-ESO-1 antigen-negative patients. Our results may also support further clinical vaccination protocols with NY-ESO-1 protein not only focused on the treatment of existing cancer, but also to prevent further development of NY-ESO-1 positive cancers in vivo. ©2010 AACR.

  8. Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

    Science.gov (United States)

    How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

    2015-07-01

    Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

  9. Prognostic value of CpG island methylator phenotype among colorectal cancer patients: a systematic review and meta-analysis.

    Science.gov (United States)

    Juo, Y Y; Johnston, F M; Zhang, D Y; Juo, H H; Wang, H; Pappou, E P; Yu, T; Easwaran, H; Baylin, S; van Engeland, M; Ahuja, N

    2014-12-01

    Divergent findings regarding the prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) patients exist in current literature. We aim to review data from published studies in order to examine the association between CIMP and CRC prognosis. A comprehensive search for studies reporting disease-free survival (DFS), overall survival (OS), or cancer-specific mortality of CRC patients stratified by CIMP is carried out. Study findings are summarized descriptively and quantitatively, using adjusted hazard ratios (HRs) as summary statistics. Thirty-three studies reporting survival in 10 635 patients are included for review. Nineteen studies provide data suitable for meta-analysis. The definition of CIMP regarding gene panel, marker threshold, and laboratory method varies across studies. Pooled analysis shows that CIMP is significantly associated with shorter DFS (pooled HR estimate 1.45; 95% confidence interval (CI) 1.07-1.97, Q = 3.95, I(2) = 0%) and OS (pooled HR estimate 1.43; 95% CI 1.18-1.73, Q = 4.03, I(2) = 0%) among CRC patients irrespective of microsatellite instability (MSI) status. Subgroup analysis of microsatellite stable (MSS) CRC patients also shows significant association between shorter OS (pooled HR estimate 1.37; 95% CI 1.12-1.68, Q = 4.45, I(2) = 33%) and CIMP. Seven studies have explored CIMP's value as a predictive factor on stage II and III CRC patient's DFS after receiving adjuvant 5-fluorouracil (5-FU) therapy: of these, four studies showed that adjuvant chemotherapy conferred a DFS benefit among CIMP(+) patients, one concluded to the contrary, and two found no significant correlation. Insufficient data was present for statistical synthesis of CIMP's predictive value among CRC patients receiving adjuvant 5-FU therapy. CIMP is independently associated with significantly worse prognosis in CRC patients. However, CIMP's value as a predictive factor in assessing whether adjuvant 5-FU therapy will confer additional survival

  10. 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)

    Milstein, Daniela; Oliveira, Mariana C; Martins, Felipe M; Matioli, Sergio R

    2008-11-07

    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. 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. 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 do not refrain intron self

  11. 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

  12. 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

  13. 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.

  14. Vliv imunizace a CPG oligodeoxynukleotidu jako adjuvans na oxidativní vzplanutí fagocytů u myší

    Czech Academy of Sciences Publication Activity Database

    Dušková, M.; Hubinka, V.; Jelínková, P.; Lojek, Antonín; Žákovská, A.; Potyšová, S.

    2006-01-01

    Roč. 8, č. 2 (2006), s. 30-30 ISSN 1212-3536. [Sjezd českých a slovenských alergologů a klinických imunologů /23./, Kongres českých a slovenských imunologů /11./. 25.10.2006-28.10.2006, Hradec Králové] Institutional research plan: CEZ:AV0Z50040507 Keywords : immunization * CpG oligodeoxynucleotide * phagocytes Subject RIV: BO - Biophysics

  15. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    Directory of Open Access Journals (Sweden)

    Kim Kyu-Tae

    2010-07-01

    Full Text Available Abstract Background DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC and Trichostatin A (TSA respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Method Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC. Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Results Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS. In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. Conclusion These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically

  16. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

    International Nuclear Information System (INIS)

    Mossman, David; Kim, Kyu-Tae; Scott, Rodney J

    2010-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes. Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR. Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated. These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of

  17. TLR9 ligand (CpG oligodeoxynucleotide induces CLL B-cells to differentiate into CD20+ antibody-secreting cells

    Directory of Open Access Journals (Sweden)

    Hussein eGhamlouch

    2014-06-01

    Full Text Available B-cell chronic lymphocytic leukemia (CLL is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5+ mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG. The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs. CD20+CD38− CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c in a two-step, seven-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19+CD20+CD27+CD38- ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA. Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1, IRF4 and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo class-switch recombination and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i identifying the normal counterpart of CLL B-cells and (ii developing novel treatment strategies in CLL.

  18. Polymorphism in the intron 20 of porcine O-linked N-acetylglucosamine transferase

    Directory of Open Access Journals (Sweden)

    Jong Gug Kim

    2017-08-01

    Full Text Available Objective O-linked N-acetylglucosamine (O-GlcNAc transferase (OGT catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone level and testes size. The objective of this study was to find the variations of OGT between European and Chinese pigs. Methods Pigs were tested initially for polymorphism in OGT among European and Chinese pigs by polymerase chain reaction and sequencing at the U.S. Meat Animal Research Center (USMARC. The polymorphism was also determined in an independent population of pigs including European and Chinese Meishan (ME breeds at the National Institute of Animal Science (NIAS, RDA, Korea. Results The intron 20 of OGT from European and Chinese pigs was 514 and 233 bp, respectively, in the pigs tested initially. They included 1 White composite (WC boar and 7 sows (2 Minzu×WC, 2 Duroc [DU]×WC, 2 ME×WC, 1 Fengzing×WC at USMARC. The 281-bp difference was due to an inserted 276-bp element and GACTT in European pigs. When additional WC and ME boars, the grandparents that were used to generate the 1/2ME×1/2WC parents, and the 84 boars of 16 litters from mating of 1/2ME×1/2WC parents were analyzed, the breeds of origin of X chromosome quantitative trait locus (QTL were confirmed. The polymorphism was determined in an independent population of pigs including DU, Landrace, Yorkshire, and ME breeds at NIAS. OGT was placed at position 67 cM on the chromosome X of the USMARC swine linkage map. Conclusion There was complete concordance with the insertion in European pigs at USMARC and NIAS. This polymorphism could be a useful marker to identify the breed of origin of X chromosome QTL in pigs produced by crossbreeding Chinese and European pigs.

  19. Mediatized Humanitarianism

    DEFF Research Database (Denmark)

    Vestergaard, Anne

    2014-01-01

    The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts to legiti......The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts...... legitimation by accountancy, legitimation by institutionalization, and legitimation by compensation. The analysis relates these changes to a problem of trust associated with mediatization through processes of mediation....

  20. Complete Chloroplast Genome of Medicinal Plant Lonicera japonica: Genome Rearrangement, Intron Gain and Loss, and Implications for Phylogenetic Studies

    Directory of Open Access Journals (Sweden)

    Liu He

    2017-02-01

    Full Text Available The complete chloroplast (cp genome of Lonicera japonica, a common ornamental and medicinal plant in North America and East Asia, was sequenced and analyzed. The length of the L. japonica cp genome is 155,078 bp, contains a pair of inverted repeat regions (IRa and IRb, of 23,774 bp each, as well as large (LSC, 88,858 bp and small (SSC, 18,672 bp single-copy regions. A total of 129 genes were identified in the cp genome, 16 of which were duplicated within the IR regions. Relative to other plant cp genomes, the L. japonica cp genome had a unique rearrangement between trnI-CAU and trnN-GUU. In L. japonica cpDNA, rps19, rpl2, and rpl23 move to the LSC region, from the IR region. The ycf1 pesudogene in the IR region is lost, and only one copy locates in the SSC region. Comparative cp DNA sequence analyses of L. japonica with other cp genomes reveal that the gene order, and the gene and intron contents, are slightly different. The introns in ycf2 and rps18 genes are found for the first time. Four genes (clpP, petB, petD, and rpl16 lost introns. However, its genome structure, GC content, and codon usage were similar to those of typical angiosperm cp genomes. All preferred synonymous codons were found to use codons ending with A/T. The AT-rich sequences were less abundant in the coding regions than in the non-coding ones. A phylogenetic analysis based on 71 protein-coding genes supported the idea that L. japonica is a sister of the Araliaceae species. This study identified unique characteristics of the L. japonica cp genome that contribute to our understanding of the cpDNA evolution. It offers valuable information for the phylogenetic and specific barcoding of this medicinal plant.

  1. Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles.

    Science.gov (United States)

    Vigentini, Ileana; De Lorenzis, Gabriella; Picozzi, Claudia; Imazio, Serena; Merico, Annamaria; Galafassi, Silvia; Piškur, Jure; Foschino, Roberto

    2012-06-15

    In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Single-dose protection against Plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron A.

    Science.gov (United States)

    Sridhar, S; Reyes-Sandoval, A; Draper, S J; Moore, A C; Gilbert, S C; Gao, G P; Wilson, J M; Hill, A V S

    2008-04-01

    Human adenovirus serotype 5 (AdH5) vector vaccines elicit strong immune responses to the encoded antigen and have been used in various disease models. We designed AdH5 vectors expressing antigen under the control of a human cytomegalovirus (HCMV) immediate-early promoter containing its intron A sequence. The transcriptional levels of antigen and immune responses to antigen for vectors with the HCMV promoter with the intron A sequence (LP) were greater than those for AdH5 vectors using the HCMV promoter sequence without intron A (SP). We compared an E1E3-deleted AdH5 adenoviral vector, which affords more space for insertion of foreign sequences, and showed it to be as immunogenic as an E1-deleted AdH5 vector. Neutralizing antibodies to AdH5 limit the efficacy of vaccines based on the AdH5 serotype, and simian adenoviral vectors offer an attractive option to overcome this problem. We constructed E1E3-deleted human and simian adenoviral vectors encoding the pre-erythrocytic-stage malarial antigen Plasmodium berghei circumsporozoite protein. We compared the immunogenicity and efficacy of AdC6, a recombinant simian adenovirus serotype 6 vector, in a murine malaria model to those of AdH5 and the poxviral vectors MVA and FP9. AdC6 induced sterile protection from a single dose in 90% of mice, in contrast to AdH5 (25%) and poxviral vectors MVA and FP9 (0%). Adenoviral vectors maintained potent CD8(+) T-cell responses for a longer period after immunization than did poxviral vectors and mainly induced an effector memory phenotype of cells. Significantly, AdC6 was able to maintain protection in the presence of preexisting immunity to AdH5.

  3. DETECTING PRESENCE OF C/T POLYMORPHISM AT POSITION 34 SECOND INTRON OF THE MYOSTATIN GENE IN RABBITS

    Directory of Open Access Journals (Sweden)

    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, exa