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Sample records for 18s rdna gene

  1. The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K.

    Wei, Wang; Hong-Lan, Yang; HuiFang, Bao; Daoyuan, Zhang; Qi-mu-ge, Shan; Woof, Andrew J

    2010-07-01

    In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml(-1) in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis. PMID:19731075

  2. Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

    Caroline Garcia

    2008-01-01

    Full Text Available Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG. The results from the application of the 18S probe confirmed the previous data obtained by silver nitrate staining, identifying a simple nucleolar organizing region system for these species. However, the labeling results from the 5S rDNA probe demonstrated a difference in the number and localization of these sites between the analyzed species. The obtained data allowed inferences on the possible processes involved in the karyotypic evolution of this genus.

  3. Localization of ribosomal genes in three Pimelodus species (Siluriformes, Pimelodidae) of the São Francisco River: 5S genes as species markers and conservation of the 18S rDNA sites

    Caroline Garcia; Orlando Moreira Filho

    2008-01-01

    Pimelodidae is one of the most representative of Neotropical catfish families. However, these fish are still poorly studied in terms of cytogenetics, especially regarding the application of more accurate techniques such as the chromosomal localization of ribosomal genes. In the present work, fluorescent in situ hybridization with 5S and 18S rDNA probes was employed for rDNA site mapping in Pimelodus sp., P. fur and P. maculatus from the São Francisco River in the Três Marias municipality - MG...

  4. Characterization of three different clusters of 18S-26S ribosomal DNA genes in the sea urchin P. lividus: Genetic and epigenetic regulation synchronous to 5S rDNA.

    Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

    2016-04-15

    We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. PMID:26789074

  5. Genetic Characterization of Nematodirella cameli Based on 18S rDNA and Cytochrome c Oxidase Subunit 1 (CO1

    Hassan SHARIFIYAZDI

    2015-01-01

    Full Text Available To determine the phylogenic position and genetic diversity of Nematodirella cameli two portions of nuclear ribosomal DNA, 18S rDNA and mitochondrial DNA gene, the subunit 1 of cytochrome C oxidase gene (CO1 were sequenced and compared with those previously reported for other nematodes in Trichostrongylina. The phylogenetic trees constructed based upon the 18S rDNA sequences, yielded strong support for close relationship between the N. cameli and Nematodirus battus, with a high bootstrap value of 100%. In the present research, the level of sequence polymorphism among N. cameli isolates was higher for CO1 with 32 polymorphic sites compared to 18S rDNA sequence. Accordingly, molecular assays based on CO1 mitochondrial marker, demonstrated the existence of at least 11 distinct haplotypes (accession nos. JX305966 to JX305976 with an intraspecific diversity of 3-7% in Iran. Whereas, all of N. cameli samples examined herein (n=11, had a unique 18S sequence (accession no. JX305977. In addition, N. cameli CO1 sequences found in this study showed maximum identities to Haemonchus (88% and Ostertagia (87% in BLAST analysis for existing Trichostrongylina sequences. Further information is necessary to infer interspecific and intraspecific phylogenetic relationships between genera and species in Trichostrongylina. This study describes for the first time the nuclear 18S rDNA and mitochondrial CO1 sequence data from Nematodirella cameli species.

  6. Identification of Chlorophyceae based on 18S rDNA sequences from Persian Gulf.

    Raheem Haddad

    2014-12-01

    Full Text Available Chlorophyceae are important constituents of marine phytoplankton. The taxonomy of Chlorophyceae was traditionally based solely on morphological characteristics. In the present research project, genetic diversity was investigated to analyze five species of Chlorophyceae from waters of the Persian Gulf.A clone library of the ribosomal small subunit RNA gene (18S rDNA in the nuclear genome was constructed by PCR, and then, after examining the clones, selected clones were sequenced. The determined clone sequences were analyzed by a similarity search of the NCBI GenBank database using BLAST.Eleven sequences were identified correctly and used for phylogenetic analysis. We identified species of Chlorophyta (Chlorella sorokiniana, Chlamydomonas sp., Neochloris aquatic, Picochlorum sp. and Nannochloris atomus without the need to conduct extensive colony isolation techniques. Therefore, this improved molecular method can be used to generate a robust database describing the species diversity of environmental samples.

  7. 18S rDNA dataset profiling microeukaryotic populations within Chicago area nearshore waters

    Daniel Searle

    2016-03-01

    Full Text Available Despite their critical role in the aquatic food web and nutrient cycling, microeukaryotes within freshwater environments are under-studied. Herein we present the first high-throughput molecular survey of microeukaryotes within Lake Michigan. Every two weeks from May 13 to August 5, 2014, we collected surface water samples from the nearshore waters of four Chicago area beaches: Gillson Park, Montrose Beach, 57th Street Beach, and Calumet Beach. Four biological replicates were collected for each sampling date and location, resulting in 112 samples. Eighty-nine of these samples were surveyed through targeted sequencing of the V7 and V8 regions of the 18S rDNA gene. Both technical and biological replicates were sequenced and are included in this dataset. Raw sequence data is available via NCBI’s SRA database (BioProject PRJNA294919.

  8. Assessment of helminth biodiversity in wild rats using 18S rDNA based metagenomics.

    Ryusei Tanaka

    Full Text Available Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.

  9. Molecular characterization of 18S rDNA partial sequence in Microcosmus (Stolidobranchiata, Pyuridae

    D. FULGIONE

    2012-12-01

    Full Text Available We present a 18S rDNA based molecular phylogeny of two species of the genus Microcosmus (M. sulcatus and M. claudicans sampled in the Mediterranean, to investigate their phylogenetic position relative to species of the order Stolidobranchiata. The analysis is based on partial sequences (739 bp of the 18S rDNA. Among the 18 variable sites found between the two species, 4 correspond to transitions (ts, 14 to transversions (tv and 4 to deletions/insertions. In the considered Stolidobranchiata, we found 4.3% overall mean number of nucleotide differences and 0.06 (S.E. ±0.01 Kimura 2-parameter distance. The mean number of nucleotide differences between Microcosmus spp. and other Stolidobranchiata species was of 6% and 0.08 (S.E. ±0.01 Kimura 2-parameter distance. A molecular phylogeny obtained by Maximum Parsimony corroborates results of the traditional taxonomy.

  10. Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbcL and 18S rDNA sequence data

    LIN Zhongheng; SHEN Songdong; CHEN Weizhou; LI Huihui

    2013-01-01

    Chlorophyta species are common in the southern and northern coastal areas of China.In recent years,frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists.In this paper,we sequenced the 18S rDNA genes,the internal transcribed spacer (ITS) regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences,1 377-1 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences.The GC base pair content was highest in the ITS regions and lowest in the rbcL genes.The sequencing results showed that the three Ulvaprolifera (or U.pertusa) gene sequences from Qingdao and Nan'ao Island were identical.The ITS,18S rDNA and rbcL genes in U.prolifera and U.pertusa from different sea areas in China were unchanged by geographic distance.U.flexuosa had the least evolutionary distance from U.californica in both the ITS regions (0.009) and the 18S rDNA (0.002).These data verified that Ulva and Enteromorpha are not separate genera.

  11. Identification of a potential fungal species by 18S rDNA for ligninases production.

    Ferhan, M; Santos, S N; Melo, I S; Yan, N; Sain, M

    2013-12-01

    Fungal species for ligninases production was investigated by 18S ribosomal DNA sequence analysis. Two primer sets were chosen to amplify a major part of the 18S rDNA, which resulted in intense PCR product of approximately 550-820 bp in size per sample. The results suggest that the 18S rDNA-based approach is a useful tool for identification of unknown potential fungal species for ligninases production. The isolated fungal species produces mainly manganese peroxidase (MnP). The enzyme oxidized a variety of the usual MnP substrates, including lignin related polyphenols. Time course studies showed that maximum production of ligninolytic enzymes MnP (64 IU L⁻¹), lignin peroxidase (26.35 IU L⁻¹), and laccase (5.44 IU L⁻¹), respectively, were achieved after 10 days of cultivation under optimum conditions. Furthermore, the biological decolorization of Remazol Brilliant Blue R dye following 10 days of cultivation was 94 %. NCBI BLAST was used to search for closest matched sequences in the GenBank database and based on sequence homology the first BLAST hit was Dothioraceae sp. LM572 with accession number EF060858.1. PMID:23744034

  12. Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus)1

    Stenger, Brianna L.S.; Clark, Mark E.; Kváč, Martin; Khan, Eakalak; Giddings, Catherine W.; Dyer, Neil W.; Schultz, Jessie L.; McEvoy, John M.

    2015-01-01

    Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequen...

  13. Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

    LIU Hongquan; YU Wengong; DAI Jixun; GONG Qianhong; SHI Xiaochong; YANG Kunfeng

    2004-01-01

    The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor-joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.

  14. Initial results on the molecular phylogeny of the Nudibranchia (Gastropoda, Opisthobranchia) based on 18S rDNA data.

    Wollscheid, E; Wägele, H

    1999-11-01

    This study investigated nudibranch phylogeny on the basis of 18S rDNA sequence data. 18S rDNA sequence data of 19 taxa representing the major living orders and families of the Nudibranchia were analyzed. Representatives of the Cephalaspidea, Anaspidea, Gymnomorpha, Prosobranchia, and Pulmonata were also sequenced and used as outgroups. An additional 28 gastropod sequences taken from GenBank were also included in our analyses. Phylogenetic analyses of these more than 50 gastropod taxa provide strong evidence for support of the monophyly of the Nudibranchia. The monophyly of the Doridoidea, Cladobranchia, and Aeolidoidea within the Nudibranchia are also strongly supported. Phylogenetic utility and information content of the 18S rDNA sequences for Nudibranchia, and Opisthobranchia in general, are examined using the program SplitsTree as well as phylogenetic reconstructions using distance and parsimony approaches. 0Results based on these molecular data are compared with hypotheses about nudibranch phylogeny inferred from morphological data. PMID:10603252

  15. Identification of cephalopod species from the North and Baltic Seas using morphology, COI and 18S rDNA sequences

    Gebhardt, Katharina; Knebelsberger, Thomas

    2015-09-01

    We morphologically analyzed 79 cephalopod specimens from the North and Baltic Seas belonging to 13 separate species. Another 29 specimens showed morphological features of either Alloteuthis mediaor Alloteuthis subulata or were found to be in between. Reliable identification features to distinguish between A. media and A. subulata are currently not available. The analysis of the DNA barcoding region of the COI gene revealed intraspecific distances (uncorrected p) ranging from 0 to 2.13 % (average 0.1 %) and interspecific distances between 3.31 and 22 % (average 15.52 %). All species formed monophyletic clusters in a neighbor-joining analysis and were supported by bootstrap values of ≥99 %. All COI haplotypes belonging to the 29 Alloteuthis specimens were grouped in one cluster. Neither COI nor 18S rDNA sequences helped to distinguish between the different Alloteuthis morphotypes. For species identification purposes, we recommend the use of COI, as it showed higher bootstrap support of species clusters and less amplification and sequencing failure compared to 18S. Our data strongly support the assumption that the genus Alloteuthis is only represented by a single species, at least in the North Sea. It remained unclear whether this species is A. subulata or A. media. All COI sequences including important metadata were uploaded to the Barcode of Life Data Systems and can be used as reference library for the molecular identification of more than 50 % of the cephalopod fauna known from the North and Baltic Seas.

  16. 18S rDNA sequences from microeukaryotes reveal oil indicators in mangrove sediment.

    Henrique F Santos

    Full Text Available BACKGROUND: Microeukaryotes are an effective indicator of the presence of environmental contaminants. However, the characterisation of these organisms by conventional tools is often inefficient, and recent molecular studies have revealed a great diversity of microeukaryotes. The full extent of this diversity is unknown, and therefore, the distribution, ecological role and responses to anthropogenic effects of microeukaryotes are rather obscure. The majority of oil from oceanic oil spills (e.g., the May 2010 accident in the Gulf of Mexico converges on coastal ecosystems such as mangroves, which are threatened with worldwide disappearance, highlighting the need for efficient tools to indicate the presence of oil in these environments. However, no studies have used molecular methods to assess the effects of oil contamination in mangrove sediment on microeukaryotes as a group. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the population dynamics and the prevailing 18S rDNA phylotypes of microeukaryotes in mangrove sediment microcosms with and without oil contamination, using PCR/DGGE and clone libraries. We found that microeukaryotes are useful for monitoring oil contamination in mangroves. Our clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the Nematoda. After contamination, a large increase in the abundance of the groups Bacillariophyta (diatoms and Biosoecida was detected. The oil-contaminated samples were almost entirely dominated by organisms related to Bacillariophyta sp. and Cafeteria minima, which indicates that these groups are possible targets for biomonitoring oil in mangroves. The DGGE fingerprints also indicated shifts in microeukaryote profiles; specific band sequencing indicated the appearance of Bacillariophyta sp. only in contaminated samples and Nematoda only in non-contaminated sediment. CONCLUSIONS

  17. Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina

    Iwanowicz, L.R.; Iwanowicz, D.D.; Pote, L.M.; Blazer, V.S.; Schill, W.B.

    2008-01-01

    Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 ?? 0.3 ??m (range 15.7-20.3) in length, and 5.4 ?? 0.1 ??m (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 ?? 1.1 ??m (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 ?? 0.1 ??m (range 5.48-7.06), while the shorter is 5.7 ?? 0.1 ??m (range 4.8-6.4) in length. Polar capsule width is 1.2 ?? 0.03 ??m (range 1.0-1.54). The total length of the spore is 60.9 ?? 1.2 ??m (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus. ?? American Society of Parasitologists 2008.

  18. Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae

    Marcelo Ricardo Vicari

    2006-01-01

    Full Text Available We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding and fluorescent in situ hybridization (FISH with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil. The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (40m + 14 sm and c-positive heterochromatin preferentially located pericentromerically in all chromosomes. The presence of partial or totally heterochromatic supernumerary chromosomes with numeric intra-individual variation was confirmed in the analyzed population. The nucleolar organizing regions (NORs were interstitially situated on the long arm of chromosome pair 4 directly beneath the centromere. The differential banding techniques and FISH revealed NOR size polymorphism due to structural events such as breaks and duplication of the larger rDNA site cluster. We also observed syntenic localization of the 5S ribosomal genes in the distal segment of the 45S cluster.

  19. Chromosome evolution in tiger beetles: Karyotypes and localization of 18S rDNA loci in Neotropical Megacephalini (Coleoptera, Cicindelidae

    Sónia J.R. Proença

    2005-12-01

    Full Text Available Four Neotropical tiger beetle species, three from the genus Megacephala and one from the genus Oxycheila, currently assigned to the tribe Megacephalini were examined cytogenetically. All three Megacephala species showed simple sex chromosome systems of the X0/XX type but different numbers of autosomal pairs (15 in M. cruciata, 14 in M. sobrina and 12 in M. rutilans, while Oxycheila tristis was inferred to have a multiple sex chromosome system with four X chromosomes (2n = 24 + X1X2X3X4Y/X1X1X2X2X3X3X4X4. Fluorescence in situ hybridization (FISH using a PCR-amplified 18S rDNA fragment as a probe revealed the presence of rDNA clusters located exclusively on the autosomes in all the Megacephala species (five clusters in M. cruciata, eight in M. sobrina and three in M. rutilans, indicating variability in the number of clusters and the presence of structural polymorphisms. The same methodology showed that O. tristis had six rDNA clusters, apparently also located on the autosomes. Although our data also show cytogenetic variability within the genus Megacephala, our findings support the most accepted hypothesis for chromosome evolution in the family Cicindelidae. The description of multiple sex chromosomes in O. tristis along with phylogenetic analyses and larval morphological characters may be assumed as an additional evidence for the exclusion of the genus Oxycheila and related taxa from the tribe Megacephalini.

  20. Phylogenetic position of the Phacotaceae within the Chlamydophyceaeas revealed by analysis of 18S rDNA and rbcL sequences.

    Hepperle, D; Nozaki, H; Hohenberger, S; Huss, V A; Morita, E; Krienitz, L

    1998-10-01

    Four genera of the Phacotaceae (Phacotus, Pteromonas, Wislouchiella, Dysmorphococcus), a family of loricated green algal flagellates within the Volvocales, were investigated by means of transmission electron microscopy and analysis of the nuclear encoded small-subunit ribosomal RNA (18S rRNA) genes and the plastid-encoded rbcL genes. Additionally, the 18S rDNA of Haematococcus pluvialis and the rbcL sequences of Chlorogonium elongatum, C. euchlorum, Dunaliella parva, Chloromonas serbinowii, Chlamydomonas radiata, and C. tetragama were determined. Analysis of ultrastructural data justified the separation of the Phacotaceae into two groups. Phacotus, Pteromonas, and Wislouchiella generally shared the following characters: egg-shaped protoplasts, a single pyrenoid with planar thylakoid double-lamellae, three-layered lorica, flagellar channels as part of the central lorica layer, mitochondria located in the central cytoplasm, lorica development that occurs in mucilaginous zoosporangia that are to be lysed, and no acid-resistant cell walls. Dysmorphococcus was clearly different in each of the characters mentioned. Direct comparison of sequences of Phacotus lenticularis, Pteromonas sp., Pteromonas protracta, and Wislouchiella planctonica revealed DNA sequence homologies of >/=98. 0% within the 18S gene and 93.9% within the rbcL gene. D. globosus was quite different from these species, with a maximum of 92.9% homology in the 18S rRNA and gene. It showed major similarities to the 18S rDNA of Dunaliella salina, with 95.3%, and to the rbcL sequence of Chlamydomonas tetragama, with 90.3% sequence homology. Additionally, the Phacotaceae sensu stricto exclusively shared 10 (rbcL: 4) characters which were present neither in other Chlamydomonadales nor in Dysmorphococcus globosus. Different phylogenetic analysis methods confirmed the hypothesis that the Phacotaceae are polyphyletic. The Phacotaceae sensu stricto form a stable cluster with affinities to the

  1. Short communication: Genetic variants of Sarcocystis cruzi in infected Malaysian cattle based on 18S rDNA.

    Ng, Yit Han; Fong, Mun Yik; Subramaniam, Vellayan; Shahari, Shahhaziq; Lau, Yee Ling

    2015-12-01

    Sarcocystis species are pathogenic parasites that infect a wide range of animals, including cattle. A high prevalence of cattle sarcocystosis has been reported worldwide, but its status is unknown in Malaysia. This study focused on utilizing 18S rDNA to identify Sarcocystis species in Malaysian cattle and to determine their genetic variants. In this study, only Sarcocystis cruzi was detected in Malaysian cattle. The intra-species S. cruzi phylogenetic tree analysis and principal coordinate analysis (PCoA), respectively displayed two minor groups among the parasite isolates. This finding was supported by high Wright FST value (FST=0.647). The definitive hosts (dogs) may play a fundamental role in the development of S. cruzi genetic variants. Additionally, the existence of microheterogeneity within the S. cruzi merozoites and/or distinct genetic variants arisen from independent merozoites in mature sarcocysts, possibly contributed to the existence of intra-species variations within the population. PMID:26679818

  2. Molecular organization of the 25S-18S rDNA IGS of Fagus sylvatica and Quercus suber: a comparative analysis.

    Inácio, Vera; Rocheta, Margarida; Morais-Cecílio, Leonor

    2014-01-01

    The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family. PMID:24893289

  3. Comparative Analysis of 18S and 28S rDNA Sequences of Schistosoma japonicum from Mainland China, the Philippines and Japan

    G.H. Zhao

    2011-01-01

    Full Text Available In the present study, a portion of the 18S and 28S ribosomal DNA (rDNA sequences of 35 Schistosoma japonicum isolates representing three geographical strains from mainland China, the Philippines and Japan were amplified and compared and phylogenetic relationships were also reconstructed by Unweighted Pair-Group Method with Arithmetic averages (UPGMA using combined 18S and 28S rDNA sequences as well as the corresponding sequences of other species belonging to the Schistosoma genus available in the public database. The results indicated that the partial 18S and 28S rDNA sequences of all S. japonicum isolates were 745 and 618 bp, respectively and displayed low genetic variation among S. japonicum strains and isolates. Phylogenetic analysis revealed that the combined 18S and 28S rDNA sequences were not able to distinguish S. japonicum isolates from three geographical origins but provided an effective molecular marker for the inter-species phylogenetic analysis and differential identification of different Schistosoma species.

  4. The spatial and temporal distribution of microalgae in the South China Sea: evidence from GIS-based analysis of 18S rDNA sequences

    LI LvYan; HUANG QiaoJuan; WU ShuHui; LIN Duan; CHEN JiaHui; CHEN YueQin

    2008-01-01

    The purpose of this study was to estimate the spatial and temporal variation of microalgae in the South China Sea and to demonstrate the environmental factors controlling the diversity of microalgae by GIS (geographic information system)-based analysis of 18S rDNA sequences. Six 18S rDNA libraries were constructed from environmental samples collected at different sites in the study area, and more than 600 18S rDNA sequences were determined. The rDNA sequence data were then analyzed by DIVA-GIS software to display the spatial and temporal variation of phytoplankton's composition. It was shown that the autotrophic eukaryotic plankton dominated over the heterotrophic cells in most of our clone libraries, and the dominating phytoplankton was Dinophyceae except for Bacillariophyta at the Xiamen harbor. The percentages of these two groups were controlled by water temperature and salinity. Our results also revealed that the species composition of Chlorophyta showed a close relationship with latitude, changing from Prasinophyceae at the high latitude to Trebouxiophyceae at the low latitude. Several newly classified picoplankton lineages were first uncovered in the South China Sea, including the pico-sized green alga Ostreococcus sp. and Picochlorum eukaryotum, and picobiliphytes, which was just discovered in 2007 with unknown affinities to other eukaryotes. Their spatial and temporal variation were also analyzed and discussed.

  5. The spatial and temporal distribution of microalgae in the South China Sea:evidence from GIS-based analysis of 18S rDNA sequences

    2008-01-01

    The purpose of this study was to estimate the spatial and temporal variation of microalgae in the South China Sea and to demonstrate the environmental factors controlling the diversity of microalgae by GIS (geographic information system)-based analysis of 18S rDNA sequences. Six 18S rDNA libraries were constructed from environmental samples collected at different sites in the study area, and more than 600 18S rDNA sequences were determined. The rDNA sequence data were then analyzed by DIVA-GIS software to display the spatial and temporal variation of phytoplankton’s composition. It was shown that the autotrophic eukaryotic plankton dominated over the heterotrophic cells in most of our clone libraries, and the dominating phytoplankton was Dinophyceae except for Bacillariophyta at the Xiamen harbor. The percentages of these two groups were controlled by water temperature and salinity. Our results also revealed that the species composition of Chlorophyta showed a close relationship with latitude, changing from Prasinophyceae at the high latitude to Trebouxiophyceae at the low latitude. Several newly classified picoplankton lineages were first uncovered in the South China Sea, including the pico-sized green alga Ostreococcus sp. and Picochlorum eukaryotum, and picobiliphytes, which was just discovered in 2007 with unknown affinities to other eukaryotes. Their spatial and temporal variation were also analyzed and discussed.

  6. Isolation, morphological and molecular characterization of phytate-hydrolysing fungi by 18S rDNA sequence analysis

    Iti Gontia-Mishra

    2013-01-01

    Full Text Available Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to efficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing fungi were isolated using phytate specific medium. Fungal isolates were selected according to their higher phytase activities. These isolates were further characterized and identified by morphological and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers. This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed additive in poultry and swine industries.

  7. Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA in the fish Prochilodus lineatus (Characiformes, Prochilodontidae)

    Marcelo Ricardo Vicari; Mara Cristina de Almeida; Luiz Antonio Carlos Bertollo; Orlando Moreira-Filho; Roberto Ferreira Artoni

    2006-01-01

    We used differential staining techniques (BSG, GTG, AgNO3, DAPI and CMA3 banding) and fluorescent in situ hybridization (FISH) with 5S and 18S probes to investigated the karyotypic and cytogenetic chracteristics of Prochilodus lineatus specimens from a population in Vila Velha state park (Parque Estadual de Vila Velha, Ponta Grossa, Paraná state, southern Brazil). The specimens studied showed the same karyotype as that found in other P. lineatus populations, i.e. 2n = 54 biarmed chromosomes (...

  8. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology.

    Giribet, G; Distel, D L; Polz, M; Sterrer, W; Wheeler, W C

    2000-09-01

    Triploblastic relationships were examined in the light of molecular and morphological evidence. Representatives for all triploblastic "phyla" (except Loricifera) were represented by both sources of phylogenetic data. The 18S ribosomal (rDNA) sequence data for 145 terminal taxa and 276 morphological characters coded for 36 supraspecific taxa were combined in a total evidence regime to determine the most consistent picture of triploblastic relationships for these data. Only triploblastic taxa are used to avoid rooting with distant outgroups, which seems to happen because of the extreme distance that separates diploblastic from triploblastic taxa according to the 18S rDNA data. Multiple phylogenetic analyses performed with variable analysis parameters yield largely inconsistent results for certain groups such as Chaetognatha, Acoela, and Nemertodermatida. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis having the least character incongruence yields the following scheme of relationships of four main clades: (1) Deuterostomia [((Echinodermata + Enteropneusta) (Cephalochordata (Urochordata + Vertebrata)))]; (2) Ecdysozoa [(((Priapulida + Kinorhyncha) (Nematoda + Nematomorpha)) ((Onychophora + Tardigrada) Arthropoda))]; (3) Trochozoa [((Phoronida + Brachiopoda) (Entoprocta (Nemertea (Sipuncula (Mollusca (Pogonophora (Echiura + Annelida)))))))]; and (4) Platyzoa [((Gnathostomulida (Cycliophora + Syndermata)) (Gastrotricha + Plathelminthes))]. Chaetognatha, Nemertodermatida, and Bryozoa cannot be assigned to any one of these four groups. For the first time, a data analysis recognizes a clade of acoelomates, the Platyzoa (sensu Cavalier-Smith, Biol. Rev. 73:203-266, 1998). Other relationships that corroborate some morphological analyses are the existence of a clade that groups Gnathostomulida + Syndermata (= Gnathifera), which is expanded to include the enigmatic phylum Cycliophora, as sister group

  9. Utility of 18S rDNA and ITS sequences as population markers for Lepeophtheirus salmonis (Copepoda: Caligidae) parasitising Atlantic salmon (Salmo salar) in Scotland

    Shinn, A.P.; Banks, B.A.; Tange, N.; Bron, J.E.; Sommerville, C.; Aoki, T.; Wootten, R.

    2000-01-01

    Genetic differentiation within the salmon louse Lepeophtheirus salmonis (Krøyer, 1837), was investigated by the sequencing of specific nucleotide regions. Partial sequences of the 18S ribosomal RNA gene and the ribosomal internal transcribed spacer (ITS-1) region from single sea lice were amplified

  10. Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus)

    Stenger, B.L.S.; Clark, M.E.; Kváč, Martin; Khan, E.; Giddings, C.W.; Dyer, N.W.; Schultz, J.L.; McEvoy, J.M.

    2015-01-01

    Roč. 32, JUN 2015 (2015), s. 113-123. ISSN 1567-1348 R&D Projects: GA MŠk(CZ) LH11061 Institutional support: RVO:60077344 Keywords : Cryptosporidium * Paralogy * 18S rRNA * 18S rDNA Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 3.015, year: 2014

  11. Localization of 18S ribosomal genes in suckermouth armoured catfishes Loricariidae (Teleostei, Siluriformes with discussion on the Ag-NOR evolution

    Anderson Alves

    2012-09-01

    Full Text Available The family Loricariidae with about 690 species divided into six subfamilies, is one of the world’s largest fish families. Cytogenetic studies conducted in the family showed that among 90 species analyzed the diploid number ranges from 2n=38 in Ancistrus sp. to 2n=96 in Hemipsilichthys gobio Luetken, 1874. In the present study, fluorescence in situ hybridization (FISH was employed to determine the chromosomal localization of the 18S rDNA gene in four suckermouth armoured catfishes: Kronichthys lacerta (Nichols, 1919, Pareiorhaphis splendens (Bizerril, 1995, Liposarcus multiradiatus (Hancock, 1828 and Hypostomus prope plecostomus (Linnaeus, 1758. All species analyzed showed one chromosome pair with 18S rDNA sequences, as observed in the previous Ag-NORs analyses. The presence of size and numerical polymorphism was observed and discussed, with proposing a hypothesis of the Ag-NOR evolution in Loricariidae.

  12. The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies

    Martins Cesar

    2010-01-01

    Full Text Available Abstract Background Diverse plant and animal species have B chromosomes, also known as accessory, extra or supernumerary chromosomes. Despite being widely distributed among different taxa, the genomic nature and genetic behavior of B chromosomes are still poorly understood. Results In this study we describe the occurrence of B chromosomes in the African cichlid fish Haplochromis obliquidens. One or two large B chromosome(s occurring in 39.6% of the analyzed individuals (both male and female were identified. To better characterize the karyotype and assess the nature of the B chromosomes, fluorescence in situ hybridization (FISH was performed using probes for telomeric DNA repeats, 18S and 5S rRNA genes, SATA centromeric satellites, and bacterial artificial chromosomes (BACs enriched in repeated DNA sequences. The B chromosomes are enriched in repeated DNAs, especially non-active 18S rRNA gene-like sequences. Conclusion Our results suggest that the B chromosome could have originated from rDNA bearing subtelo/acrocentric A chromosomes through formation of an isochromosome, or by accumulation of repeated DNAs and rRNA gene-like sequences in a small proto-B chromosome derived from the A complement.

  13. Characterization of Hydrocortisone Biometabolites and 18S rRNA Gene in Chlamydomonas reinhardtii Cultures

    Seyed Bagher Mosavi-Azam

    2008-10-01

    Full Text Available A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1. This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25ºC for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17b-Dihydroxyandrost-4-en-3-one (2, 11b-hydroxyandrost-4-en-3,17-dione (3, 11b,17a,20b,21-tetrahydroxypregn-4-en-3-one (4 and prednisolone (5 were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

  14. Intragenomic sequence variation at the ITS1 - ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: mollusca)

    Hoy, Marshal S.; Rodriguez, Rusty J.

    2013-01-01

    Molecular genetic analysis was conducted on two populations of the invasive non-native New Zealand mud snail (Potamopyrgus antipodarum), one from a freshwater ecosystem in Devil's Lake (Oregon, USA) and the other from an ecosystem of higher salinity in the Columbia River estuary (Hammond Harbor, Oregon, USA). To elucidate potential genetic differences between the two populations, three segments of nuclear ribosomal DNA (rDNA), the ITS1-ITS2 regions and the 18S and 28S rDNA genes were cloned and sequenced. Variant sequences within each individual were found in all three rDNA segments. Folding models were utilized for secondary structure analysis and results indicated that there were many sequences which contained structure-altering polymorphisms, which suggests they could be nonfunctional pseudogenes. In addition, analysis of molecular variance (AMOVA) was used for hierarchical analysis of genetic variance to estimate variation within and among populations and within individuals. AMOVA revealed significant variation in the ITS region between the populations and among clones within individuals, while in the 5.8S rDNA significant variation was revealed among individuals within the two populations. High levels of intragenomic variation were found in the ITS regions, which are known to be highly variable in many organisms. More interestingly, intragenomic variation was also found in the 18S and 28S rDNA, which has rarely been observed in animals and is so far unreported in Mollusca. We postulate that in these P. antipodarum populations the effects of concerted evolution are diminished due to the fact that not all of the rDNA genes in their polyploid genome should be essential for sustaining cellular function. This could lead to a lessening of selection pressures, allowing mutations to accumulate in some copies, changing them into variant sequences.                   

  15. Molecular dissection of the rDNA array and of the 5S rDNA gene in Meloidogyne artiellia: phylogenetic and diagnostic implications.

    Veronico, Pasqua; De Luca, Francesca; De Giorgi, Carla

    2004-06-01

    The sequence of a 13.423 nucleotide genomic fragment has been determined for the plant parasitic nematode Meloidogyne artiellia. It contains an entire rDNA cluster, the bordering intergenic regions and portions of the flanking coding regions. The sequence analysis of the rDNA repeats suggests homogeneity in M. artiellia, thus providing a further indication of the usefulness of these genes for the diagnostic identification of this species. The comparison of the secondary structures of the internal transcribed spacer 2 region in several Meloidogyne species indicates that RNA folding predictions can be used as a tool of potential diagnostic relevance. The other ribosomal gene, 5S rDNA, has been demonstrated to be functional and located near the trans-spliced leader sequences, in the same arrangement found in the distantly related nematode Caenorhabditis elegans but never in other Meloidogyne thus providing species-specific markers for the identification of several Thylenchida parasitic nematodes. PMID:15135452

  16. Assessing Fungal Population in Soil Planted with Cry1Ac and CPTI Transgenic Cotton and Its Conventional Parental Line Using 18S and ITS rDNA Sequences over Four Seasons

    Qi, Xiemin; Liu, Biao; Song, Qinxin; Zou, Bingjie; Bu, Ying; Wu, Haiping; Ding, Li; Zhou, Guohua

    2016-01-01

    Long-term growth of genetically modified plants (GMPs) has raised concerns regarding their ecological effects. Here, FLX-pyrosequencing of region I (18S) and region II (ITS1, 5.8S, and ITS2) rDNA was used to characterize fungal communities in soil samples after 10-year monoculture of one representative transgenic cotton line (TC-10) and 15-year plantation of various transgenic cotton cultivars (TC-15mix) over four seasons. Soil fungal communities in the rhizosphere of non-transgenic control (CC) were also compared. No notable differences were observed in soil fertility variables among CC, TC-10, and TC-15mix. Within seasons, the different estimations were statistically indistinguishable. There were 411 and 2 067 fungal operational taxonomic units in the two regions, respectively. More than 75% of fungal taxa were stable in both CC and TC except for individual taxa with significantly different abundance between TC and CC. Statistical analysis revealed no significant differences between CC and TC-10, while discrimination of separating TC-15mix from CC and TC-10 with 37.86% explained variance in PCoA and a significant difference of Shannon indexes between TC-10 and TC-15mix were observed in region II. As TC-15mix planted with a mixture of transgenic cottons (Zhongmian-29, 30, and 33B) for over 5 years, different genetic modifications may introduce variations in fungal diversity. Further clarification is necessary by detecting the fungal dynamic changes in sites planted in monoculture of various transgenic cottons. Overall, we conclude that monoculture of one representative transgenic cotton cultivar may have no effect on fungal diversity compared with conventional cotton. Furthermore, the choice of amplified region and methodology has potential to affect the outcome of the comparison between GM-crop and its parental line. PMID:27462344

  17. ITS-2 and 18S rRNA gene phylogeny of Aplysinidae (Verongida, Demospongiae).

    Schmitt, Susanne; Hentschel, Ute; Zea, Sven; Dandekar, Thomas; Wolf, Matthias

    2005-03-01

    18S ribosomal DNA and internal transcribed spacer 2 (ITS-2) full-length sequences, each of which was sequenced three times, were used to construct phylogenetic trees with alignments based on secondary structures, in order to elucidate genealogical relationships within the Aplysinidae (Verongida). The first poriferan ITS-2 secondary structures are reported. Altogether 11 Aplysina sponges and 3 additional sponges (Verongula gigantea, Aiolochroia crassa, Smenospongia aurea) from tropical and subtropical oceans were analyzed. Based on these molecular studies, S. aurea, which is currently affiliated with the Dictyoceratida, should be reclassified to the Verongida. Aplysina appears as monophyletic. A soft form of Aplysina lacunosa was separated from other Aplysina and stands at a basal position in both 18S and ITS-2 trees. Based on ITS-2 sequence information, the Aplysina sponges could be distinguished into a single Caribbean-Eastern Pacific cluster and a Mediterranean cluster. The species concept for Aplysina sponges as well as a phylogenetic history with a possibly Tethyan origin is discussed. PMID:15871043

  18. Molecular organization of the 5S rDNA gene type II in elasmobranchs.

    Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

    2016-04-01

    The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS. PMID:26488198

  19. Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA in the Asteraceae family

    Garcia Sònia

    2010-08-01

    Full Text Available Abstract Background In flowering plants and animals the most common ribosomal RNA genes (rDNA organisation is that in which 35S (encoding 18S-5.8S-26S rRNA and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae, a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing, gene copy number (quantitative PCR and chromosomal position (FISH of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups. Results Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases, tribe Gnaphalieae (100% and in the "Heliantheae alliance" (23%. The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Conclusions Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic

  20. Gene cloning of the 18S rRNA of an ancient viable moss from the permafrost of northeastern Siberia

    Marsic, Damien; Hoover, Richard B.; Gilichinsky, David A.; Ng, Joseph D.

    1999-12-01

    A moss plant dating as much as 40,000 years old was collected from the permafrost of the Kolyma Lowlands of Northeastern Siberia. The plant tissue was revived and cultured for the extraction of its genomic DNA. Using the polymerase chain reaction technique, the 18S ribosomal RNA gene was cloned and its sequence studied. Comparative sequence analysis of the cloned ribosomal DNA to other known 18S RNA showed very high sequence identity and was revealed to be closest to the moss specie, Aulacomnium turgidum. The results of this study also show the ability of biological organisms to rest dormant in deep frozen environments where they can be revived and cultured under favorable conditions. This is significant in the notion that celestial icy bodies can be media to preserve biological function and genetic material during long term storage or transport.

  1. The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes.

    Torczynski, R; Bollon, A P; Fuke, M

    1983-01-01

    The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined. A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts. (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat). GC pairs were inserted preferentially over AT pair...

  2. Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland.

    Liu, Qin; Meli, Marina L; Zhang, Yi; Meili, Theres; Stirn, Martina; Riond, Barbara; Weibel, Beatrice; Hofmann-Lehmann, Regina

    2016-05-15

    A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genus-specific probe. Of these, two hybridized also to the Theileria equi-specific probe. The other five positive samples did not hybridize to any of the species-specific probes, suggesting the presence of unrecognized Theileria variants or genotypes. The 18S rRNA gene of the latter five samples were sequenced and found to be closely related to T. equi isolated from horses in Spain (AY534822) and China (KF559357) (≥98.4% identity). Four of the seven horses that tested positive had a documented travel history (France, Italy, and Spain) or lived abroad (Hungary). The present study adds new insight into the presence and sequence heterogeneity of T. equi in Switzerland. The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi. Of note, none of the seven positive horses were suspected of having Theileria infection at the time of presentation to the clinic. Clinicians should be aware of the possibility of equine piroplasma infections outside of endemic areas and in horses without signs of piroplasmosis. PMID:27084467

  3. PCR amplification of a multi-copy mitochondrial gene (cox3) improves detection of Cytauxzoon felis infection as compared to a ribosomal gene (18S).

    Schreeg, Megan E; Marr, Henry S; Griffith, Emily H; Tarigo, Jaime L; Bird, David M; Reichard, Mason V; Cohn, Leah A; Levy, Michael G; Birkenheuer, Adam J

    2016-07-30

    Cytauxzoon felis is a tick-transmitted protozoan parasite that infects felids. Clinical disease caused by acute C. felis infection rapidly progresses in domestic cats, leading to high morbidity and mortality. Accurately diagnosing cytauxzoonosis as soon as possible during acute infection would allow for earlier initiation of antiprotozoal therapy which could lead to higher survival rates. Molecular detection of parasite rRNA genes (18S) by PCR has previously been shown to be a sensitive method of diagnosing C. felis infections. Based on evidence from related apicomplexan species, we hypothesized that C. felis mitochondrial genes would exist at higher copy numbers than 18S and would be a more sensitive diagnostic target. In this study we have designed a PCR assay targeting the C. felis mitochondrial gene cytochrome c oxidase subunit III (cox3). Herein we demonstrate that (1) the cox3 PCR can detect as low as 1 copy of DNA target and can detect C. felis in samples with known mitochondrial sequence heterogeneity, (2) cox3 copy number is increased relative to 18S in blood and tissue samples from acutely infected cats, and (3) the cox3 PCR is more sensitive than 18S PCR for detection of C. felis during early infections. PMID:27369587

  4. Evolutionary History of the Chaetognaths Inferred from Actin and 18S-28S rRNA Paralogous Genes

    J.P. Casanova

    2006-01-01

    Full Text Available The chaetognaths constitute a small and enigmatic phylum of marine invertebrates whose phylogenetic affinities remain uncertain. Our phylogenetical investigations inferred from partial paralogous 18S-28S rRNA genes suggest that the event resulting in the presence of two classes of rRNA genes would have occurred at approximately 300-400 million years and prior to the radiation of extant chaetognath, whereas the taxon, according to both molecular and paleontological data, would be dated from at least the Early Cambrian. These divergent rRNA genes could be the result of a whole ribosomal cluster duplication or of an allopolyploid event during a crisis period, since, the fossil are lacking posterioly to the post-Carboniferous period (c.a., 300 million years. In addition, actin phylogeny evidenced that the cytoplasmic chaetognath actin clustered with the cytoplasmic insect actins, while the muscular chaetognath actins are placed basal to all muscular vertebrate actins. The present study suggests that the gene conversion mechanisms could be inefficient in this taxon; this could explain the conservation of extremely divergent paralogous sequences in the chaetognath genomes which could be correlated to the difficulties to identify a sister group between chaetognaths and other taxa among metazoans.

  5. The comparison of yeast-like symbionts in the brown planthopper,Nilaparvata lugens St(a)l with different virulence based on partial 18S rDNA sequence%褐飞虱不同致害性种群体内共生菌 18S rDNA部分序列比较

    张珏锋; 吕仲贤; 陈法军; 陈建明; 郑许松; 徐红星; 陈列忠; 俞晓平

    2006-01-01

    分离纯化了褐飞虱3种不同致害性种群体内类酵母共生菌(yeast-like symbionts,YLS),并对其18S rDNA基因序列进行了比较.结果表明,褐飞虱3种不同致害性种群体内类酵母共生菌18S rDNA均扩增出600 bp左右的片断.依据获得的18S rDNA特异性序列,结合已知真菌的18S rDNA部分序列,构建了不同宿主的YLS分子系统树.结果显示,褐飞虱3种不同致害性种群体内的YLS同属子囊菌亚门(Ascomycotina)的核菌纲(Pyrenomycetes),并与此纲中的Hypomyces chrysospermus亲缘关系相对最近.

  6. Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences.

    Yan, Ping; Pang, Qi-Hua; Jiao, Xu-Wen; Zhao, Xuan; Shen, Yan-Jing; Zhao, Shu-Jin

    2008-10-01

    FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it. PMID:18759218

  7. Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in Oreochromis mossambicus, O. urolepis hornorum and their hybrid

    Hua Ping Zhu; Mai Xin Lu; Feng Ying Gao; Zhang Han Huang; Li Ping Yang; Jain Fang Gui

    2010-08-01

    In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female × O. u. hornorum male. An identical karyotype (($2n = 44$, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.

  8. 基于核18S rDNA和叶绿体trnK序列鉴定姜黄属植物%Authentication of Curcuma species (Zingiberaceae)based on nuclear 18S rDNA and plastid trnK sequences

    曹晖; 佐佐木阳平; 伏见裕利; 小松かつ子

    2010-01-01

    姜科(Zingiberaceae)姜黄属(Curcuma)多种植物的根茎或块根如莪术(Curcumae Rhizoma)、姜黄(Curcumae Longae Rhizoma)、郁金(Curcumae Radix)等为常用中药,莪术为蓬莪术Curcuma phaeocaulis Val.、广西莪术C. kwangsinensis S.G Lee et C.F. Liang和温莪术C. wenyujin Y.H.Chen et C.Ling根茎,具有行气破血、消积化食等功效;姜黄为姜黄C. longa L.根茎,具有行气破血、通经止痛等功效;郁金为莪术及姜黄块根,具有活血化瘀、行气止痛、利胆等功效.此3类药材常有混用,而且姜黄属植物鉴别主要基于新鲜状态,依赖腊叶标本较难鉴别.本研究通过分子系统学和生物信息学手段对姜黄属18种植物核糖体RNA小亚基基因(18S rRNA)和叶绿体赖氨酸tRNA基因(trnK)进行PCR直接测序,获得这2个基因的完整序列,以1种姜花属(Hedychium)和2种姜属(Zingiber)植物为外类群分析比较其序列变异,用PAUP法建立了18种姜黄属植物的亲缘关系.所得结果显示,18S rDNA序列长度1810 bp,trnK为2696~2703 bp.姜黄属植物基因种间变异范围为0~0.05%(18S rDNA)和0~0.19%(trnK),在种一级水平单系分化上得到100%bootstrap确证.18种姜黄属植物18S rDNA序列仅有1个变异位点,在系统树上广西莪术C. kwangsiensis和莪术C. zedoaria日本居群与其他16种形成分化.trnK基因序列可变区在2个trnK外显子与matK内含子之间,共有3个DNA插入/缺失,包括1个8-bp的缺失重复序列和1个4-bp、1个14-bp插入重复序列;5个单核苷酸多态性(SNPs)位点,trnK序列存在明显的种间基因条形码(DNAbarcoding).研究结果表明分子系统学可以作为姜黄属植物亲缘关系研究的重要手段,为部分姜黄属植物的种属归并提供了分子依据,trnK基因序列可变区作为DNA条形码候选基因可用于鉴定姜黄属植物及其药材.

  9. 灰飞虱类酵母型共生菌18S rDNA序列变异及系统发生%Phylogenetic Relationships and Evolution of Yeast-like Symbionts of the Small Brown Planthoppers Based on Partial 18S rDNA Sequences

    严健; 邓可京; 曹清玉; 邵红光; 沈大棱

    2000-01-01

    分离纯化了云南楚雄、宁夏银川、北京通县、上海七宝镇、四川西昌5个地区的灰飞虱体内类酵母型共生菌(Yeast-like Symbionts, YLS),并对其18S rDNA的约600bp的序列进行了测定.结合已有的序列,构建了不同宿主的YLS的分子系统树.结果表明,灰飞虱的YLS属于子囊菌亚门Pyrenomycetes纲,与此纲的H. chrysospermus亲缘关系最近.我国各地区及日本的灰飞虱体内YLS可能是同一种的不同地理群体,不同的YLS在其宿主分化后才选择了不同的宿主,之后与其宿主长期共同进化、共同适应.

  10. A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH)

    Ke BI; James P.BOGART; Jinzhong FU

    2009-01-01

    The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution [Current Zoology 55(2):145-149,2009].

  11. A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents.

    Silva, Sheila O S; Richtzenhain, Leonardo J; Barros, Iracema N; Gomes, Alessandra M M C; Silva, Aristeu V; Kozerski, Noemila D; de Araújo Ceranto, Jaqueline B; Keid, Lara B; Soares, Rodrigo M

    2013-11-01

    Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Paraná State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III

  12. Characterization of ribosomal DNA (rDNA in Drosophila arizonae

    Francisco Javier Tovar

    2000-06-01

    Full Text Available Ribosomal DNA (rDNA is a multigenic family composed of one or more clusters of repeating units (RU. Each unit consists of highly conserved sequences codifying 18S, 5.8S and 28S rRNA genes intercalated with poorly conserved regulatory sequences between species. In this work, we analyzed the rDNA of Drosophila arizonae, a member of the mulleri complex (Repleta group. Using genomic restriction patterns, cloning and mapping of some representative rDNA fragments, we were able to construct a representative restriction map. RU in this species are 13.5-14 kb long, restriction sites are completely conserved compared with other drosophilids and the rDNA has an R1 retrotransposable element in some RU. We were unable to detect R2 elements in this species.O DNA ribossômico (rDNA é uma família multigênica composta de um ou mais aglomerados de unidades de repetição (RU. Cada unidade consiste de seqüências altamente conservadas que codificam os rRNAs 18S, 5.8S e 28S, intercaladas com seqüências regulatórias pouco conservadas entre as espécies. Neste trabalho analisamos o rDNA de Drosophila arizonae, um membro do complexo mulleri (grupo Repleta. Usando padrões de restrição genômicos, clonagem e mapeamento de alguns fragmentos de rDNA representativos, estabelecemos um mapa de restrição do rDNA representativo desta espécie. Neste drosofilídeo, a RU tem um tamanho médio de 13.5-14 kb e os sítios de restrição estão completamente conservados com relação a outras drosófilas. Além disto, este rDNA possui um elemento transponível tipo R1 presente em algumas unidades. Neste trabalho não tivemos evidências da presença de elementos R2 no rDNA desta espécie.

  13. Phylogenetic analysis of Thai oyster (Ostreidae) based on partial sequences of the mitochondrial 16S rDNA gene

    Bussarawit, Somchai; Gravlund, Peter; Glenner, Henrik;

    2006-01-01

    Ten oyster species of the family Ostreidae (Subfamilies Crassostreinae and Lophinae) from Thailand were studied using morphological data and mitochondrial 16S rDNA gene sequences. Additional sequence data from five specimens of Ostreidae and one specimen of Tridacna gigas were downloaded from Gen......Bank (T. gigas was used as outgroup). Some specimens were found to be genetically identical despite obvious morphological differences (e.g. four specimens of Crassostrea iredalei from east and west sides of the Malaysian peninsula and Saccostrea forskali and S. cf. malabonensis, both from Thai waters...

  14. Isolamento e caracterização parcial de sequências homólogas a genes ribossomais (rDNA em Blastocladiella emersonii - DOI: 10.4025/actascibiolsci.v25i2.2037 Isolation and partial characterization of homologous sequences of ribosomal genes (rDNA in Blastocladiella emersonii

    Luiz Carlos Correa

    2003-04-01

    Full Text Available A definição e a caracterização de regiões de origens de replicação nos eucariotos superiores são ainda controversas. A iniciação da replicação é sítio-específica em alguns sistemas e, em outros, parece estar contida em regiões extensas. Regiões rDNA são modelos atrativos para o estudo de origens de replicação pela sua organização in tandem, reduzindo a área de estudo para o espaço restrito que codifica uma unidade de transcrição. Neste trabalho nós isolamos e caracterizamos parcialmente um clone que contém uma sequência ribossomal do fungo aquático Blastocladiella emersonii, Be97M20. Southern blots mostraram diversos sítios para enzimas de restrição Eco RI, HindIII e SalI. Northern blot de RNA total hibridado contra uma sonda feita com Be97M20 confirmou a sua homologia com o gene ribossomal 18S. A caracterização detalhada, incluindo o mapeamento de restrição completo, subclonagem, sequenciamento e análise em géis bidimensionais proverão informações adicionais importantes sobre a estrutura e dinâmica desta regiãoThe definition and the characterization of replication origins regions in higher eukaryotes are still controversial. The initiation of the replication is site-specific in some systems but seems to occur in large regions in others. Because of its in tandem organization, reducing the area to the restricted space that codifies an unit of transcription, rDNA regions are attractive models to study replication origins. In this work we isolated and started to characterize a clone that contains a ribosomal sequence from the aquatic fungus B. emersonii, Be97M20. Southern blots showed several sites for the restrition enzymes Eco RI, HindIII and SalI. A northern blot of total RNA, hybridized against a probe made from Be97M20, confirmed its homology with the ribosomal 18S gene. The detailed characterization, including complete restriction map, subcloning, sequence and analysis on bidimensional gels will

  15. Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil

    Lara, Enrique; Berney, Cédric; Harms, Hauke; Chatzinotas, Antonis

    2010-01-01

    Using cultivation-independent methods the ciliate communities of a clay-rich soil with a 90-year record of pollution by polycyclic aromatic hydrocarbons (PAH) (4.5 g kg−1 PAH) were compared with that of a nonpolluted soil collected in its vicinity and with similar properties. A ciliate-specific set of 18S rRNA gene targeting primers was designed and used to amplify DNA extracted from both soils (surface and 20 cm depth). Four clone libraries were generated with PCR products that covered an 18...

  16. Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

    LI Haitao; YANG Guanpin; SUN Ying; WU Suihan; ZHANG Xiufang

    2007-01-01

    The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA (SSU rDNA) and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅡ contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineage Ⅰ was significantly higher than that of other microalgae species (P < 0.01). These results suggested the existence of cryptic species within C. closterium.

  17. 罗氏沼虾18S rRNA基因生物素标记探针的制备及应用%Preparation and application of the biotin-labeled probe of 18S rRNA gene in Macrobrachium rosenbergii

    高风英; 叶星; 白俊杰; 吴锐全; 劳海华; 简清; 罗建仁

    2005-01-01

    Probes are essential for study of gene expression and regulation. In this study, a method was established to prepare the biotin-labeled probe for 18S rRNA gene of freshwater prawn, Macrobrachium rosenbergii. And the labeled method was used to produce a lysozyme gene probe, then applied in analysis of lysozyme gene expression. Primers were designed according to the nucleotide sequences of 18S rRNA of Decalxxta in order to isolate the 18S rRNA gene sequences of M. rosenbergii. Total genomic DNA was isolated from hepatopancreas of the freshwater prawn. A specific DNA fragment with desired size was amplified by PCR using the total DNA as templates. The DNA fragment was inserted into pGEM-T Easy vector and sequenced. The result of BLAST and alignment analysis confirmed that the DNA fragment isolated was the 18S rRNA gene of M. rosenbergii, which was 418 nt in length.Biotin-labeled probe of the 18S rRNA was then produced by PCR using the recombinant plasmid as templates. The biotin-21-dTTP and the non-labeled dNTP were added to the PCR reaction system. Ratio of the biotin-21-dTTP and the non-labeled dTFP was 3 to 1.The yield of the labeled probe is 300 ng·μL-1. The detection limit of the probe is 60 pg. A biotin-labeled probe of lysozyme gene was prepared by the same label method, and the yield of the lysozyme gene probe is 500 ng·μL-1. These biotin-labeled probes were applied in Northern dot blotting analysis of tissue distribution of lysoyzme mRNA of M. rosenbergii. Signals were scanned and quantified by Analysis System of Biology Image. The signal intensity ratio of the lysozyme to 18S rRNA represents the relative expression level of lysozyme mRNA. The results showed that the lysozyme mRNA existed in all the tissues checked, including eye,muscle, gill, hepatopancreas, haemocytes and intestine. But lysoyzme mRNA levels varied among different tissues. The highest level was found in the intestine, and the second was in the hepatopancreas and the lowest was in the

  18. The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.

    Cooper, Madalyn K; Phalen, David N; Donahoe, Shannon L; Rose, Karrie; Šlapeta, Jan

    2016-01-30

    Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations. PMID:26801593

  19. Molecular analysis of 18S rRNA gene of Cryptosporidium parasites from patients living in Iran, Malawi, Nigeria and Vietnam.

    Ghaffari, Salman; Kalantari, Narges

    2012-01-01

    Cryptosporidium species are one of the most common causes of gastrointestinal infection in humans around the world. This study has aimed to investigate the hyper variable region of the 18S rRNA gene in Cryptosporidium for exact parasite identification. DNA was extracted from 26 fecal samples from which initially Cryptosporidium oocysts were identified by Ziehl-Neelsen acid-fast , Auramine phenol and ELISA techniques. Nested PCR, targeting the most polymorphic region of the 18S rRNA gene and genotyping was performed by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing and phylogenic analysis. Among 26 isolates analyzed, three species of Cryptosporidium were identified; 38.5% of the isolates were C. hominis while 53.8% of the isolates were C. parvum and 7.7% of the isolates were C. meleagridis, which the last two species have the potentially zoonotic transmission. The only 11T subtype of C. hominis was demonstrated. These strains clustered distinctly into either human or animal origin regardless of the geographical origin, age, or immunity status of the patients. In summary, this work is the first report of C. meleagridis infecting human in Iran. Moreover, it suggested that multi-locus study of Cryptosporidium species in developing countries would be necessary to determine the extent of transmission of cryptosporidiosis in the populations. PMID:24551771

  20. Phylogenetic analysis of the spider mite sub-family Tetranychinae (Acari: Tetranychidae based on the mitochondrial COI gene and the 18S and the 5' end of the 28S rRNA genes indicates that several genera are polyphyletic.

    Tomoko Matsuda

    Full Text Available The spider mite sub-family Tetranychinae includes many agricultural pests. The internal transcribed spacer (ITS region of nuclear ribosomal RNA genes and the cytochrome c oxidase subunit I (COI gene of mitochondrial DNA have been used for species identification and phylogenetic reconstruction within the sub-family Tetranychinae, although they have not always been successful. The 18S and 28S rRNA genes should be more suitable for resolving higher levels of phylogeny, such as tribes or genera of Tetranychinae because these genes evolve more slowly and are made up of conserved regions and divergent domains. Therefore, we used both the 18S (1,825-1,901 bp and 28S (the 5' end of 646-743 bp rRNA genes to infer phylogenetic relationships within the sub-family Tetranychinae with a focus on the tribe Tetranychini. Then, we compared the phylogenetic tree of the 18S and 28S genes with that of the mitochondrial COI gene (618 bp. As observed in previous studies, our phylogeny based on the COI gene was not resolved because of the low bootstrap values for most nodes of the tree. On the other hand, our phylogenetic tree of the 18S and 28S genes revealed several well-supported clades within the sub-family Tetranychinae. The 18S and 28S phylogenetic trees suggest that the tribes Bryobiini, Petrobiini and Eurytetranychini are monophyletic and that the tribe Tetranychini is polyphyletic. At the genus level, six genera for which more than two species were sampled appear to be monophyletic, while four genera (Oligonychus, Tetranychus, Schizotetranychus and Eotetranychus appear to be polyphyletic. The topology presented here does not fully agree with the current morphology-based taxonomy, so that the diagnostic morphological characters of Tetranychinae need to be reconsidered.

  1. Avian malaria in captive psittacine birds: detection by microscopy and 18S rRNA gene amplification.

    Belo, N O; Passos, L F; Júnior, L M C; Goulart, C E; Sherlock, T M; Braga, E M

    2009-03-01

    A cross-sectional survey was conducted to estimate the occurrence of malaria infection among captive psittacine birds (n=127) from three zoological gardens in Brazil. Malaria infection was evaluated by the association of direct examination of blood smears with amplification of the 18SSU rRNA gene of the Plasmodium genus, demonstrating an overall occurrence of 36%. Most infected bird species were Amazona aestiva (28/73), Ara ararauna (6/10), and Amazona amazonica (3/10). The low parasitemias observed among the infected birds suggest a chronic infection. The sequence analyses of 10 isolates indicate a potential occurrence of four distinct Plasmodium lineages. These findings provide new data on malarial infection in captive psittacine birds, and emphasize the need for better control of importation and exportation of these birds. PMID:18937986

  2. Protist 18S rRNA gene Sequence Analysis Reveals Multiple Sources of Organic Matter Contributing to Turbidity Maxima of the Columbia River Estuary

    Herfort, Lydie; Peterson, Tawnya D.; McCue, Lee Ann; Zuber, Peter A.

    2011-10-05

    The Columbia River estuary is traditionally considered a detritus-based ecosystem fueled in summer by organic matter (OM) from expired freshwater diatoms. Since Estuarine Turbidity Maxima (ETM) are sites of accumulation and transformation of this phytoplankton-derived OM, to further characterize the ETM protist assemblage, we collected in August 2007 bottom waters throughout an ETM event, as well as surface water during the peak of bottom turbidity, and performed biogeochemical, microscopic and molecular (18S rRNA gene clone libraries) analyses. These data confirmed that the majority of the particulate OM in ETMs is derived from chlorophyll a-poor particulate organic carbon tagged by DNA too damaged to be detected by molecular analysis.

  3. Fungal community analysis in the deep-sea sediments of the Pacific Ocean assessed by comparison of ITS, 18S and 28S ribosomal DNA regions

    Xu, Wei; Luo, Zhu-Hua; Guo, Shuangshuang; Pang, Ka-Lai

    2016-03-01

    We investigated the diversity of fungal communities in 6 different deep-sea sediment samples of the Pacific Ocean based on three different types of clone libraries, including internal transcribed spacer (ITS), 18S rDNA, and 28S rDNA regions. A total of 1978 clones were generated from 18 environmental clone libraries, resulting in 140 fungal operational taxonomic units (OTUs), including 18 OTUs from ITS, 44 OTUs from 18S rDNA, and 78 OTUs from 28S rDNA gene primer sets. The majority of the recovered sequences belonged to diverse phylotypes of the Ascomycota and Basidiomycota. Additionally, our study revealed a total of 46 novel fungal phylotypes, which showed low similarities (<97%) with available fungal sequences in the GenBank, including a novel Zygomycete lineage, suggesting possible new fungal taxa occurring in the deep-sea sediments. The results suggested that 28S rDNA is an efficient target gene to describe fungal community in deep-sea environment.

  4. Phylogeny and classification of the Litostomatea (Protista, Ciliophora), with emphasis on free-living taxa and the 18S rRNA gene.

    Vd'ačný, Peter; Bourland, William A; Orsi, William; Epstein, Slava S; Foissner, Wilhelm

    2011-05-01

    The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of species ranging from aerobic, free-living predators to anaerobic endocommensals. This is traditionally reflected by classifying the Litostomatea into the subclasses Haptoria and Trichostomatia. The morphological classifications of the Haptoria conflict with the molecular phylogenies, which indicate polyphyly and numerous homoplasies. Thus, we analyzed the genealogy of 53 in-group species with morphological and molecular methods, including 12 new sequences from free-living taxa. The phylogenetic analyses and some strong morphological traits show: (i) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea and (ii) three distinct lineages (subclasses): the Rhynchostomatia comprising Tracheliida and Dileptida; the Haptoria comprising Lacrymariida, Haptorida, Didiniida, Pleurostomatida and Spathidiida; and the Trichostomatia. The curious Homalozoon cannot be assigned to any of the haptorian orders, but is basal to a clade containing the Didiniida and Pleurostomatida. The internal relationships of the Spathidiida remain obscure because many of them and some "traditional" haptorids form separate branches within the basal polytomy of the order, indicating one or several radiations and convergent evolution. Due to the high divergence in the 18S rRNA gene, the chaeneids and cyclotrichiids are classified incertae sedis. PMID:21333743

  5. Molecular characterization of Cryptosporidium xiaoi in goat kids in Bangladesh by nested PCR amplification of 18S rRNA gene

    AMAM; Zonaed; Siddiki; Sohana; Akter; Mina; Zinat; Farzana; Bibi; Ayesa; Rasel; Das; Mohammad; Alamgir; Hossain

    2015-01-01

    Objective:To investigate the prevalence of Cryptosporidium spp.in goat kids in selected areas of Bangladesh and to elucidate the potential zoonotic hazards.Methods:In the present study,we have used Ziehl-Neelsen staining and nested PCR approach to identify and characterize the Cryptosporidium sp.from diarrhoeic feces of goat kids.A total of 100 diarrhoeic feces samples were collected from Chittagong region in Southern Bangladesh.For nested PCR analysis,specific primers for amplification of 581 base pair fragments of 18 S rRNA gene were used.Results:A total of 15%and 3%samples were found positive in microscopic study and in nested PCR analysis respectively.Phylogenetic analysis of sequence data showed similarity with that of Cryptosporidium xiaoi recorded from sheep and goat.Conclusions:To our knowledge,this is the first report of Cryptosporidium xiaoi responsible for diarrhoea in goat kids in Bangladesh.Further study can highlight their zoonotic significance along with genetic diversity in other host species inside the country.

  6. Sequence and Taxonomy Analysis of Arctium lappa 18S rRNA Gene%牛蒡18S核糖体RNA基因分析和分类学研究

    蔡侃; 孔文刚; 夏红剑; 侯进慧

    2011-01-01

    Arctium lappa 18S rRNA gene was amplified,and a 1636bp DNA were sequenced with its Genbank accession number JF509958.The gene sequence of Arctium lappa 18Sr RNA was analyzed with related species in GenBank.The result shows,Arctium lappa 18S rRNA gene has a high homology with many families within Dicotyledoneae,such as Asteraceae and Caprifoliaceae.This study provides reference for further study of Arctium lappa in molecular level.%扩增牛蒡18S rRNA基因,测序获得1 636bp的DNA序列,GenBank登录号是JF703098。利用牛蒡18S rDNA序列和GenBank相关序列构建系统发育树,结果表明,牛蒡18S rRNA基因与双子叶纲的菊科、忍冬科的一些物种序列相似度高。对在分子水平上牛蒡的研究提供了资料。

  7. Localização física dos genes ribossomais 5S e 18S em "Ancistrus" sp. da bacia dos rios costeiros no Rio de Janeiro (Loricariidae: Ancistrini. DOI: 10.7902/ecb.v3i1.13

    Dinaíza Abadia Rocha Reis

    2012-10-01

    Full Text Available Ancistrus é considerado um gênero diversificado na tribo Ancistrini e corresponde a peixes conhecidos popularmente por “cascudos”.  Os DNAs ribossômicos mais estudados são o rDNA 18S, que participa da formação de Regiões Organizadoras de Nucléolo,  e o rDNA 5S. O objetivo deste trabalho é determinar a localização física dos sítios de DNAr 18S e 5S em Ancistrus sp. da região de Angra dos Reis-RJ, bacia dos rios Costeiros. Através de Hibridação Fluorescente in situ (FISH foram identificados 4 sítios de rDNA 18S localizados em quatro diferentes cromossomos (pares 3 e 14 e 2 sítios de rDNA 5S, localizados em dois cromossomos (par 13, todos em região terminal. Os resultados deste estudo podem ser aplicados em abordagens conservacionistas, podendo auxiliar no entendimento da evolução cromossômica desta espécie e da tribo Ancistrini.

  8. 3-Nitropropionic acid modifies neurotrophin mRNA expression in the mouse striatum:18S-rRNA is a reliable control gene for studies of the striatum

    S.Espíndola; A Vilches-Flores; E.Hernández-Echeagaray

    2012-01-01

    Objective The aim of the present study was to determine the changes in the mRNA levels ofneurotrophins and their receptors in the striatal tissue of mice treated with 3-nitropropionic acid (3-NP).Methods At 1 and 48 h after the last drug administration,the mRNA expression of nerve growth factor,brain-derived neurotrophic factor,neurotrophin-3 and neurotrophin-4/5 as well as their receptors p75,TrkA,TrkB and TrkC,was evaluated using semi-quantitative (semi-Q) and real-time RT-PCR.β-actin mRNA and ribosomal 18S (18S rRNA) were tested as internal controls.Results 3-NP treatment did not affect mRNA expression of all neurotrophins and their respective receptors equally.Also,differences in neurotrophin and receptor mRNA expression were observed between semi-Q and real-time RT-PCR.Real-time RT-PCR was more accurate in evaluating the mRNA expression of the neurotrophins than semi-Q,and 18S rRNA was more reliable than β-actin as an internal control.Conclusion Neurotrophins and their receptors expression is differentially affected by neuronal damage produced by inhibition of mitochondrial respiration with 3-NP treatment in low,sub-chronic doses in vivo.

  9. Inhibition of deoxyribonucleic acid transcription by ultraviolet irradiation in mammalian cells: determination of the transcriptional linkage of the 18S and 28S ribosomal ribonucleic acid genes

    The inhibition of deoxyribonucleic acid (DNA) transcription in mammalian cells by ultraviolet irradiation has been studied. The reduction in the rates and the amounts of total ribonucleic acid (RNA) synthesis and of 18S, 28S, and 45S ribosomal RNA (rRNA) synthesis, in tissue cultured mouse L cells, were examined as functions of ultraviolet dose and time after ultraviolet irradiation. Total RNA synthesis in the ultraviolet irradiated L cell was found to decrease as a function of ultraviolet dose. The rates of synthesis for the 18S and 28S rRNAs and the 45S precursor RNA decreased exponentially with ultraviolet dose; the respective D37 values were 310 erg/mm2, 130 erg/mm2, and 90 erg/mm2. Ultraviolet inactivation kinetics of rRNA synthesis in HeLa cells indicated that, as in L cells, each 45S rRNA transcriptional unit has its own promotor, and that the 18S rRNA cistron is promotor proximal and the 28S rRNA cistron is promotor distal. All of the above findings support the hypothesis that irradiation of mammalian cells with ultraviolet light causes the formation of lesions on the DNA templates which result in premature termination of transcription. (U.S.)

  10. Polygenic analysis of ammonia-oxidizing bacteria using 16S rDNA, amoA, and amoB genes

    Calvó Perxas, Laia; Cortey Marqués, Martí; García Marín, José Luis; Garcia-Gil, L. J.

    2005-01-01

    Finding a unique molecular marker capable of quickly providing rigorous and useful phylogenetic information would facilitate assessing the diversity of ammonia-oxidizing bacteria in environmental samples. Since only one of several available markers can be used at a time in these kinds of studies, the 16S rDNA, amoA and amoB genes were evaluated individually and then compared in order to identify the one that best fits the information provided by the composite dataset. Distance-based neighbor-...

  11. Nematode 18S rRNA gene is a reliable tool for environmental biosafety assessment of transgenic banana in confined field trials.

    Nakacwa, R; Kiggundu, A; Talwana, H; Namaganda, J; Lilley, C; Tushemereirwe, W; Atkinson, H

    2013-10-01

    Information on relatedness in nematodes is commonly obtained by DNA sequencing of the ribosomal internal transcribed spacer region. However, the level of diversity at this locus is often insufficient for reliable species differentiation. Recent findings suggest that the sequences of a fragment of the small subunit nuclear ribosomal DNA (18S rRNA or SSU), identify genera of soil nematodes and can also distinguish between species in some cases. A database of soil nematode genera in a Ugandan soil was developed using 18S rRNA sequences of individual nematodes from a GM banana confined field trial site at the National Agricultural Research Laboratories, Kawanda in Uganda. The trial was planted to evaluate transgenic bananas for resistance to black Sigatoka disease. Search for relatedness of the sequences gained with entries in a public genomic database identified a range of 20 different genera and sometimes distinguished species. Molecular markers were designed from the sequence information to underpin nematode faunal analysis. This approach provides bio-indicators for disturbance of the soil environment and the condition of the soil food web. It is being developed to support environmental biosafety analysis by detecting any perturbance by transgenic banana or other GM crops on the soil environment. PMID:23661261

  12. 多浆旱生植物霸王18SrRNA基因的克隆及序列分析%Cloning and sequence analysis of 18S rRNA gene fragment from succulent xerophyte Zygophyllum xanthoxylum

    胡静; 谢俊仁; 王锁民

    2012-01-01

    In order to reveal the relationship between succulent xerophyte Zygophyllum xanthoxylum and other plants and to provide evidences for the biologically evolution, total DNA was extracted from leaves of Z. xanthoxylurn seedlings, and the 18S rRNA gene was cloned by PCR using general primers and cloned into pGEM-T vector. The positive clone identified by PCR was sequenced. The sequencing result revealed that the 18S rRNA gene fragment from Z. xanthoxylum contains 1808 bp. Homology comparison with other plants 18S rRNA gene sequences in the GenBank showed that it shared over 96% nucleotide sequence homology, so it is concluded that 18S rRNA is very conservative gene in plants. However, Homology matrix and Blast showed that Z. xanthoxylurn shared high similarity (98%) with the identified 18S rRNA in Galearia fili formis , Cnidoscolus aconiti folius and Hevea brasiliensis. Phylogenetic tree analysis indicated that Z. xanthoxylum and Panax notoginseng were most consanguineously grouped.%为探讨多浆旱生植物霸王(Zygophyllum xanthoxylum)的生物进化历程及与其他植物的亲缘关系,本研究以霸王叶基因组DNA为模板,使用通用引物扩增其18SrRNA基因片段,并克隆到pGEM—T载体,阳性克隆经鉴定后进行测序。核苷酸序列分析结果表明,该片段长1808bp,所得序列与GenBank中注册的18SrRNA基因序列的同源性均在96%以上。可见,高等植物18SrRNA的基因非常保守。同源性分析与Blast比较结果表明,霸王与小盘木(Galearia filiformis)、驱虫苋(Cnidoscolus aconitifolius)及橡胶树(Herera brasiliensis)同源性最高。系统进化树分析表明,霸王与三七(Panax notoginseng)的亲缘关系最近。

  13. The phylogenetic position of Lygodactylus angularis and the utility of using the 16S rDNA gene for delimiting species in Lygodactylus (Squamata, Gekkonidae

    Riccardo Castiglia

    2011-06-01

    Full Text Available The African genus Lygodactylus Gray, is composed of roughly 60 species of diurnal geckos that inhabit tropical and temperate Africa, Madagascar, and South America. In this study, we assessed the phylogenetic position of L. angularis, for which molecular data were so far lacking, by means of sequence analysis of the mitochondrial 16S rDNA gene. We also compared intraspecific vs. interspecific genetic divergences using an extended data set (34 species, 153 sequences, to determine whether a fragment of this gene can be useful for species identification and to reveal the possible existence of new cryptic species in the genus. The analysis placed L. angularis in a monophyletic group together with members of “fischeri” and “picturatus” groups. Nevertheless, the independence of the “angularis” lineage is supported by the high genetic divergence. Comparison of intraspecific vs. interspecific genetic distances highlights that, assuming an equal molecular rate of evolution among the studied species for the used gene, the threshold value useful for recognising a candidate new species can be tentatively placed at 7%. We identified four species that showed an intraspecific divergence higher than, or close to, the 7% threshold: L. capensis (8.7%, L. gutturalis (9.3%, L. madagascariensis (6.5% and L. picturatus (8.1%. Moreover, two species, L. mombasicus and L. verticillatus, are paraphyletic in terms of gene genealogy. Thus, the study shows that a short fragment of the 16S rDNA gene can be an informative tool for species-level taxonomy in the genus Lygodactylus.

  14. Identification of novel spp. of rice and wheat endophytic diazotrophs by 16S rDNA gene and FTIR analysis

    Mohammad Javad Mehdipour Moghaddam

    2012-06-01

    Full Text Available In this research, six isolates, including three from three rice roots (PxR1, PxR2 and StR1 and three from three wheat roots (PxW1, PxW2 and PxW3 were isolated as endophytic bacteria and except for StR1, all the isolates were identified as Pseudoxanthomonas based on phenotypic analysis including FTIR and PCR amplification of 16S rDNA. The results showed that PxR1, PxR2, PxW1 and PxW2 were all similar and belonged to a novel species of Pseudoxanthomonas, but PxW3 was from different species. StR1 belonged to a novel species of Stenotrophomonas. Two strains including Azospirillum brasiliense Sp7 (S1 and Azospirillum lipoferum (S2 were selected as standard strains and compared with those isolates however, phenotypic and genotypic analysis verified that those isolates were not Azospirillum. For the first time, it was indicated that Pseudoxanthomonas existed as an endophytic bacterium in rice root.

  15. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repeti...

  16. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5 ' UTRs and its implications for eukaryotic gene translation regulation

    Pánek, J. (Josef); Kolář, M. (Michal); Vohradský, J; Valášek, L. (Leoš)

    2013-01-01

    There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computat...

  17. Microbial diversities (16S and 18S rDNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

    Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, e.g. Legionella pneumophila, via parasitization of free-living amoebae such as Acanthamoebae. Yet knowledge about the microbial composition of DW biofilms developed on common in-premise pl...

  18. The evolutionary history of the genus Timarcha (Coleoptera, Chrysomelidae) inferred from mitochondrial COII gene and partial 16S rDNA sequences.

    Gómez-Zurita, J; Juan, C; Petitpierre, E

    2000-02-01

    The apterous genus Timarcha consists of three subgenera and more than 100 species in its Palearctic distribution, with specialized feeding on few plant families. Fifty-four sequences sampled from 31 taxa of the genus plus three outgroup leaf beetles were studied for their complete cytochrome oxidase II (COII) and a fragment of 16S rDNA mitochondrial genes, representing a total of about 1200 bp. Phylogenetic analyses using maximum-parsimony and distance methods for each gene separately and for the combined data set gave compatible topologies. The subgenus Metallotimarcha consistently appears in a basal position and is well differentiated from the remaining Timarcha, but no clear monophyletic grouping of Timarchostoma and Timarcha s. str. subgenera can be deduced from our analysis. Calibration of the molecular clock has been done using the opening of the Gibraltar Strait after the Messinian salinity crisis (about 5.5 MYA) as the biogeographic event causing disjunction of two particular taxa. Accordingly, the COII evolutionary rate has been estimated to be of 0.76 x 10(-8) substitution/site/year in Timarcha. Relation between phylogeny and host-plant use indicates widening of trophic regime as a derived character in Timarcha. PMID:10679162

  19. Cloning and sequence analysis of the 18S rRNA gene of Trichinella from cat in Heilongjiang province%黑龙江省猫旋毛虫18S rRNA基因分子克隆及序列分析

    李冬梅; 王秀荣; 董小波; 路义鑫; 宋铭忻

    2007-01-01

    本文利用GenBank中发表的( Trichinella spiralis )18S rRNA序列为参考设计引物,对分离自黑龙江省猫体内的旋毛虫及本地毛形线虫( Trichinella nativa )的18S rRNA基因进行扩增,克隆后测序,序列分析结果表明:猫旋毛虫与旋毛形线虫基因同源性更高.

  20. Molecular species identification of Central European ground beetles (Coleoptera: Carabidae using nuclear rDNA expansion segments and DNA barcodes

    Raupach Michael J

    2010-09-01

    Full Text Available Abstract Background The identification of vast numbers of unknown organisms using DNA sequences becomes more and more important in ecological and biodiversity studies. In this context, a fragment of the mitochondrial cytochrome c oxidase I (COI gene has been proposed as standard DNA barcoding marker for the identification of organisms. Limitations of the COI barcoding approach can arise from its single-locus identification system, the effect of introgression events, incomplete lineage sorting, numts, heteroplasmy and maternal inheritance of intracellular endosymbionts. Consequently, the analysis of a supplementary nuclear marker system could be advantageous. Results We tested the effectiveness of the COI barcoding region and of three nuclear ribosomal expansion segments in discriminating ground beetles of Central Europe, a diverse and well-studied invertebrate taxon. As nuclear markers we determined the 18S rDNA: V4, 18S rDNA: V7 and 28S rDNA: D3 expansion segments for 344 specimens of 75 species. Seventy-three species (97% of the analysed species could be accurately identified using COI, while the combined approach of all three nuclear markers provided resolution among 71 (95% of the studied Carabidae. Conclusion Our results confirm that the analysed nuclear ribosomal expansion segments in combination constitute a valuable and efficient supplement for classical DNA barcoding to avoid potential pitfalls when only mitochondrial data are being used. We also demonstrate the high potential of COI barcodes for the identification of even closely related carabid species.

  1. Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of the lichen genus Usnea (Parmeliaceae, Ascomycota).

    Truong, Camille; Divakar, Pradeep K; Yahr, Rebecca; Crespo, Ana; Clerc, Philippe

    2013-08-01

    In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data, and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also susceptible to causing bias in the generation of the alignment. We compared several methods of alignment of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy) improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution was even better when using protein-coding genes, especially RPB1 although it is less variable. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. However, characters that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions. PMID:23603312

  2. The phylogenetic position of Lygodactylus angularis and the utility of using the 16S rDNA gene for delimiting species in Lygodactylus (Squamata, Gekkonidae)

    Riccardo Castiglia; Flavia Annesi

    2011-01-01

    The African genus Lygodactylus Gray, is composed of roughly 60 species of diurnal geckos that inhabit tropical and temperate Africa, Madagascar, and South America. In this study, we assessed the phylogenetic position of L. angularis, for which molecular data were so far lacking, by means of sequence analysis of the mitochondrial 16S rDNA gene. We also compared intraspecific vs. interspecific genetic divergences using an extended data set (34 species, 153 sequences), to determine whether a fra...

  3. Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic analyses of ITS rDNA and actin gene sequences.

    Park, Mi-Jeong; Choi, Young-Joon; Hong, Seung-Beom; Shin, Hyeon-Dong

    2010-01-01

    Ampelomyces quisqualis complex is well known as the most common and widespread hyperparasite of the family Erysiphaceae, the cause of powdery mildew diseases. As commercial biopesticide products it is widely used to control the disease in field and plastic houses. Although genetic diversity within Ampelomyces isolates has been previously recognized, a single name A. quisqualis is still applied to all pycnidial intracellular hyperparasites of powdery mildew fungi. In this study, the phylogenetic relationships among Ampelomyces isolates originating from various powdery mildew fungi in Korea were inferred from Bayesian and maximum parsimony analyses of the sequences of ITS rDNA region and actin gene. In the phylogenetic trees, the Ampelomyces isolates could be divided into four distinct groups with high sequence divergences in both regions. The largest group, Clade 1, mostly accommodated Ampelomyces isolates originating from the mycohost Podosphaera spp. (sect. Sphaerotheca). Clade 2 comprised isolates from several genera of powdery mildews, Golovinomyces, Erysiphe (sect. Erysiphe), Arthrocladiella, and Phyllactinia, and was further divided into two subclades. An isolate obtained from Podosphaera (sect. Sphaerotheca) pannosa was clustered into Clade 3, with those from powdery mildews infecting rosaceous hosts. The mycohosts of Ampelomyces isolates in Clade 4 mostly consisted of species of Erysiphe (sect. Erysiphe, sect. Microsphaera, and sect. Uncinula). The present phylogenetic study demonstrates that Ampelomyces hyperparasite is indeed an assemblage of several distinct lineages rather than a sole species. Although the correlation between Ampelomyces isolates and their mycohosts is not obviously clear, the isolates show not only some degree of host specialization but also adaptation to their mycohosts during the evolution of the hyperparasite. PMID:20943134

  4. Aligned 18S for Zoraptera (Insecta) : Phylogenetic position and molecular evolution

    Yoshizawa, Kazunori; Johnson, Kevin P.

    2005-01-01

    The order Zoraptera (angel insects) is one of the least known insect groups, containing only 32 extant species. The phylogenetic position of Zoraptera is poorly understood, but it is generally thought to be closely related to either Paraneoptera (hemipteroid orders: booklice, lice, thrips, and bugs), Dictyoptera (blattoid orders: cockroaches, termites, and mantis), or Embioptera (web spinners). We inferred the phylogenetic position of Zoraptera by analyzing nuclear 18S rDNA sequences, which w...

  5. Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

    Garcia, S.; Panero, J.L.; Široký, Jiří; Kovařík, Aleš

    2010-01-01

    Roč. 10, č. 176 (2010), s. 1-18. ISSN 1471-2229 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : organization of rDNA unit * intergenic spacer * Asteraceae Subject RIV: BO - Biophysics Impact factor: 4.085, year: 2010

  6. The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

    Bishop, R P; Hemmink, J.D.; Morrison, W.I.; Weir, W.; TOYE, P. G.; Sitt, T.; Spooner, P.R.; Musoke, A.J.; Skilton, R. A.; Odongo, D.O.

    2015-01-01

    African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle ...

  7. Male meiosis, heterochromatin characterization and chromosomal location of rDNA in Microtomus lunifer (Berg, 1900 (Hemiptera: Reduviidae: Hammacerinae

    María Poggio

    2011-05-01

    Full Text Available In the present work, we analysed the male meiosis, the content and distribution of heterochromatin and the number and location of nucleolus organizing regions in Microtomus lunifer (Berg, 1900 by means of standard technique, C- and fluorescent bandings, and fluorescent in situ hybridization with an 18S rDNA probe. This species is the second one cytogenetically analysed within the Hammacerinae. Its male diploid chromosome number is 31 (2n=28+X1X2Y, including a minute pair of m-chromosomes. The diploid autosomal number and the presence of m-chromosomes are similar to those reported in M. conspicillaris (Drury, 1782 (2n=28+XY. However, M. lunifer has a multiple sex chromosome system X1X2Y (male that could have originated by fragmentation of the ancestral X chromosome. Taking into account that M. conspicillaris and M. lunifer are the only two species within Reduviidae that possess m-chromosomes, the presence of this pair could be a synapomorphy for the species of this genus. C- and fluorescent bandings showed that the amount of heterochromatin in M. lunifer was small, and only a small CMA3 bright band was observed in the largest autosomal pair at one terminal region. FISH with the 18S rDNA probe demonstrated that ribosomal genes were terminally placed on the largest autosomal pair. Our present results led us to propose that the location of rDNA genes could be associated with variants  of the sex chromosome systems in relation with a kind of the sex chromosome systems within this family. Furthermore, the terminal location of NOR in the largest autosomal pair allowed us to use it as a chromosome marker and, thus, to infer that the kinetic activity of both ends is not a random process, and there is an inversion of this activity.

  8. Regulation of rDNA stability by sumoylation

    Eckert-Boulet, Nadine; Lisby, Michael

    2009-01-01

    Repair of DNA lesions by homologous recombination relies on the copying of genetic information from an intact homologous sequence. However, many eukaryotic genomes contain repetitive sequences such as the ribosomal gene locus (rDNA), which poses a risk for illegitimate recombination. Therefore, t...... review is the regulation of recombinational DNA repair at the rDNA locus by sumoylation and the Smc5-Smc6 complex in S. cerevisiae....

  9. rDNA Genetic Imbalance and Nucleolar Chromatin Restructuring Is Induced by Distant Hybridization between Raphanus sativus and Brassica alboglabra

    Long, Hong; Chen, Chunli; Bing WANG; Feng, Yanni

    2015-01-01

    The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional ...

  10. Variation in rDNA locus number and position among legume species and detection of 2 linked rDNA loci in the model Medicago truncatula by FISH.

    Abirached-Darmency, Mona; Prado-Vivant, Emilce; Chelysheva, Liudmila; Pouthier, Thomas

    2005-06-01

    Within Fabaceae, legume species have a variable genome size, chromosome number, and ploidy level. The genome distribution of ribosomal genes, easily detectable by fluorescent in situ hybridization (FISH), is a good tool for anchoring physical and genetic comparative maps. The organisation of 45S rDNA and 5S loci was analysed by FISH in the 4 closely related species: Pisum sativum, Medicago truncatula, Medicago sativa (2 diploid taxa), and Lathyrus sativus. The 2 types of rDNA arrays displayed interspecific variation in locus number and location, but little intraspecific variation was detected. In the model legume, M. truncatula, the presence of 2 adjacent 45S rDNA loci was demonstrated, and the location of the rDNA loci was independent of the general evolution of the genome DNA. The different parameters relative to clustering of the rDNA loci in specific chromosome regions and the possible basis of rDNA instability are discussed. PMID:16121252

  11. Identificarion of contaminant bacteria in cachaça yeast by 16s rDNA gene sequencing Identificação de bactérias contaminantes de fermento de cachaça por seqüenciamento do gene 16s rDNA

    Osmar Vaz de Carvalho-Netto

    2008-01-01

    Full Text Available Cachaça is a typical Brazilian liquor produced from the distillation of fermented sugarcane juice mainly by Saccharomyces cerevisiae. Most of the domestic production is artisanal, and producers usually are not concerned regarding microbiological control of the fermentation. This study aimed to characterize the contaminant bacterial community of the yeast used in the production of cachaça in an artisanal still. Four samples were collected, of which one (NA was used for comparison purposes and was collected one year earlier. The remaining samples were collected at three different periods: at the end of the first day of fermentation (NP, after fifteen days (NS, and thirty days after the same yeast was used (NT. Five hundred and eighty-seven sequences were analyzed from the partial sequencing of the 16S rDNA gene. Sequence analyses revealed the presence of 170 operational taxonomic units (OTUs. Of these, only one was shared among three samples and seventeen were shared between two samples. The remaining 152 OTUs were identified only once in distinct samples indicating that the contaminant bacterial population is highly dynamic along the fermentation process. Statistical analyses revealed differences in bacterial composition among samples. Undescribed species in the literature on yeasts of cachaça were found, such as Weissella cibaria, Leuconostoc citreum, and some species of Lactobacillus, in addition to some unknown bacteria. The community of bacteria in the fermentation process is much more complex than it was previously considered. No previous report is known regarding the use of this technique to determine bacterial contaminants in yeast for the production of cachaça.A cachaça é uma bebida típica brasileira produzida a partir da destilação do caldo de cana-de-açúcar fermentado principalmente por Saccharomyces cerevisiae. Grande parte da produção nacional é artesanal, e não há uma preocupação por parte dos produtores quanto ao

  12. Filling a gap in the phylogeny of flatworms: relationships within the Rhabdocoela (Platyhelminthes), inferred from 18S ribosomal DNA sequences

    Willems, Wim; Walberg, A.; Jondelius, U.; Littlewood, D.; Backeljau, T.; Schockaert, Ernest; Artois, Tom

    2006-01-01

    The phylogeny of the Rhabdocoela, a species-rich taxon of free-living flatworms, is reconstructed based on complete 18S rDNA sequences. The analysis includes 62 rhabdocoels and 102 representatives of all major flatworm taxa. In total, 46 new sequences are used, 41 of them from rhabdocoel species, five from proseriates. Phylogenetic analysis was performed using maximum parsimony and Bayesian inference. Clade support was evaluated with parsimony jackknifing, Bremer support indice...

  13. Physical mapping of 5S and 18S ribosomal DNA in three species of Agave (Asparagales, Asparagaceae

    Victor Manuel Gomez-Rodriguez

    2013-08-01

    Full Text Available Agave Linnaeus, 1753 is endemic of America and is considered one of the most important crops in Mexico due to its key role in the country’s economy. Cytogenetic analysis was carried out in A. tequilana Weber, 1902 ‘Azul’, A. cupreata Trelease et Berger, 1915 and A. angustifolia Haworth, 1812. The analysis showed that in all species the diploid chromosome number was 2n = 60, with bimodal karyotypes composed of five pairs of large chromosomes and 25 pairs of small chromosomes. Furthermore, different karyotypical formulae as well as a secondary constriction in a large chromosome pair were found in all species. Fluorescent in situ hybridization (FISH was used for physical mapping of 5S and 18S ribosomal DNA (rDNA. All species analyzed showed that 5S rDNA was located in both arms of a small chromosome pair, while 18S rDNA was associated with the secondary constriction of a large chromosome pair. Data of FISH analysis provides new information about the position and number of rDNA loci and helps for detection of hybrids in breeding programs as well as evolutionary studies.

  14. Phylogeny of the sundews, Drosera (Droseraceae), based on chloroplast rbcL and nuclear 18S ribosomal DNA Sequences.

    Rivadavia, Fernando; Kondo, Katsuhiko; Kato, Masahiro; Hasebe, Mitsuyasu

    2003-01-01

    The sundew genus Drosera consists of carnivorous plants with active flypaper traps and includes nearly 150 species distributed mainly in Australia, Africa, and South America, with some Northern Hemisphere species. In addition to confused intrageneric classification of Drosera, the intergeneric relationships among the Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda, are problematic. We conducted phylogenetic analyses of DNA sequences of the chloroplast rbcL gene for 59 species of Drosera, covering all sections except one. These analyses revealed that five of 11 sections, including three monotypic sections, are polyphyletic. Combined rbcL and 18S rDNA sequence data were used to infer phylogenetic relationships among Drosera, Dionaea, and Aldrovanda. This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, suggesting that the snap traps of Aldrovanda and Dionaea are homologous despite their morphological differences. MacClade reconstructions indicated that multiple episodes of aneuploidy occurred in a clade that includes mainly Australian species, while the chromosome numbers in the other clades are not as variable. Drosera regia, which is native to South Africa, and most species native to Australia, were clustered basally, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that Australian species expanded their distribution to South America and then to Africa. Expansion of distribution to the Northern Hemisphere from the Southern Hemispere occurred in a few different lineages. PMID:21659087

  15. The phylogenetic position of Lygodactylus angularis and the utility of the 16S rDNA gene for delimiting species in Lygodactylus (Squamata, Gekkonidae

    Riccardo Castiglia

    2011-06-01

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    The African genus Lygodactylus Gray, is composed of about 60 species of diurnal geckos inhabiting tropical and temperate Africa, Madagascar and South America. In this paper we analysed, by means of the mitochondrial 16S rDNA gene

  16. Population distribution of 45S and 5S rDNA in golden mahseer, Tor putitora: population-specific FISH marker

    Mamta Singh; Ravindra Kumar; N. S. Nagpure; Basdeo Kushwaha; Indra Mani; U. K. Chauhan; W. S. Lakra

    2009-12-01

    Chromosomal locations of major 45S and minor 5S ribosomal DNAs (rDNAs) and organization of 5S rRNA genes were analysed in five different populations of golden mahseers (Tor putitora) using fluorescence in situ hybridization (FISH) and Southern blot hybridization. All five populations of T. putitora ($2n = 100$) showed a similar type of macro-karyotype composed of 12 metacentric, 22 submetacentric, 14 subtelocentric and 52 telocentric chromosomes. Analysis of active nucleolar organizer regions (NORs) by silver staining did not show any differences in number and chromosomal position in different populations. But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on two different chromosome pairs in Kosi river population contain non-transcribed spacers (NTS) of same length. In the present study, simultaneous localization of 45S and 5S rDNA by in situ hybridization helped us to develop the discrete population-specific markers in different geographically isolated populations of T. putitora.

  17. Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

    缪炜; 余育和; 沈韫芬; 张锡元

    2002-01-01

    Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceolata, E. chrysemydis, and E. wenrichi was consistent with variations in the thickness of the peristomial lip. We concluded that the macronucleus and peristomial lip might be the important phylogenetic characteristics within the genus Epistylis.

  18. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  19. Efficiency of the V3 region of 16S rDNA and the rpoB gene for bacterial community detection in Thai traditional fermented shrimp (Kung-Som using PCR-DGGE techniques

    Chatthaphisuth Sanchart

    2015-06-01

    Full Text Available Kung-Som is one of several Thai traditional fermented shrimp products, that is especially popular in the southern part of Thailand. This is the first report to reveal the bacterial communities in the finished product of Kung-Som. Ten Kung-Som samples were evaluated using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE methodology combined with appropriate primers to study the dynamics of the bacterial population. Two primer sets (V3; 341f(GC- 518r and rpoB; rpoB1698f(GC-rpoB2014r primers were considered as a possible tool for the differentiation of bacteria and compared with respect to their efficiency of 16S rDNA and rpoB gene amplification. PCR-DGGE analysis of both the V3-region and rpoB amplicon was successfully applied to discriminate between lactic acid bacteria and Gram positive strains in the bacterial communities of Kung-Som. In conclusion, the application of these two primer sets using PCR-DGGE techniques is a useful tool for analyzing the bacterial diversity in Kung-Som. Moreover, these preliminary results provide useful information for further isolation of desired bacterial strains that could be used as a starter culture in order to improve the quality of Kung-Som.

  20. Isolation and Identification of Lactobacilli From Cheese, Yoghurt and Silage by 16S rDNA Gene and Study of Bacteriocin and Biosurfactant Production

    Sharareh Peymanfar

    2012-09-01

    Full Text Available Background: Background: The Lactobacilli belong to lactic acid bacteria, whose primary fermentation end product from sugars is lactic acid and that is why foods are conserved. Lactic acid bacteria have been used for millennia in the production of silage. Therefore, they are an indispensable part of intestinal microflora in human and animals. .Objectives: This research meant to isolate lactic acid bacteria with significant effects from different environments. .Materials and Methods: In this study, heterofermentative LAB were isolated from cheese, yoghurt and corn silage in Broujerd ,Iran. The standard biochemical methods were applied. Acid tolerance was studied by exposure to acidic PBS and growth in bile salt was measured by the spectrophotometric method. The isolated bacteria were studied for antagonistic effects on environment isolated E.coli, bacteriocin and biosurfactant production. Bacterial DNA was extracted, and amplified by PCR method. .Results: The 3 isolates from cheese, yoghurt and silage were effective against isolated E.coli and could produce biosurfactants. Phylogenic relationships of the 3 potential candidates were determined comparing the 16Sr DNA gene sequences, they were found to be as 3 isolates of Lactobacillus buchneri, L.brevis and L.kefiri that were effective on the isolated E.coli from environment. .Conclusions: It was found that the isolated bacteria produced biosurfactants that had a great potential for different industries.

  1. Identidade molecular dos fitoplasmas associados aos enfezamentos do tomateiro e da berinjela com base na análise do gene 16S rDNA Molecular identity of the phytoplasma associated to stunting of tomato and eggplant on the basis of analyses of the 16S rDNA

    Ana Paula de Oliveira Amaral Mello

    2007-09-01

    , shortening internodes, reduced size of leaves, flowers and fruits were observed. In nested PCR with primers R16 mF1/mR2 e R16 F2n/R2, fragments of 1.2kb in size were amplified from symptomatic samples demonstrating the presence of phytoplasmas in plant tissues. By using especific primers pairs it was demonstrated that the phytoplasmas were affiliated to group 16SrIII. RFLP analysis using the restriction enzymes AluI, HpaII, KpnI, MboI, MseI, and RsaI confirmed that the phytoplasmas were representatives of group 16SrIII. Amplified DNA fragments were cloned in Escherichia coli, sequenced and compared by sequence similarities among themselves and with sequences belonging to phytoplasmas of group 16SrIII. Sequence similarities greater than 95% were found when the phytoplamas detected in tomato and eggplant were compared to the representatives of group 16SrIII. Values of 98-99% were obtained when sequences of phytoplasmas found in tomato and eggplant were compared among themselves. The results evidenced that tomato and eggplant stunting were associated with the same phytoplasma based upon the sequencing of the 16S rDNA gene.

  2. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples.

    Guo, Yi; Yang, Jing-Xian; Liang, Guo-Wei

    2016-06-01

    The prevalence of Candida in bloodstream infections (BSIs) has increased. To date, the identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations. Therefore, a real-time PCR assay for the detection of Candida from whole blood is presented. The unique primers/probe system was designed on 5.8S rRNA gene (5.8S rDNA) of Candida genus. The analytical sensitivity was determined by numbers of positive PCRs in 12 repetitions. At the concentration of 10(1) CFU/ml blood, positive PCR rates of 100 % were obtained for C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. The detection rate for C. glabrata was 75 % at 10(1) CFU/ml blood. The reaction specificity was 100 % when evaluating the assay using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit were 1.22 and 2.22 %, respectively. To assess the clinical applicability, 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100 % using as gold standard blood culture, and specificity was 98.4 %. Our data suggest that the developed assay can be used in clinical laboratories as an accurate and rapid screening test for the Candida from whole blood. Although further evaluation is warranted, our assay holds promise for earlier diagnosis of candidemia. PMID:26687075

  3. CTD kinase I is required for the integrity of the rDNA tandem array

    Grenetier, Sabrina; Bouchoux, Céline; Goguel, Valérie

    2006-01-01

    The genomic stability of the rDNA tandem array is tightly controlled to allow sequence homogenization and to prevent deleterious rearrangements. In this report, we show that the absence of the yeast CTD kinase I (CTDK-I) complex in null mutant strains leads to a decrease in the number of tandem rDNA repeats. Reintroduction of the missing gene induces an increase of rDNA repeats to reach a copy number similar to that of the original strain. Interestingly, while expansion is dependent on Fob1, ...

  4. rDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

    Long, Hong; Chen, Chunli; Wang, Bing; Feng, Yanni

    2015-01-01

    The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins. PMID:25723542

  5. Ribosomal genes of Histoplasma capsulatum var. duboisii and var. farciminosum.

    Okeke, C N; Kappe, R; Zakikhani, S; Nolte, O; Sonntag, H G

    1998-11-01

    A total of 1704 basepairs of the 18S rDNA of Histoplasma capsulatum var. duboisii (HCD, strain CBS175.57) and H. capsulatum var. farciminosum (HCF, strain CBS478.64) were sequenced (EMBL accession no. Z75306 and no. Z75307). The 18S rDNA of HCD was 100% identical to a published sequence of H. capsulatum var. capsulatum (HCC). The 18S rDNA of HCF showed one transversional point mutation at the nucleotide position 114 (ref. Saccharomyces cerevisiae). Hybridization confirmed that, in the 18S rDNA of two out of five strains of HCF, guanine was substituted for cytosine at the nucleotide position 114. Furthermore, identical group 1C1 introns (403 bp) were found to be inserted after position 1165 in four out of five strains of HCF, including the two strains with point mutations in the 18S rDNA, and a slightly different group 1C1 intron (408 bp) was detected in one strain of HCC without this point mutation. Intraspecific sequence variability in the highly conserved 18S rDNA because of occurrence of introns and mutations as a possible source of error in molecular diagnostics is discussed. In addition, internal transcribed spacer regions between the 18S rDNA and the 5.8S rDNA (ITS1) of three strains of HCF, and one strain each of HCC and HCD showed significant sequence variability between varieties and strains of H. capsulatum. PMID:9916456

  6. Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP.

    Fliegerová, K; Mrázek, J; Voigt, K

    2006-01-01

    The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology. PMID:17007423

  7. Identification of organic matter sources in sulfidic late Holocene Antarctic fjord sediments from fossil rDNA sequence analysis

    Coolen, M.J.L.; Volkman, J.K.; Abbas, B.; Muyzer, G; Schouten, S.; Sinninghe Damsté, J. S.

    2007-01-01

    The 18S ribosomal DNA (rDNA) isolated from sulfidic Holocene sediments and particulate organic matter in the water column of the stratified Small Meromictic Basin (SMB) in Ellis Fjord (eastern Antarctica) was analyzed to identify possible biological sources of organic matter. Previous work had shown that the sediments contained numerous diatom frustules and high contents of a highly branched isoprenoid (HBI) C25:2 alkene (which is a specific biomarker of certain species of the diatom genera N...

  8. The 5S rDNA in two Abracris grasshoppers (Ommatolampidinae: Acrididae): molecular and chromosomal organization.

    Bueno, Danilo; Palacios-Gimenez, Octavio Manuel; Martí, Dardo Andrea; Mariguela, Tatiane Casagrande; Cabral-de-Mello, Diogo Cavalcanti

    2016-08-01

    The 5S ribosomal DNA (rDNA) sequences are subject of dynamic evolution at chromosomal and molecular levels, evolving through concerted and/or birth-and-death fashion. Among grasshoppers, the chromosomal location for this sequence was established for some species, but little molecular information was obtained to infer evolutionary patterns. Here, we integrated data from chromosomal and nucleotide sequence analysis for 5S rDNA in two Abracris species aiming to identify evolutionary dynamics. For both species, two arrays were identified, a larger sequence (named type-I) that consisted of the entire 5S rDNA gene plus NTS (non-transcribed spacer) and a smaller (named type-II) with truncated 5S rDNA gene plus short NTS that was considered a pseudogene. For type-I sequences, the gene corresponding region contained the internal control region and poly-T motif and the NTS presented partial transposable elements. Between the species, nucleotide differences for type-I were noticed, while type-II was identical, suggesting pseudogenization in a common ancestor. At chromosomal point to view, the type-II was placed in one bivalent, while type-I occurred in multiple copies in distinct chromosomes. In Abracris, the evolution of 5S rDNA was apparently influenced by the chromosomal distribution of clusters (single or multiple location), resulting in a mixed mechanism integrating concerted and birth-and-death evolution depending on the unit. PMID:27106499

  9. Phylogenetic study of Class Armophorea (Alveolata, Ciliophora based on 18S-rDNA data

    Thiago da Silva Paiva

    2013-01-01

    Full Text Available The 18S rDNA phylogeny of Class Armophorea, a group of anaerobic ciliates, is proposed based on an analysis of 44 sequences (out of 195 retrieved from the NCBI/GenBank database. Emphasis was placed on the use of two nucleotide alignment criteria that involved variation in the gap-opening and gap-extension parameters and the use of rRNA secondary structure to orientate multiple-alignment. A sensitivity analysis of 76 data sets was run to assess the effect of variations in indel parameters on tree topologies. Bayesian inference, maximum likelihood and maximum parsimony phylogenetic analyses were used to explore how different analytic frameworks influenced the resulting hypotheses. A sensitivity analysis revealed that the relationships among higher taxa of the Intramacronucleata were dependent upon how indels were determined during multiple-alignment of nucleotides. The phylogenetic analyses rejected the monophyly of the Armophorea most of the time and consistently indicated that the Metopidae and Nyctotheridae were related to the Litostomatea. There was no consensus on the placement of the Caenomorphidae, which could be a sister group of the Metopidae + Nyctorheridae, or could have diverged at the base of the Spirotrichea branch or the Intramacronucleata tree.

  10. Unusual dispersion of histone repeats on the whole chromosomal complement and their colocalization with ribosomal genes in Rachycentron canadum (Rachycentridae, Perciformes).

    Costa, Gideão W W F; Cioffi, Marcelo B; Bertollo, Luiz A C; Molina, Wagner F

    2014-01-01

    Rachycentron canadum, the only representative of the family Rachycentridae, has been the focus of biotechnological interest due to its significant potential in marine fish farming. The chromosome set of this species has been widely investigated with respect to the location of genes and multigene families. A FISH analysis was performed using 4 multigene families as probes, represented by 5S and 18S ribosomal genes and histones H2B-H2A and H3. Earlier data suggested that differential replication of heterochromatin could be partially associated with functional genes. Indeed, our results showed that the DNA contained in heterochromatic regions of R. canadum contains 5S and 18S ribosomal genes as well as the gene sequences of histones H2B-H2A and H3, which were colocalized. The distribution of H3 sequences in all heterochromatic regions, except in 13q, could indicate an important evolutionary role for this class of repetitive sequences. Besides, the presence of chromosome regions bearing multifunctional repetitive sequences formed by H2B-H2A/H3/18S rDNA and H2B-H2A/H3/5S rDNA clusters was demonstrated for the first time in fishes. The implications of differential histone gene extension and its functionality in the karyotype of R. canadum remain unknown. PMID:25341625

  11. Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses.

    Nakada, Takashi; Misawa, Kazuharu; Nozaki, Hisayoshi

    2008-07-01

    The taxonomy of Volvocales (Chlorophyceae, Chlorophyta) was traditionally based solely on morphological characteristics. However, because recent molecular phylogeny largely contradicts the traditional subordinal and familial classifications, no classification system has yet been established that describes the subdivision of Volvocales in a manner consistent with the phylogenetic relationships. Towards development of a natural classification system at and above the generic level, identification and sorting of hundreds of sequences based on subjective phylogenetic definitions is a significant step. We constructed an 18S rRNA gene phylogeny based on 449 volvocalean sequences collected using exhaustive BLAST searches of the GenBank database. Many chimeric sequences, which can cause fallacious phylogenetic trees, were detected and excluded during data collection. The results revealed 21 strongly supported primary clades within phylogenetically redefined Volvocales. Phylogenetic classification following PhyloCode was proposed based on the presented 18S rRNA gene phylogeny along with the results of previous combined 18S and 26S rRNA and chloroplast multigene analyses. PMID:18430591

  12. Nucleolar dominance and maternal control of 45S rDNA expression.

    Michalak, Katarzyna; Maciak, Sebastian; Kim, Young Bun; Santopietro, Graciela; Oh, Jung Hun; Kang, Lin; Garner, Harold R; Michalak, Pawel

    2015-12-01

    Using a system of interspecies hybrids, trihybrids, and recombinants with varying proportions of genomes from three distinct Xenopus species, we provide evidence for de novo epigenetic silencing of paternal 45 S ribosomal ribonucleic acid (rRNA) genes and their species-dependent expression dominance that escapes transcriptional inactivation after homologous recombination. The same pattern of imprinting is maintained in the offspring from mothers being genetic males (ZZ) sex-reversed to females, indicating that maternal control of ribosomal deoxyribonucleic acid (rDNA) expression is not sex-chromosome linked. Nucleolar dominance (nucleolus underdevelopment) in Xenopus hybrids appears to be associated with a major non-Mendelian reduction in the number of 45 S rDNA gene copies rather than a specific pattern of their expression. The loss of rRNA gene copies in F1 hybrids was non-random with respect to the parental species, with the transcriptionally dominant variant preferentially removed from hybrid zygotes. This dramatic disruption in the structure and function of 45 S rDNA impacts transcriptome patterns of small nucleolar RNAs and messenger RNAs, with genes from the ribosome and oxidative stress pathways being among the most affected. Unorthodoxies of rDNA inheritance and expression may be interpreted as hallmarks of genetic conflicts between parental genomes, as well as defensive epigenetic mechanisms employed to restore genome integrity. PMID:26645200

  13. Toxicity analysis of pesticides on cyanobacterial species by 16S rDNA molecular characterization

    J. I. Nirmal Kumar

    2013-06-01

    Full Text Available Damaging effects of endosulfan on native structure of DNA, evident as a result of PCR based assay such as 16S rDNA amplification and sequencing, led to formation of gaps, mismatching of base pairs and dissimilarities in entire 16S rDNA sequences of treated cultures. Endosulfan was the most fatal to Westiellopsis prolifica of 16S rDNA region at 40ppm insecticide induced series of mispairing, and other lesions amounting up to 20% dissimilarity and 7% gaps. Whereas, 16S rDNA region of Anabaena fertilissima was comparatively less influenced with 18% dissimilarity and 7% gaps in response to 12ppm endosulfan, while 16S rDNA gene of Aulosira fertilissima was the least prone to changes with 17% dissimilarity, and 5% gaps under 60ppm endosulfan stress by the end of 16 days. On the other side, impact of fungicide tebuconazole after 16 days reflected identities up to 78% and 8% gaps for 30ppm treated A. fertilissima, while 60ppm treatment instilled 79% similarities with 10% gaps in W. prolifica and 83% identities with 5% gaps of Aulosira fertilissima after 16 days.

  14. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  15. Molecular epidemiology of Plasmodium species prevalent in Yemen based on 18 s rRNA

    A Azazy Ahmed

    2010-11-01

    Full Text Available Abstract Background Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000 population per year and 43,000 disability adjusted life years lost. Although malaria in Yemen is caused mainly by Plasmodium falciparum and Plasmodium vivax, there are no sequence data available on the two species. This study was conducted to investigate the distribution of the Plasmodium species based on the molecular detection and to study the molecular phylogeny of these parasites. Methods Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA gene was amplified using nested PCR. From the 86 positive blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. Results Malaria was detected by PCR in 86 samples (16.8%. The majority of the single infections were caused by P. falciparum (80.3%, followed by P. vivax (5.8%. Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of P. falciparum. Conclusions Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.

  16. 山羊奇异变形杆菌的分离鉴定及16SrDNA和ZapA基因的PCR-RFLP分析%Isolation and identification of Proteus mirabilis from goat and the analysis of 16S rDNA and ZapA gene by PCR-RFLP

    王豪举; 倪莉; 杨红军; 赵俊; 徐宗可; 徐强; 徐丽敏

    2011-01-01

    Five strains of pathogen were isolated from the infected goats. Swarming behaviour assays, biochemical, animal and antibiotic resistance tests were carried out to identify its properties. Then 16S rDNA and ZapA were cloned and sequenced and both 16S rDNA and ZapA were analyzed with PCR-PFLP after digestion by specific restriction enzymes. The data showed the field strains had the swarming behaviour,which were sensitive to amikacin and for- turn,the LDs0 in mice was from 2. 500 0× 10^7 CFU/mL to 4. 445 3× 10^7CFU/mL. The DNA sequencing showed 99. 5%-99.8% similarity with Proteus mirabilis for 16S rDNA and 95. 5% for ZapA gene; the segments from PCRRFLP analysis displayed no disparity with reference strain in number and size. The results indicate that the field strains are a member of Proteus rnirabilis with no polymorphism.%从发病山羊分离到5株病原菌,对分离株进行游散行为分析,生化试验、动物试验、药敏试验及16SrDNA和ZapA基因克隆测序,选用限制性内切酶对分离株16SrDNA和ZapA基因进行PCR-RFLP分析。结果表明分离株出现游散生长现象,对丁胺卡那霉素和复达欣敏感,小鼠LD5。为2.5000×10 ^7CFU/mL-4.4453×10^7CFU/mL;分离株16SrDNA与奇异变形杆菌的同源率为99.5%~99.8%,ZapA基N与奇异变形杆菌的同源率为99.5%;PCR—RFLP分析发现,分离株酶切片段数目和大小与标准株相同。结果表明分离株是奇异变形杆菌,多态性较为单一。

  17. Detection of 16S rDNA and PIA genes of Neisseria isolated from the patients with male genitourinary tract infections%男性泌尿生殖道感染患者分离奈瑟菌的16S rDNA和PIA基因检测

    姜敏敏; 王涛; 王和

    2012-01-01

    目的 检测男性泌尿生殖道分离奈瑟菌的16S rDNA和PIA基因,探讨奈瑟菌属菌种的基因鉴定及其致病机理.方法 用PCR扩增和核苷酸序列分析方法分别检测11例男性泌尿生殖道感染患者泌尿生殖道分离的14株奈瑟菌属菌种的16S rDNA和PIA基因及其序列.结果 14株奈瑟菌属菌种经16S rDNA检测鉴定分别为淋病奈瑟菌2株,黏液奈瑟菌3株,灰色奈瑟菌5株,微黄奈瑟菌2株,干燥奈瑟菌1株,嗜乳糖奈瑟菌及多糖奈瑟菌各1株;与常规细菌学方法鉴定的符合率为85.7%.非淋球菌奈瑟菌未检出淋病奈瑟菌毒力相关的PIA核苷酸序列.结论 常规细菌学方法与染色体16S rDNA检测及其序列分析方法的联合使用,可提高奈瑟菌属菌种感染的实验室诊断准确率;PIA基因对于奈瑟菌属的男性生殖道致病性无关.%Objective To detect the 16S rDNA and PIA genes of Neisseria isolated from the patients with male genitourinary tract infections and investigate the gene identification and pathogenesis of Neisseria species. Methods The 16S rRNA and PIA genes and their sequences of 14 Neisseria species isolated from reproductive tract of 11 patients with male genitourinary tract infections were identified by PCR amplification and nucleotide sequencing. Results Fourteen Neisseria strains contained 2 strains of Neisseria gonorrhoeae, 3 strains of Neisseria mucosa, 5 strains of Neisseria cinerea, 2 strain of Neisseria subflava, 1 strain of Neisseria sicca,1 strain of Neisseria lactamica and 1 strain of Neisseria polysaccharea. The accordance rate of the results reported by 16S rDNA and routine bacteriological method was 85. 7%. The nucleotide sequence of virulence-associated gene PIA of Neisseria gonorrhoeae was not found in non-gonococcal strains of Neisseria. Conclusion The accuracy rate of laboratory diagnosis for the infection caused by Neisseria can be improved by combined use of routine bacteriological method and 16S rDNA

  18. Aberrant DNA Methylation of rDNA and PRIMA1 in Borderline Personality Disorder.

    Teschler, Stefanie; Gotthardt, Julia; Dammann, Gerhard; Dammann, Reinhard H

    2016-01-01

    Borderline personality disorder (BPD) is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5'-external transcribed spacer/5'ETS) and the promoter of the proline rich membrane anchor 1 gene (PRIMA1) in peripheral blood samples of 24 female patients (mean age (33 ± 11) years) diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7) years). A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5'ETS were significantly lower (0.9-fold) in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5'ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD. PMID:26742039

  19. Aberrant DNA Methylation of rDNA and PRIMA1 in Borderline Personality Disorder

    Stefanie Teschler

    2016-01-01

    Full Text Available Borderline personality disorder (BPD is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5′-external transcribed spacer/5′ETS and the promoter of the proline rich membrane anchor 1 gene (PRIMA1 in peripheral blood samples of 24 female patients (mean age (33 ± 11 years diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7 years. A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5′ETS were significantly lower (0.9-fold in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5′ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD.

  20. Limitations of metazoan 18S rRNA sequence data : implications for reconstructing a phylogeny of the animal kingdom and inferring the reality of the cambrian explosion

    Abouheif, Ehab; Zardoya, Rafael; Meyer, Axel

    1998-01-01

    We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of am...

  1. Clinorotation influences rDNA and NopA100 localization in nucleoli

    Sobol, M. A.; González-Camacho, F.; Rodríguez-Vilariño, V.; Kordyum, E. L.; Medina, F. J.

    The nucleolus is the transcription site of rRNA genes as well as the site of processing and initial packaging of their transcripts. The plant nucleolin homologue NopA100 is involved in the regulation of r-chromatin condensation/expansion and rDNA transcription as well as in rRNA processing. We have investigated with immunogold electron microscopy the location of nucleolar DNA and NopA100 in cress root meristematic cells grown under slow horizontal clinorotation, reproducing an important feature of microgravity, namely the absence of an orienting action of a gravity vector, compared to control conditions. We demonstrate redistribution of both rDNA and NopA100 in nucleolar subcomponents induced by clinorotation. Ribosomal DNA concentrated predominantly in fibrillar centers in the form of condensed r-chromatin inclusions and internal non condensed fibrils, redistributing from the dense fibrillar component and the transition zone between fibrillar centers and the dense fibrillar component, recognized as the loci of rDNA transcription. The content of NopA100 was much higher in the inner space of fibrillar centers and reduced in the dense fibrillar component as compared to the control. Based on these data, an effect of slow horizontal clinorotation in lowering the level of rDNA transcription as well as rRNA processing is suggested.

  2. Diversity of thraustochytrid protists isolated from brown alga, Sargassum cinereum using 18S rDNA sequencing and their morphological response to heavy metals

    Damare, V.S.

    nutrients such as polyunsaturated fatty acids (Damare et al., 2013).They have been found to be endolithic in mollusk shell fragments (Porter & Lingle, 1992) and have been isolated from surface mucus of hermatypic corals (Harel et al., 2008). They are also... to protocol described by Washington State University (http://sbs.wsu.edu/fmic/protocol/fixindex/sem_standard_fix.html). In brief, the cells were washed with sterile seawater, primarily fixed in 1 % glutaraldehyde and filtered over pretreated 0.4 µm white...

  3. 18S-rDNA SEQUENCING, ENZYME PATTERNS AND MORPHOLOGICAL CHARACTERIZATION OF TRICHOPHYTON ISOLATES

    Nascimento Adriana Mendes do

    2001-01-01

    Full Text Available Dermatophytes, capable to use keratin of the host for nutrition, belong to one of the major groups of pathogenic fungi. Since dermatophytes are a closely related group they share various common features, and the morphology of isolates of a given species can be atypical, making species identification and differentiation even more difficult. Many methods have been explored in attempts to distinguish dermatophytes, but the combined use of different approaches for the investigation of the intraspecific and interspecific variability of Trichophyton continues to be scarce. Some studies have shown that amplified fragments of the small ribosomal DNA subunit 18S contains variable regions which can be used to discriminate between medically relevant yeast species, indicating that these regions could also be used for differentiation between dermatophytes. In our study, sequence analysis of the 18S-rDNA gene was combined with morphological and biochemical criteria in order to detect genetic differences between seven Trichophyton isolates and estimate their phylogenetic relationships. The results show that the isolates investigated belong to the Trichophyton group, which potentially contains the Trichophyton rubrum cluster.

  4. Utility of internally transcribed spacer region of rDNA (ITS) and β-tubulin gene sequences to infer genetic diversity and migration patterns of Colletotrichum truncatum infecting Capsicum spp.

    Rampersad, Kandyce; Ramdial, Hema; Rampersad, Sephra N

    2016-01-01

    Anthracnose is among the most economically important diseases affecting pepper (Capsicum spp.) production in the tropics and subtropics. Of the three species of Colletotrichum implicated as causal agents of pepper anthracnose, C. truncatum is considered to be the most destructive in agro-ecosystems worldwide. However, the genetic variation and the migration potential of C. truncatum infecting pepper are not known. Five populations were selected for study and a two-locus (internally transcribed spacer region, ITS1-5.8S-ITS2, and β-tubulin, β-TUB) sequence data set was generated and used in the analyses. Sequences of the ITS region were less informative than β -tubulin gene sequences based on comparisons of DNA polymorphism indices. Trinidad had the highest genetic diversity and also had the largest effective population size in pairwise comparisons with the other populations. The Trinidad population also demonstrated significant genetic differentiation from the other populations. AMOVA and STRUCTURE analyses both suggested significant genetic variation within populations more so than among populations. A consensus Maximum Likelihood tree based on β-TUB gene sequences revealed very little intraspecific diversity for all isolates except for Trinidad. Two clades consisting solely of Trinidad isolates may have diverged earlier than the other isolates. There was also evidence of directional migration among the five populations. These findings may have a direct impact on the development of integrated disease management strategies to control C. truncatum infection in pepper. PMID:26843942

  5. Systematics of Chaetognatha under the light of molecular data, using duplicated ribosomal 18S DNA sequences.

    Papillon, Daniel; Perez, Yvan; Caubit, Xavier; Le Parco, Yannick

    2006-03-01

    While the phylogenetic position of Chaetognatha has became central to the question of early bilaterian evolution, the internal systematics of the phylum are still not clear. The phylogenetic relationships of the chaetognaths were investigated using newly obtained small subunit ribosomal RNA nuclear 18S (SSU rRNA) sequences from 16 species together with 3 sequences available in GenBank. As previously shown with the large subunit ribosomal RNA 28S gene, two classes of Chaetognatha SSU rRNA gene can be identified, suggesting a duplication of the whole ribosomal cluster; allowing the rooting of one class of genes by another in phylogenetic analyses. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the molecular data, and statistical tests showed (1) that there are three main monophyletic groups: Sagittidae/Krohnittidae, Spadellidae/Pterosagittidae, and Eukrohniidae/Heterokrohniidae, (2) that the group of Aphragmophora without Pterosagittidae (Sagittidae/Krohnittidae) is monophyletic, (3) the Spadellidae/Pterosagittidae and Eukrohniidae/Heterokrohniidae families are very likely clustered, (4) the Krohnittidae and Pterosagittidae groups should no longer be considered as families as they are included in other groups designated as families, (5) suborder Ctenodontina is not monophyletic and the Flabellodontina should no longer be considered as a suborder, and (6) the Syngonata/Chorismogonata and the Monophragmophora/Biphragmophora hypotheses are rejected. Such conclusions are considered in the light of morphological characters, several of which are shown to be prone to homoplasy. PMID:16434216

  6. Hot spots of DNA double-strand breaks in human rDNA units are produced in vivo.

    Tchurikov, Nickolai A; Yudkin, Dmitry V; Gorbacheva, Maria A; Kulemzina, Anastasia I; Grischenko, Irina V; Fedoseeva, Daria M; Sosin, Dmitri V; Kravatsky, Yuri V; Kretova, Olga V

    2016-01-01

    Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer genomics(1,2). There are nine hot spots of DSBs located in human rDNA units(3-6). Here we describe that the profiles of these hot spots coincide with the profiles of γ-H2AX or H2AX, strongly suggesting a high level of in vivo breakage inside rDNA genes. The data were confirmed by microscopic observation of the largest γ-H2AX foci inside nucleoli in interphase chromosomes. In metaphase chromosomes, we observed that only some portion of rDNA clusters possess γ-H2AX foci and that all γ-H2AX foci co-localize with UBF-1 binding sites, which strongly suggests that only active rDNA units possess the hot spots of DSBs. Both γ-H2AX and UBF-1 are epigenetically inherited and thus indicate the rDNA units that were active in the previous cell cycle. These results have implications for diverse fields, including epigenetics and cancer genomics. PMID:27160357

  7. PFR²: a curated database of planktonic foraminifera 18S ribosomal DNA as a resource for studies of plankton ecology, biogeography and evolution.

    Morard, Raphaël; Darling, Kate F; Mahé, Frédéric; Audic, Stéphane; Ujiié, Yurika; Weiner, Agnes K M; André, Aurore; Seears, Heidi A; Wade, Christopher M; Quillévéré, Frédéric; Douady, Christophe J; Escarguel, Gilles; de Garidel-Thoron, Thibault; Siccha, Michael; Kucera, Michal; de Vargas, Colomban

    2015-11-01

    Planktonic foraminifera (Rhizaria) are ubiquitous marine pelagic protists producing calcareous shells with conspicuous morphology. They play an important role in the marine carbon cycle, and their exceptional fossil record serves as the basis for biochronostratigraphy and past climate reconstructions. A major worldwide sampling effort over the last two decades has resulted in the establishment of multiple large collections of cryopreserved individual planktonic foraminifera samples. Thousands of 18S rDNA partial sequences have been generated, representing all major known morphological taxa across their worldwide oceanic range. This comprehensive data coverage provides an opportunity to assess patterns of molecular ecology and evolution in a holistic way for an entire group of planktonic protists. We combined all available published and unpublished genetic data to build PFR(2), the Planktonic foraminifera Ribosomal Reference database. The first version of the database includes 3322 reference 18S rDNA sequences belonging to 32 of the 47 known morphospecies of extant planktonic foraminifera, collected from 460 oceanic stations. All sequences have been rigorously taxonomically curated using a six-rank annotation system fully resolved to the morphological species level and linked to a series of metadata. The PFR(2) website, available at http://pfr2.sb-roscoff.fr, allows downloading the entire database or specific sections, as well as the identification of new planktonic foraminiferal sequences. Its novel, fully documented curation process integrates advances in morphological and molecular taxonomy. It allows for an increase in its taxonomic resolution and assures that integrity is maintained by including a complete contingency tracking of annotations and assuring that the annotations remain internally consistent. PMID:25828689

  8. The African buffalo parasite Theileria. sp. (buffalo can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

    R.P. Bishop

    2015-12-01

    Full Text Available African Cape buffalo (Syncerus caffer is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo, which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo, using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo. This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo. Collectively, the data provides further evidence that T. sp. (buffalo. is a distinct species from T. parva.

  9. An 18S ribosomal DNA barcode for the study of Isomermis lairdi, a parasite of the blackfly Simulium damnosum s.l.

    Crainey, J L; Wilson, M D; Post, R J

    2009-09-01

    The mermithid parasite, Isomermis lairdi Mondet, Poinar & Bernadou (Nematoda: Mermithidae), is known to have a major impact on populations of Simulium damnosum s.l. Theobald (Diptera: Simuliidae) and on their efficiency as vectors of Onchocerca volvulus (Leuckart) (Nematoda: Filarioidea). However, the value of I. lairdi and other mermithid parasites as potential means of integrated vector control has not been fully realized. This is partly because traditional taxonomic approaches have been insufficient for describing and analysing important aspects of their biology and host range. In total, rDNA barcode sequences have been obtained from over 70 I. lairdi mermithids found parasitizing S. damnosum s.l. larvae in three different rivers. No two sequences were found to vary by more than 0.5%, and cytospecies identification of mermithid hosts revealed that I. lairdi with identical rDNA barcodes can parasitize multiple cytoforms of the S. damnosum complex, including S. squamosum (Enderlein). Phylogenetic analysis using a partial sequence from the 18S ribosomal DNA barcode, grouped I. lairdi in a monophyletic group with Gastromermis viridis Welch (Nematoda: Mermithidae) and Isomermis wisconsinensis Welch (Nematoda: Mermithidae). PMID:19712154

  10. A phylogeny of the damselfly genus calopteryx (Odonata) using mitochondrial 16S rDNA markers.

    Misof, B; Anderson, C L; Hadrys, H

    2000-04-01

    We seek to reconstruct the phylogenetic relationships of the damselfly genus Calopteryx, for which extensive behavioral and morphological knowledge already exists. To date, analyses of the evolutionary pathways of different life history traits have been hampered by the absence of a robust phylogeny based on morphological data. In this study, we concentrate on establishing phylogenetic information from parts of the 16S rDNA gene, which we sequenced for nine Calopteryx species and five outgroup species. The mt 16S rDNA data set did not show signs of saturated variation for ingroup taxa, and phylogenetic reconstructions were insensitive to variation of outgroup taxa. Parsimony, neighbor-joining, and maximum-likelihood reconstructions agreed on parts of the tree. A consensus tree summarizes the significant results and indicates problematic nodes. The 16S rDNA sequences support monophyly of the genera Mnais, Matrona, and Calopteryx. However, the genus Calopteryx may not be monophyletic, since Matrona basilaris and Calopteryx atrata are sister taxa under every parameter setting. The North American and European taxa each appear as monophyletic clades, while the Asian Calopteryx atrata and Calopteryx cornelia are not monophyletic. Our data implies a different paleobiogeographic history of the Eurasian and North American species, with extant Eurasian species complexes shaped by glacial periods, in contrast to extant North American species groups. PMID:10764530

  11. Experimental Conditions: SE18_S1_M1_D1 [Metabolonote[Archive

    Full Text Available liver and brain by Orbitrap MS and automated search engine Lipid Search SE18_S1 Mouse liver SE18_S1_M1 34.1...phy. SE18_MS1 Preparation of lipid extract and ESI negative detection by LC-MS analysis SE18_DS1 Identification of phospholipids with Lipid Search default ...

  12. Experimental Conditions: SE18_S2_M1_D1 [Metabolonote[Archive

    Full Text Available liver and brain by Orbitrap MS and automated search engine Lipid Search SE18_S2 Mouse brain SE18_S2_M1 10.8...phy. SE18_MS1 Preparation of lipid extract and ESI negative detection by LC-MS analysis SE18_DS1 Identification of phospholipids with Lipid Search default ...

  13. An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

    Shenkar Noa

    2009-08-01

    suggest a sister-group relationship between Salpida and Pyrosomatida within Thaliacea. Conclusion An updated phylogenetic framework for tunicates is provided based on phylogenetic analyses using the most realistic evolutionary models currently available for ribosomal molecules and an unprecedented taxonomic sampling. Detailed analyses of the 18S rRNA gene allowed a clear definition of the major tunicate groups and revealed contrasting evolutionary dynamics among major lineages. The resolving power of this gene nevertheless appears limited within the clades composed of Phlebobranchia + Thaliacea + Aplousobranchia and Pyuridae + Styelidae, which were delineated as spots of low resolution. These limitations underline the need to develop new nuclear markers in order to further resolve the phylogeny of this keystone group in chordate evolution.

  14. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

    Forouzandeh-Moghadam, M; MB Rokni; Dalimi, A; M Mahami-Oskouei

    2011-01-01

    Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, I...

  15. Investigation of molluscan phylogeny on the basis of 18S rRNA sequences.

    Winnepenninckx, B; Backeljau, T; De Wachter, R

    1996-12-01

    The 18S rRNA sequences of 12 molluscs, representing the extant classes Gastropoda, Bivalvia, Polyplacophora, Scaphopoda, and Caudofoveata, were determined and compared with selected known 18S rRNA sequences of Metazoa, including other Mollusca. These data do not provide support for a close relationship between Platyhelminthes (Turbellaria) and Mollusca, but rather suggest that the latter group belongs to a clade of eutrochozoan coelomates. The 18S rRNA data fail to recover molluscan, bivalve, or gastropod monophyly. However, the branching pattern of the eutrochozoan phyla and classes is unstable, probably due to the explosive Cambrian radiation during which these groups arose. Similarly, the 18S rRNA data do not provide a reliable signal for the molluscan interclass relationships. Nevertheless, we obtained strong preliminary support for phylogenetic inferences at more restricted taxonomic levels, such as the monophyly of Polyplacophora, Caenogastropoda, Euthyneura, Heterodonta, and Arcoida. PMID:8952075

  16. Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.

    Black, W C; Piesman, J

    1994-01-01

    Ticks are parasitiform mites that are obligate hematophagous ectoparasites of amphibians, reptiles, birds, and mammals. A phylogeny for tick families, subfamilies, and genera has been described based on morphological characters, life histories, and host associations. To test the existing phylogeny, we sequenced approximately 460 bp from the 3' end of the mitochondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick species; a mesostigmatid mite, Dermanyssus gallinae, was used as an outgroup. P...

  17. Type I-like intervening sequences are found in the rDNA of the nematode Ascaris lumbricoides.

    Neuhaus, H; Müller, F.; Etter, A; Tobler, H

    1987-01-01

    The intervening sequences in the large ribosomal RNA gene of Ascaris lumbricoides var. suum show many similarities to the type I insertions, previously found only in some insect species. They include structural features, but also a presumed transcriptional inactivity in vivo: No transcript of the rDNA intervening sequence in A. lumbricoides could be detected in Northern and dot blot hybridizations. However, the primary structure of the Pol I promoter region is well conserved in interrupted an...

  18. Sharp switches between regular and swinger mitochondrial replication: 16S rDNA systematically exchanging nucleotides AT+CG in the mitogenome of Kamimuria wangi.

    Seligmann, Hervé

    2016-07-01

    Swinger DNAs are sequences whose homology with known sequences is detected only by assuming systematic exchanges between nucleotides. Nine symmetric (XY, i.e. AC) and fourteen asymmetric (X->Y->Z, i.e. A->C->G) exchanges exist. All swinger DNA previously detected in GenBank follow the AT+CG exchange, while mitochondrial swinger RNAs distribute among different swinger types. Here different alignment criteria detect 87 additional swinger mitochondrial DNAs (86 from insects), including the first swinger gene embedded within a complete genome, corresponding to the mitochondrial 16S rDNA of the stonefly Kamimuria wangi. Other Kamimuria mt genome regions are "regular", stressing unanswered questions on (a) swinger polymerization regulation; (b) swinger 16S rDNA functions; and (c) specificity to rDNA, in particular 16S rDNA. Sharp switches between regular and swinger replication, together with previous observations on swinger transcription, suggest that swinger replication might be due to a switch in polymerization mode of regular polymerases and the possibility of swinger-encoded information, predicted in primordial genes such as rDNA. PMID:25865623

  19. 16S/18S ribosomal DNA clone library analysis of rumen microbial diversity

    The rumen contains a complex ecosystem where billions of bacteria, archaea, protozoa and fungi reside. This diverse microbiota is well adapted to live in the rumen and play an important role in the digestion of feed and nutrient supply to the host in the form of microbial protein and volatile fatty acids. It is estimated that the rumen microbial population consists of about 106 protozoa/ml, 103-107 fungi/ml, 1010 bacteria/ml, and 109 methanogens/ml. To better understand the complex relationships in the rumen, it is necessary to gain an insight into the diversity of the rumen microbes and how the quantity and composition of rumen micro-organisms are altered by a number of different host factors such as age, genetics and diet. In the past, the diversity of micro-organisms from the digestive tracts of domesticated ruminants has been identified by classical microbiological techniques. However, given the fastidious growth requirements of rumen micro-organisms, it is reasonable to concede that the culture-dependent methods may select against some species, or taxonomic groups, leading researchers to underestimate the microbial diversity that is actually present in the rumen. In fact, it has been speculated that 90% of micro-organisms in nature have escaped traditional cultivation methods. Therefore, a major challenge in microbial ecology has been to assess the diversity and structure of natural microbial communities. The field of molecular biology has advanced with many innovative technological breakthroughs. The ability to extract and to isolate high-molecular weight DNA from rumen digesta, PCR amplify genes from specific microbial groups and obtain gene sequence data is now a routine event. The small subunit ribosomal RNA (SSU-rRNA) gene, called 16S in prokaryotes and 18S in eukaryotes, is the most widely used molecular marker to presumptively identify morphologically indistinguishable species, to infer their phylogenetic relationships, and to elucidate microbial

  20. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

    Wörheide Gert

    2008-02-01

    Full Text Available Abstract Background The cytoplasmic ribosomal small subunit (SSU, 18S ribosomal RNA (rRNA is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836, a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes. Results We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges and Calcarea (calcareous sponges. We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early

  1. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

    2008-01-01

    Background The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa. Here, we explore SSU rRNA secondary structures from the three extant classes of Phylum Porifera (Grant, 1836), a pivotal, but largely unresolved taxon of early branching Metazoa. This is the first phylogenetic study of poriferan SSU rRNA data to date that includes detailed comparative secondary structure information for all three sponge classes. Results We found base compositional and structural differences in SSU rRNA among Demospongiae, Hexactinellida (glass sponges) and Calcarea (calcareous sponges). We showed that analyses of primary rRNA sequences, including secondary structure-specific evolutionary models, in combination with reconstruction of the evolution of unusual structural features, reveal a substantial amount of additional information. Of special note was the finding that the gene tree topologies of marine haplosclerid demosponges, which are inconsistent with the current morphology-based classification, are supported by our reconstructed evolution of secondary structure features. Therefore, these features can provide alternative support for sequence-based topologies and give insights into the evolution of the molecule itself. To encourage and facilitate the application of rRNA models in phylogenetics of early metazoans, we present 52 SSU r

  2. Phylogenetic position of the yeast-like symbiotes of Tagosodes orizicolus (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences.

    Xet-Mull, Ana M; Quesada, Tania; Espinoza, Ana M

    2004-09-01

    Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus. PMID:17361570

  3. [An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae)].

    Cruz, V P; Oliveira, C; Foresti, F

    2015-01-01

    5S rDNA genes of the stingray Potamotrygon motoro were PCR replicated, purified, cloned and sequenced. Two distinct classes of segments of different sizes were obtained. The smallest, with 342 bp units, was classified as class I, and the largest, with 1900 bp units, was designated as class II. Alignment with the consensus sequences for both classes showed changes in a few bases in the 5S rDNA genes. TATA-like sequences were detected in the nontranscribed spacer (NTS) regions of class I and a microsatellite (GCT) 10 sequence was detected in the NTS region of class II. The results obtained can help to understand the molecular organization of ribosomal genes and the mechanism of gene dispersion. PMID:26107907

  4. The Evolutionary Dynamics of Ribosomal Genes, Histone H3, and Transposable Rex Elements in the Genome of Atlantic Snappers.

    Costa, Gideão Wagner Werneck Félix da; Cioffi, Marcelo de Bello; Bertollo, Luiz Antonio Carlos; Molina, Wagner Franco

    2016-03-01

    Lutjanidae is a family of primarily marine and carnivorous fishes distributed in the Atlantic, Indian, and Pacific oceans, with enormous economic and ecological importance. In order to better clarify the conservative chromosomal evolution of Lutjanidae, we analyzed the evolutionary dynamics of 5 repetitive DNA classes in 5 Lutjanus and in 1 Ocyurus species from the Western Atlantic. The ribosomal 18S sites were generally located in a single chromosome pair, except for L. jocu and L. alexandrei where they are found in 2 pairs. In turn, the 5S rDNA sites are unique, terminal and nonsyntenic with the 18S rDNA sites. In 3 species analyzed, H3 hisDNA genes were found in 1 chromosomal pair. However, while L. jocu presented 2 H3 sites, O. chrysurus showed a noteworthy dispersion of this gene in almost all chromosomes of the karyotype. Retrotransposons Rex1 and Rex3 do not exhibit any association with the explosive distribution of H3 sequences in O. chrysurus. The low compartmentalization of Rex elements, in addition to the general nondynamic distribution of ribosomal and H3 genes, corroborate the karyotype conservatism in Lutjanidae species, also at the microstructural level. However, some "disturbing evolutionary waves" can break down this conservative scenario, as evidenced by the massive random dispersion of H3 hisDNA in the genome of O. chrysurus. The implication of the genomic expansion of H3 histone genes and their functionality remain unknown, although suggesting that they have higher evolutionary dynamics than previously thought. PMID:26792596

  5. Phylogenetic relationships within the Mysidae (Crustacea, Peracarida, Mysida) based on nuclear 18S ribosomal RNA sequences

    Remerie, T.; Bulckaen, B.; Calderon, J; Deprez, T.; Mees, J.; J. Vanfleteren; Vanreusel, A.; Vierstraete, A; Vincx, M.; Wittmann, K.J.; Wooldridge, T.

    2005-01-01

    Species of the order Mysida (Crustacea, Peracarida) are shrimp-like animals that occur in vast numbers in coastal regions of the world. The order Mysida comprises 1053 species and 165 genera. The present study covers 25 species of the well-defined Mysidae, the most speciose family within the order Mysida. 18S rRNA sequence analysis confirms that the subfamily Siriellinae is monophyletic. On the other hand the subfamily Gastrosaccinae is paraphyletic and the subfamily Mysinae, represented in t...

  6. 18S rRNA suggests that Entoprocta are protostomes, unrelated to Ectoprocta

    Mackey, L.Y.; Winnepenninckx, B.; Wachter, R.; Backeljau, T.; Emschermann, P.; Garey, J.R.

    1996-01-01

    The Ento- and Ectoprocta are sometimes placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes. However, Entoprocta have also been allied to the pseudocoelomates, while Ectoprocta are often united with the Brachiopoda and Phoronida in the (super)phylum Lophophorata. Hence, the phylogenetic relationships of these taxa are still much debated. We determined complete 18S rRNA sequences of two entoprocts, an ectoproct, an inarticulate brachiopod, a phoronid, t...

  7. Details of gastropod phylogeny inferred from 18S rRNA sequences.

    Winnepenninckx, B; Steiner, G; Backeljau, T; De Wachter, R

    1998-02-01

    Some generally accepted viewpoints on the phylogenetic relationships within the molluscan class Gastropoda are reassessed by comparing complete 18S rRNA sequences. Phylogenetic analyses were performed using the neighbor-joining and maximum parsimony methods. The previously suggested basal position of Archaeogastropoda, including Neritimorpha and Vetigastropoda, in the gastropod clade is confirmed. The present study also provides new molecular evidence for the monophyly of both Caenogastropoda and Euthyneura (Pulmonata and Opisthobranchia), making Prosobranchia paraphyletic. The relationships within Caenogastropoda and Euthyneura data turn out to be very unstable on the basis of the present 18S rRNA sequences. The present 18S rRNA data question, but are insufficient to decide on, muricacean (Neogastropoda), neotaenioglossan, pulmonate, or stylommatophoran monophyly. The analyses also focus on two systellommatophoran families, namely, Veronicellidae and Onchidiidae. It is suggested that Systellommatophora are not a monophyletic unit but, due to the lack of stability in the euthyneuran clade, their affinity to either Opisthobranchia or Pulmonata could not be determined. PMID:9479694

  8. Application of EMA, Fluorescent Staining and FISH of rDNA in Analysis of Aloe vera (L.) Burm. f. Chromosomes

    JHA, Timir Baran; Yamamoto, Masashi; ヤマモト, マサシ; 山本, 雅

    2012-01-01

    Fluorescent banding patterns and the locations of the ribosomal RNA gene (rDNA) of Indian Aloe vera (L.) Burm. f. chromosomes were determined. Root tips were pretreated in 2 mM 8- hydroxyquinoline at 10 to 12℃ for 3h. The chromosome samples were prepared by the enzymatic maceration and air-drying method (EMA). The best preparation, with all chromosomes relatively extended and well spread without cytoplasm, was obtained following enzymatic condition of 0.67% Cellulase Onozuka RS, 0.5% Macerozy...

  9. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  10. The linked units of 5S rDNA and U1 snDNA of razor shells (Mollusca: Bivalvia: Pharidae).

    Vierna, J; Jensen, K T; Martínez-Lage, A; González-Tizón, A M

    2011-08-01

    The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at -25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinant. PMID:21364693

  11. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

    Paolo Gaibani; Mara Mariconti; Gloria Bua; Sonia Bonora; Davide Sassera; Maria Paola Landini; Patrizia Mulatto; Stefano Novati; Claudio Bandi; Vittorio Sambri

    2013-01-01

    Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole b...

  12. Species markers for equine strongyles detected in intergenic rDNA by PCR-RFLP.

    Gasser, R B; Stevenson, L A; Chilton, N B; Nansen, P; Bucknell, D G; Beveridge, I

    1996-10-01

    Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosomal DNA was amplified from genomic DNA by polymerase chain reaction (PCR) using conserved primers, digested separately with six restriction endonucleases (AluI, BfaI, CfoI, Hae III, VSpI and XbaI) and the fragments separated by agarose gel electrophoresis. The PCR products of the three Strongylus species were approx. 90-100 bp smaller in size compared with those of the other 13 species. The PCR-RFLP analysis of the rDNA region spanning the first and second internal transcribed spacers plus the 5.85 rDNA gene (ITS+) produced characteristic patterns for each of the 16 species examined, and no variation in RFLP patterns was detected within the species Cy. catinatum, where multiple isolates were analysed. The study demonstrates that the internal transcribed spacer sequences provide genetic markers for the species identification of a range of equine strongyles. These markers will be of use for the identification of egg and larval stages, where morphological characters alone are unreliable. The results also indicate that the spacer sequences will be of use to study the systematics of equine strongyles. PMID:8910892

  13. 河南猪株旋毛虫18S rRNA基因的同源性序列分析%18S rRNA sequence analysis and construction of phylogenetic tree of Trichinella from swine in Henan Province

    王丽娜; 路国兵; 杨晓东; 高云; 陈晓宁

    2011-01-01

    目的 通过分析18S rRNA基因序列同源性,对河南猪株旋毛虫进行分子鉴定及分类. 方法 收集河南猪株旋毛虫成虫,提取总RNA,反转录合成cDNA,经特异引物扩增获得18S rRNA基因片段.将此目的基因与pMD18-T载体连接,转化大肠埃希菌感受态细胞,阳性克隆经PCR及酶切鉴定后进行序列测定及分析,构建系统发育树. 结果 构建的重组质粒酶切片段大小分别为2 700和1 800 bp,与预期值相符.根据18S rRNA碱基序列构建系统发生树,河南猪株旋毛虫与虫株Trichinella nativa (AY487254.1)的亲缘关系较近,同源性为99.1%. 结论 河南猪株旋毛虫归属于T2.%Objective To identify and classify Trichinella from swine in Henan Province at the molecular level by sequence homology analysis of the 18S rRNA gene. Methods Total RNA was extracted from adult Trichinella collected from swine in Henan. cDNA was obtained by reverse transcription. The 18S rRNA gene was amplified with a specific primer. The fragments of PCR products were ligated to pMD18-T. This was then transformed into E. Coli competent cells. After identification by PCR and restrictive endonuclease digestion, the positive clone was sequenced and analyzed and then a phylogenetic tree was constructed. Results The fragments of the constructed recombinant plasmid were a-bout 2 700 bp and 1 800 bp, which were consistent with expected values. In the phylogenetic tree based on the base sequence of the 18S rRNA gene, Trichinella from swine in Henan Province was the closest relative to T. Nativa (AY487254. 1) with sequence similarity of more than 99. 1%. Conclusion Trichinella from swine in Henan Province was Trichinella nativa (T2).

  14. Methylation pattern of the intergenic spacer of rRNA genes in excised cotyledons of Cucurbita pepo L. (Zucchini) after hormone treatment

    High molecular mass genomic DNA was isolated from excised marrow cotyledons (Cucurbita pepo L. zucchini) treated with 6-benzyladenine (BA) of methyl ester of jasmonic acid (MeJA) for 24 h in darkness. DNA purified from contaminating polysaccharides with Celite column was completely digested with the restriction enzyme Eco RI and the changes in the methylation pattern of the intergenic spacer (IGS) of r RNA genes were studied after subsequent digestion with the couple of restriction enzymes-isoschizomers MSP I and Hpa II by the method of 'indirect end labelling'. As rDNA units probe a cloned 32P-labelled Eco RI 2.1 kb fragment spanning in the most part of 18S r RNA gene from flax rDNA was used. Results showed heavy methylation of the rRNA genes. As judged from the almost total lack of digestion with HPA II, there were no methylation free regions in repeated rDNA units or little if any were observed. A hypo methylated Hps II site was detected near the promoter region in some of the repeats. Digestion with Msp I affected nearly 50% of the repeating units. The Msp digestion fragments of the 6.2 kb Eco RI fragment of r DNA were few in number and large in size (0.5 - 2.5 kb). This suggested that in addition with -CpG- sequences, methylation in -CpNpG- might not be random. Methylation pattern in IGS was not changed upon treatment of the cotyledons in vivo with BA and MeJA. Thus, previously observed hormone-mediated effects on the eactivity of rRNA gene expression were not accompanied by any significant changes of the methylation pattern in IGS. (authors)

  15. Length polymorphisms for intergenic spacer regions of 16S-23S rDNA in members of the new hydrogen-producing bacteria

    2007-01-01

    A method based on PCR amplification of the 16S rRNA gene (rDNA) -23S rDNA intergenic spacer regions (ISR) was developed for the identification of species within the novel group hydrogen-producing anaerobes. The sizes of the PCR products varied from 1264 to 398 bp. Strain of isolate Rennanqilyf 3 was characterized as having products of 1262, 398, 638, 437 and 436 bp. The isolate Rennanqilyf 1 had product of 1264 bp. The isolate Rennanqilyf 13 had products of 1261, 579 and 485 bp. Of the 3 species of the novel group hydrogenproducing anaerobes examined, no one was indistinguishable. Two environmental isolates were identified as hydrogen-producing bacteria, which were new species in present taxon. Rennanqilyf 3 could not be associated With any Clostridium sp. Studied. Rennanqilyf 1 could be classified into Clostridium genus. The combination between 16S rDNA equencing and length polymorphisms of IRS in 16S-23S rDNA is a better method for determining species of the hydrogen-producing bacteria.

  16. Magic wavelengths for the $5s-18s$ transition in rubidium

    Goldschmidt, E A; Koller, S B; Wyllie, R; Brown, R C; Porto, J V; Safronova, U I; Safronova, M S

    2015-01-01

    Magic wavelengths, for which there is no differential ac Stark shift for the ground and excited state of the atom, allow trapping of excited Rydberg atoms without broadening the optical transition. This is an important tool for implementing quantum gates and other quantum information protocols with Rydberg atoms, and reliable theoretical methods to find such magic wavelengths are thus extremely useful. We use a high-precision all-order method to calculate magic wavelengths for the $5s-18s$ transition of rubidium, and compare the calculation to experiment by measuring the light shift for atoms held in an optical dipole trap at a range of wavelengths near a calculated magic value.

  17. Preliminary phylogeny of the thrips parasitoids of Turkey based on some morphological scales and 28S D2 rDNA, with description of a new species

    DOĞANLAR, Oğuzhan; Doğanlar, Mikdat; Frary, Anne

    2010-01-01

    Species of the Ceranisus thrips-attacking genus are difficult to distinguish morphologically. The phylogenetic relationships within the Ceranisus species were explored using nucleotide sequences of the 28S D2 expansion region of the rDNA gene. Bayesian, maximum likelihood, and parsimony inference methods were employed to construct the phylogenetic relationships. Principal component analysis on the Turkish species of Ceranisus, namely antalyacus, menes, bozovaensis, hirsutus, planitianus (a ne...

  18. Inferring a classification of the Adenophorea (Nematoda) from nucleotide sequences of the D3 expansion segment (26/28s rDNA)

    Litvaitis, M.K.; Bates, J.W.; Hope, W. D.; Moens, T.

    2000-01-01

    Nucleotide sequences of the D3 expansion segment of the 28S rDNA gene were used to reconstruct evolutionary relationships within the Adenophorea. Neighbor-joining and parsimony analyses of representatives of most major taxa revealed a paraphyletic Adenophorea (p = 0.0005). Within Adenophorea, the Enoplia, Enoplida, and Enoplina were paraphyletic (p = 0.0024, 0.0014, and 0.0120, respectively). A major division was evident within the Enoplida, with one lineage consisting of a basal Thoracostomo...

  19. The phylogenetic position of the Loimoidae Price, 1936 (Monogenoidea: Monocotylidea) based on analyses of partial rDNA sequences and morphological data.

    Boeger, W A; Kritsky, D C; Domingues, M V; Bueno-Silva, M

    2014-06-01

    Phylogenetic analyses of partial sequences of 18S and 28S rDNA of some monogenoids, including monocotylids and a specimen of Loimosina sp. collected from a hammerhead shark off Brazil, indicated that the Loimoidae (as represented by the specimen of Loimosina sp.) represents an in-group taxon of the Monocotylidae. In all analyses, the Loimoidae fell within a major monocotylid clade including species of the Heterocotylinae, Decacotylinae, and Monocotylinae. The Loimoidae formed a terminal clade with two heterocotyline species, Troglocephalus rhinobatidis and Neoheterocotyle rhinobatis, for which it represented the sister taxon. The following morphological characters supported the clade comprising the Loimoidae, Heterocotylinae, Decacotylinae and Monocotylinae: single vagina present, presence of a narrow deep anchor root, and presence of a marginal haptoral membrane. The presence of cephalic pits was identified as a putative synapomorphy for the clade (Loimoidae (T. rhinobatidis, N. rhinobatis)). Although rDNA sequence data support the rejection of the Loimoidae and incorporating its species into the Monocotylidae, this action was not recommended pending a full phylogenetic analysis of morphological data. PMID:24491371

  20. Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt.

    Gjerde, Bjørn; Hilali, Mosaad; Mawgood, Sahar Abdel

    2015-09-01

    A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15

  1. 'Think before you buy under-18s drink': evaluation of a community alcohol intervention.

    Kypri, Kypros; Dean, Johanna; Kirby, Sandra; Harris, Jennifer; Kake, Tai

    2005-01-01

    Hazardous consumption of alcohol by teenagers is a significant public health problem in New Zealand. Concern about supply of alcohol to minors motivated 'Think before you buy under-18s drink', a campaign to reduce alcohol-related harm by discouraging inappropriate supply of alcohol by adults. Two intervention districts and a comparison district, in the South Island of New Zealand, were selected for the purpose of evaluating the campaign. Primary outcome measures were changes in the prevalence of parent supply to their teenager (13-17 years) for unsupervised drinking (SUD), and changes in the prevalence of binge drinking among teenagers. At baseline, 49% of teenagers reported a recent episode of binge drinking. SUD in the past month was reported by 36% of teenagers. Recent purchases of alcohol by under-18s were common (bottle shops: 16%; pubs/bars: 11%). In contrast to teenagers, only 2% of parents reported SUD in the past month. Levels of binge drinking decreased in all three districts. Analysis of data from 474 teenagers who completed questionnaires, at baseline and follow-up, showed decreased SUD in Ashburton and Waitaki relative to Clutha, although this was not significant (OR=0.73; 95% CI: 0.43, 1.25). Discrepancies between teenager and parent reports of SUD may be due to the latter providing a socially desirable survey response and to differences in the interpretation of what constitutes adult supervision. The lack of a significant association between changes in SUD and binge drinking may be a consequence of teenagers obtaining relatively small amounts of alcohol from their parents and larger quantities from other sources, e.g. peers (some of whom may be able to purchase alcohol legally) and from licensed premises. PMID:16191716

  2. Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

    McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

    2015-07-01

    The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland. PMID:25631743

  3. Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools

    Oldach, David W.; Delwiche, Charles F.; Jakobsen, Kjetill S.; Tengs, Torstein; Brown, Ernest G.; Kempton, Jason W.; Schaefer, Eric F.; Bowers, Holly A; Glasgow, Howard B.; Burkholder, JoAnn M.; Steidinger, Karen A.; Rublee, Parke A.

    2000-01-01

    The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing...

  4. FISH and AgNor mapping of the 45S and 5S rRNA genes in wild and cultivated species of Capsicum (Solananceae).

    Scaldaferro, Marisel A; da Cruz, M Victoria Romero; Cecchini, Nicolás M; Moscone, Eduardo A

    2016-02-01

    Chromosome number and position of rDNA were studied in 12 wild and cultivated species of the genus Capsicum with chromosome numbers x = 12 and x = 13 (22 samples). For the first time in these species, the 5S and 45S rRNA loci were localized and physically mapped using two-color fluorescence in situ hybridization and AgNOR banding. We focused on the comparison of the results obtained with both methods with the aim of accurately revealing the real functional rRNA genes. The analyzes were based on a previous work that reported that the 18S-5.8S-25S loci mostly coincide with GC-rich heterochromatic regions and likely have given rise to satellite DNAs, which are not active genes. These data show the variability of rDNA within karyotypes of the genus Capsicum, providing anchor points for (comparative) genetic maps. In addition, the obtained information might be useful for studies on evolution of repetitive DNA. PMID:26853884

  5. Complete sequence of the extrachromosomal rDNA molecule from the ciliate Tetrahymena thermophila strain B1868VII

    Engberg, J; Nielsen, Henrik

    1990-01-01

    The recent development of rDNA vectors for transformation of Tetrahymena combined with improved microinjection technology should lead to a renewed interest in this organism. In particular, the rDNA itself constitutes an attractive system for biochemical studies. The rDNA is amplified to a level of...

  6. Nucleolar dominance in triticales: control by unlinked genes

    Neves, N.; Silva, M; Heslop-Harrison, J. S.; Viegas, W.

    1997-01-01

    Hybrid plants and animals often show suppression of activity of ribosomal genes (rDNA) originating from one of the parental or ancestral species. In the wheat6rye amphiploid triticale, containing 28 chromosomes of wheat origin and 14 from rye, rDNA of rye origin (on chromosome 1R) is not normally expressed, while the 1B- and 6B-origin rDNA from wheat shows strong expression. Expression of rDNA can be accurately assessed by the silver staining method, which stains bo...

  7. Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control

    Shaoning Chen; Hao Gu; Xiaoming Wang; Jishuang Chen; Weimin Zhu

    2011-01-01

    A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV),using 18S rRNA as an internal control.Species- and subgroups-specific primers designed to differentiate CMV subgroups Ⅰ and Ⅱ, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum).Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parmeters were optimized and a multiplex RT-PCR procedure was established.Six fragments of 704, 593, 512, 421,385, 255 bp, specific to CMV subgroup ll, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were sinultaneously amplified.The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA).This method was successfully used for field detection.Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found.The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.

  8. Molecular phylogeny of endophytic isolates of Ampelomyces from Iran based on rDNA ITS sequences.

    Jamali, Samad

    2015-01-01

    During 2012, five isolates of pycnidial fungi were recovered from roots of tomato (Solanum lycopersicum) plants in Iran. Based on morphological characteristics the presence of Ampelomyces was documented. To confirm morphological identification and clarify the placement of endophytic isolates of Ampelomyces, DNA was extracted from isolates using a genomic DNA purification Kit. Region of internal transcribed spacers 1, 2 and 5.8S genes of rDNA were amplified using ITS4 and ITS1 universal primer set. Amplicons were purified, sequenced and submitted to the GenBank. The resulting sequence (600 bp) was submitted to a BLAST search to find most similar sequences in GenBank. The ITS sequences of isolates obtained in Iran were compared to those of other related authentic sequences obtained from GenBank. Iranian endophytic isolates had 100 % similarity of among themselves, while all isolates of Ampelomyces sequences analyzed had an average of 95.2 % (range 87-100 %) similarity. When Ampelomyces ITS sequences were analyzed by both distance-based and maximum parsimony methods, the Ampelomyces isolates were segregate into 11 distinct clades. The ITS sequences of endophytic isolates obtained in Iran were identical with endophytic isolates from other country including USA, Australia, Hungary and Spain. Our analyses of phylogenetic data showed that endophytic isolates from Iran and other countries are distinct group. The high ITS sequence-divergence values and the phylogenetic analysis suggested the isolates of Ampelomyces in the clades are not closely related and indeed a problematic species complex. PMID:25245955

  9. Angiosperm phylogeny inferred from sequences of four mitochondrial genes

    Yin-Long QIU; Zhi-Duan CHEN; Libo LI; Bin WANG; Jia-Yu XUE; Tory A. HENDRY; Rui-Qi LI; Joseph W. BROWN; Yang LIU; Geordan T. HUDSON

    2010-01-01

    An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.

  10. Phylogenetic Analyses of Three Genes of Pedinomonas noctilucae, the Green Endosymbiont of the Marine Dinoflagellate Noctiluca scintillans, Reveal its Affiliation to the Order Marsupiomonadales (Chlorophyta, Pedinophyceae) under the Reinstated Name Protoeuglena noctilucae.

    Wang, Lu; Lin, Xin; Goes, Joaquim I; Lin, Senjie

    2016-04-01

    In the last decade, field studies in the northern Arabian Sea showed a drastic shift from diatom-dominated phytoplankton blooms to thick and widespread blooms of the green dinoflagellate, Noctiluca scintillans. Unlike the exclusively heterotrophic red form, which occurs widely in tropical to temperate coastal waters, the green Noctiluca contains a large number of endosymbiotic algal cells that can perform photosynthesis. These symbiotic microalgae were first described under the genus Protoeuglena Subrahmanyan and further transferred to Pedinomonas as P. noctilucae Sweeney. In this study, we used the 18S rDNA, rbcL and chloroplast 16S rDNA as gene markers, in combination with the previously reported morphological features, to re-examine the phylogenetic position of this endosymbiotic algal species. Phylogenetic trees inferred from these genes consistently indicated that P. noctilucae is distantly related to the type species of Pedinomonas. The sequences formed a monophyletic clade sister to the clade of Marsupiomonas necessitating the placement of the algal symbionts as an independent genus within the family Marsupiomonadaceae. Based on the phylogenetic affiliation and ecological characteristics of this alga as well as the priority rule of nomenclature, we reinstate the genus Protoeuglena and reclassify the endosymbiont as Protoeuglena noctilucae. PMID:27033730

  11. Phylogeny of parasitic parabasalia and free-living relatives inferred from conventional markers vs. Rpb1, a single-copy gene.

    Shehre-Banoo Malik

    Full Text Available BACKGROUND: Parabasalia are single-celled eukaryotes (protists that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA, while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms. PRINCIPAL FINDINGS: Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1 in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas. CONCLUSIONS/SIGNIFICANCE: The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive

  12. Self-organizing maps: A tool to ascertain taxonomic relatedness based on features derived from 16S rDNA sequence

    D V Raje; H J Purohit; Y P Badhe; S S Tambe; B D Kulkarni

    2010-12-01

    Exploitation of microbial wealth, of which almost 95% or more is still unexplored, is a growing need. The taxonomic placements of a new isolate based on phenotypic characteristics are now being supported by information preserved in the 16S rRNA gene. However, the analysis of 16S rDNA sequences retrieved from metagenome, by the available bioinformatics tools, is subject to limitations. In this study, the occurrences of nucleotide features in 16S rDNA sequences have been used to ascertain the taxonomic placement of organisms. The tetra- and penta-nucleotide features were extracted from the training data set of the 16S rDNA sequence, and was subjected to an artificial neural network (ANN) based tool known as self-organizing map (SOM), which helped in visualization of unsupervised classification. For selection of significant features, principal component analysis (PCA) or curvilinear component analysis (CCA) was applied. The SOM along with these techniques could discriminate the sample sequences with more than 90% accuracy, highlighting the relevance of features. To ascertain the confidence level in the developed classification approach, the test data set was specifically evaluated for Thiobacillus, with Acidiphilium, Paracocus and Starkeya, which are taxonomically reassigned. The evaluation proved the excellent generalization capability of the developed tool. The topology of genera in SOM supported the conventional chemo-biochemical classification reported in the Bergey manual.

  13. Inhibition of DNA methylation alters chromatin organization, nuclear positioning and activity of 45S rDNA loci in cycling cells of Q. robur.

    Bočkor, Vedrana Vičić; Barišić, Darko; Horvat, Tomislav; Maglica, Željka; Vojta, Aleksandar; Zoldoš, Vlatka

    2014-01-01

    Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2'-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization. PMID:25093501

  14. Estimation of divergence times in litostomatean ciliates (Ciliophora: Intramacronucleata), using Bayesian relaxed clock and 18S rRNA gene.

    Vďačný, Peter

    2015-08-01

    The class Litostomatea comprises a diverse assemblage of free-living and endosymbiotic ciliates. To understand diversification dynamic of litostomateans, divergence times of their main groups were estimated with the Bayesian molecular dating, a technique allowing relaxation of molecular clock and incorporation of flexible calibration points. The class Litostomatea very likely emerged during the Cryogenian around 680 Mya. The origin of the subclass Rhynchostomatia is dated to about 415 Mya, while that of the subclass Haptoria to about 654 Mya. The order Pleurostomatida, emerging about 556 Mya, was recognized as the oldest group within the subclass Haptoria. The order Spathidiida appeared in the Paleozoic about 442 Mya. The three remaining haptorian orders evolved in the Paleozoic/Mesozoic periods: Didiniida about 419 Mya, Lacrymariida about 269 Mya, and Haptorida about 194 Mya. The subclass Trichostomatia originated from a spathidiid ancestor in the Mesozoic about 260 Mya. A further goal of this study was to investigate the impact of various settings on posterior divergence time estimates. The root placement and tree topology as well as the priors of the rate-drift model, birth-death process and nucleotide substitution rate, had no significant effect on calculation of posterior divergence time estimates. However, removal of calibration points could significantly change time estimates at some nodes. PMID:26204556

  15. Taxonomy of the genus Rhexinema (Ulvophyceae) based on phylogeny of the 18S rRNA and morphology

    Caisová, Lenka

    2009-01-01

    Roč. 48, č. 4 (2009), s. 15-15. ISSN 0031-8884. [International Phycological Congress /9./. 02.08.2009-08.08.2009, Tokyo] Institutional research plan: CEZ:AV0Z60050516 Keywords : Rhexinema * 18S rRNA * morphology Subject RIV: EF - Botanics

  16. Interplay of RNA Pol IV and ROS1 during post-embryonic 5S rDNA chromatin remodeling.

    Douet, Julien; Blanchard, Bertrand; Cuvillier, Claudine; Tourmente, Sylvette

    2008-12-01

    We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features. PMID:18845569

  17. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. PMID:22510214

  18. Nucleotide sequence, genomic organization and chromosome localization of 5S rDNA in two species of Curimatidae (Teleostei, Characiformes

    Lessandra Viviane de Rosa Santos

    2006-01-01

    Full Text Available The 5S ribosomal DNA (5S rDNA of higher eukaryotes is organized in repeat units of tandem arrays composed of a 5S rDNA coding region, conserved even among non-related taxa, and a variable non-transcribed spacer sequence (NTS. To contribute to knowledge on the organization and evolution of vertebrate 5S rDNA we used PCR, nucleotide sequencing, Southern blot hybridization and chromosome fluorescence in situ hybridization (FISH to investigate 5S rDNA tandem repeats in the South American Curimatidae fish Steindachnerina insculpta and Cyphocharax modesta. 5S rDNA repeats of 180 base pairs (bp from both species were PCR-generated and sequenced evidencing the shortest 5S rDNA monomer so far described in eukaryote species. Southern blotting revealed that both species contained two tandem 5S rDNA classes, the PCR amplified fragment composed of 180 bp monomers and a class of 1600 bp monomers not detected by PCR. Chromosome mapping of the 5S rDNA repeats identified a major locus in both species and a second minor locus only in C. modesta. The Southern blot and chromosome mapping data indicate the presence of different types of 5S rDNA tandem repeats in the Curimatidae genome.

  19. Analysis of 5S rDNA Arrays in Arabidopsis thaliana: Physical Mapping and Chromosome-Specific Polymorphisms

    Cloix, C.; Tutois, S; O. Mathieu; Cuvillier, C.; Espagnol, M C; G. Picard; Tourmente, S

    2000-01-01

    A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning ∼1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units ex...

  20. Phylogeny and genetic diversity of Bridgeoporus nobilissimus inferred using mitochondrial and nuclear rDNA sequences

    Redberg, G.L.; Hibbett, D.S.; Ammirati, J.F., Jr.; Rodriguez, R.J.

    2003-01-01

    The genetic diversity and phylogeny of Bridgeoporus nobilissimus have been analyzed. DNA was extracted from spores collected from individual fruiting bodies representing six geographically distinct populations in Oregon and Washington. Spore samples collected contained low levels of bacteria, yeast and a filamentous fungal species. Using taxon-specific PCR primers, it was possible to discriminate among rDNA from bacteria, yeast, a filamentous associate and B. nobilissimus. Nuclear rDNA internal transcribed spacer (ITS) region sequences of B. nobilissimus were compared among individuals representing six populations and were found to have less than 2% variation. These sequences also were used to design dual and nested PCR primers for B. nobilissimus-specific amplification. Mitochondrial small-subunit rDNA sequences were used in a phylogenetic analysis that placed B. nobilissimus in the hymenochaetoid clade, where it was associated with Oxyporus and Schizopora.

  1. Molecular Detection of Toxoplasmosis Using Specific Primers P30, B1, and rDNA

    Wisnu Nurcahyo

    2014-04-01

    Full Text Available Study in order to develop molecular techniques using specific primers for the early diagnosis oftoxoplasmosis have been conducted. Detection of Toxoplasma gondii genome was performed usingpolymerase chain reaction (PCR technique. The primers used in this study were rDNA, P30, and B1. ThePCR products were further run using gel electrophoresis (gel 1.5% – 2.0% and the band was documented.Toxoplasma was detected at 500 bp and 600 bp using primer P30 and B1, respectively. Whereas usingprimer rDNA no band was observed. It was assumed that primer rDNA was not sensitive since the targetamplification was 88 bp.

  2. Multicolor FISH analysis of rDNA and telomere on spinach

    Tianying LAN; Bo LIU; Fengping DONG; Ruiyang CHEN; Xiulan LI; Chengbin CHEN

    2008-01-01

    In this study,multicolor fluorescence in situ hybridization (FISH) analysis on metaphase chromosomes of spinach with biotin-labeled 25S rDNA,DIG-labeled telomere sequences and biotin-labeled and DIG-labeled 5S rDNA was performed.There were six 25S rDNA loci located on the satellites of the third,the fifth and the sixth chromosomes,and four 5S rDNA loci located on the long arms of the third and the fifth chromosomes.The telomere loci were located on the end of the sixth chromosome and also on both the end and centromeric regions of other chromosomes.This study is an important complement to both traditional karyotype analysis and FISH karyotype analysis in spinach.

  3. Intraspecific diversity within Diaporthe helianthi: evidence from rDNA intergenic spacer (IGS) sequence analysis.

    Pecchia, Susanna; Mercatelli, Elisabetta; Vannacci, Giovanni

    2004-04-01

    Diaporthe helianthi is the causal agent of sunflower stem canker, a serious pathogen of sunflower in Europe but recorded sporadically in Italy. The genetic diversity of D. helianthi isolates from different geographic origins (Argentina, France, Italy, Yugoslavia, Romania) was investigated using IGS sequences. A 400 bp fragment of the portion of the IGS region flanking the 5' end of the 18S gene was amplified from each isolate. The aligned nucleotide sequences showed intraspecific sequence homology from 99-100% among French/Yugoslavian isolates to 95-100% among Italian isolates. French/Yugoslavian isolates shared 90-92% sequence homology with Italian isolates. The phylogenetic tree obtained from the aligned data revealed three separate groups. Group 1 included all isolates from France and former Yugoslavia and one isolate from Argentina; Group 2 included all Italian isolates and one isolate from Argentina. The most distantly related isolate was that from Romania (Group 3). The average genetic distances among isolates within Group 1 and within Group 2 were 0.22 and 3.29 respectively. The analysis showed that all isolates originating from countries where severe outbreaks of the disease are reported annually (France and former Yugoslavia) form a well defined taxon characterized by relatively low variability. This group is distinct from the group formed by isolates originating from Italy, whose variability is relatively much higher. Results obtained revealed a marked differentiation among pathogen isolates, and members of Group 1 seem not yet to have spread into Italian sunflower-growing areas. PMID:15180160

  4. An intramolecular recombination mechanism for the formation of the rRNA gene palindrome of Tetrahymena thermophila.

    Butler, D. K.; Yasuda, L E; Yao, M C

    1995-01-01

    Large palindromic DNAs are found in a wide variety of eukaryotic cells. In Tetrahymena thermophila, a large palindrome is formed from a single rRNA gene (rDNA) during nuclear differentiation. We present evidence that a key step in the formation of the rDNA palindrome of T. thermophila involves homologous intramolecular recombination. Heteroduplex micronuclear rDNA molecules were constructed in vitro and microinjected into developing macronuclei, where they formed palindromes. Analysis of the ...

  5. Phylogenetic Analysis of Geographically Diverse Radopholus similis via rDNA Sequence Reveals a Monomorphic Motif

    Kaplan, D. T.; Thomas, W. K.; Frisse, L. M.; Sarah, J. L.; Stanton, J. M.; Speijer, P. R.; Marin, D. H.; Opperman, C. H.

    2000-01-01

    The nucleic acid sequences of rDNA ITS1 and the rDNA D2/D3 expansion segment were compared for 57 burrowing nematode isolates collected from Australia, Cameroon, Central America, Cuba, Dominican Republic, Florida, Guadeloupe, Hawaii, Nigeria, Honduras, Indonesia, Ivory Coast, Puerto Rico, South Africa, and Uganda. Of the 57 isolates, 55 were morphologically similar to Radopholus similis and seven were citrus-parasitic. The nucleic acid sequences for PCR-amplified ITS1 and for the D2/D3 expans...

  6. Tsetse fly rDNA: an analysis of structure and sequence.

    CROSS, N.C.; Dover, G A

    1987-01-01

    A genomic library of Glossina morsitans morsitans (tsetse fly) has been constructed in the phage vector EMBL 4 and a complete rDNA unit isolated by using a D. melanogaster rDNA clone as a probe. The overall organisation is typical of higher eukaryotes, including an intergenic spacer consisting of a subrepeating structure. Atypically, however, the 45S precursor RNA promoter was shown to lie within the last subrepeat by S1 mapping; i.e. the last subrepeat extends 90 bp into the ETS. The sequenc...

  7. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

    Wörheide Gert; Erpenbeck Dirk; Voigt Oliver

    2008-01-01

    Abstract Background The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by...

  8. Complete Sequence Construction of the Highly Repetitive Ribosomal RNA Gene Repeats in Eukaryotes Using Whole Genome Sequence Data.

    Agrawal, Saumya; Ganley, Austen R D

    2016-01-01

    The ribosomal RNA genes (rDNA) encode the major rRNA species of the ribosome, and thus are essential across life. These genes are highly repetitive in most eukaryotes, forming blocks of tandem repeats that form the core of nucleoli. The primary role of the rDNA in encoding rRNA has been long understood, but more recently the rDNA has been implicated in a number of other important biological phenomena, including genome stability, cell cycle, and epigenetic silencing. Noncoding elements, primarily located in the intergenic spacer region, appear to mediate many of these phenomena. Although sequence information is available for the genomes of many organisms, in almost all cases rDNA repeat sequences are lacking, primarily due to problems in assembling these intriguing regions during whole genome assemblies. Here, we present a method to obtain complete rDNA repeat unit sequences from whole genome assemblies. Limitations of next generation sequencing (NGS) data make them unsuitable for assembling complete rDNA unit sequences; therefore, the method we present relies on the use of Sanger whole genome sequence data. Our method makes use of the Arachne assembler, which can assemble highly repetitive regions such as the rDNA in a memory-efficient way. We provide a detailed step-by-step protocol for generating rDNA sequences from whole genome Sanger sequence data using Arachne, for refining complete rDNA unit sequences, and for validating the sequences obtained. In principle, our method will work for any species where the rDNA is organized into tandem repeats. This will help researchers working on species without a complete rDNA sequence, those working on evolutionary aspects of the rDNA, and those interested in conducting phylogenetic footprinting studies with the rDNA. PMID:27576718

  9. Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells.

    Kwasniewska, Jolanta; Grabowska, Marta; Kwasniewski, Miroslaw; Kolano, Bozena

    2012-06-01

    We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment. PMID:22556029

  10. FISH Loci of 18-26s rDNA in Four Gossypium Species%四个棉种18-26s rDNA荧光原位杂交

    Kunbo WANG; Chunying WANG; Shu BIE; Guoli SONG; Maoxue LI

    2002-01-01

    @@ Detection of specific nucleic acid sequences such as RNA or DNA in chromosomes by in situ hybridization has important applications in many areas of biology. The genes encoding 18-26s rRNA are located nucleus organizer regions (NORs) in plant chromosomes. Fluorescent in situ hybridization ( FISH ) with 18-26s rDNA as probe to somatic chromosomes may directly provide insight into genetic mapping and then,by comparisons with karyotypes, physical loci of NORs of the genome.

  11. Systematics of Penicillium simplicissimum based on rDNA sequences, morphology and secondary metabolites

    Tuthill, D.E.; Frisvad, Jens Christian; Christensen, M.

    2001-01-01

    supported by differences in micromorphological characters, particularly of the conidia and phialides, and the production of distinct profiles of secondary metabolites by each species. Group-I introns, located in the SSU rDNA, were identified in six of the 21 isolates; their presence was used to test...

  12. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2

    M Forouzandeh-Moghadam

    2011-09-01

    Full Text Available Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and ana-lyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifi-cally of Fasciola species. Results: The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.Conclusion: The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.

  13. Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

    Mahami-Oskouei, M; Dalimi, A; Forouzandeh-Moghadam, M; Rokni, MB

    2011-01-01

    Background In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species. Methods The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Results The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates. Conclusion The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates. PMID:22347295

  14. Phylogeographic structure of cotton pest Adelphocoris suturalis (Hemiptera: Miridae): strong subdivision in China inferred from mtDNA and rDNA ITS markers.

    Zhang, Lijuan; Li, Hu; Li, Shujuan; Zhang, Aibing; Kou, Fei; Xun, Huaizhu; Wang, Pei; Wang, Ying; Song, Fan; Cui, Jianxin; Cui, Jinjie; Gouge, Dawn H; Cai, Wanzhi

    2015-01-01

    Phylogeographic patterns of some extant plant and vertebrate species have been well studied; however, they are poorly understood in the majority of insects. The study documents analysis of mitochondrial (COI, CYTB and ND5) and nuclear (5.8S rDNA, ITS2 and 28S rDNA) data from 419 individuals of Adelphocoris suturalis, which is one of the main cotton pests found in the 31 locations in China and Japan involved in the study. Results show that the species is highly differentiated between populations from central China and peripheral China regions. Analysis of molecular variance showed a high level of geographical differentiation at different hierarchical levels. Isolation-by-distance test showed no significant correlation between genetic distance and geographical distance among A. suturalis populations, which suggested gene flow is not restricted by distance. In seven peripheral populations, the high levels of genetic differentiation and the small Nem values implied that geographic barriers were more likely restrict gene flow. Neutrality tests and the Bayesian skyline plot suggested population expansion likely happened during the cooling transition between Last Interglacial and Last Glacial Maximum. All lines of evidence suggest that physical barriers, Pleistocene climatic oscillations and geographical heterogeneity have affected the population structure and distribution of this insect in China. PMID:26388034

  15. Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

    Planý Matej

    2016-06-01

    Full Text Available Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season and one traditional (artisanal, most valued, produced in May Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively. They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

  16. Effects of altered gravity on a distribution of rDNA and nucleolar proteins and the expression of nucleolar proteins in plants

    Sobol, Margaryta; Kordyum, Elizabeth; Medina, Francisco Javier

    The nucleolus is an inner nuclear organelle originated from the activity of hundreds of rRNA genes, typically spanning several megabases. It morphologically reflects the functional events leading to ribosome biogenesis, from the transcription of rDNA through the processing of nascent pre-rRNA to the assembly of pre-ribosomes. A typical nucleolus consists of three major elements, namely fibrillar centers (FCs), the dense fibrillar component (DFC), and granular component (GC). The rate of ribosome biosynthesis and the subnucleolar structure are reliable monitors of the general level of cell metabolism and, consequently, of the rate of cellular growth, being influenced with many external factors, among which altered gravity could be included. Thus, we can hypothesize that the structural organization of the nucleolar subcomponents and the level, distribution and quantitative/qualitative characteristics of the nucleolar proteins would be changed under conditions of altered gravity. To confirm our hypothesis, we applied parallel procedures, such as cytochemistry, immunofluorescence, confocal laser microscopy, immunogold electron microscopy, monoand bi-dimensional electrophoresis and immunoblotting in root meristematic cells from two-day cress seedlings grown under slow horizontal clinorotation (2 rpm) and in stationary control. The complex model of the ultrastructural organization and functions of the nucleolus was created based on the location of rDNA and the nucleolar proteins fibrillarin, NhL90 and NhL68, these latter being cress nucleolin homologues. The principal stages of ribosome biogenesis, namely ribosomal gene activation, rDNA transcription and pre-rRNA processing were reflected in this model. Compared to the pattern shown in control ground gravity conditions, we found firstly a redistribution of both rDNA and nucleolar proteins in nucleolar subcomponents, induced by clinorotation. Under the conditions of altered gravity, nucleolar DNA concentrated

  17. Karyotyping of Brassica oleracea L.based on rDNA and Cot-1 DNA fluorescence in situ hybridization

    WANG Taixia; WU Chunhong; HUANG Jinyong; WEI Wenhui

    2007-01-01

    To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.

  18. Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections.

    Yang, Rongchang; Palermo, Cindy; Chen, Linda; Edwards, Amanda; Paparini, Andrea; Tong, Kaising; Gibson-Kueh, Susan; Lymbery, Alan; Ryan, Una

    2015-12-15

    Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n=14), Cryptosporidium huwi (n=8), piscine genotype 2 (n=4), piscine genotype 3-like (n=1), piscine genotype 4 (n=2), piscine genotype 5 (n=13), piscine genotype 5-like (n=1) and five novel genotypes (n=5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected. PMID:26527238

  19. Structure of 5S rDNA sequence of the gibel carp (Carassius gibelio with 2n=100 chromosomes

    Lech Kirtiklis

    2015-11-01

    Our preliminary research has related to diploid individuals of gibel carp (Carassius gibelio with 2n=100 chromosomes. The PCR reaction has resulted in product 340 bp. Sequencing has yielded data on the exact nucleotide sequence and length of 5S rDNA fragment. Studies have shown no difference in length and nucleotide composition of the 5S sequence between males and females. Some study has revealed the influence of polyploidy on the organization and evolution of the 5S rDNA in teleosts fishes, therefore, then we will have studied the 5S rDNA sequences in triploid individuals.

  20. Chromosomal distribution of rDNA in Pan paniscus, Gorilla gorilla beringei, and Symphalangus syndactylus: comparison to related primates

    Henderson, A.S.; Atwood, K.C.; Warburton, D.

    1976-01-01

    Hybridization in situ was used to identify rDNA in chromosomes of the pygmy chimpanzee, mountain gorilla, and siamang gibbon. In contrast to other Pongids, and man, the gorilla has only two pairs of rDNA-containing chromosomes. The single pair in the siamang bears no resemblance to the nucleolar chromosome of the closely related lar gibbon. Pan paniscus and P. troglodytes have the same rDNA distribution, and similar karyotypes except in the structure of chromosome 23p. Grain counts over unbanded preparations show that the human, orangutan, and both chimpanzees have about the same total rDNA multiplicity.

  1. Systematics of Penicillium simplicissimum based on rDNA sequences, morphology and secondary metabolites

    Tuthill, D.E.; Frisvad, Jens Christian; Christensen, M.

    2001-01-01

    supported by differences in micromorphological characters, particularly of the conidia and phialides, and the production of distinct profiles of secondary metabolites by each species. Group-I introns, located in the SSU rDNA, were identified in six of the 21 isolates; their presence was used to test...... the P. simplicissimum group, here referred to as Penicillium spp. I, II and III. The name P. simplicissimum should be used only for isolates which closely match the neotype and NRRL 902 in all characters.......Penicillium simplicissimum is a broadly circumscribed taxon often encountered on soil community lists and in biotechnological reports. Phylogenetic analysis of the ITS1- 5.8S-ITS2 rDNA region indicated that P. simplicissimum s.l. includes several species. Recognition of these species is further...

  2. Transcription of rDNA correlates with a tendency towards intergenomic homogenization in Nicotiana polyploids

    Nešpor Dadejová, Martina; Lim, K.Y.; Součková Skalická, Kamila; Matyášek, Roman; Leitch, A.; Kovařík, Aleš

    Heidelberg, 2007. s. 80-80. [EMBO Conference on Chromatin and Epigenetics. 03.05.2007-06.05.2007, Heidelberg] R&D Projects: GA ČR(CZ) GD204/05/H505; GA ČR(CZ) GA204/05/0687; GA ČR(CZ) GA521/07/0116; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507 Keywords : polyploidy * rDNA homogenization * silencing Subject RIV: BO - Biophysics

  3. Uniqueness of the Gossypium mustelinum Genome Revealed by GISH and 45S rDNA FISH

    WU Qiong; STELLY David; SONG Guo-li; WANG Kun-bo; WANG Chun-ying; LIU Fang; LI Shao-hui; ZHANG Xiang-di; WANG Yu-hong; LIU San-hong

    2008-01-01

    @@ Gossypium mustelinum [-(AD)4"] is one of five tetraploid species in Gossypium.Three pairs of nucleolar organizer regions (NOR) in (AD)4 were detected by FISH with 45S rDNA as a probe,they also were observed with genomic DNA (gDNA) from Gossypium D genome species as probes.Of the three NORs or GISH-NORs,one was super-major and other two were minor,which was distinctly different from other tetraploid cottons.

  4. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    Ekwan Nofa Wiratno; Suharjono; Agustin Krisna Wardani

    2016-01-01

    High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g...

  5. Characterizing contrasting soils and sediments with semispecific actinomycete r-DNA PCR primers

    Hill, P.; Krištůfek, Václav; Caballero, S.; Kroetsch, D. J.; Rauch, Ota

    České Budějovice: Institute of Soil Biology AS CR, 2004, s. 88-92. ISBN 80-86525-03-1. [Present methods for investigation of microbial community biodiversity in soils and substrates. Methodological workshop /9./. České Budějovice (CZ), 02.03.2004-03.03.2004] Institutional research plan: CEZ:AV0Z6066911 Keywords : contrasting soils * sediments * semispecific actinomycete r-DNA PCR primers Subject RIV: EH - Ecology, Behaviour

  6. Plant rDNA database: ribosomal DNA loci information goes online

    Garcia, S.; Garnatje, T.; Kovařík, Aleš

    2012-01-01

    Roč. 121, č. 4 (2012), s. 389-394. ISSN 0009-5915 R&D Projects: GA ČR(CZ) GAP501/10/0208; GA ČR GBP501/12/G090 Institutional research plan: CEZ:AV0Z50040702 Keywords : rDNA loci * FISH * database Subject RIV: BO - Biophysics Impact factor: 3.340, year: 2012

  7. Visualization of rDNA spacer transcription in Xenopus oocytes treated with fluorouridine

    Rungger, M.; Crippa, M; Trendelenburg, M F; Scheer, Ulrich; Franke, Werner W

    2009-01-01

    Under the intluence of 5-tluoro-uridine, the ultrastructure of the rDNA transcription units in Xenopus oocytes is altered. Whereas part of the matrix units maintains anormal aspect or shows various degrees of inhibition, in a strong proportion of the transcription units the alternating pattern of matrix units and fibril-free spacer regions is no longer recognized. Transcriptional complexes are found along the entire DNP axis, including the regions of the spacers. These observations support bi...

  8. Secondary structure of expansion segment D1 in LSU rDNA from Arachnida and its phylogenetic application in Eriophyoid mites and in Acari.

    Wang, Zheng-Hang; Zhao, Ya-E; Xu, Yang; Hu, Li; Chen, Yi-Meng

    2015-12-01

    An increasing number of researchers have applied secondary-structure based multiple alignments of rDNA genes in phylogeny. These studies mostly depended on a few valuable divergent domains in LSU and SSU rDNA. Yet other divergent domains, e.g. D1, were poorly investigated and rarely used. However, these domains might contain additional evolutionary data and play a vital role in DNA-based phylogenetic study. Here, we investigated all available D1 sequences of Arachnida taxa and predicted corresponding secondary structures to help identify homologous positions in the D1 region. Long insertions were found exclusive to Eriophyoidea and folded into three newly proposed helices. Non-Acari taxa were all GC rich. In Acari, most Trombidiformes and all Mesostigmata (Parasitiformes) taxa were AT rich and Ixodida (Parasitiformes) GC rich; however there was no consistent base bias in Sarcoptiformes sequences. For Eriophyoid mites, genera Cecidophyopsis and Aceria were both well supported in MP, NJ, ME and ML tress based on D1 sequences, and clusters of Cecidophyopsis species were identical with former study. This demonstrated that the D1 region could act as a valuable molecular marker in phylogenetic reconstruction of Eriophyoidea. Additionally, D1 has been proven suitable in phylogenetic analysis at the family and genus level in Acari, but not in Opiliones. PMID:26420464

  9. The world in a river? A preliminary analysis of the 16S rDNA variability of Tubifex species (Clitellata: Tubificidae) from the Lambro River.

    Crottini, Angelica; Marotta, Roberto; Barbuto, Michela; Casiraghi, Maurizio; Ferraguti, Marco

    2008-09-01

    Tubifex tubifex Müller, 1774 is a cosmopolitan freshwater tubificid widely used as a model in ecotoxicology, population dynamics and developmental biology. It is traditionally recognized as a polytypic species and in Lambro River (Milano, Northern Italy) it occurs in two of the three recognized forms, named "tubifex" and "blanchardi", alternatively considered as ecological forms or distinct species. To investigate the genetic differentiation of the populations occurring in the Lambro River we sequenced a fragment of the 16S rDNA mitochondrial gene. T. blanchardi, characterized by a low genetic diversity, was genetically segregated from the other sympatric T. tubifex. The ancestral state reconstruction was used to define the morphological traits that support its distinctness. On the contrary, the other T. tubifex from the Lambro community, although morphologically indistinguishable, revealed an astonishing degree of genetic variability, both between and within the three identified clades that proved to be genetically isolated. Using samples from the mixed Lambro River community and from other countries around the world we present an overview of the species complex' 16S rDNA variability. Our results show that the genetic variability did not sensibly increase widening the data set, suggesting that the Lambro River populations meet the species' worldwide genetic variability. PMID:18625325

  10. Phylogeny of hard- and soft-tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA sequences.

    Black, W C; Piesman, J

    1994-01-01

    Ticks are parasitiform mites that are obligate hematophagous ectoparasites of amphibians, reptiles, birds, and mammals. A phylogeny for tick families, subfamilies, and genera has been described based on morphological characters, life histories, and host associations. To test the existing phylogeny, we sequenced approximately 460 bp from the 3' end of the mitochondrial 16S rRNA gene (rDNA) in 36 hard- and soft-tick species; a mesostigmatid mite, Dermanyssus gallinae, was used as an outgroup. Phylogenies derived using distance, maximum-parsimony, or maximum-likelihood methods were congruent. The existing phylogeny was largely supported with four exceptions. In hard ticks (Ixodidae), members of Haemaphysalinae were monophyletic with the primitive Amblyomminae and members of Hyalomminae grouped within the Rhipicephalinae. In soft ticks (Argasidae), the derived phylogeny failed to support a monophyletic relationship among members of Ornithodorinae and supported placement of Argasinae as basal to the Ixodidae, suggesting that hard ticks may have originated from an Argas-like ancestor. Because most Argas species are obligate bird octoparasites, this result supports earlier suggestions that hard ticks did not evolve until the late Cretaceous. PMID:7937832

  11. The phylogeny of native and exotic scallops cultured in China based on 16S rDNA sequences

    LIU Baozhong; DONG Bo; XIANG Jianhai; WANG Zaizhao

    2007-01-01

    Scallops of the Family Pectinidae are a valuable resource in marine industry of the world.Understanding the phylogeny of the family is important for the development of the industry. In this study,partial 16S mitochondrial rDNA genes were obtained from 8 scallop species that are commonly cultured indigenous and transplanted species in China. Phylogenetic relationships of Pectinidae were analyzed based on the 8 sequences and other 5 published ones in GenBank, representing 9 genera of the family. The molecular phylogeny trees were constructed using 3 methods with software PHYLIP. The results showe that total 13 species of scallops clustered in 4 clades. Pecten maximus joins P. jacobaeus then Amusium pleuronectes in cluster, indicating close relationship of genus Amusium with Pecten in evolution. P. yessoensis is close to Chlamysfarreri and C. islandica. No enough material was available to single out genus Patinopecten as an independent monophyletic subfamily. The position ofAdamussium colbecki indicates that it is far from genus Pecten but near to genus Chlamys in evolution.

  12. The phylogeny of native and exotic scallops cultured in China based on 16S rDNA sequences

    Liu, Baozhong; Dong, Bo; Xiang, Jianhai; Wang, Zaizhao

    2007-01-01

    Scallops of the Family Pectinidae are a valuable resource in marine industry of the world. Understanding the phylogeny of the family is important for the development of the industry. In this study, partial 16S mitochondrial rDNA genes were obtained from 8 scallop species that are commonly cultured indigenous and transplanted species in China. Phylogenetic relationships of Pectinidae were analyzed based on the 8 sequences and other 5 published ones in GenBank, representing 9 genera of the family. The molecular phylogeny trees were constructed using 3 methods with software PHYLIP. The results showe that total 13 species of scallops clustered in 4 clades. Pecten maximus joins P. jacobaeus then Amusium pleuronectes in cluster, indicating close relationship of genus Amusium with Pecten in evolution. P. yessoensis is close to Chlamys farreri and C. islandica. No enough material was available to single out genus Patinopecten as an independent monophyletic subfamily. The position of Adamussium colbecki indicates that it is far from genus Pecten but near to genus Chlamys in evolution.

  13. Taiwanese Trichogramma of Asian Corn Borer: Morphology, ITS-2 rDNA Characterization, and Natural Wolbachia Infection

    Wu, Li-Hsin; Hoffmann, Ary A.; Thomson, Linda J.

    2016-01-01

    Egg parasitoids of the genus Trichogramma are natural enemies of many lepidopteran borers in agricultural areas around the world. It is important to identify the correct species and ideally focus on endemic Trichogramma for pest control in particular crops. In this study, Trichogramma wasps were collected from parasitized eggs of Asian corn borer in Southwestern Taiwan. Three Trichogramma species, Trichogramma ostriniae Pang and Chen, Trichogramma chilonis Ishii, and T. sp. y, were identified based on morphology and the nucleotide sequence of the internal transcribed spacer 2 (ITS-2) region of rDNA. Although T. ostriniae and T. sp. y appear to be morphologically similar, ITS-2 identity between these two taxa is only 89%. Surprisingly, a commercially released Trichogramma colony thought to be T. chilonis possessed 99% identity (ITS-2) with the field T. sp. y individuals. This suggests past contamination leading to subsitution of the laboratory-reared T. chilonis colony by T. sp. y. Natural populations of all three Trichogramma species were found to be infected by a single Wolbachia strain which was identified using a wsp gene sequence. PMID:26896674

  14. Wide genetic variations at 18S ribosomal RNA locus of Cyclospora cayetanensis isolated from Egyptian patients using high resolution melting curve.

    Hussein, Eman M; El-Moamly, Amal A; Mahmoud, Moushira A; Ateek, Nayera S

    2016-07-01

    A variable clinical picture of cyclosporiasis including gastrointestinal tract (GIT) symptomatic or asymptomatic beside extraintestinal consequences suggests a possibility of heterogenicity of Cyclospora cayetanensis. The present work aimed to explore the possibility of genetic variation of C. cayetanensis using high-resolution melting (HRM) curve of polymerase chain reaction (PCR) amplified 18S rRNA genes. DNAs extracted from the stool samples of 70 cyclosporiasis patients were amplified and scanned by PCR/HRM curve. The results showed that there are four different genotypic profiles of C. cayetanensis with presence of mixed ones. Although Tm of all profiles was within the same range, they were discerned by plotting of the temperature-shifted florescence difference between normalized melting curves (dF/dT). Genotypic profile I was found alone in 40 % of patients and mixed with genotypic profile II and/or III in 25.7 % of patients, followed by genotypic profile II in 14.3 % then genotypic profile III and IV (10 % each). A significant relation was found between genotypic profiles and GIT symptomatic status as profile I and profile II were mostly detected in patients with acute GIT symptoms without or with chronic illness, respectively, while profile IV cases only were GIT asymptomatic. Statistical significance relations between genotypic profiles and age, gender, residence and oocyst shape index were determined. In conclusion, PCR/HRM proved a wide variation on C. cayetanensis genes that could be reflected on its pathogenic effects and explaining the variability of the clinical manifestations presented by cyclosporiasis patients. PMID:27041342

  15. Oligonucleotide Fingerprinting of rRNA Genes for Analysis of Fungal Community Composition

    Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

    2002-01-01

    Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones d...

  16. Isolation and identification of a marine killer yeast strain YF07b and cloning of the gene encoding killer toxin from the yeast

    2007-01-01

    It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculatus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.

  17. Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

    Fouts Derrick E

    2012-08-01

    Full Text Available Abstract Background Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI. Asymptomatic bacteriuria (ABU represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. Methods A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. Results A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein

  18. FastGroupII: A web-based bioinformatics platform for analyses of large 16S rDNA libraries

    McNairnie Pat

    2006-02-01

    Full Text Available Abstract Background High-throughput sequencing makes it possible to rapidly obtain thousands of 16S rDNA sequences from environmental samples. Bioinformatic tools for the analyses of large 16S rDNA sequence databases are needed to comprehensively describe and compare these datasets. Results FastGroupII is a web-based bioinformatics platform to dereplicate large 16S rDNA libraries. FastGroupII provides users with the option of four different dereplication methods, performs rarefaction analysis, and automatically calculates the Shannon-Wiener Index and Chao1. FastGroupII was tested on a set of 16S rDNA sequences from coral-associated Bacteria. The different grouping algorithms produced similar, but not identical, results. This suggests that 16S rDNA datasets need to be analyzed in multiple ways when being used for community ecology studies. Conclusion FastGroupII is an effective bioinformatics tool for the trimming and dereplication of 16S rDNA sequences. Several standard diversity indices are calculated, and the raw sequences are prepared for downstream analyses.

  19. Epigeneitc silencing of ribosomal RNA genes by Mybbp1a

    Tan Bertrand

    2012-06-01

    Full Text Available Abstract Background Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. Methods and Results Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. Conclusions Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters.

  20. Gene

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  1. Genomic and Haplotype Comparison of Butanol Producing Bacteria Based on 16S rDNA

    Ekwan Nofa Wiratno

    2016-04-01

    Full Text Available High butanol demand for transportation fuel triggers butanol production development. Exploration of butanolproducing bacteria using genomic comparison and biogeography will help to develop butanol industry. The objectives of this research were butanol production, genome comparison and haplotype analysis of butanolproducing bacteria from Ranu Pani Lake sediment using 16S rDNA sequences. The highest butanol concentrations were showed by Paenibacillus polymyxa RP 2.2 isolate (10.34 g.L-1, followed by Bacillus methylotrophicus RP 3.2 and B. methylotrophicus RP 7.2 isolate (10.11 g.L-1 and 9.63 g.L-1 respectively. Paenibacillus polymyxa RP 2.2 showed similarity in nucleotide composition (ATGC with B. methylotrophicus RP 3.2, B. methylotrophicus RP 7.2, P. polymyxa CR1, Bacillus amyloliquefaciens NELB-12, and Paenibacillus polymyxa WR-2. Clostridium acetobutylicum ATCC 824 showed similarity in nucleotide composition (ATGC with Clostridium saccharoperbutylacetonicum N1-4, and Clostridium saccharobutylicum Ox29. The lowest G+C content was C. saccharobutylicum Ox29 (51.35%, and the highest was B. methylotrophicus RP 7.2 (55.33%. Conserved region of 16S rDNA (1044 bp were consisted of 17 conserved sequences. The number of Parsimony Informative Site (PIS was 319 spot and single tone was 48 spot. We found in this study that all of butanolproducing bacterial DNA sequences have clustered to 8 haplotypes. Based on the origin of sample, there were three haplotype groups. Bacteria from group A were could produce butanol 8.9-10.34 g.L-1, group B 9.2-14.2 g.L-1 and group C was could produce butanol 0.47 g.L-1. The haplotype analysis of bacteria based on 16S rDNA sequences in this study could predict capability of butanol production.

  2. Structure and function of the nontranscribed spacer regions of yeast rDNA.

    Skryabin, K G; Eldarov, M A; Larionov, V L; Bayev, A A; Klootwijk, J; de Regt, V C; Veldman, G M; Planta, R J; Georgiev, O I; Hadjiolov, A A

    1984-01-01

    The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined. The NTS sequences of both species were found to be 93% homologous. The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs. Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1). The NTS between the starts...

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

    Nielsen, Henrik; Engberg, J

    1985-01-01

    We have studied the sequence variation of the rDNA intron among six species of Tetrahymena. From these data, the intron appears to be relatively well conserved in evolution. We have evaluated the sequence variations among the most distant of these species in relation to the secondary structure...... involving compensating base pair changes. Interestingly, one of these is found in a region that is known to be dispensable in the in vitro splicing reaction suggesting differences between the in vivo and in vitro reactions. One of the single nucleotide deletions is found in the so-called "internal guide...

  4. [Gene cloning, expression and characterization of two cold-adapted lipases from Penicillium sp. XMZ-9].

    Zheng, Xiaomei; Wu, Ningfeng; Fan, Yunliu

    2012-04-01

    Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids, cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry. PMID:22803398

  5. Localization of 5S and 25S rRNA genes on somatic and meiotic chromosomes in Capsicum species of chili pepper.

    Kwon, Jin-Kyung; Kim, Byung-Dong

    2009-02-28

    The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, annuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense, frutescens, and chinense, and four in baccatum, with the exceptions that 'CM334' of annuum had three loci and 'tabasco' of frutescens had one locus. 'CM334'-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from 'CM334' plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili. PMID:19277503

  6. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice.

    Allen, Julie M; Burleigh, J Gordon; Light, Jessica E; Reed, David L

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  7. Karyotype analysis and physical mapping of 45S rDNA in eight species of Sophora,Robinia,and Amorpha

    LIU Bo; CHEN Chengbin; LI Xiulan; QI Liwang; HAN Suying

    2006-01-01

    The karyotype analysis and physical locations of 45S rDNA were carried out by means of fluorescence in situ hybridization in three species,and two forms of Sophora,two species of Robina,and one species of Amorpha.S.japonica L.,S.japonica L.f.oligophylla Franch.,S.japonica L.f.pendula Loud.,and S.xanthantha C.Y.Ma.are all tetraploids with 2n=28.There were four 45S rDNA sites in pericentromeric regions of two Pairs of chromosomes in each of them.S.rubriflora Tsoong.is a triploid with 2n=21,and three sites were located in each satellite of group 5 chromosomes.In R.pseudoacacia L.(2n=2x=22),we examined four intensive signals in telomeric regions of two pairs of satellite chromosomes.In R.hispida L.(2n=2x=30),there were four other signals in centromeric regions besides those like in R.pseudoacacia.Amorpha fruticosa L.has most chromosomes(2n=40)among the eight materials,however,there were only six 45S rDNA loci and they laid in centromeric regions,and satellites of three pairs of chromosomes.45S rDNA is a valuable chromosomal landmark in karyotype analysis.The distribution and genomic organization Of rDNA in the three genera were also discussed.

  8. Outside-in recrystallization of ZnS-Cu1.8 S hollow spheres with interdispersed lattices for enhanced visible light solar hydrogen generation.

    Zhu, Ting; Nuo Peh, Connor Kang; Hong, Minghui; Ho, Ghim Wei

    2014-09-01

    For the first time an earth-abundant and nontoxic ZnS-Cu(1.8) S hybrid photocatalyst has been engineered with well-defined nanosheet hollow structures by a template-engaged method. In contrast to conventional surface coupling and loading, the unique outside-in recrystallization promotes co-precipitation of ZnS and Cu(1.8) S into homogeneous interdispersed lattices, hence forming a hybrid semiconductor with visible responsive photocatalytic activity. The as-derived ZnS-Cu(1.8) S semiconductor alloy is tailored into a hierarchical hollow structure to provide readily accessible porous shells and interior spaces for effective ion transfer/exchange. Notably, this synergistic morphology, interface and crystal lattice engineering, aim towards the design of novel nanocatalysts for various sustainable environmental and energy applications. PMID:25043270

  9. Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA

    Yueh, Andrew; Schneider, Robert J.

    2000-01-01

    Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m7GpppN), mediated by initiation factor eIF4F. 40S eukaryotic ribosomes and initiation factors undergo 5′ scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5′ noncoding region of mRNAs. We demonstrate that the 5′ noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to ...

  10. Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

    Aline R. Rabello

    2008-01-01

    Full Text Available The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distinguished from the BR individuals. A phylogenetic tree based on ITS1 rDNA sequence was constructed. This is the first report of the ITS1 rDNA sequence of Bemisia tuberculata and of the BR biotype of B. tabaci.

  11. Specific Detection of Peronospora tabacina by PCR-amplified rDNA Sequences

    Jwu-Guh Tsay; Chishih Chu; Ya-Han Chuang; Ruey-Shyang Chen

    2006-01-01

    Peronospora tabacina Adam, a downy mildew fungus, is a devastating disease of tobacco and a pathogen of import prohibition in Taiwan. For quarantine purpose, we developed a nested PCR method to detect this pathogen. With universal primer pair ITS1/ITS4, internal transcribed spacer (ITS) region of pathogen`s rDNA was amplified. Specific PCR primers (PT1/PT2) were designed based on ITS sequence and used to amplify a 493-bp rDNA fragments from P. tabacina. In order to increase the sensitivity, a...

  12. Sequence analysis of the rDNA intergenic spacer of Metarhizium strains isolated in Brazil

    Fabiana Y. Yanaka-Schäfer

    2008-01-01

    Full Text Available To assess the extent of genetic variability of rDNA intergenic spacer (IGS in Metarhizium sp., 34 strains (27 isolated in Brazil were sequenced and analyzed together with an additional 20 Metarhizium anisopliae var. anisopliae sequences retrieved from GenBank. Overall, the global nucleotide diversity for the region under study was of 0.090, while for the Brazilian isolates it was only 0.016. Phylogenetic analyses showed four well-supported groups (A, B, C, and D, one of which (D has not been previously identified. All but one of the Brazilian strains cluster in this novel D phylogroup, suggesting that the genetic variation found in Brazil is a subset of the worldwide M. anisopiliae var. anisopliae variation.

  13. rRNA genes from the lower chordate Herdmania momus: structural similarity with higher eukaryotes.

    Degnan, B M; Yan, J.; Hawkins, C J; Lavin, M F

    1990-01-01

    Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence...

  14. CORE: a phylogenetically-curated 16S rDNA database of the core oral microbiome.

    Ann L Griffen

    Full Text Available Comparing bacterial 16S rDNA sequences to GenBank and other large public databases via BLAST often provides results of little use for identification and taxonomic assignment of the organisms of interest. The human microbiome, and in particular the oral microbiome, includes many taxa, and accurate identification of sequence data is essential for studies of these communities. For this purpose, a phylogenetically curated 16S rDNA database of the core oral microbiome, CORE, was developed. The goal was to include a comprehensive and minimally redundant representation of the bacteria that regularly reside in the human oral cavity with computationally robust classification at the level of species and genus. Clades of cultivated and uncultivated taxa were formed based on sequence analyses using multiple criteria, including maximum-likelihood-based topology and bootstrap support, genetic distance, and previous naming. A number of classification inconsistencies for previously named species, especially at the level of genus, were resolved. The performance of the CORE database for identifying clinical sequences was compared to that of three publicly available databases, GenBank nr/nt, RDP and HOMD, using a set of sequencing reads that had not been used in creation of the database. CORE offered improved performance compared to other public databases for identification of human oral bacterial 16S sequences by a number of criteria. In addition, the CORE database and phylogenetic tree provide a framework for measures of community divergence, and the focused size of the database offers advantages of efficiency for BLAST searching of large datasets. The CORE database is available as a searchable interface and for download at http://microbiome.osu.edu.

  15. Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms.

    Cloix, C; Tutois, S; Mathieu, O; Cuvillier, C; Espagnol, M C; Picard, G; Tourmente, S

    2000-05-01

    A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3. Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin. The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units. PMID:10810091

  16. Homology-dependent repair is involved in 45S rDNA loss in plant CAF-1 mutants

    Muchová, V.; Amiard, S.; Mozgová, I.; Dvořáčková, Martina; Gallego, M.E.; White, C.; Fajkus, Jiří

    2015-01-01

    Roč. 81, č. 2 (2015), s. 198-209. ISSN 0960-7412 R&D Projects: GA ČR(CZ) GP13-11563P Institutional support: RVO:68081707 Keywords : DNA repair * genome instability * 45S rDNA Subject RIV: BO - Biophysics Impact factor: 5.972, year: 2014

  17. Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae

    Liu Bo

    2011-11-01

    Full Text Available Abstract Background The genus Fragaria comprises species at ploidy levels ranging from diploid (2n = 2x = 14 to decaploid (2n = 10x = 70. Fluorescence in situ hybridization with 5S and 25S rDNA probes was performed to gather cytogenetic information that illuminates genomic divergence among different taxa at multiple ploidy levels, as well as to explore the evolution of ribosomal RNA genes during polyploidization in Fragaria. Results Root tip cells of diploid taxa were typified by two 5S and six 25S rDNA hybridization signals of varying intensities, providing a baseline for comparisons within the genus. In three exceptional diploid genotypes, F. nilgerrensis (CFRA 1358 and CFRA 1825 and F. vesca 'Yellow Wonder', two 5S but only four 25S rDNA sites were found but with differing site losses. The numbers of 5S and 25S rDNA signals, respectively were three and nine in a triploid F. ×bifera accession, and were four and twelve in three tetraploids, thus occurring in proportional 1.5× and 2× multiples of the typical diploid pattern. In hexaploid F. moschata, a proportional multiple of six 5S rDNA sites was observed, but the number of 25S rDNA sites was one or two less than the proportionate prediction of eighteen. This apparent tendency toward rDNA site loss at higher ploidy was markedly expanded in octoploids, which displayed only two 5S and ten 25S rDNA sites. In the two decaploids examined, the numbers of 5S and 25S rDNA signals, respectively, were four and fifteen in F. virginiana subsp. platypetala, and six and twelve in F. iturupensis. Conclusions Among diploid Fragaria species, a general consistency of rDNA site numbers implies conserved genomic organization, but highly variable 25S signal sizes and intensities and two instances of site loss suggest concurrent high dynamics of rDNA copy numbers among both homologs and non-homologs. General conservation of rDNA site numbers in lower ploidy, but marked site number reductions at higher ploidy

  18. Sequence analysis of 16S rDNA derived from a high yield lipase strain%一株高产脂肪酶产生菌16S rDNA的序列分析

    孙新城; 马淑玲; 张玲丽; 张浩; 景建洲

    2012-01-01

    [目的]对分离获得的高产脂肪酶菌株进行鉴定,为其改造和更好利用奠定基础.[方法]对从食堂下水道中分离获得的一株高效产脂肪酶细菌(JLΠ-4)进行培养,提取其基因组DNA.设计16S rDNA通用引物,扩增16S rDNA基因片段,并连接到pUC19-T载体上,转化大肠杆菌DH5X,经PCR鉴定的阳性克隆摇菌培养后测序.[结果]提取获得较高质量的基因组DNA,扩增获得新分离菌株16S rDNA基因片段,长度为1528 bp,BLAST相似性比对分析结果表明,其与伯克霍尔德氏菌16S rDNA序列相似性达97%,是一株与伯克霍尔德氏菌最近的革兰氏阴性菌.[结论]初步将高产脂肪酶细菌JTΠ-4鉴定为唐菖蒲伯克霍尔德菌.%[Objective]The present study was conducted to identify the separated high yield lipase strain to provide theoretical research references and to enhance the high yield lipase strain's transformation and application. [Method]A strain of bacterium with high yield lipase JLIT-4 was isolated from the sewage of a canteen, then its genomic DNA was extracted. The gene fragments of 16S rDNA were amplified using 16S rDNA universal primers and connected to pUC19-T vector; the fragments are then transformed into E. Coli DH5X. The positive clones identified by the PCR method were cultured and sequenced. [Result]Quality genome DNA was successfully extracted. The 16S rDNA gene fragments of newly isolated strain were amplified with the length of 1528 bp. According to comparison analysis of BLAST, 16S rDNA sequence similarity between the strains and Burkholderia(DQ355168) were 97%, so the lipase producing strains were identified as gram-negative bacteria that were most similar to the structures of Burkholderia. [ Conclusion ]The high-yield lipase JLΠ-4 was primarily identified as Burkholderia gladioli.

  19. SEQUENCE ANALYSIS OF SSU rDNA VARIABLE REGION OF DIFFERENT LEISHMANIA ISOLATES OF CHINA%利什曼原虫中国分离株SSU rDNA多变区序列分析

    卜玲毅; 胡孝素; 敬保迁; 马莹; 易桃林

    2001-01-01

    Aim  To analyze the sequence of the SSU rDNA variable region of Leishmania isolates from desert and hill foci of China. Methods  Specific SSU rDNA fragments from nuclear DNA of two Leishmania isolates were amplified by PCR and then cloned into pGEMR-T Easy Vector.After that, the specific fragments were sequenced by the automated DNA sequencer. Results Sequence analysis showed that the amplified DNA fragments of two Leishmania isolates(L.d.XJ771 and L.d.SC6) were all 392bp in length, point mutations were located in the two unique sequence (UQ-Ⅰ and UQ-Ⅱ),and no insertion/deletion found. The identities of comparison of Leishmania in GeneBank were more than 98%.Conclusion Sequence difference of the SSU rDNA variable region existed among Leishmania isolates from desert and hill foci; The sequences of the SSU rDNA variable region of L.d.XJ771 isolate and L. d.DD8 were identical.%目的分析我国荒漠、山丘疫区利什曼原虫分离株的 SSU rDNA多变区序列差异。方法 nDNA进行PCR扩增,将扩增出的SSU rDNA基因的特异片段克隆于pGEMR-T Easy Vector上,采用通用引物M13进行PCR扩增,全自动测序仪测序。结果序列分析显示本文报道的荒漠、山丘疫区的2株利什曼原虫(L.d.XJ771、L.d.SC6)的SSU rDNA序列大小均为392bp;序列差异发生在两个独特序列区(UQ-Ⅰ和UQ-Ⅱ),无移码突变;与GeneBank中的利什曼原虫比较分析,同源性在98%以上。结论我国荒漠、山丘疫区利什曼原虫分离株之间的SSU rDNA多变区的碱基序列有差异;荒漠疫区分离株L.d.XJ771与国际标准株L.d. DD8的SSU rDNA多变区的碱基序列完全相同。

  20. The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

    Jeng, R S; Svircev, A M; Myers, A L; Beliaeva, L; Hunter, D M; Hubbes, M

    2001-02-01

    The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. PMID:11166101

  1. Comparative Analysis of Sequences of the 5S rDNA NTS in 7 Wheat Species from Xinjiang of China%新疆7个小麦种5S rDNA及其NTS序列分析

    米日古丽·马木提; 玉山江·麦麦提; 托乎提·艾买提; 李继洋; 何丹; 赵奇

    2014-01-01

    本研究参照GeneBank中禾本科植物大麦5S rDNA序列,利用PCR技术扩增获得新疆7个小麦种5S rDNA部分序列,进一步与禾本科植物大麦5S rRNA序列比对,得到了5S rDNA结构和NTS边界范围。序列分析发现,不同类型小麦5S rDNA序列保守程度不同,其中大片段保守性较高,小片段相对较低,7个小麦种5S rDNA序列均存在不同程度的插入和缺失序列,同种不同类型和不同种5S rDNA非转录间隔区(NTS)长度和存在位置均呈现不同程度差异。利用MEGA4.0软件,采用邻接法(NJ)和最大简约法(MP)构建分子进化树并计算种间遗传距离。以7个小麦种进化关系的分析结果为依据,利用5S rDNA两种类型片段建立了两种不同的亲缘关系分类依据,旨在为新疆7个小麦种亲缘关系分析提供一定理论依据,以期为后期种间同源关系分析,建立序列集合,推导系统进化与发育关系,重建发育史和遗传育种奠定一定理论依据。%Referring GeneBank in barley grasses 5S rDNA sequences, partial 5S rDNA sequences from several accessions of 7 wheat species from Xinjiang were obtained by polymerase chain reaction, the 5S rDNA structure and NTS boundaries were obtained by further alignments with barley grasses 5S rRNA sequence. Sequence analysis revealed that the degree of the nontranscribed spacer of the different unit classes was different. The nontranscribed spacer of long repeat classes was less variable than that of short repeat classes. Different degrees of insertion and deletion in 5S rDNA sequences of 7 wheat species from Xinjiang was presented and the different degree of difference was presented in 5S rDNA non-transcribed spacer (NTS) length and position exists in different repeats of one species and different species. Molecular phylogenetic tree was constructed and genetic distance between species was calculated by using the MEGA4.0 software, the neighbor-joining method (NJ) and

  2. Copy Number of the Transposon, Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

    LeRiche, Kaitlynn; Eagle, Shannon H. C.; Crease, Teresa J

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL)...

  3. Performance Based Logistics (PBL) for the FA-18/S-3/P-3/C-2 auxiliary power unit (APU) at Honeywell: an applied analysis

    Landreth, Clifford J.; Corporon, Laura L.; Wilhelm, Richard H.

    2005-01-01

    The purpose of this MBA project is to evaluate and assess the metrics, incentives and other terms and conditions of the Performance Based Logistics (PBL) contract between Naval Aviation Inventory Control Point (NAVICP) and Honeywell in support of FA-18/S-3/P-3/C-2 Auxiliary Power Unit (APU) to determine if the contractual terms and conditions established are effective in facilitating and encouraging the full potential of PBL savings and improved performance. PBL is an acquisition reform initi...

  4. First record of Bothriocephalus acheilognathi (Cestoda: Pseudophyllidea) in the Rio Grande with comparative analysis of ITS2 and V4-18S rRNA gene sequences

    Bean, M. G.; Škeříková, Andrea; Bonner, T. H.; Scholz, Tomáš; Huffman, D. G.

    2007-01-01

    Roč. 19, č. 2 (2007), s. 71-76. ISSN 0899-7659 R&D Projects: GA ČR GA524/04/0342; GA MŠk LC522 Institutional research plan: CEZ:AV0Z60220518 Keywords : Cestoda * Bothriocephalus * fish parasite Subject RIV: EG - Zoology Impact factor: 1.178, year: 2007

  5. An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5 ' UTRs and its implications for eukaryotic gene translation regulation

    Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Valášek, Leoš

    2013-01-01

    Roč. 41, č. 16 (2013), s. 7625-7634. ISSN 0305-1048 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:61388971 ; RVO:68378050 Keywords : 80S RIBOSOME * UNTRANSLATED REGION * ANGSTROM RESOLUTION Subject RIV: CE - Biochemistry Impact factor: 8.808, year: 2013

  6. Sequence and organization of 5S ribosomal RNA-encoding genes of Arabidopsis thaliana.

    Campell, B R; Song, Y; Posch, T E; Cullis, C A; Town, C D

    1992-03-15

    We have isolated a genomic clone containing Arabidopsis thaliana 5S ribosomal RNA (rRNA)-encoding genes (rDNA) by screening an A. thaliana library with a 5S rDNA probe from flax. The clone isolated contains seven repeat units of 497 bp, plus 11 kb of flanking genomic sequence at one border. Sequencing of individual subcloned repeat units shows that the sequence of the 5S rRNA coding region is very similar to that reported for other flowering plants. Four A. thaliana ecotypes were found to contain approx. 1000 copies of 5S rDNA per haploid genome. Southern-blot analysis of genomic DNA indicates that 5S rDNA occurs in long tandem arrays, and shows the presence of numerous restriction-site polymorphisms among the six ecotypes studied. PMID:1348233

  7. The effect of secondary compounds on the rumen microbial population structure measured by 16S rRNA and 18S rRNA

    Full text: Plant secondary compounds in the forages have an important role in determining forage quality. A method for evaluating their effects on microbial population structure was carried out using the in vitro gas syringe system followed by extraction of RNA and gel separation of 16S rRNA and 18S rRNA. Quantification of 16S rRNA and 18S rRNA bands indicated the prokaryote and eukaryote populations, respectively. Five types of plant materials, i.e. Nothopanax scutellarium (Mangkokan) leaves, Morinda citrifolia (Mengkudu) fruit, Sapindus rarak (lerak) fruit and two types of Sesbania sesban leaves (hgh saponin and low saponin) were tested and Pennisetum purpureum (rumput gajah, Indonesian name) was used as a control roughage. Presence of saponin in these plant materials was determined qualitatively by thin layer chromatography. Eukaryote population was found to be significantly affected by the above plant materials. Both types of S. sesban leaves caused total elimination of eukaryotes. S. rarak reduced both eukaryote and prokaryote populations. The observed inhibition of eukaryote population might be due to the presence of saponin in these plant materials. In another experiment, a methanol extract of S. rarak which contained saponin was included and its effect on in vitro fermentation of P. purpureum was evaluated. The results showed that at higher levels of inclusion of S. rarak methanol extract, eukaroytes were totally eliminated. Comparison was made between microbial mass calculated based on difference between apparent undigested residue and true undigested residue and microbial mass calculations based on 16S rRNA and 18S rRNA. Microbial mass calculated by difference method was much higher than the microbial mass calculated on the basis of 16S rRNA and 18S rRNA. The quantification of RNA can be a useful and rapid technique for an accurate assessment of the effect of new forage materials on the microbial population structure. Other parameters from in vitro

  8. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  9. Comparative molecular analysis of Herbaspirillum strains by RAPD, RFLP, and 16S rDNA sequencing

    Soares-Ramos Juliana R.L.

    2003-01-01

    Full Text Available Herbaspirillum spp. are endophytic diazotrophic bacteria associated with important agricultural crops. In this work, we analyzed six strains of H. seropedicae (Z78, M2, ZA69, ZA95, Z152, and Z67 and one strain of H. rubrisubalbicans (M4 by restriction fragment length polymorphism (RFLP using HindIII or DraI restriction endonucleases, random amplified polymorphic DNA (RAPD, and partial sequencing of 16S rDNA. The results of these analyses ascribed the strains studied to three distinct groups: group I, consisting of M2 and M4; group II, of ZA69; and group III, of ZA95, Z78, Z67, and Z152. RAPD fingerprinting showed a higher variability than the other methods, and each strain had a unique electrophoretic pattern with five of the six primers used. Interestingly, H. seropedicae M2 was found by all analyses to be genetically very close to H. rubrisubalbicans M4. Our results show that RAPD can distinguish between all Herbaspirillum strains tested.

  10. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription

  11. Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis Diversidade bacteriana de solo sob eucaliptos obtida por seqüenciamento do 16S rDNA

    Érico Leandro da Silveira; Rodrigo Matheus Pereira; Denilson César Scaquitto; Eliamar Aparecida Nascimbém Pedrinho; Silvana Pómpeia Val-Moraes; Ester Wickert; Lúcia Maria Carareto-Alves; Eliana Gertrudes Macedo Lemos

    2006-01-01

    Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversi...

  12. Effects of coal ash pollution on the genetic diversity of Brachionus calyciflorus based on rDNA ITS sequences

    Xinli Wen; Xianling Xiang; Xin Hu; Yinghao Xue; Yilong Xi; Gen Zhang

    2010-01-01

    In this study, rDNA ITS sequences were analyzed to compare the genetic diversity of Brachionus calyciflorus from the coal ash contaminated (Lake Hui) and two uncontaminated lakes (Lake Tingtang and Lake Fengming). The results showed that two sibling species in Brachionus calyciflorus species complex were defined in both Lake Tingtang and Lake Fengming, but only one sibling species was found in Lake Hui. The coal ash pollution decreased the number of sibling species. Based on the sequences of ...

  13. Molecular phylogeny of the stingless bees (Apidae, Apinae, Meliponini) inferred from mitochondrial 16S rDNA sequences

    Costa, Marco; Marco A. Del Lama,; Melo,Gabriel; Sheppard, Walter

    2003-01-01

    Sequence data from the mitochondrial 16S rDNA of 34 species from 22 genera of stingless bees plus outgroup sequences from 11 species of other corbiculate bees were used to investigate the phylogenetic relationships among the Meliponini. Equally weighted parsimony and maximum likelihood analyses were performed. Four main clades were recognized in the parsimony consensus tree: (A) Hypotrigona, (B) Austroplebeia, (C) remaining African genera (Plebeina, Meliplebeia, and Axestotrigona) plus the tw...

  14. Bacterial diversity in the Uranium mill-tailings Gittersee as estimated via a 16S rDNA approach

    Bacterial diversity in a soil sample collected from uranium mill-tailings called Gittersee and situated near the city of Dresden, Germany, was analysed by using a culture-independent 16S rDNA approach exploiting PCR amplification primers 7F and 1513R. The results were compared with those obtained earlier analysing the same sample by using another primer pair, namely 43F-1404R. The two 16S rDNA approaches demonstrated that Proteobacteria were the most predominant group in the sample, followed by Cytophaga/Flavobacterium/ Bacteroidesand by Gram positive bacteria with low and also with high G+C content too. A large number of 16S rDNA sequences from two libraries were identical or almost identical. However, the ratio between the bacterial groups represented in them significantly differed. 7F-1513R primer set retrieved in addition to the above mentioned sequences, also 16S rRNA of green non-sulphur bacteria and representatives of the AD1 and the OP11 divisions. The latter indicates that the 7F-1513R primer set seems to be more reliable in analyses of bacterial diversity. (authors)

  15. Molecular characterization of Stictodora tridactyla (Trematoda: Heterophyidae) from Kuwait Bay using rDNA ITS and mtCO1.

    Al-Kandari, Wafa Y; Alnaqeeb, Majed A; Isaac, Asha M; Al-Bustan, Suzanne A

    2015-11-01

    Stictodora tridactyla is an intestinal fluke in the family Heterophyidae that parasitizes shorebirds and mammals, including humans. Its metacercarial cyst stage was reported in the Arabian killifish, Aphanius dispar, at Kuwait Bay. In the present study, Cerithidea cingulata was found to serve as the first intermediate host of S. tridactyla. In order to establish the snail-fish link in the life cycle of S. tridactyla, complete sequences of ribosomal DNA internal transcribed spacer region 1 and 2 (rDNA ITS1 and ITS2) and partial sequence of cytochrome oxidase subunit 1 were obtained for metacercarial cysts isolated from the fish A. dispar and rediae isolated from the snail C. cingulata. Sequence alignment demonstrated that these larval stages belong to the same heterophyid species, S. tridactyla. Phylogenetic analysis based on rDNA ITS1, ITS2, and mtCO1 confirmed the position of S. tridactyla within the Heterophyidae and found it to cluster with Haplorchis spp. The present study represents the first molecular study correlating the larval stages of S. tridactyla using rDNA ITS1, ITS2, and mtCO1 and examining the phylogenetic relationships of S. tridactyla with different heterophyid species. PMID:26268569

  16. C-banding and fluorescent in situ hybridization with rDNA sequences in chromosomes of Cycloneda sanguinea Linnaeus (Coleoptera, Coccinellidae

    Eliane Mariza Dortas Maffei

    2004-01-01

    Full Text Available The aim of this study was to describe mitotic and meiotic chromosomes of Cycloneda sanguinea using C-banding, fluorescent in situ hybridization (FISH rDNA probes, and sequential FISH/Ag-NOR staining. The chromosome number was 2n = 18 + XX for females and 2n = 18 + Xy for males. The X chromosome was metacentric and the Y chromosome was very small. During meiosis, the karyotypic meioformula was n = 9 + Xy p, and sex chromosomes configured a parachute at metaphase I. At the beginning of pachytene, bivalents were still individualized, and sex chromosomes were associated end-to-end through the heteropycnotic region of the X chromosome. Later in pachytene, further condensation led to the formation of a pseudo-ring by the sex bivalent. All chromosomes showed pericentromeric heterochromatin. FISH and sequential FISH/Ag-NOR staining evidenced the location of the nucleolar organizer region in one pair of autosomes (at spermatogonial metaphase. During meiosis, these genes were mapped to a region outside the sex vesicle by FISH, although Xy p was deeply stained with silver at metaphase I. These results suggest that these argyrophilic substances are of a nucleolar protein nature, and seem to be synthesized by a pair of autosomes and imported during meiosis (prophase I to the sex pair, during the association of the sex chromosomes.

  17. Evaluation of direct 16S rDNA sequencing as a metagenomics-based approach to screening bacteria in bottled water.

    Hansen, Trine; Skånseng, Beate; Hoorfar, Jeffrey; Löfström, Charlotta

    2013-09-01

    Deliberate or accidental contamination of food, feed, and water supplies poses a threat to human health worldwide. A rapid and sensitive detection technique that could replace the current labor-intensive and time-consuming culture-based methods is highly desirable. In addition to species-specific assays, such as PCR, there is a need for generic methods to screen for unknown pathogenic microorganisms in samples. This work presents a metagenomics-based direct-sequencing approach for detecting unknown microorganisms, using Bacillus cereus (as a model organism for B. anthracis) in bottled water as an example. Total DNA extraction and 16S rDNA gene sequencing were used in combination with principle component analysis and multicurve resolution to study detection level and possibility for identification. Results showed a detection level of 10(5) to 10(6) CFU/L. Using this method, it was possible to separate 2 B. cereus strains by the principal component plot, despite the close sequence resemblance. A linear correlation between the artificial contamination level and the relative amount of the Bacillus artificial contaminant in the metagenome was observed, and a relative amount value above 0.5 confirmed the presence of Bacillus. The analysis also revealed that background flora in the bottled water varied between the different water types that were included in the study. This method has the potential to be adapted to other biological matrices and bacterial pathogens for fast screening of unknown bacterial threats in outbreak situations. PMID:23971801

  18. Morphology and SSU rDNA sequence analysis of two hypotrichous ciliates (Protozoa, Ciliophora, Hypotrichia) including the new species Metaurostylopsis parastruederkypkeae n. sp.

    Lu, Borong; Wang, Chundi; Huang, Jie; Shi, Yuhong; Chen, Xiangrui

    2016-05-01

    The morphology and phylogeny of two hypotrichous ciliates, Metaurostylopsis parastruederkypkeae n. sp. and Neourostylopsis flavicana (Wang et al., 2011) Chen et al., 2013 were investigated based on morphology, infraciliature and the small subunit (SSU) ribosomal RNA gene (rRNA) sequence. The new species, M. parastruederkypkeae n. sp. was identified according to its characteristics: body shape ellipsoidal, size about (165-200) × (45-60) μm in vivo, cell color reddish; two types of cortical granules including wheat grain-like and yellow-greenish larger ones along the marginal cirri rows and dorsal kineties and dot-like and reddish smaller ones, grouped around marginal cirri on ventral side and arranged in short lines on dorsal side; 26-41 adoral membranelles; three frontal and one parabuccal, five to seven frontoterminal, one buccal, and three to six transverse cirri; seven to thirteen midventral pairs; five to nine unpaired ventral cirri, five to seven left and three to five right marginal rows; and three complete dorsal kineties. Phylogenetic analysis based on SSU rDNA sequences showed that both Metaurostylopsis and Neourostylopsis are monophyletic. As the internal relationship between and within both genera are not clear, further studies on the species in these two genera are necessary. The key characteristics of all known twelve Metaurostylopsis-Apourostylopsis-Neourostylopsis species complex were updated.

  19. Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts

    CHENYING; BOZHANG; 等

    1997-01-01

    The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects.However,the distribution pattern of DNA in these nuclei remains unknown.We introduced rDNA from the macronuclei of Tetrahymena into Xenopus cellfree extracts to examine the association of specific DNA sequences with nuclear matrix(NM) in the nuclei assembled in vitro.Our previous works showed the 5'NTS(nontranscription sequences) of the rDNA specifically bind to the NM system in the macronuclei.We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system.When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system,we found that the 5'NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the estracts of Xenopus eggs.

  20. 复发性外阴阴道念珠菌病菌种的26S rDNA序列分析%Analysis on recurrent vulvovaginal candidiasis monilia germs of 26S rDNA sequence

    迟绍琴; 许瑞环; 李康; 黄兴国; 陈亦微

    2012-01-01

    目的 探讨基于核糖体基因26S rDNA D1/D2区序列分析法在临床酵母菌菌种鉴定中的应用.方法 收集来源于复发性外阴阴道念珠菌病分泌物标本93株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行序列测定和分析,并与基因库中的基因序列进行同源性比对.结果 所有菌株均鉴定到种,同源性达99%和100%,同属于真菌双核亚界、子囊菌门、酵母菌科的3个属,89株为candida,3株为Kodamaea,1株为Pichia.其中candida中有7个种,38株candida glabrata,23株 candida albicans,16株candida parapsolisis,9株candida metapdilosis,1株candida orthopsilisis,1株 candida tropicalis,1株candida nivariensis;3株Kodamaea ohmeri;1株Pichia kudriavzevii.结论 复发性外阴阴道念珠菌病的病原体主要为candida属的candida glabrata、candida albicans 和candida parapsolisis,非candida albicans占75.27%是其特征;26SrDNA D1/D2区序列分析为临床酵母菌的分子水平鉴定提供了一种准确、可行的方法.%Objective To explore the ribosomes gene based on 26S rDNA D1/D2 area sequence analysis method in the clinical application of yeast strain identification. Methods To collect from recurrent genital vaginal moniiiosis secretion specimens of 93 plants,PCR amplification its 26S rDNA D1/D2 area,the amplification products scries were determined and analyzed,and the genetic sequences with gene pool than homology. Results All strains arc identification to the kind,the homology of 99% and 100%, belong to the fung, dikarya, ascomycota, saccharomycctaccac of this three genera,89 strains for Candida,3 strains for Kodamaca,for Pichia 1 strain. One of seven kinds of Candida,38 strains Candida glabrata, Candida albicans 23 strains, 16 strains Candida parapsolisis, 9 of Candida mctapdilosis, Candida orthopsilisis 1 strain,Candida tropicalis 1 strain, 1 strain Candida nivaricnsis;3 strains Kodamaca ohmcri;Pichia kudriavzevii 1 strain. Conclusion Recurrent genital vaginal moniliosis pathogen of

  1. Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes.

    Rossi, Anna Rita; Milana, Valentina; Hett, Anne Kathrin; Tancioni, Lorenzo

    2012-12-01

    Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA(3)-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a "leuciscine" cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes. PMID:23238894

  2. Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection.

    Robledo, G; Lavia, G I; Seijo, G

    2009-05-01

    Section Arachis of the homonymous genus includes 29 wild diploid species and two allotetraploids (A. monticola and the domesticated peanut, A. hypogaea L.). Although, three different genomes (A, B and D) have been proposed for diploid species with x = 10, they are still not well characterized. Moreover, neither the relationships among species within each genome group nor between diploids and tetraploids (AABB) are completely resolved. To tackle these issues, particularly within the A genome, in this study the rRNA genes (5S and 18S-26S) and heterochromatic bands were physically mapped using fluorescent in situ hybridization (FISH) in 13 species of Arachis. These molecular cytogenetic landmarks have allowed individual identification of a set of chromosomes and were used to construct detailed FISH-based karyotypes for each species. The bulk of the chromosome markers mapped revealed that, although the A genome species have a common karyotype structure, the species can be arranged in three groups (La Plata River Basin, Chiquitano, and Pantanal) on the basis of the variability observed in the heterochromatin and 18S-26S rRNA loci. Notably, these groups are consistent with the geographical co-distribution of the species. This coincidence is discussed on the basis of the particular reproductive traits of the species such as autogamy and geocarpy. Combined with geographic distribution of the taxa, the cytogenetic data provide evidence that A. duranensis is the most probable A genome ancestor of tetraploid species. It is expected that the groups of diploid species established, and their relation with the cultigen, may aid to rationally select wild species with agronomic traits desirable for peanut breeding programs. PMID:19234686

  3. A local role for the small ribosomal subunit primary binder rpS5 in final 18S rRNA processing in yeast.

    Andreas Neueder

    Full Text Available In vivo depletion of the yeast small ribosomal subunit (SSU protein S5 (rpS5 leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3' processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head-platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3' end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3' processing phenotypes observed in many eukaryotic SSU head assembly mutants.

  4. Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella-positive environmental samples.

    Grimm, D; Ludwig, W F; Brandt, B C; Michel, R; Schleifer, K H; Hacker, J; Steinert, M

    2001-04-01

    Aquatic protozoa are natural hosts of the human pathogen Legionella pneumophila. The fluorescence labeled 16S rRNA-targeted oligonucleotide probe LEGPNE1 has recently been shown to specifically detect extracellular legionellae as well as intracellular legionellae parasitizing protozoa. In this study we designed oligonucleotide probes which are complementary to distinct regions of the 18S rRNA of the Legionella host organisms of the genera Hartmannella and Naegleria. The specificity of the probes, HART498 and NAEG1088, was tested by in situ hybridization of various laboratory reference strains. In order to evaluate the fluorescent probes for environmental studies three selected Legionella-positive cold water habitats were examined for the presence of these protozoa. Traditional culture methods followed by morphological identification revealed an almost consistent presence of Naegleria spp. in cold water habitats. Other protozoa species including Acanthamoeba spp., Echinamoeba spp., Hartmannella spp., Platyamoeba placida, Saccamoeba spp., Thecamoeba quadrilineata, and Vexillifera spp. were found sporadically. Concomitant analysis of the pH, conductivity and temperature of the water samples revealed no preference of Legionella or the respective protozoa for certain environmental conditions. The specificity of the newly designed 18S rRNA probes demonstrates that they are valuable and rapid tools for the identification of culturable environmental protozoa. PMID:11403402

  5. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia.

    Joseph Edward Ironside

    Full Text Available Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual

  6. Bacterial Identification Using 16S rDNA Gene Sequencing and Antibiogram Analysis on Biofield Treated Pseudomonas fluorescens

    Mahendra Kumar Trivedi; Gopal Nayak; Alice Branton; Dahryn Trivedi

    2016-01-01

    Biofield therapies have been reported to improve the quality of life as compared to other energy medicine. The aim of the study was to evaluate the impact of Mr. Trivedi’s biofield energy treatment on Pseudomonas fluorescens (P. fluorescens) for antimicrobial sensitivity, minimum inhibitory concentration (MIC), biochemical reactions, and biotype number. P. fluorescens cells were procured from MicroBioLogics Inc., USA in sealed packs bearing the American Type Culture Collection (ATCC 498...

  7. Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

    Sebastian Pita

    2013-05-01

    Full Text Available In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae. The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome or both sex chromosomes (X and Y chromosomes. This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

  8. Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of Community Fingerprints

    Heuer, Holger; Hartung, Kathrin; Wieland, Gabriele; Kramer, Ina; Smalla, Kornelia

    1999-01-01

    Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in ...

  9. Regulation of Arabidopsis thaliana 5S rRNA Genes.

    Vaillant, Isabelle; Tutois, Sylvie; Cuvillier, Claudine; Schubert, Ingo; Tourmente, Sylvette

    2007-05-01

    The Arabidopsis thaliana genome comprises around 1,000 copies of 5S rRNA genes encoding both major and minor 5S rRNAs. In mature wild-type leaves, the minor 5S rRNA genes are silent. Using different mutants of DNA methyltransferases (met1, cmt3 and met1 cmt3), components of the RNAi pathway (ago4) or post-translational histone modifier (hda6/sil1), we show that the corresponding proteins are needed to maintain proper methylation patterns at heterochromatic 5S rDNA repeats. Using reverse transcription-PCR and cytological analyses, we report that a decrease of 5S rDNA methylation at CG or CNG sites in these mutants leads to the release of 5S rRNA gene silencing which occurred without detectable changes of the 5S rDNA chromatin structure. In spite of severely reduced DNA methylation, the met1 cmt3 double mutant revealed no increase in minor 5S rRNA transcripts. Furthermore, the release of silencing of minor 5S rDNAs can be achieved without increased formation of euchromatic loops by 5S rDNA, and is independent from the global heterochromatin content. Additionally, fluorescence in situ hybridization with centromeric 180 bp repeats confirmed that these highly repetitive sequences, in spite of their elevated transcriptional activity in the DNA methyltransferase mutants (met1, cmt3 and met1 cmt3), remain within chromocenters of the mutant nuclei. PMID:17412735

  10. Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing.

    Eastman, Alexander W; Yuan, Ze-Chun

    2014-01-01

    Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID

  11. Structure and transcription of the spinach chloroplast rDNA leader region.

    Briat, J F; Dron, M; Loiseaux, S; Mache, R

    1982-01-01

    A cloned fragment of spinach chloroplast DNA carrying 140 bp of the 16S rRNA gene and 691 bp upstream this gene has been analysed by DNA sequencing, by in vitro transcription, by S1 mapping with chloroplast RNAs and purified 16S rRNA from 30S ribosomal subunits. A tRNAVal gene has been located between the position 394 and 465. Crude chloroplast RNA polymerase has been purified by heparin sepharose chromatography of a 80 000 g supernatant from pure lysed spinach plastids and used to transcribe...

  12. Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

    Rabello, Aline R; Paulo R. Queiroz; Kenya C.C. Simões; Hiragi, Cássia O.; Luzia H.C. Lima; Maria Regina V. Oliveira; Angela Mehta

    2008-01-01

    The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distin...

  13. Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

    Kamimura, K; Wakai, S; Sugio, T

    2001-01-01

    The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis. PMID:11414499

  14. rDNA mapping, heterochromatin characterization and AT/GC content of Agapanthus africanus (L. Hoffmanns (Agapanthaceae

    ARYANE C. REIS

    2016-01-01

    Full Text Available ABSTRACT Agapanthus (Agapanthaceae has 10 species described. However, most taxonomists differ respect to this number because the great phenotypic plasticity of the species. The cytogenetic has been an important tool to aid the plant taxon identification, and to date, all taxa of Agapanthus L'Héritier studied cytologically, presented 2n = 30. Although the species possess large chromosomes, the group is karyologically little explored. This work aimed to increase the cytogenetic knowledge of Agapanthus africanus (L. Hoffmanns by utilization of chromosome banding techniques with DAPI / CMA3 and Fluorescent in situ Hybridization (FISH. In addition, flow cytometry was used for determination of DNA content and the percentage of AT / GC nitrogenous bases. Plants studied showed 2n = 30 chromosomes, ranging from 4.34 - 8.55 µm, with the karyotype formulae (KF = 10m + 5sm. Through FISH, one 45S rDNA signal was observed proximally to centromere of the chromosome 7, while for 5S rDNA sites we observed one signal proximally to centromere of chromosome 9. The 2C DNA content estimated for the species was 2C = 24.4 with 59% of AT and 41% of GC. Our data allowed important upgrade for biology and cytotaxonomy of Agapanthus africanus (L. Hoffmanns.

  15. TURKEY FECAL MICROBIAL COMMUNITY STRUCTURE AND ECOLOGICAL FUNCTIONS REVEALED BY 16S RDNA AND METAGENOME SEQUENCES

    Turkey feces are an important source of fecal waste in the United States. With the exception of isolated studies on bacterial pathogens, little is known about the type of bacteria inhabiting the turkey gut. In order to understand the microbial diversity and functional genes assoc...

  16. Effect of secondary compounds in forages on rumen micro-organisms quantified by 16S and 18S rRNA

    A gas syringe method was used to evaluate the effect of secondary compounds from plant materials on in vitro fermentation products and microbial biomass. The experiment used Pennisetum purpureum, Morinda citrifolia fruit, Nothopanax scutellarium leaves, Sesbania sesban LS (low saponins type), Sesbania sesban HS (high saponins type) and Sapindus rarak fruit as substrates. The incubation was conducted with and without polyethylene glycol 6000 (PEG) addition for 24 hours. Gas production and short-chain fatty acids (SCFA) were analysed. Prokaryotic and eukaryotic concentrations were measured by quantifying 16S and 18S rRNA. The percentage increase in gas production due to PEG was very small (<5%) for all plant materials, which indicated that the biological effect of tannin in these plant materials is limited. TLC analysis revealed that all materials contained saponin, but only S. rarak, followed by S. sesban, contained a high diversity of saponins. S. sesban gave the highest (234 ml/g) while S. rarak gave the lowest gas production (115 ml/g). S. rarak gave the lowest SCFA production (3.57 mmole/g) and also the lowest ratio of acetate to propionate (1.76), indicating a change in pattern of SCFA production. Total elimination of eukaryotic concentration was evident from the absence of the 18S rRNA band when S. rarak and S. sesban were used as sole substrates. S. rarak also reduced the prokaryotic concentration. To use S. rarak as a defaunating agent without affecting prokaryotes, a crude saponin extract was prepared from S. rarak for further experiment. Different concentrations of crude saponins in a methanol extract of S. rarak fruit dissolved in rumen buffer were added to a substrate consisting of elephant grass and wheat bran (7:3 w/w). Microbial biomass yield was quantified by gravimetry and using rRNA as a marker. Addition of crude saponin extract from S. rarak to a high-roughage diet increased microbial biomass (MB) yield to 1.07 and 1.14 times MB yield of the

  17. Molecular Characterization of Stool Microbiota in HIV-Infected Subjects by Panbacterial and Order-Level 16S Ribosomal DNA (rDNA) Quantification and Correlations with Immune Activation

    Ellis, Collin L.; Ma, Zhong-Min; Mann, Surinder K.; Li, Chin-Shang; Wu, Jian; Knight, Thomas H.; Yotter, Tammy; Hayes, Timothy L.; Maniar, Archana H.; Troia-Cancio, Paolo V.; Overman, Heather A; Torok, Natalie J.; Albanese, Anthony; Rutledge, John C.; Miller, Christopher J.; Pollard, Richard B.; Asmuth, David M.

    2011-01-01

    Background The relationship between gut microbial community composition at the higher-taxonomic order-level and local and systemic immunologic abnormalities in HIV disease may provide insight into how bacterial translocation impacts HIV disease. Methods Antiretroviral (ART)-naive HIV patients underwent upper endoscopy before and nine months after starting ART. Duodenal tissue was paraffin-embedded for immunohistochemical analysis (IHC) and digested for FACS for T-cell subsets and immune activation (CD38+/HLA-DR+) enumeration. Stool samples were provided from patients and controls for comparison. Metagenomic microbial DNA was extracted from feces for optimized 16S ribosomal RNA gene (rDNA) real-time qPCR assays designed to quantify panbacterial loads and the relative abundances of proinflammatory Enterobacteriales order, and the dominant Bacteroidales and Clostridiales orders. Results Samples from 10 HIV-subjects prior to initiating, and from 6 subjects receiving, ART were available for analysis. There was a trend for a greater proportion of Enterobacteriales in HIV-positive subjects compared to controls (p=0.099). There were significant negative correlations between total bacterial load and duodenal CD4+ and CD8+ T-cell activation levels (r= −0.74, p= 0.004 and r= −0.67, p=0.013, respectively). The proportions of Enterobacteriales and Bacteroidales were significantly correlated with duodenal CD4+ T-cell depletion and peripheral CD8+ T-cell activation, respectively. Conclusions These data represent the first report of quantitative molecular and cellular correlations between total/universal and order-level gut bacterial populations and GALT levels of immune activation in HIV-infected subjects. The correlations between lower overall 16S rDNA levels and tissue immune activation suggest that the gut microbiome may contribute to immune activation and influence HIV progression. PMID:21436711

  18. Karyotype conservation in 2 populations of the parthenogenetic scorpion Tityus serrulatus (Buthidae): rDNA and its associated heterochromatin are concentrated on only one chromosome.

    Schneider, Marielle C; Cella, Doralice M

    2010-01-01

    Within the order Scorpiones, the parthenogenetic mode of reproduction has been described for 11 species, 6 of which belong to the genus Tityus. In this work, an investigation of the chromosome characteristics of 2 populations of Tityus serrulatus, the first scorpion species known to be thelytokously parthenogenetic, is described. An analysis of 40 individuals revealed holocentric chromosomes of large, medium, and small sizes and an invariable diploid number of 2n = 12. In addition to the conserved macrokaryotype structure, specific chromosome regions also appeared unchanged within and between the samples studied; that is, each sample displayed only one chromosome carrier of the active nucleolar organizer region containing ribosomal genes (5.8S, 18S, and 28S) and AT-rich heterochromatin associated with the ribosomal DNA. The high conservation of the chromosomal features observed in T. serrulatus differed from that verified in certain species of other groups of animals that possess both holocentric chromosomes and parthenogenetic reproduction. Moreover, the cytogenetic results obtained herein permit us to suggest how the eggs of T. serrulatus develop, whether by apomixis or automixis. PMID:20231264

  19. Comparative evaluation of microscopy, OptiMAL®and 18S rRNAgene based multiplex PCR for detection of Plasmodium falciparum & Plasmodium vivax from field isolates of Bikaner, India

    Deepak Pakalapati; Jyoti Acharya; Dhanpat Kumar Kochar; Ashis Das; Shilpi Garg; She Middha; Abhishek Kochar; Amit Kumar Subudhi; Boopathi Pon Arunachalam; Sanjay Kumar Kochar; Vishal Saxena; Pareek RP

    2013-01-01

    Objective: To evaluate microscopy, OptiMAL®and multiplex PCR for the identification of Plasmodium falciparum (P. falciparum) and Plasmodium vivax (P. vivax) from the field isolates of Bikaner, Rajasthan (Northwest India). Methods: In this study, a multiplex PCR (P. falciparum and P. vivax) was further developed with the incorporation of Plasmodium malariae (P. malariae) specific primer and also a positive control. The performance of microscopy, plasmodium lactate dehydrogenase (pLDH) based malaria rapid diagnostic test OptiMAL®and 18S rRNA gene based multiplex PCR for the diagnosis of P. falciparum and P. vivax was compared. Results: The three species multiplex PCR (P. falciparum, P. vivax and P. malariae) with an inbuilt positive control was developed and evaluated. In comparison with multiplex PCR, which showed the sensitivity and specificity of 99.36% (95% CI, 98.11%-100.00%) and 100.00% (95% CI, 100.00%-100.00%), the sensitivity and specificity of microscopy was 90.44% (95% CI, 88.84%-95.04%) and 99.22% (95%CI, 97.71%-100.00%), and OptiMAL®was 93.58% (95% CI, 89.75%-97.42%) and 97.69% (95% CI, 95.10%-100.00%). The efficiencies were 99.65%, 95.10% and 95.45% for multiplex PCR, microscopy and OptiMAL®, respectively. Conclusions: Our results raise concerns over the overall sensitivities of microscopy and OptiMAL®, when compared to the multiplex PCR and thus stress the need for new molecular interventions in the accurate detection of the malarial parasites. This further highlights the fact that further developments are needed to improve the performance of rapid diagnostic tests at field level.

  20. Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences

    Kapley A; Raje DV; Purohit HJ

    2003-01-01

    Abstract Background Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation ...

  1. TBP-TAF Complex SL1 Directs RNA Polymerase I Pre-initiation Complex Formation and Stabilizes Upstream Binding Factor at the rDNA Promoter*||

    Friedrich, J. Karsten; Panov, Kostya I.; Cabart, Pavel; Russell, Jackie; Zomerdijk, Joost C. B. M.

    2005-01-01

    Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports o...

  2. Phylogeographic structure of cotton pest Adelphocoris suturalis (Hemiptera: Miridae): strong subdivision in China inferred from mtDNA and rDNA ITS markers

    Lijuan Zhang; Hu Li; Shujuan Li; Aibing Zhang; Fei Kou; Huaizhu Xun; Pei Wang; Ying Wang; Fan Song; Jianxin Cui; Jinjie Cui; Gouge, Dawn H.; Wanzhi Cai

    2015-01-01

    Phylogeographic patterns of some extant plant and vertebrate species have been well studied; however, they are poorly understood in the majority of insects. The study documents analysis of mitochondrial (COI, CYTB and ND5) and nuclear (5.8S rDNA, ITS2 and 28S rDNA) data from 419 individuals of Adelphocoris suturalis, which is one of the main cotton pests found in the 31 locations in China and Japan involved in the study. Results show that the species is highly differentiated between populatio...

  3. The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

    Meili Shen

    Full Text Available The promoters of poised rRNA genes (rDNA are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

  4. FISH-mapping of the 5S rDNA locus in chili peppers (Capsicum-Solanaceae).

    Aguilera, Patricia M; Debat, Humberto J; Scaldaferro, Marisel A; Martí, Dardo A; Grabiele, Mauro

    2016-03-01

    We present here the physical mapping of the 5S rDNA locus in six wild and five cultivated taxa of Capsicum by means of a genus-specific FISH probe. In all taxa, a single 5S locus per haploid genome that persistently mapped onto the short arm of a unique metacentric chromosome pair at intercalar position, was found. 5S FISH signals of almost the same size and brightness intensity were observed in all the analyzed taxa. This is the first cytological characterization of the 5S in wild taxa of Capsicum by using a genus-derived probe, and the most exhaustive and comprehensive in the chili peppers up to now. The information provided here will aid the cytomolecular characterization of pepper germplasm to evaluate variability and can be instrumental to integrate physical, genetic and genomic maps already generated in the genus. PMID:26959315

  5. Isolation and 16s rdna sequence analysis of bacteria from dieback affected mango orchards in southern pakistan

    A broad range of microorganisms are involved in various mango plant diseases such as fungi, algae and bacteria. In order to study the role of bacteria in mango dieback, a survey of infected mango plants in southern Pakistan was carried out. A number of bacterial isolates were obtained from healthy looking and infected mango trees, and their characterization was undertaken by colony PCR and subsequent sequence analysis of 16S rDNA. These analyses revealed the presence of various genera including Acinetobacter, Bacillus, Burkholderia, Cronobacter, Curtobacterium, Enterobacter, Erwinia, Exiguobacterium, Halotelea, Lysinibacillus, Micrococcus, Microbacterium, Pantoea, Pseudomonas, Salmonella and Staphylococcus. It is noteworthy that several members of these genera have been reported as plant pathogens. The present study provided baseline information regarding the phytopathogenic bacteria associated with mango trees in southern Pakistan. (author)

  6. Sequence analysis of the rDNA internal transcribed spacer 2 of five species of South American human malaria mosquitoes.

    Fritz, G N

    1998-03-01

    The rDNA internal transcribed spacer 2 (ITS2) was sequenced for 5 species of mosquitoes that may be important vectors of human malaria in certain regions of South America and are difficult to distinguish by morphology: Anopheles evansae, An. nuneztovari, An. rangeli, An. strodei and An. trinkae. ITS2 sequences from samples collected in Ecuador, Bolivia, Venezuela and Brazil were aligned and compared in order to determine the usefulness of this spacer for the elaboration of species specific primers and DNA probes. The ITS2 was found to be different in size (ranging from 333 to 397 bp) and sequence between all pairs of species. Highly variable regions were found primarily at the 3' end of the spacer and were interspersed with relatively conserved sites. Instraspecific sequence variation was limited to a single transversion between specimens of An. rangeli from distant geographic locations suggesting concerted evolution and homogenization of the ITS2. PMID:10520449

  7. Description of the male, redescription of the female and 16S rDNA sequence of Ixodes aulacodi (Ixodidae).

    Chiţimia-Dobler, Lidia; D'Amico, Gianluca; Yao, Patrick Kouassi; Kalmár, Zsuzsa; Gherman, Călin Mircea; Mihalca, Andrei Daniel; Estrada-Peña, Agustin

    2016-04-01

    Ixodes (Afrixodes) aulacodiArthur, 1956 is a poorly known species that has been recorded predominantly in the wet countries of western and central Africa, mainly associated to the greater cane rat Thryonomys swinderianus (Temmink). We herein redescribe the female, describe the male (ascribed to the species from specimens found in copula) and provide the 16S rDNA sequence. We also provide complete illustrations of the adults based on specimens found on greater cane rats in Ivory Coast. Ixodes aulacodi is included in the group of species of the subgenus Afrixodes that have horseshoe shaped anal groove, and which lack auriculae and cornua. The female is easily separated when compared with other species because of a unique combination of characters: All the coxae have internal spurs, coxa II has two external spurs, syncoxae are absent, and trochanters I-III have one spur each. The male has a notched hypostome and lacks syncoxae, auriculae and cornua. PMID:26803353

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

    Chao Xu

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

  9. Cell wall degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA species

    Sanz L; Hermosa M R; González F J; Monte E

    2004-01-01

    @@ Species of the fungus Trichoderma, a genus of Hyphomycetes, are ubiquitous in the environment, but especially in soil. They have been used in a wide range of commercial applications including the production of hydrolases and in the biological control of plant diseases. A fundamental part of the Trichoderma antifungal system consists of a series of genes coding for a surprising variety of extracellular cell wall degrading enzymes (CWDE).Characterisation and identification of strains at the species level is the first step in utilizing the full potential of fungi in specific applications. One aim when isolating Trichoderma strains is to identify those which can be used in new agricultural and industrial applications. In the past it was not uncommon that biocontrol strains were defined as T. harzianum Rifai, due to the limited classification system of the genus Trichoderma. In recent years, several PCR-based molecular techniques have been used to detect and discriminate among microorganisms. Sequence analysis of the ITS regions of the ribosomal DNA and gene fragments as those corresponding to tef1 gene have been helpful in the neotypification, description and characterization of species in the genus Trichoderna.Another useful method for the identification of Trichoderma strains is the randomly amplified polymorphic DNA (RAPD) technique.Isozyme polymorphisms evaluation of five putative extracellular lytic enzymes loci (β-1,3-glucanase, β-1,6-glucanase, cellulase, chitinase and protease antivities) were carried out using representative strains of defined molecular groups. CWDE groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location and antifungal activities.Compiling morphological, biochemical and sequence information data into a common database would provide a useful resource that could be used to accurately name new haplotypes identified in the future and correctly place them within the genus Trichoderma.

  10. 45S rDNA在多种植物中期染色体上的定位%Physical Mapping of 45S rDNA on Metaphase Chromosomes in Several Plant Species

    刘博; 陈成彬; 李秀兰; 陈瑞阳; 宋文芹

    2006-01-01

    应用荧光原位杂交技术首次确定了日本小檗(Berberis thunbergii DC)、车前(Plantago major L.)、野芹菜(Sanicula lamelligera Hance)、荔枝(Litchi chinensis Sonn.)、槭树(Acer buergerianum Miq.)、天目琼花(Viburnum sargentii Koehne.)、丹参(Salvia miltorrhiza Bunge.)、榆树(Ulmus pumila L.)中45S rDNA在中期染色体上的位置.根据rDNA的位点数和位置的变化,分为四种类型:①在日本小檗、车前和野芹菜中,荧光信号正好位于随体染色体的次缢痕或端部;②荔枝和槭树,分别有1对和3对染色体具随体,但荧光原位杂交却检测到3对和5对染色体上具有杂交信号;③天目琼花,具有4对随体染色体,但仅在其中一对随体上显示了杂交信号;④在丹参和榆树中,有的杂交信号位于着丝粒部位或长臂的末端,杂交信号的数目成奇数.黄瓜(Cucumis sativus L.)的染色体45S rDNA信号正好位于6条染色体的着丝粒部位,这与Dal-Hoe和Hoshi等人的结果是一致的.上述结果表明:45S rDNA可以作为染色体的一个识别指标,对识别染色体的个体性具有一定的参考价值.另外还对45S rDNA位点分布的多态性进行了讨论.%The genomic distribution of ribosomal RNA genes has been determined for the first time by fluo rescence in situ hybridization (FISH) in Berberis thunbergii DC. , Plantago major L. , Sanicula lamelligera Hance, Litchi chinensis Sonn. , Acer buergerianum Miq. , Viburnum sargentii Koehne. , Salvia miltiorrhiza Bunge. , and Ulmus pumila L.. These species could be divided into four groups based on the difference on the number and sites of their rDNA loci: the fluorescence signals lay in the secondary constrictions or the terminal regions of SAT-chromosomes in B. thunbergii, P. major, and S. lamelligera; 3 and 5 pairs of signals were de tected in L. chinensis and A. buergerianum, respectively which had 1 and 3 pairs of satellites respectively ; there were 4 pairs of SAT-chromosomes in V

  11. Randomly detected genetically modified (GM maize (Zea mays L. near a transport route revealed a fragile 45S rDNA phenotype.

    Nomar Espinosa Waminal

    Full Text Available Monitoring of genetically modified (GM crops has been emphasized to prevent their potential effects on the environment and human health. Monitoring of the inadvertent dispersal of transgenic maize in several fields and transport routes in Korea was carried out by qualitative multiplex PCR, and molecular analyses were conducted to identify the events of the collected GM maize. Cytogenetic investigations through fluorescence in situ hybridization (FISH of the GM maize were performed to check for possible changes in the 45S rDNA cluster because this cluster was reported to be sensitive to replication and transcription stress. Three GM maize kernels were collected from a transport route near Incheon port, Korea, and each was found to contain NK603, stacked MON863 x NK603, and stacked NK603 x MON810 inserts, respectively. Cytogenetic analysis of the GM maize containing the stacked NK603 x MON810 insert revealed two normal compact 5S rDNA signals, but the 45S rDNA showed a fragile phenotype, demonstrating a "beads-on-a-string" fragmentation pattern, which seems to be a consequence of genetic modification. Implications of the 45S rDNA cluster fragility in GM maize are also discussed.

  12. Bacterial diversity in a soil sample from Uranium mining waste pile as estimated via a culture-independent 16S rDNA approach

    Bacterial diversity was studied in a soil sample collected from a uranium mining waste pile situated near the town of Johanngeorgenstadt, Germany. As estimated by ICP-MS analysis the studied sample was highly contaminated with Fe, Al, Mn, Zn, As, Pb and U. The 16S rDNA retrieval, applied in this study, demonstrated that more than the half of the clones of the constructed 16S rDNA library were represented by individual RFLP profiles. This indicates that the composition of the bacterial community in the sample was very complex. However, several 16S rDNA RFLP groups were found to be predominant and they were subjected to a sequence analysis. The most predominant group, which represented about 13% of the clones of the 16S rDNA library, was affiliated with the Holophaga/Acidobacterium phylum. Significant was also the number of the proteobacterial sequences which were distributed in one predominant α-proteobacterial cluster representing 11% of the total number of clones and in two equal-sized β- and γ-proteobacterial clusters representing each 6% of the clones. Two smaller groups representing both 2% of the clones were affiliated with Nitrospira and with the novel division WS3. Three of the analysed sequences were evaluated as a novel, not yet described lineage and one as a putative chimera. (authors)

  13. Phylogenetic analysis of encapsulated and non-encapsulated Trichinella species by studying the 5S rDNA tandemly repeated intergenic region.

    van der Giessen, J W B; Fonville, M; Briels, I; Pozio, E

    2005-09-01

    The identification of sequence regions in the genomes of pathogens which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular, and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identified as a good target to distinguish among eight Trichinella species and genotypes. The recent discovery of two non-encapsulated species in this genus, Trichinella papuae and Trichinella zimbabwensis, which can infect both mammals and reptiles, has suggested analyzing their 5S rDNA. Amplification of the tandem repeats of the 5S rDNA intergenic region of encapsulated species of Trichinella shows a 751bp fragment, whereas the three non-encapsulated species show a fragment of 800bp with T. pseudospiralis showing an additional fragment of 522bp. Although the size of the 800bp PCR fragments of T. papuae and T. zimbabwensis are similar to that of T. pseudospiralis, there are differences in the 5S rDNA intergenic regions among the three non-encapsulated species. Phylogenetic analysis of the 5S rDNA intergenic regions shows a clustering together of the three non-encapsulated Trichinella species that is well separated from the encapsulated ones. In addition, a single PCR-based method allows distinguishing non-encapsulated and encapsulated species. PMID:16076532

  14. Chromosomal localization of 45S rDNA, sex-specific C values, and heterochromatin distribution in Coccinia grandis (L.) Voigt.

    Bhowmick, Biplab Kumar; Yamamoto, Masashi; Jha, Sumita

    2016-01-01

    Coccinia grandis is a widely distributed dioecious cucurbit in India, with heteromorphic sex chromosomes and X-Y sex determination mode. The present study aids in the cytogenetic characterization of four native populations of this plant employing distribution patterns of 45S rDNA on chromosomes and guanine-cytosine (GC)-rich heterochromatin in the genome coupled with flow cytometric determination of genome sizes. Existence of four nucleolar chromosomes could be confirmed by the presence of four telomeric 45S rDNA signals in both male and female plants. All four 45S rDNA sites are rich in heterochromatin evident from the co-localization of telomeric chromomycin A (CMA)(+ve) signals. The size of 45S rDNA signal was found to differ between the homologues of one nucleolar chromosome pair. The distribution of heterochromatin is found to differ among the male and female populations. The average GC-rich heterochromatin content of male and female populations is 23.27 and 29.86 %, respectively. Moreover, the male plants have a genome size of 0.92 pg/2C while the female plants have a size of 0.73 pg/2C, reflecting a huge genomic divergence between the genders. The great variation in genome size is owing to the presence of Y chromosome in the male populations, playing a multifaceted role in sexual divergence in C. grandis. PMID:25795278

  15. 16s-23S rDNA: polymorphisms and their use for detection and identification of Xylella fastidiosa strains 16S-23S rDNA: polimorfismos e sua aplicação na detecção e identificação de linhagens de Xylella fastidiosa

    Juliana Camargo Martinati

    2007-03-01

    Full Text Available Strains of Xylella fastidiosa from several hosts (coffee, citrus, grape, almond, oleander, peach, plum, etc. were characterized by analyzing the content of the nucleotide sequences of 16S-23S rDNA (coding for a small subunit ribosomal RNA spacer region (ITS. Current methods for sequencing the ITS region yields partial sequences that do not contribute with significant information. According to this fact, new primers have been designed in order to obtain a complete sequence and facilitate the sequencing. The complete 16S-23S sequences from 08 strains were amplified through PCR using the primers designed in our lab. The 16S-23S sequences obtained were compared with 52 others sequences entries in GenBank database. The results revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.79-1.00. The dendogram based on similarity data revealed 5 main groups. This spacer sequence contains two genes for tRNA (tRNAala and tRNAile. The sequence analysis of the tRNA content showed a conserved region with a few differences in the nucleotide composition.Linhagens de Xylella fastidiosa de diferentes hospedeiros (café, uva, amêndoa, ameixa, etc. foram caracterizados analisando as sequências de nucleotídeos do espaço intergênico 16S-23S (ITS. Os métodos atuais para o sequenciamento da região ITS produzem fragmentos parciais das sequências que não contribuem com informações significantes. Em vista disso, novos primers foram desenhados a fim de obter uma sequência completa e facilitar o sequenciamento. A sequencia completa da região ITS de 08 linhagens de X. Fastidiosa foram amplificadas via PCR, sequenciadas e comparadas com outras 52 sequencias depositadas no GenBank. Os resultados revelaram um alto nível de variação sendo maior que os níveis encontrados quando se utiliza o gene 16S para este tipo de análise, com valores variando entre 0.79 a 1.00. O dendograma baseado em dados de

  16. Application of Faecalibacterium 16S rDNA genetic marker for accurate identification of duck faeces.

    Sun, Da; Duan, Chuanren; Shang, Yaning; Ma, Yunxia; Tan, Lili; Zhai, Jun; Gao, Xu; Guo, Jingsong; Wang, Guixue

    2016-04-01

    The aim of this study was to judge the legal duty of pollution liabilities by assessing a duck faeces-specific marker, which can exclude distractions of residual bacteria from earlier contamination accidents. With the gene sequencing technology and bioinformatics method, we completed the comparative analysis of Faecalibacterium sequences, which were associated with ducks and other animal species, and found the sequences unique to duck faeces. Polymerase chain reaction (PCR) and agarose gel electrophoresis techniques were used to verify the reliability of both human and duck faeces-specific primers. The duck faeces-specific primers generated an amplicon of 141 bp from 43.3 % of duck faecal samples, 0 % of control samples and 100 % of sewage wastewater samples that contained duck faeces. We present here the initial evidence of Faecalibacterium-based applicability as human faeces-specificity in China. Meanwhile, this study represents the initial report of a Faecalibacterium marker for duck faeces and suggests an independent or supplementary environmental biotechnology of microbial source tracking (MST). PMID:26743644

  17. Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

    Ahmed Marroki

    2011-03-01

    Full Text Available Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21 and one strain of L. rhamnosus (LbMF25 have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.

  18. Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.

    Hebb, Jennifer K; Cohen, Craig R; Astete, Sabina G; Bukusi, Elizabeth A; Totten, Patricia A

    2004-12-15

    Although Chlamydia trachomatis and Neisseria gonorrhoeae are established causes of salpingitis, the majority of cases have no known etiology. We used broad-range 16S rDNA polymerase chain reaction to identify novel, possibly uncultivable, bacteria associated with salpingitis and identified bacterial 16S sequences in Fallopian-tube specimens from 11 (24%) of 45 consecutive women with laparoscopically confirmed acute salpingitis (the case patients) and from 0 of 44 women seeking tubal ligations (the control subjects) at Kenyatta National Hospital, Nairobi, Kenya. Bacterial phylotypes most closely related to Leptotrichia spp. were detected as the sole phylotypes in 1, and mixed with other bacterial phylotypes in 2, specimens. Novel bacterial phylotypes and those associated with bacterial vaginosis, including Atopobium vaginae, were identified in 3 specimens. N. gonorrhoeae and Streptococcus pyogenes were identified in 2 and 1 specimens, respectively. The finding of novel phylotypes associated with salpingitis has important implications for the etiology, pathogenesis, and treatment of this important reproductive-tract disease syndrome. PMID:15551209

  19. Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast TRNT-TRNF region and nuclear rDNA.

    Hamzeh, Mona; Dayanandan, Selvadurai

    2004-09-01

    The species of the genus Populus, collectively known as poplars, are widely distributed over the northern hemisphere and well known for their ecological, economical, and evolutionary importance. The extensive interspecific hybridization and high morphological diversity in this group pose difficulties in identifying taxonomic units for comparative evolutionary studies and systematics. To understand the evolutionary relationships among poplars and to provide a framework for biosystematic classification, we reconstructed a phylogeny of the genus Populus based on nucleotide sequences of three noncoding regions of the chloroplast DNA (intron of trnL and intergenic regions of trnT-trnL and trnL-trnF) and ITS1 and ITS2 of the nuclear rDNA. The resulting phylogenetic trees showed polyphyletic relationships among species in the sections Tacamahaca and Aigeiros. Based on chloroplast DNA sequence data, P. nigra had a close affinity to species of section Populus, whereas nuclear DNA sequence data suggested a close relationship between P. nigra and species of the section Aigeiros, suggesting a possible hybrid origin for P. nigra. Similarly, the chloroplast DNA sequences of P. tristis and P. szechuanica were similar to that of the species of section Aigeiros, while the nuclear sequences revealed a close affinity to species of the section Tacamahaca, suggesting a hybrid origin for these two Asiatic balsam poplars. The incongruence between phylogenetic trees based on nuclear- and chloroplast-DNA sequence data suggests a reticulate evolution in the genus Populus. PMID:21652373

  20. Physical Mapping of the 5S Ribosomal RNA Gene in Citreae of Aurantioideae Species using Fluorescence in situ Hybridization

    Yamamoto, Masashi; Asad Asadi Abkenar; Matsumoto, Ryoji; KUBO, Tatsuya; TOMINAGA, Shigeto; ヤマモト, マサシ; マツモト, リョウジ; クボ, タツヤ; トミナガ, シゲト; 山本, 雅史; 松本, 亮司; 久保, 達也; 冨永, 茂人

    2009-01-01

    The location of the 5S ribosomal RNA gene (rDNA) in species from six genera of the Citreae of Aurantioideae was determined using fluorescence in situ hybridization (FISH). A 5S rDNA probe was labeled with biotin-16-dUTP. The probe was detected using a fluorescein isothiocyanate (FITC)-avidin conjugate with chromosomes counterstained with propidium iodide (PI). When the chromosomes were observed under a G filter, PI-stained chromosomes were classified into the following five types based on the...

  1. 我国内脏利什曼病不同疫区病原体小亚基核糖体DNA可变区序列差异%Difference in DNA sequences in SSU rDNA variable regions among pathogens isola ted from different epidemic foci of visceral leishmaniasis in China

    胡孝素; 卜玲毅; 马莹; 王雅静; 敬保迁; 易桃林

    2002-01-01

    目的 试验证实我国黑热病不同疫区分离出的5个L.d分离株的小亚基核糖体DNA可变区序列存在点突变.方法将7个利什曼原虫种/株的nDNA进行PCR扩增,将扩增出的SSU rDNA基因的特异片段克隆于pGEMR-T Easy Vector上,采用通用引物M13进行PCR扩增,全自动测序仪测序。结果序列分析显示,扩增的7种/株利什曼原虫SSU rDNA序列大小为392 bp,5个点突变均发生在两个独特序列区(UQ-I和UQ-II);无移码突变。经与基因库中利什曼原虫序列类似性比较,同源性在98%以上。结论首次报道我国黑热病不同疫区的5个L.d.分离株的SSU rDNA可变区存在5个点突变。%Objective To confirm the existence of point mutations I n the SSU rDNA variable regions of 5 Leishmania donovani (L-d) isolates from different epidemic foci in ChinaMethods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEMR-T Easy Vectors After that, the specific fragments were sequenced by an auto mated DNA sequencerResults Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 po int mutations were located in two unique sequence blocks (UQ-Ⅰ and UQ-Ⅱ), a nd no insertions or deletions were found The dentities of comparison of Leish mania in GeneBank were more than 98%.Conclusion Five point mutations exist in the ssu rDNA variable region of 5 L.d.isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences differences of the SSU rDNA variable region exist among L.d.isolates from different foci.

  2. Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

    Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

    2004-06-15

    The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples. PMID:15183874

  3. Molecular confirmation of the genomic constitution of Douglasdeweya (Triticeae: Poaceae): demonstration of the utility of the 5S rDNA sequence as a tool for haplome identification.

    Baum, Bernard R; Johnson, Douglas A

    2008-06-01

    A new genus Douglasdeweya containing the two species, Douglasdeweya deweyi and D. wangii was published in 2005 by Yen et al. based upon the results of cytogenetical and morphological findings. The genome constitution of Douglasdeweya-PPStSt-allowed its segregation from the genus Pseudoroegneria which contains the StSt or StStStSt genomes. Our previous work had demonstrated the utility of using 5S rDNA units, especially the non-transcribed spacer sequence variation, for the resolution of genomes (haplomes) previously established by cytology. Here, we show that sequence analysis of the 5S DNA units from these species strongly supports the proposed species relationships of Yen et al. (Can J Bot 83:413-419, 2005), i.e., the PP genome from Agropyron and the StSt genome from Pseudoroegneria. Analysis of the 5S rDNA units constitutes a powerful tool for genomic research especially in the Triticeae. PMID:18421479

  4. 16S rDNA sequence analysis of bacterial isolates from biodeteriorated mural paintings in the Servilia tomb (Necropolis of carmona, Seville, Spain).

    Heyrman, J; Swings, J

    2001-11-01

    Bacteria were isolated from damaged mural paintings of the Servilia tomb (necropolis of Carmona, Seville, Spain). Selected strains, representative for different clusters of isolates with similar fatty acid profiles, were analysed by 16S rDNA sequence analysis. Bacillus is the dominant genus among the isolates: members of the rRNA species complexes of B. megaterium, B. pumilus and B. firmus were found as well as several other Bacillus species. One group of halotolerant isolates falls in the Bacillus sensu lato group, with closest relatedness to the genera Salibacillus and Virgibacillus. Other genera found are Artbrobacter, Micrococcus, Streptomyces, Sphingomonas, Paenibacillus, and a genus closely related to Paracraurococcus. Many isolates showed low 16S rDNA sequence similarities with the closest related database entries, a strong indication for the presence of several new species among the isolates. PMID:11822679

  5. Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

    Chun Chen

    2016-03-01

    Full Text Available Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S, 28S rRNA(28S and elongation factor 1 alpha-like protein (EF1, were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae, and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189, 28S (1.414 and EF1 (3. The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

  6. Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

    Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

    2016-01-01

    The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis. PMID:26887253

  7. Neospora caninum in bovine fetuses of Minas Gerais, Brazil: genetic characteristics of rDNA Neospora caninum em fetos bovinos de Minas Gerais, Brasil: características genéticas do rDNA

    Domingos Sávio dos Santos

    2011-12-01

    Full Text Available Neosporosis is an important cause of abortion in cattle and information on their genetics and host parasite relationships are desirable. Neospora caninum samples obtained from 24 bovine fetuses from Minas Gerais, were genetically analyzed in part of the rDNA region, coding for rRNAs. Previously, brain, heart, liver, skeletal muscle, lung, kidney, spleen, thymus, lymph nodes, ovary or testis, uterus and skin of the ear were analyzed by conventional histopathology and immunohistochemistry. Of these, eight had lesions compatible in the brain and heart and two also in skeletal muscle and liver. Three brains showed antigen identification in immunohistochemistry. Brain and heart tissues were subjected to DNA extraction for PCR, whose product of 588 bp of ITS-1 region was sequenced in three samples. We obtained 96% similarity with dozens of sequences N. caninum deposited in GenBank. The phylogenetic tree showed great conservation among isolates of N. caninum in this study and those deposited in GenBank, while well-defined and specific branches were generated against other Apicomplexa. PCR for this region is useful as a diagnosis, with good analytical specificity, but the ITS-1 region is not suitable for genetic differentiation intra species because the sequences obtained were identical to the others analyzed.A neosporose é uma importante causa de abortos em bovinos, e informações sobre sua genética e relação parasito-hospedeiro são desejáveis. Amostras de Neospora caninum, obtidas de 24 conceptos bovinos oriundos de Minas Gerais, foram analisadas geneticamente em parte da região rDNA, codificadora de rRNAs. Previamente, cérebro, coração, fígado, músculo esquelético, pulmão, rim, baço, timo, linfonodos, ovário ou testículo, útero e pele da orelha foram analisados por histopatologia convencional e imuno-histoquímica. Dessas, oito apresentaram lesões compatíveis no encéfalo e coração e dois também no músculo esquelético e f

  8. Molecular identification of Dunaliella viridis Teod. strain MSV-1 utilizing rDNA ITS sequences and its growth responses to salinity and copper toxicity

    Ali Moradshahi; Hajar Zamani; Mansour Kharati-KoupaeI

    2012-01-01

    In addition to biochemical, physiological and morphological analysis, molecular studies provide additional information for establishing phylogenetic relationships among different species and strains of the genus Dunaliella. In the present study, based on neighbor- joining analysis of the nuclear rDNA ITS sequence, a novel strain of the green algae Dunaliella viridis was identified from Maharlu salt lake in Shiraz, Iran. The phylogenetic tree shows that the new strain is part of a clade contai...

  9. In situ chromosomal localization of rDNA sites in "Safed Musli" Chlorophytum ker-gawl and their physical measurement by fiber FISH.

    Lavania, U C; Basu, S; Srivastava, S; Mukai, Y; Lavania, S

    2005-01-01

    Fluorescence In Situ Hybridization (FISH) technique has been applied on somatic chromosomes and extended DNA fibers in the medicinally important species of Chlorophytum to elucidate physical localization and measurement of the rDNA sites using two rRNA multigene families homologous to 45S and 5S rDNA. The two species of Chlorophytum, namely C. borivillianum and C. comosum, both with 2n = 28, reveal diversity for copy number and localization of rDNA sites. C. borivillianum is comprised of five 45S-rDNA sites:one each in the secondary constriction region of chromosomes 7, 8, 9; one in the subtelomeric region of the short arm of chromosome 2 and the telomeric region of the short arm of chromosome 12; and one 5S-rDNA site in the subtelomeric region of the long arm of chromosome 1. In C. comosum, there are three 45S-rDNA sites (one each in the short arm of chromosomes 12, 13, and 14) and two 5S-rDNA sites (in the secondary constriction regions of chromosomes 2 and 13). Fiber FISH analysis conducted on extended DNA fibers revealed variation in the size of continuous tandem strings for the two r-DNA families. Taking the standard value of native B DNA equivalent to 3.27 kb for 1 mum, it was estimated that the physical size of continuous DNA strings is of the order of approximately 90 kb, 180 kb, and 300 kb for 45S-rDNA and of the order of 60 kb, 150 kb for 5S-rDNA in C. comosum, grossly in correspondence to their respective physical sizes at metaphase. PMID:15618304

  10. Systematics and DNA barcoding of free-living marine nematodes with emphasis on tropical desmodorids using nuclear SSU rDNA and mitochondrial COI sequences

    Armenteros, M.; Rojas-Corzo, A; Ruiz-Abierno, A.; Derycke, S.; Backeljau, T.; Decraemer, W.

    2014-01-01

    The diversity and phylogenetic relationships of the Desmodoridae, a widespread tropical family of free-living marine nematodes, is hitherto poorly known both from molecular and taxonomic points of view. We performed a molecular phylogenetic analysis of marine nematodes to: i) disentangle relationships among tropical desmodorid species; and ii) compare the performance of the nuclear SSU rDNA and mitochondrial COI nucleotide sequences in 42 and 45 nominal species, respectively, to identify spec...

  11. Wild Termitomyces Species Collected from Ondo and Ekiti States Are More Related to African Species as Revealed by ITS Region of rDNA

    Victor Olusegun Oyetayo

    2012-01-01

    Molecular identification of eighteen Termitomyces species collected from two states, Ondo and Ekiti in Nigeria was carried out using the internal transcribed spacer (ITS) region. The amplicons obtained from rDNA of Termitomyces species were compared with existing sequences in the NCBI GenBank. The results of the ITS sequence analysis discriminated between all the Termitomyces species (obtained from Ondo and Ekiti States) and Termitomyces sp. sequences obtained from NCBI GenBank. The degree of...

  12. Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies.

    Beckers, Bram; Op De Beeck, Michiel; Thijs, Sofie; Truyens, Sascha; Weyens, Nele; Boerjan, Wout; Vangronsveld, Jaco

    2016-01-01

    Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. PMID:27242686

  13. Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies

    Beckers, Bram; Op De Beeck, Michiel; Thijs, Sofie; Truyens, Sascha; Weyens, Nele; Boerjan, Wout; Vangronsveld, Jaco

    2016-01-01

    Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high-resolution community profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant-associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula × Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non-target DNA and retrieval of bacterial OTUs. PMID:27242686

  14. Polyphyletic origin of the genus Physarum (Physarales, Myxomycetes revealed by nuclear rDNA mini-chromosome analysis and group I intron synapomorphy

    Nandipati Satish CR

    2012-08-01

    Full Text Available Abstract Background Physarales represents the largest taxonomic order among the plasmodial slime molds (myxomycetes. Physarales is of particular interest since the two best-studied myxomycete species, Physarum polycephalum and Didymium iridis, belong to this order and are currently subjected to whole genome and transcriptome analyses. Here we report molecular phylogeny based on ribosomal DNA (rDNA sequences that includes 57 Physarales isolates. Results The Physarales nuclear rDNA sequences were found to be loaded with 222 autocatalytic group I introns, which may complicate correct alignments and subsequent phylogenetic tree constructions. Phylogenetic analysis of rDNA sequences depleted of introns confirmed monophyly of the Physarales families Didymiaceae and Physaraceae. Whereas good correlation was noted between phylogeny and taxonomy among the Didymiaceae isolates, significant deviations were seen in Physaraceae. The largest genus, Physarum, was found to be polyphyletic consisting of at least three well supported clades. A synapomorphy, located at the highly conserved G-binding site of L2449 group I intron ribozymes further supported the Physarum clades. Conclusions Our results provide molecular relationship of Physarales genera, species, and isolates. This information is important in further interpretations of comparative genomics nd transcriptomics. In addition, the result supports a polyphyletic origin of the genus Physarum and calls for a reevaluation of current taxonomy.

  15. A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

    Gokhman, Vladimir E; Bolsheva, Nadezhda L; Govind, Shubha; Muravenko, Olga V

    2016-06-01

    Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed. PMID:27150102

  16. Who are the active players of the Iberian Margin deep biosphere? Microbial diversity of borehole U1385 through analysis of 16S rDNA and rRNA

    Russell, J. A.; Orsi, W.; Edgcomb, V. P.; Biddle, J.

    2013-12-01

    Microbial community structure and activity in marine deep subsurface environments across the globe have been assayed using various molecular biology tools including 16S rDNA sequencing, microarrays, FISH/CARD-FISH, and metagenomics. Many studies involving these techniques are DNA-based. This limits study of microbial function in these environments as DNA does not degrade as quickly as RNA and may lead to misinterpreting relic microbial genes as important for present-day activity. In this study, the diversity of bacteria and archaea from sediments of the Iberian Margin IODP borehole U1385 was analyzed from bulk extracted DNA and RNA at seven different depths ranging from 10 to 123 meters below seafloor (mbsf). Presented data suggests that the picture of microbial diversity obtained from DNA is markedly different from that seen through analysis of RNA. IODP borehole U1385 offers a great comparison to ODP Site 1229, a well characterized borehole on the Peru Margin. Similar sediment depositional history and geochemistry will allow exploration of what represents a 'typical' continental margin sediment microbial community or if microbial endemism is established despite similar conditions. This study represents the first molecular exploration of sediment microbial communities from the Iberian Margin IODP Site U1385.

  17. PCR-RFLP and AP-PCR of rbcL and ITS of rDNA Show That × Taxodiomeria peizhongii (Taxodium x Cryptomeria) Is not an Intergeneric Hybrid

    2006-01-01

    ×Taxodiomeria peizhongiiZ. J. Ye, J. J. Zhang et S. H. Pan was regarded as a new intergeneric hybrid between Taxodium mucronatum Tenore (as the female donor) and Cryptomeria fortunei Hooibrenk ex Otto et Dietr (as the male donor). To confirm the authenticity of the intergeneric hybrid, we analyzed the rbcL gene and the internal transcribed spacer (ITS) of 26S-18S ribosomal RNA gene of the three species using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and arbitrarily primed PCR (AP-PCR), and obtained the following results: i) Taxodiomeria peizhongii had the same RFLP maps of the rbcL gene and the ITS as Taxodium mucronatum, but was different from C. fortunei; ii) a 311-bp PCR amplification product was obtained in C. fortunei by AP-PCR of ITS, but was not found in Taxodiomeria peizhongii. Our results have demonstrated that C. fortunei did not provide any genome for Taxodiomeria peizhongii, implying that T. peizhongii is not an intergeneric hybrid between the two species.

  18. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

    Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination. This study validated RPL29 and RPL29-B2M as the best single reference

  19. Evaluation of amplified rDNA restriction analysis (ARDRA for the identification of cultured mycobacteria in a diagnostic laboratory

    Rottiers Sylvianne

    2002-03-01

    Full Text Available Abstract Background The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only. Results During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern. Conclusions In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days. However, culture is needed after all to assess the antibiotic susceptibility of the strains.

  20. Genes and Gene Therapy

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  1. Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural features and evolution of piscine 5S rRNA genes and nontranscribed intergenic spacers.

    Pasolini, Paola; Costagliola, Domenico; Rocco, Lucia; Tinti, Fausto

    2006-05-01

    The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. PMID:16612546

  2. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    Fengquin, X; Nielsen, Henrik; Zhen, W;

    1993-01-01

    The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA genes (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of < or = 1/2 x 10(4) base pairs (bp) are distributed equall...

  3. Karyotypic diversity in four species of the genus Gymnotus Linnaeus, 1758 (Teleostei, Gymnotiformes, Gymnotidae: physical mapping of ribosomal genes and telomeric sequences

    Priscilla Scacchetti

    2011-08-01

    Full Text Available Conventional (Giemsa, C-Banding, Ag-NORs, CMA3 and molecular (5S rDNA, 18S rDNA, telomeric sequences cytogenetic studies were carried out in specimens of ten distinct fish populations of the genus Gymnotus (G. sylvius Albert and Fernandes-Matioli, 1999, G. inaequilabiatus Valenciennes, 1839, G. pantherinus Steindachner, 1908, and G. cf. carapo Linnaeus, 1758 from different Brazilian hydrographic basins. G. sylvius presented a diploid number of 40 chromosomes (22m+12sm+6st, G. pantherinus presented 52 chromosomes (32m+18sm+2st, while G. inaequilabiatus (42m+10sm+2a and G. cf. carapo (38m+12sm+4st presented 54 chromosomes. The C-banding technique revealed centromeric marks in all chromosomes of all species. Besides that, conspicuous blocks of heterochromatin were found interstitially on the chromosomes of G. inaequilabiatus, G. cf. carapo, and G. pantherinus. All four species showed single nucleolus organizing regions confirmed by results obtained through Ag-NORs and FISH experiments using 18S rDNA probes, which showed the NORs localized on the first chromosome pair in G. inaequilabiatus, G. cf. carapo, and G. pantherinus, and on pair 2 in G. sylvius. CMA3 staining revealed additional unrelated NORs marks in G. sylvius and G. pantherinus. The 5S rDNA probes revealed signals on one pair in G. sylvius and two pairs in G. pantherinus; G. inaequilabiatus had about seventeen pairs marked, and G. cf. carapo had about fifteen pairs marked. It is considered that the high amount of heterochromatin identified in the chromosomes of G. inaequilabiatus and G. cf. carapo could have facilitated the dispersion of 5S rDNA in these species. Interstitial signals were detected on the first metacentric pair of G. sylvius by telomeric probes (TTAGGGn indicating the possible occurrence of chromosomal fusions in this species. The present study reveals valuable cytotaxonomic markers for this group and allows a more precise evaluation of the processes involved in the

  4. Selection of reliable reference genes for gene expression studies in peach using real-time PCR

    Zhou Jun

    2009-07-01

    Full Text Available Abstract Background RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch. Therefore, the present study was conducted to identify suitable reference gene(s for normalization of gene expression in peach. Results In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT, cyclophilin (CYP2, RNA polymerase II (RP II, phospholipase A2 (PLA2, ribosomal protein L13 (RPL13, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 18S ribosomal RNA (18S rRNA, tubblin beta (TUB, tubblin alpha (TUA, translation elongation factor 2 (TEF2 and ubiquitin 10 (UBQ10. All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results. Conclusion Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls

  5. Phylogenetic utility of ribosomal genes for reconstructing the phylogeny of five Chinese satyrine tribes (Lepidoptera, Nymphalidae

    Mingsheng Yang

    2015-03-01

    Full Text Available Satyrinae is one of twelve subfamilies of the butterfly family Nymphalidae, which currently includes nine tribes. However, phylogenetic relationships among them remain largely unresolved, though different researches have been conducted based on both morphological and molecular data. However, ribosomal genes have never been used in tribe level phylogenetic analyses of Satyrinae. In this study we investigate for the first time the phylogenetic relationships among the tribes Elymniini, Amathusiini, Zetherini and Melanitini which are indicated to be a monophyletic group, and the Satyrini, using two ribosomal genes (28s rDNA and 16s rDNA and four protein-coding genes (EF-1α, COI, COII and Cytb. We mainly aim to assess the phylogenetic informativeness of the ribosomal genes as well as clarify the relationships among different tribes. Our results show the two ribosomal genes generally have the same high phylogenetic informativeness compared with EF-1α; and we infer the 28s rDNA would show better informativeness if the 28s rDNA sequence data for each sampling taxon are obtained in this study. The placement of the monotypic genus Callarge Leech in Zetherini is confirmed for the first time based on molecular evidence. In addition, our maximum likelihood (ML and Bayesian inference (BI trees consistently show that the involved Satyrinae including the Amathusiini is monophyletic with high support values. Although the relationships among the five tribes are identical among ML and BI analyses and are mostly strongly-supported in BI analysis, those in ML analysis are lowly- or moderately- supported. Therefore, the relationships among the related five tribes recovered herein need further verification based on more sampling taxa.

  6. The karyotype and 5S rRNA genes from Spanish individuals of the bat species Rhinolophus hipposideros (Rhinolophidae; Chiroptera).

    Puerma, Eva; Acosta, Manuel J; Barragán, Maria José L; Martínez, Sergio; Marchal, Juan Alberto; Bullejos, Mónica; Sánchez, Antonio

    2008-11-01

    The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair. PMID:18066670

  7. The Chemistry behind DNA Isolation from Orange Juice and Detection of 16S rDNA of Candidatus Liberibacter asiaticus by qPCR

    J. Bai; Baldwin, E. A.; Liao, H.; Kostenyuk, I.; Burns, J.; Irey, M.

    2014-01-01

    The current standard to diagnose Huanglongbing (HLB) for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and apply quantitative real-time PCR (qPCR) test to detect 16S rDNA of Candidatus Liberibacter asiaticus (CLas), the putative causal pathogen. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high pectin concentration, low pH and possible existence of an inhibitor to DNA amplification. The objective of th...

  8. Gastrointestinal Bacterial and Methanogenic Archaea Diversity Dynamics Associated with Condensed Tannin-Containing Pine Bark Diet in Goats Using 16S rDNA Amplicon Pyrosequencing

    Min, Byeng R.; Sandra Solaiman; Raymon Shange; Jong-Su Eun

    2014-01-01

    Eighteen Kiko-cross meat goats (n=6) were used to collect gastrointestinal (GI) bacteria and methanogenic archaea for diversity measures when fed condensed tannin-containing pine bark (PB). Three dietary treatments were tested: control diet (0% PB and 30% wheat straw (WS); 0.17% condensed tannins (CT) dry matter (DM)); 15% PB and 15% WS (1.6% CT DM), and 30% PB and 0% WS (3.2% CT DM). A 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing technique was used to characterize and elucida...

  9. Phylogenetic analysis of Rhizoctonia solani AG-4 isolates from common beans in Black Sea coastal region, Turkey, based on ITS-5.8S rDNA

    KILIÇOĞLU, Melike ÇEBİ; ÖZKOÇ, İbrahim

    2013-01-01

    In this study, 114 Rhizoctonia solani isolates were obtained from diseased common bean (Phaseolus vulgaris L.) plants from the Black Sea coastal region of Turkey. Genetic variation was determined among R. solani anastomosis group 4 (AG-4) subgroups (AG-4 HG-I and AG-4 HG-II). ITS-5.8 rDNA sequences of these isolates were aligned with other known R. solani sequences from GenBank, and distance and parsimony analysis were used to determine phylogenetic relationships. The R. solani AG-4 isolates ...

  10. Applicability of the 16S-23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

    Baudoin, Ezékiel; Couillerot, O.; Spaepen, S.; Moenne-Loccoz, Y.; Nazaret, S.

    2010-01-01

    Aims: To assess the applicability of the 16S-23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Methods and Results: Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400-550 bp) from Azo...

  11. The nucleolar size is associated to the methylation status of ribosomal DNA in breast carcinomas

    There is a body of evidence that shows a link between tumorigenesis and ribosome biogenesis. The precursor of mature 18S, 28S and 5.8S ribosomal RNAs is transcribed from the ribosomal DNA gene (rDNA), which exists as 300–400 copies in the human diploid genome. Approximately one half of these copies are epigenetically silenced, but the exact role of epigenetic regulation on ribosome biogenesis is not completely understood. In this study we analyzed the methylation profiles of the rDNA promoter and of the 5’ regions of 18S and 28S in breast cancer. We analyzed rDNA methylation in 68 breast cancer tissues of which the normal counterpart was partially available (45/68 samples) using the MassARRAY EpiTYPER assay, a sensitive and quantitative method with single base resolution. We found that rDNA locus tended to be hypermethylated in tumor compared to matched normal breast tissues and that the DNA methylation level of several CpG units within the rDNA locus was associated to nuclear grade and to nucleolar size of tumor tissues. In addition we identified a subgroup of samples in which large nucleoli were associated with very limited or absent rDNA hypermethylation in tumor respect to matched normal tissue. In conclusion, we suggest that rDNA is an important target of epigenetic regulation in breast tumors and that rDNA methylation level is associated to nucleolar size

  12. Characterization of Bacterial Community Structure and Diversity in Rhizosphere Soils of Three Plants in Rapidly Changing Salt Marshes Using 16S rDNA

    2007-01-01

    The structure and diversity of the bacterial communities in rhizosphere soils of native Phragmites australis and Scirpus mariqueter and alien Spartina alterniflora in the Yangtze River Estuary were investigated by constructing 16S ribosomal DNA (rDNA) clone libraries. The bacterial diversity was quantified by placing the clones into operational taxonomic unit (OTU) groups at the level of sequence similarity of > 97%. Phylogenetic analysis of the resulting 398 clone sequences indicated a high diversity of bacteria in the rhizosphere soils of these plants. The members of Alphaproteobacteria,Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria of the phylum Proteobacteria were the most abundant in rhizobacteria. Chao 1 nonparametric diversity estimator coupled with the reciprocal of Simpson's index (1/D) was applied to sequence data obtained from each library to evaluate total sequence diversity and quantitatively compare the level of dominance. The results showed that Phragmites, Scirpus, and Spartina rhizosphere soils contained 200, 668, and 382 OTUs, respectively. The bacterial communities in the Spartina and Phragmites rhizosphere soils displayed species dominance revealed by 1/D, whereas the bacterial community in Scirpus rhizosphere soil had uniform distributions of species abundance. Overall, analysis of 16S rDNA clone libraries from the rhizosphere soils indicates that the changes in bacterial composition may occur concomitantly with the shift of species composition in plant communities.

  13. Molecular analysis of the genus Mitragyna existing in Thailand based on rDNA ITS sequences and its application to identify a narcotic species: Mitragyna speciosa.

    Sukrong, Suchada; Zhu, Shu; Ruangrungsi, Nijsiri; Phadungcharoen, Thatree; Palanuvej, Chanida; Komatsu, Katsuko

    2007-07-01

    In Thailand, there are four Mitragyna species; M. speciosa, M. hirsuta, M. diversifolia, and M. rotundifolia. One, M. speciosa, is a narcotic plant and has medicinal importance for its opium-like effect. Since the use of M. speciosa has been forbidden in Thailand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, accurate authentication of M. speciosa is essential for both medicinal and forensic purposes. The nucleotide sequences of internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA (rDNA) of the Mitragyna species were analyzed. The whole length of ITS1-5.8S-ITS2 region was 608 bp in M. speciosa, 607 bp in the other species. Nineteen sites of nucleotide substitutions and 3 sites of 1-bp indels were observed, and M. speciosa showed specific sequence differed from the others. Based on the ITS sequences, a distinctive site recognized by a restriction enzyme XmaI in M. speciosa was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established to differentiate M. speciosa from the others. By the method, a 409-bp PCR fragment of ITS1-5.8S (partial) rDNA region from M. speciosa was cleaved into two fragments of 119 bp and 290 bp while the other species remained undigested. This method provides an effective and accurate identification of M. speciosa. PMID:17603168

  14. Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Dinka eMandakovic

    2016-05-01

    Full Text Available The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS, a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination and fish samples (coinfection, aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants. Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies.

  15. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  16. A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae)

    Reblánová, M.; Špakulová, M.; Orosová, Martina; Kraľová-Hromadová, I.; Bazsalovicsová, E.; Rajský, D.

    2011-01-01

    Roč. 109, č. 4 (2011), s. 2021-1028. ISSN 0932-0113 Institutional research plan: CEZ:AV0Z60220518 Keywords : SCHISTOSOMA-MANSONI * DIFFERENTIATION * FISH * ITS2 * HYBRIDIZATION * EPIDEMIOLOGY * LOCALIZATION * CYTOGENETICS * SHEEP Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 2.149, year: 2011

  17. Expression of 5 S rRNA genes linked to 35 S rDNA in plants, their epigenetic modification and regulatory element divergence

    Garcia, S.; Crhák Khaitová, Lucie; Kovařík, Aleš

    2012-01-01

    Roč. 12, JUN 20 (2012), ID 95. ISSN 1471-2229 R&D Projects: GA ČR GAP501/10/0208; GA ČR GBP501/12/G090 Institutional support: RVO:68081707 Keywords : ARABIDOPSIS-THALIANA * NUCLEOTIDE-SEQUENCES * DNA METHYLATION * GENUS ARTEMISIA Subject RIV: BO - Biophysics Impact factor: 4.354, year: 2012

  18. Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima

    Boutte, J.; Aliaga, B.; Lima, O.; de Carvalho, J.F.; Ainouche, A.; Macas, Jiří; Rousseau-Gueutin, M.; Coriton, O.; Ainouche, M.; Salmon, A.

    2016-01-01

    Roč. 6, č. 1 (2016), s. 29-40. ISSN 2160-1836 Institutional support: RVO:60077344 Keywords : poaceae * duplication * paralogy * bioinformatics * polyploidy Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.198, year: 2014

  19. Evaluation of reference genes for quantitative real-time PCR analysis of gene expression during early development processes of the tongue sole (Cynoglossus semilaevis)

    MA Qian; ZHUANG Zhimeng; FENG Wenrong; LIU Shufang; TANG Qisheng

    2015-01-01

    Differential expression of genes is crucial to growth and development of fish. To select the appropriate genes for gene normalization duringCynoglossus semilaevis early developmental process, eight candidate reference genes (ACTB, B2M, EF1A, GADPH, RPL7, TUBA, UBCE and 18S) were tested for their adequacy by using quantitative real-time PCR. The results showed that the expression of all the examined genes exhibited tissue dependent variations in the matureC.semilaevis. EF1A was listed as the most stable reference among the 14 tissues by RefFinder. Furthermore, the recommended comprehensive ranking of the stability determined by RefFinder showed that 18S was the most stable gene during the early developmental stages (from oosphere to 90 days old) in this study. However, when divided theCt value data of the above mentioned early developmental stages into two separate periods (embryo and post-hatching periods), TUBA and 18S represented the most stable references of these two developmental periods, respectively. Consequently, the reference gene should be carefully and accurately chosen even for studies of the same species at various developmental processes. The relevant data may help in selecting appropriate reference genes for mRNA expression analysis, and is of great value in the studies of fish growth and development.

  20. Molecular characterization of a deep-sea methanotrophic mussel symbiont that carries a RuBisCO gene

    Elsaied, Hosam Easa; Kaneko, Ryo; Naganuma, Takeshi

    2006-01-01

    In our previous investigation on the genes of 1,5-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further revealed the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA), particul...

  1. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  2. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription

  3. Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

    Siala, Mariam; Gdoura, Radhouane; Fourati, Hela; Rihl, Markus; Jaulhac, Benoit; Younes, Mohamed; Sibilia, Jean; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Sghir, Abdelghani; Hammami, Adnane

    2009-01-01

    Introduction Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. Methods We examined the SF samples from a total of 27 patients consisting of patients with rea...

  4. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae.

    Meng Sun

    Full Text Available The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA, elongation factor 1 (EF1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ribosomal protein S13 (RPS13, ribosomal protein S20 (RPS20, tubulin (TUB, and β-actin (ACTB were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1 were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands. 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults. 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C. To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83 was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  5. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR

    Wang Nian

    2009-03-01

    Full Text Available Abstract Background Citrus Huanglongbing (HLB is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. Results We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Conclusion Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

  6. Molecular analysis of the rRNA genes of Babesia spp and Ehrlichia canis detected in dogs from RibeirÃo Preto, Brazil

    de Oliveira, L. P.; Cardozo, G.P.; E.V. Santos; Mansur, M.A.B.; I.A.N. Donini; Zissou, V.G.; P.G. Roberto; M. Marins

    2009-01-01

    The partial DNA sequences of the 18S rRNA gene of Babesia canis and the 16S rRNA gene of Ehrlichia canis detected in dogs from Ribeirão Preto, Brazil, were compared to sequences from other strains deposited in GenBank. The E. canis strain circulating in Ribeirão Preto is identical to other strains previously detected in the region, whereas the subspecies Babesia canis vogeli is the main Babesia strain circulating in dogs from Ribeirão Preto.As sequências parciais dos genes RNAr 18S de Babesia...

  7. Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

    Knut Rudi

    2003-01-01

    Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

  8. lncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress

    Zhongliang Zhao

    2016-03-01

    Full Text Available The activity of rRNA genes (rDNA is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3 and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional “off” position. Thus, PAPAS exerts a “stress-tailored” dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning.

  9. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    Qian, Pei-Yuan; Wang, Yong; Lee, On On; Lau, Stanley C K; Yang, Jiangke; Lafi, Feras F.; Al-Suwailem, Abdulaziz; Wong, Tim YH

    2010-01-01

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversit...

  10. Community Analysis of Arbuscular Mycorrhizal Fungi in Roots of Poncirus trifoliata and Citrus reticulata Based on SSU rDNA

    Wang, Peng; Wang, Yin

    2014-01-01

    Morphological observation of arbuscular mycorrhizal fungi (AMF) species in rhizospheric soil could not accurately reflect the actual AMF colonizing status in roots, while molecular identification of indigenous AMF colonizing citrus rootstocks at present was rare in China. In our study, community of AMF colonizing trifoliate orange (Poncirus trifoliata L. Raf.) and red tangerine (Citrus reticulata Blanco) were analyzed based on small subunit of ribosomal DNA genes. Morphological observation sh...

  11. Population studies for STR loci (D3S1358, D5S818, D7S820, D18S51 and FGA) in NWFP and Sindhi populations of Pakistan for forensic use.

    Saqib Shahzad, M; Abbas Bokhari, S Yassir; Rao, Abdul Qayyum; Raza, M Hashim; Ullah, Obaid; Zia-Ur-rahman; Shahid, A Ali; Ahmad, Zahoor; Riazuddin, S

    2004-01-01

    CEMB's Forensic DNA typing project is directed towards the introduction of DNA typing technique in Pakistan's criminal justice system so as to exonerate an innocent and wrongly accused person and incriminates the culprit. The present study is a part of the project of CEMB to analyze Sindhi and NWFP (North West Frontier Province) populations for five STR (Short Tandem Repeat) loci out of 13 CODIS (Combined DNA Index System) loci. Allelic frequencies and heterozygosity for STR markers D3S1358, D5S818, D7S820, D18S51 and FGA (FIBRA) were determined. Samples from unrelated individuals were amplified by multiplex PCR using the unlabelled primers for these markers followed by denaturing Polyacrylamide Gel Electrophoresis (PAGE). Statistical analysis was performed to determine the allelic frequencies and was evaluated using the Chi Square Test. PMID:15782779

  12. Study of endophytic Xylariaceae in Thailand: diversity and taxonomy inferred from rDNA sequence analyses with saprobes forming fruit bodies in the field

    Okane, Izumi; Srikitikulchai, Prasert; Toyama, Kyoko; Læssøe, Thomas; Sivichai, Somsak; Hywell-Jones, Nigel; Nakagiri, Akira; Potacharoen, Wanchern; Suzuki, Ken-ichiro

    2008-01-01

    A study of the diversity, taxonomy, and ecology of endophytic Xylariaceae (Ascomycota) was carried out. In this study, we obtained isolates of Xylariaceae from healthy, attached leaves and teleomorphic stromata on decayed plant materials in a permanent plot at Khao Yai National Park (Thailand). In...... addition, strains deposited beforehand were selected in which both endophytic strains isolated from living plant tissues and saprobic strains from fruit bodies were included. Consequently, 405 strains of Xylariaceae (273 endophytic and 132 saprobic strains, including identified strains) were studied to...... reveal the diversity and taxonomy of endophytes and the relationships between those endophytes and saprobic Xylariaceae in Thailand that have been recorded according to fruit-body formation on decayed plant materials. Analysis of 28S rDNA D1/D2 sequences revealed 21 xylariaceous species inhabiting...

  13. Molecular characterization of the first internal transcribed spacer of rDNA of Parabronema skrjabini for the first time in sheep.

    Hasheminasab, Seyed Sajjad

    2015-01-01

    Parabronema skrjabini is a spirurid nematode of the family Habronematidae that lives in the abomasum of ruminants such as sheep and goats. The purpose of this study was to investigate the molecular aspects of Parabronema skrjabini in sheep. The worms were collected from sheep in Sanandaj (west of Iran). The first internal transcribed spacer (ITS) of ribosomal DNA (rDNA) nucleotide fragments of Parabronema skrjabini were amplified by polymerase chain reaction (PCR) using two pairs of specific primers (Para-Ir-R and Para-Ir-F). ITS1 homology in the sequence of this study was 69% compared with the sequence data in GenBank. To our knowledge, this is the first study in the world exploring the genetic diversity of P. skrjabini in sheep based on ITS1. PMID:26878620

  14. Heritability and Variability in Ribosomal RNA Genes of Vicia faba

    Rogers, Scott O; Bendich, Arnold J.

    1987-01-01

    We have compared the restriction patterns and copy numbers of ribosomal RNA genes (rDNA) between and within individuals of Vicia faba . While the EcoRI blot-hybridization patterns changed only after one to two generations, copy number changes were found among different tissues of the same plant. Copy number differences among individuals in the population were as great as 95-fold, whereas as much as a 12-fold variation was seen among tissues of the same plant. Among individual F1 progeny from ...

  15. Accumulation of pharmaceuticals, Enterococcus, and resistance genes in soils irrigated with wastewater for zero to 100 years in central Mexico.

    Philipp Dalkmann

    Full Text Available Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin, 4.3 µg/kg (sulfamethoxazole, and 5.4 µg/kg (carbamazepine in soils irrigated for 19-28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15 × 10(-3 ± 0.22 × 10(-3 copies/16S rDNA than in non-irrigated soils (4.35 × 10(-5± 1.00 × 10(-5 copies/16S rDNA, while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61 × 10(-4 ± 0.59 × 10(-4 versus 2.99 × 10(-5 ± 0.26 × 10(-5 copies/16S rDNA. Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a

  16. Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae

    Larisa Gunderina

    2015-05-01

    Full Text Available Chromosomal localization of ribosomal RNA coding genes has been studied by using FISH (fluorescence in situ hybridization in 21 species from the genus Chironomus Meigen, 1803. Analysis of the data has shown intra- and interspecific variation in number and location of 5.8S rDNA hybridization sites in 17 species from the subgenus Chironomus and 4 species from the subgenus Camptochironomus Kieffer, 1914. In the majority of studied species the location of rDNA sites coincided with the sites where active NORs (nucleolus organizer regions were found. The number of hybridization sites in karyotypes of studied chironomids varied from 1 to 6. More than half of the species possessed only one NOR (12 out of 21. Two rDNA hybridization sites were found in karyotypes of five species, three – in two species, and five and six sites – in one species each. NORs were found in all chromosomal arms of species from the subgenus Chironomus with one of them always located on arm G. On the other hand, no hybridization sites were found on arm G in four studied species from the subgenus Camptochironomus. Two species from the subgenus Chironomus – Ch. balatonicus Devai, Wuelker & Scholl, 1983 and Ch. “annularius” sensu Strenzke, 1959 – showed intraspecific variability in the number of hybridization signals. Possible mechanisms of origin of variability in number and location of rRNA genes in the karyotypes of species from the genus Chironomus are discussed.

  17. Application of gene sequencing directly to identify the pathogens in specimens

    LU Xin-xin; YUAN Liang; WAN Xiao-hua; GENG Jia-jing

    2010-01-01

    Background Accurate identification of bacterial isolates is an essential task in clinical microbiology. This study compared culturing to analyzing 16S rRNA gene sequences as methods to identify bacteria in clinical samples. We developed a key technique to directly identify bacteria in clinical samples via nucleic acid sequences, thus improving the ability to confirm pathogens.Methods We obtained 225 samples from Beijing Tongran Hospital and examined them by conventional culture and 16S rDNA sequencing to identify pathogens. This study made use of a modified sample pre-treatment technique which came from our laboratory to extract DNA. 16S rDNA was amplified by PCR. The amplified product was sequenced on a CEQ8000 capillary sequencer. Sequences were uploaded to the GenBank BLAST database for comparison.Results Among the positively cultivated bacterial strains, seven strains were identified differently by Vitek32 and by 16S rDNA sequencing. Twelve samples that were negative by standard culturing were determined to have pathogens by sequence analysis.Conclusion The use of 16S rRNA gene sequencing can improve clinical microbiology by providing better identification of unidentified bacteria or providing reference identification of unusual strains.

  18. Reference genes for quantitative gene expression studies in multiple avian species.

    Olias, Philipp; Adam, Iris; Meyer, Anne; Scharff, Constance; Gruber, Achim D

    2014-01-01

    Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species. PMID:24926893

  19. Reference genes for quantitative gene expression studies in multiple avian species.

    Philipp Olias

    Full Text Available Quantitative real-time PCR (qPCR rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ on different tissues of the mallard (Anas platyrhynchos, domestic chicken (Gallus gallus domesticus, common crane (Grus grus, white-tailed eagle (Haliaeetus albicilla, domestic turkey (Meleagris gallopavo f. domestica, cockatiel (Nymphicus hollandicus, Humboldt penguin (Sphenicus humboldti, ostrich (Struthio camelus and zebra finch (Taeniopygia guttata, spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

  20. Phylogenetic Relationships of Tribes Within Harpalinae (Coleoptera: Carabidae) as Inferred from 28S Ribosomal DNA and the Wingless Gene

    Ober, Karen A; Maddison, David R.

    2008-01-01

    Harpalinae is a large, monophyletic subfamily of carabid ground beetles containing more than 19,000 species in approximately 40 tribes. The higher level phylogenetic relationships within harpalines were investigated based on nucleotide data from two nuclear genes, wingless and 28S rDNA. Phylogenetic analyses of combined data indicate that many harpaline tribes are monophyletic, however the reconstructed trees showed little support for deeper nodes. In addition, our results suggest that the Le...

  1. 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

    Matioli Sergio R.; Martins Felipe M; Oliveira Mariana C; Milstein Daniela

    2008-01-01

    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 Bra...

  2. Preliminary evaluation of the use of soil bacterial 16S rDNA DNA markers in sediment fingerprinting in two small endorheic lagoons in southern Spain

    Gomez, Jose Alfonso; Landa del Castillo, Blanca; Guzman, Gema; Petticrew, Ellen L.; Owens, Phillip N.

    2016-04-01

    bulk community of DNA was extracted from 250 mg of soil samples (three replicates per sample) using the procedure described in Landa et al. (2014). The bacterial 16S rRNA gene V1-V2 hypervariable regions were amplified in polymerase chain reaction (PCR). The sequencing procedure was performed according to the manufacturer's recommendations using MiSeq Reagent Kit v2 for 300 cycles on MiSeq desktop sequencer. The raw dataset for each sample consisted of the number of counts for each of the 6640 operational taxonomic units (OTU) analyzed. All the screening and analysis was performed independently for each lagoon. Given the large number of OTUs, a first screening was made discarding any OTU that did not presented at least five samples with counts >20 for that OTU. This lowered the number of OTUs to 205 in Dulce and 217 in Zoñar. Because of the limited number of samples, we did not perform independent analysis for each soil depth. All the analyses were performed twice; one with the original number of counts and another with the normalized number of counts. We screened the OTU following a 4-step method to determine those with the best ability to discriminate among the three potential source areas. These steps were: 1) eliminate OTUs with no readings or very few, that could be experimental noise; 2) keep only OTUs that are different among source areas; 3) eliminate OTUs that range outside of feasible solutions to explain average values found in sediment; and 4) eliminate OTUs with the largest variability. Afterwards, several over-determined mixing models were solved considering different combinations of OTUs using limSolve (Soetaert et al., 2014) in R. Preliminary results show that 0.2 to 0.6 % of the searched OTUs (i.e. 14 to 42) had the potential for use in the mixing models after the four-step screening process. The results indicate a large variability in the number of counts among the samples from different areas within the subcatchments ranging, on average, from 49 to

  3. A yeast transcription system for the 5S rRNA gene.

    Keulen, H.; Thomas, D. Y.

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA gen...

  4. Variation in Stability of Endogenous Reference Genes in Fallopian Tubes and Endometrium from Healthy and Ectopic Pregnant Women

    Konje, Justin C.; Willets, Jonathon M; Marczylo, Emma L.; Amoako, Akwasi A.; Gebeh, Alpha K.

    2012-01-01

    RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes an...

  5. GENETIC DIFFERENTIATION BETWEEN NOBLE CRAYFISH, ASTACUS ASTACUS (L., POPULATIONS DETECTED BY MICROSATELLITE LENGTH VARIATION IN THE RDNA ITS1 REGION

    EDSMAN L.

    2002-07-01

    Full Text Available The Internal Transcribed Spacer (ITS region of the nuclear ribosomal repeat was investigated in the search for a suitable genetic marker for population studies of the noble crayfish Astacus astacus (L.. DNA sequencing revealed the presence of two microsatellite insertions in ITS1. By designing highly specific PCR primers, adjacent to one of the insertions, we were able to use fragment analysis to explore the variation of the insertion in 642 specimens from 17 populations of crayfish from Sweden and former Yugoslavia. A new statistical test, the Population Divergence Test, was developed to assess statistical significance of divergence between populations. This test does not assume Mendelian inheritance. Our results demonstrate that different populations often produce characteristic fragment patterns, and that most, but not all, populations are genetically distinct, with high significance. The populations that cannot be significantly differentiated are situated in close geographic proximity to each other and belong to the same main river system, probably reflecting that these populations have had recent contact and that gene flow has occurred.

  6. Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing

    Qian, Peiyuan

    2010-07-29

    The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (2 and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea. © 2011 International Society for Microbial Ecology All rights reserved.

  7. Community Analysis of Arbuscular Mycorrhizal Fungi in Roots of Poncirus trifoliata and Citrus reticulata Based on SSU rDNA

    Peng Wang

    2014-01-01

    Full Text Available Morphological observation of arbuscular mycorrhizal fungi (AMF species in rhizospheric soil could not accurately reflect the actual AMF colonizing status in roots, while molecular identification of indigenous AMF colonizing citrus rootstocks at present was rare in China. In our study, community of AMF colonizing trifoliate orange (Poncirus trifoliata L. Raf. and red tangerine (Citrus reticulata Blanco were analyzed based on small subunit of ribosomal DNA genes. Morphological observation showed that arbuscular mycorrhizal (AM colonization, spore density, and hyphal length did not differ significantly between two rootstocks. Phylogenetic analysis showed that 173 screened AMF sequences clustered in at least 10 discrete groups (GLO1~GLO10, all belonging to the genus of Glomus Sensu Lato. Among them, GLO1 clade (clustering with uncultured Glomus accounting for 54.43% clones was the most common in trifoliate orange roots, while GLO6 clade (clustering with Glomus intraradices accounting for 35.00% clones was the most common in red tangerine roots. Although, Shannon-Wiener indices exhibited no notable differences between both rootstocks, relative proportions of observed clades analysis revealed that composition of AMF communities colonizing two rootstocks varied severely. The results indicated that native AMF species in citrus rhizosphere had diverse colonization potential between two different rootstocks in the present orchards.

  8. Selection of Reference Genes for Gene Expression Analysis in Nilaparvata lugens with Different Levels of Virulence on Rice by Quantitative Real-Time PCR

    WANG Wei-xia; LAI Feng-xiang; LI Kai-long; FU Qiang

    2014-01-01

    The brown planthopperNilaparvata lugens Stål (Homoptera: Delphacidae) can cause hopperburn by feeding on rice and also can transmit the grassy stunt disease. Resistant rice varieties have been developed, but severalN. lugens strains can recover their virulence to these resistant rice varieties. In the present study, reference genes with stable expression levels inN. lugens populations showed different levels of virulence to susceptible and resistant rice varieties. The expression of six candidate reference genes inN. lugens feeding on susceptible and resistant rice varieties was analyzed. These genes were evaluated for their potential use in the analysis of differential gene expression. Polymerase chain reaction data was generated fromN. lugens, including two different treatments (resistant or susceptible rice) and three virulentN. lugens populations. Three software programs (BestKeeper, Normfinder and geNorm) were used to assess the candidate reference genes. Both geNorm and Normfinder identified the genes18S,β-ACT,β-TUB andα-TUB as the most stable reference genes. BestKeeper identifiedETIF1 as the optimal reference gene with the least overall variation, whereas18S andα-TUB were the second and third most stably expressed genes, respectively. Therefore, we concluded that the genes18S andα-TUB were the most suitable reference genes inN. lugens. These results will facilitate future transcript profiling studies onN. lugens populations that show variation in virulence levels on different rice varieties.

  9. Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

    LIU Yong; YANG Guanpin; WANG Hualei; CHEN Jixiang; SHI Xianming; ZOU Guiwei; WEI Qiwei; SUN Xiuqin

    2006-01-01

    The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene (rDNA) fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.

  10. Partial characterization of nif genes from the bacterium Azospirillum amazonense

    D.P. Potrich

    2001-09-01

    Full Text Available Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense.

  11. Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

    Levy Finn Olav

    2010-03-01

    Full Text Available Abstract Background Quantitative real-time RT-PCR (RT-qPCR is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s. In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI heart failure and across developmental stages in fetal and neonatal rat myocardium. Results The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions. Conclusions The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy. The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

  12. Ichthyophonus parasite phylogeny based on ITS rDNA structure prediction and alignment identifies six clades, with a single dominant marine type

    Gregg, Jacob; Thompson, Rachel L.; Purcell, Maureen; Friedman, Carolyn S.; Hershberger, Paul

    2016-01-01

    Despite their widespread, global impact in both wild and cultured fishes, little is known of the diversity, transmission patterns, and phylogeography of parasites generally identified as Ichthyophonus. This study constructed a phylogeny based on the structural alignment of internal transcribed spacer (ITS) rDNA sequences to compare Ichthyophonus isolates from fish hosts in the Atlantic and Pacific oceans, and several rivers and aquaculture sites in North America, Europe, and Japan. Structure of the Ichthyophonus ITS1–5.8S–ITS2 transcript exhibited several homologies with other eukaryotes, and 6 distinct clades were identified within Ichthyophonus. A single clade contained a majority (71 of 98) of parasite isolations. This ubiquitous Ichthyophonus type occurred in 13 marine and anadromous hosts and was associated with epizootics in Atlantic herring, Chinook salmon, and American shad. A second clade contained all isolates from aquaculture, despite great geographic separation of the freshwater hosts. Each of the 4 remaining clades contained isolates from single host species. This study is the first to evaluate the genetic relationships among Ichthyophonus species across a significant portion of their host and geographic range. Additionally, parasite infection prevalence is reported in 16 fish species.

  13. Cytogenetic analysis on geographically distant parthenogenetic populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones, Buthidae: karyotype, constitutive heterochromatin and rDNA localization

    Renzo Sebastián Adilardi

    2014-03-01

    Full Text Available Tityus trivittatus Kraepelin, 1898 is the most medically important scorpion species of Argentina, and parthenogenetic populations are present in the major cities of this country. We performed a detailed cytogenetic analysis of specimens of three synanthropic parthenogenetic populations, all distant about 900 km from each other, using Ag-NOR, C-banding, DAPI/CMA3 staining and FISH with autologous 28S rDNA probes. The karyotype of females and embryos from the three populations showed 2n=6, with two large and four middle-sized holokinetic chromosomes. Constitutive heterochromatin was found in terminal and interstitial location and its pattern allowed the identification of three chromosome pairs. NORs were found on the terminal heterochromatic region of one pair of middle-sized chromosomes. The use of fluorochromes to characterize heterochromatin showed the absence of GC-rich heterochromatin and a low and variable number of AT-rich heterochromatic regions. We propose that a possible explanation for the lack of karyotypic variation between these geographically distant populations could be a recent colonization of urban areas by human means of synanthropic specimens from a single lineage of northeastern Argentina.

  14. Cytogenetic analysis on geographically distant parthenogenetic populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones, Buthidae): karyotype, constitutive heterochromatin and rDNA localization.

    Adilardi, Renzo Sebastián; Affilastro, Andrés Alejandro Ojanguren; Martí, Dardo Andrea; Mola, Liliana María

    2014-01-01

    Tityus trivittatus Kraepelin, 1898 is the most medically important scorpion species of Argentina, and parthenogenetic populations are present in the major cities of this country. We performed a detailed cytogenetic analysis of specimens of three synanthropic parthenogenetic populations, all distant about 900 km from each other, using Ag-NOR, C-banding, DAPI/CMA3 staining and FISH with autologous 28S rDNA probes. The karyotype of females and embryos from the three populations showed 2n=6, with two large and four middle-sized holokinetic chromosomes. Constitutive heterochromatin was found in terminal and interstitial location and its pattern allowed the identification of three chromosome pairs. NORs were found on the terminal heterochromatic region of one pair of middle-sized chromosomes. The use of fluorochromes to characterize heterochromatin showed the absence of GC-rich heterochromatin and a low and variable number of AT-rich heterochromatic regions. We propose that a possible explanation for the lack of karyotypic variation between these geographically distant populations could be a recent colonization of urban areas by human means of synanthropic specimens from a single lineage of northeastern Argentina. PMID:25147621

  15. Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate.

    Vasaitis, Rimvydas; Menkis, Audrius; Lim, Young Woon; Seok, Soonja; Tomsovsky, Michal; Jankovsky, Libor; Lygis, Vaidotas; Slippers, Bernard; Stenlid, Jan

    2009-03-01

    The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100%. Within each cluster, the ITS rDNA sequence variation was below 3%. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed. PMID:19073254

  16. IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis

    Howell Paul I

    2006-12-01

    Full Text Available Abstract Background Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps. Methods Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: (1 an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; (2 detection of the Mopti and Savanna rDNA types; and (3 an assay to distinguish resistance to dieldrin (Rdl alleles in Anopheles arabiensis. Results Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands. Conclusion The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.

  17. Community analysis of ammonia oxidizer in the oxygen-limited nitritation stage of OLAND system by DGGE of PCR amplified 16S rDNA Fragments and FISH

    ZHANG Dan; ZHANG De-min; LIU Yao-ping; CAO Wen-wei; CHEN Guan-xiong

    2004-01-01

    OLAND(oxygen limited autotrophic nitrification and denitrification) nitrogen removal system was constructed by coupling with oxygen limited nitritation stage and anaerobic ammonium oxidation stage. Ammonia oxidizer, as a kind of key bacteria in N cycle, plays an important role at the oxygen limited nitritation stage of OLAND nitrogen removal system. In this study, specific amplification of 16S rDNA fragment of ammonia oxidizer by nested PCR, separation of mixed PCR samples by denaturing gradient gel electrophoresis(DGGE), and the quantification of ammonia oxidizer by Fluorescence in situ hybridization(FISH) were combined to investigate the shifts of community composition and quantity of ammonia oxidizer of the oxygen limited nitritation stage in OLAND system. It showed that the community composition of ammonia oxidizer changed drastically when dissolved oxygen was decreased gradually, and the dominant ammonia oxidizer of the steady nitrite accumulation stage were completely different from that of the early stage of oxygen limited nitritation identified by DGGE . It was concluded that the Nitrosomonas may be the dominant genus of ammonia oxidizer at the oxygen limited nitritation stage of OLAND system characterized by nested PCR-DGGE and FISH, and the percentage of Nitrosomonas was 72.5% ( 0.8% of ammonia oxidizer at the steady nitrite accumulation stage detected by FISH.

  18. [Phylogenetic relations of Salix L. subg. Salix species (Salicaceae) according to sequencing data of intergenic spacers of chloroplasic genomes and ITS rDNA].

    Barkalov, V Iu; Kozyrenko, M M

    2014-08-01

    A phylogenetic analysis based on a comparison of nucleotide sequences of six regions from cpDNA and ITS rDNA (petN-psbM, trnD-trnT, trnC-petN, psaA-ycf3, petG-trnP, and rpoB-trnC) allowed for elucidating the relationship among species and sections belonging to the Salix subgenus and, more generally, to the Salix genus, as well as revealing the relations of the Chosenia genus. The definition of the subgenera of Pleuradenia (including the Urbanianea genus and the Chosenia genus), Salix (without the Triandrae section), Triandrae, and Longifoliae is essentially consistent with current classification schemes of the Salix genus. The previously defined genera of Chosenia and Toisusu (Urbanianea) are not only joined with the Salix genus but are also closely related between themselves. The Protitea subgenus only corresponds to the American species of the Humboldtianae section (S. humboldtiana, S. amygdaloides, S. gooddingii). The relationship of S. chaenomeloides, which is a nomenclatural type of this subgenus, as well as the relationship of the Wilsonia section, remains unresolved. The Humboldtianae section should be interpreted more narrowly, apparently, separating the Acmophillae and Tetraspermae sections from it. The monotypic American Floridanae section is related to the Salix, Salicaster, Tetraspermae, and Wilsonia. PMID:25731023

  19. A molecular phylogeny of the Dactylogyridae sensu Kritsky & Boeger (1989) (Monogenea) based on the D1-D3 domains of large subunit rDNA.

    Simková, A; Matejusová, I; Cunningham, C O

    2006-07-01

    Phylogenetic analyses based on the partial large subunit rDNA (LSU) sequences of polyonchoinean monogeneans belonging to the Dactylogyridea and Monocotylidea were generated to investigate relationships among various subfamilies of the Dactylogyridae sensu Kritsky & Boeger, 1989. Monophyly of the Dactylogyridae was supported by all analyses performed. Status of the Ancyrocephalidae sensu Bychowsky & Nagibina, 1978 and Ancyrocephalinae sensu Kritsky & Boeger, 1989 was revised based on the present data. All phylogenetic analyses indicated polyphyletic origins of the Ancyrocephalidae and Ancyrocephalinae. Freshwater species of Ancyrocephalinae (Actinocleidus, Ancyrocephalus, Cleidodiscus and Urocleidus) and Ancylodiscoidinae (Thaparocleidus) collected from the fish in European waters were positioned at the base of the Dactylogyridae. The Dactylogyrinae formed a monophyletic group, sister to a clade including the Pseudodactylogyrinae and the tropical and subtropical Ancyrocephalinae. Analyses including only data set on Dactylogyridea were focused on relationships between representatives of the Asian and European Dactylogyrus species. Dactylogyrus species formed a monophyletic group, and the parasite colonization appeared to follow the dispersal history of the Cyprinidae from Asia to Europe. Three lineages of Dactylogyrus species were recognized: the first including species specific to hosts of Asian origin, the second by Dactylogyrus species from Chinese fish hosts, and the third included Dactylogyrus species from European cyprinids and one species from a percid host. The position of D. cryptomeres from Gobio gobio seems to be unresolved. PMID:16515727

  20. Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

    Mariana Ferreira Leal

    Full Text Available The anterior cruciate ligament (ACL is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1 injured ACL tears and controls, and (2 ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

  1. A multi-gene transcriptional profiling approach to the discovery of cell signature markers

    Wada, Youichiro; Li, Dan; Merley, Anne; Zukauskas, Andrew; Aird, William C.; Dvorak, Harold F.; Shih, Shou-Ching

    2010-01-01

    A profile of transcript abundances from multiple genes constitutes a molecular signature if the expression pattern is unique to one cell type. Here we measure mRNA copy numbers per cell by normalizing per million copies of 18S rRNA and identify 6 genes (TIE1, KDR, CDH5, TIE2, EFNA1 and MYO5C) out of 79 genes tested as excellent molecular signature markers for endothelial cells (ECs) in vitro. The selected genes are uniformly expressed in ECs of 4 different origins but weakly or not expressed ...

  2. Systematic Method for Determining an Ideal Housekeeping Gene for Real-Time PCR Analysis

    Mane, Viraj P.; Heuer, Melissa A.; Hillyer, Philippa; Navarro, Maria B.; Rabin, Ronald L

    2008-01-01

    To standardize the amount of biological material between samples (e.g., number of cells or amount of tissue) for quantitative real-time reverse transcriptase PCR (qRT-PCR), the cycle of the target gene at which expression is detected (the cycle threshold, or Ct) is divided by the Ct of a gene either thought to be unaffected by experimental conditions or similarly expressed among donors. Genes that maintain cellular structure or homeostasis, referred to as housekeeping genes, or 18S ribosomal ...

  3. Bacterial diversity in Philippine fermented mustard (burong mustasa) as revealed by 16S rRNA gene analysis.

    Larcia, L L H; Estacio, R C; Dalmacio, L M M

    2011-12-01

    Previous studies on the bacterial profile of burong mustasa, a traditional Philippine fermented food, had been conducted using culture-dependent techniques. Since these methods may underestimate the total microbiota of a sample, a culture-independent study was done to determine the bacterial diversity in burong mustasa through molecular biology techniques. Bacterial DNA was isolated from fermented mustard samples at different stages of fermentation. The isolated genomic DNA was amplified by PCR using specific primers for the 16S ribosomal RNA gene (16S rDNA). The 1.5 kb amplicons obtained were subjected to nested PCR using primers for the internal variable region of the 16S rDNA. The 585 bp nested PCR amplicons were then subjected to denaturing gradient gel electrophoresis (DGGE) to separate the different bacteria present in each sample. Distinct and unique bands in the DGGE profile were excised, reamplified, purified and sequenced for bacterial identification. Molecular cloning of the 1.5 kb 16S rDNA was also performed using the pGEM-T Easy Vector System. The cloned gene was sequenced for bacterial identification. The identified microbiota in burong mustasa at different stages of fermentation include lactic acid bacteria and several uncultured bacteria (initial up to the final stages); acetic acid bacteria (middle stage); and Streptobacillus and Fusobacterium species (initial stage). The potential probiotic bacteria found in burong mustasa are Weissella and Lactobacillus. PMID:22146686

  4. Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression

    Ji, Hong; Wang, Jianfa; Liu, Juxiong; Guo, Jingru; Wang, Zhongwei; Zhang, Xu; Guo, Li; Yang, Huanmin

    2013-01-01

    Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), β-actin (ACTB), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese. PMID:25049806

  5. Identification of a novel nucleolin related protein (NRP gene expressed during rat spermatogenesis

    Rao MRS

    2009-07-01

    Full Text Available Abstract Background Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells. Results By RT PCR and western blot analysis we found that nucleolin is strongly down regulated in meiotic spermatocytes and haploid germ cells. We have identified a new nucleolin related protein (NRP gene in the rat genome, which is over expressed in the testis and is up regulated several fold in meiotic spermatocytes and haploid germ cells. The NRP protein lacks the acidic stretches in its N terminal domain, and it is encoded in rat chromosome 15 having a different genomic organization as compared to nucleolin gene present on chromosome 9. We have also found NRP genes encoded in genomes of other mammalian species. We performed run-on transcription assay where we have observed that rDNA is transcribed at much lower level in meiotic spermatocytes and haploid spermatids as compared to diploid cells. By siRNA knock down experiments we could also demonstrate that NRP can support rDNA transcription in the absence of nucleolin. Conclusion We have identified a new nucleolin variant over expressed in germ cells in rat and analyzed its domain structure. We attribute that the transcriptional activity of rDNA genes in the late spermatogenesis is due to the presence of this variant NRP. The expression of this variant in the germ cells in the absence of nucleolin, could have additional functions in the mammalian spermatogenesis which needs to be investigated further.

  6. Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

    Lee Yeon-Su

    2010-05-01

    Full Text Available Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. Methods We assessed the suitability of six possible reference genes, beta-actin (ACTB, glyceraldehydes-3-phosphate dehydrogenase (GAPDH, hypoxanthine phosphoribosyl transferase 1 (HPRT1, beta-2-microglobulin (B2M, ribosomal subunit L29 (RPL29 and 18S ribosomal RNA (18S rRNA in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. Results This RT-qPCR study showed that there are statistically significant (p Conclusion This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

  7. A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

    Aundy Kumar; Thekkan Puthiyaveedu Prameela; Rajamma Suseelabhai

    2013-06-01

    Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

  8. Evaluation of reference genes for gene expression studies in human brown adipose tissue.

    Taube, Magdalena; Andersson-Assarsson, Johanna C; Lindberg, Kristin; Pereira, Maria J; Gäbel, Markus; Svensson, Maria K; Eriksson, Jan W; Svensson, Per-Arne

    2015-01-01

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT. PMID:26451284

  9. Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

    Wu, Shuang; Zhou, Jiannan; Cao, Xupeng; Xue, Song

    2016-02-01

    Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under nitrogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction (qPCR) provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin (ACT), α-tubulin (TUA), ß-tubulin (TUB), ubiquitin (UBI), 18S rRNA (18S) and one target gene, diacylglycerol acyltransferase (DGAT), were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed I. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experimental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of I. zhangjiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

  10. Non-random distribution of methyl-CpG sites and non-CpG methylation in the human rDNA promoter identified by next generation bisulfite sequencing.

    Pietrzak, Maciej; Rempala, Grzegorz A; Nelson, Peter T; Hetman, Michal

    2016-07-01

    A next generation bisulfite sequencing (NGBS) was used to study rDNA promoter methylation in human brain using postmortem samples of the parietal cortex. Qualitative analysis of patterns of CpG methylation was performed at the individual rDNA unit level. CpG site-specific differences in methylation frequency were observed with the core promoter harboring three out of four most methylated CpGs. Moreover, there was an overall trend towards co-methylation for all possible pairs of 26 CpG sites. The hypermethylated CpGs from the core promoter were also most likely to be co-methylated. Finally, although rare, non-CpG (CpH) methylation was detected at several sites with one of them confirmed using the PspGI-qPCR assay. Similar trends were observed in samples from control individuals as well as patients suffering of Alzheimer's disease (AD), mild cognitive impairment (MCI) or ataxia telangiectasia (AT). Taken together, while some methyl-CpG sites including those in the core promoter may have relatively greater inhibitory effect on rRNA transcription, co-methylation at multiple sites may be required for full and/or long lasting silencing of human rDNA. PMID:27008990

  11. A Toolbox for Quantitative Gene Expression in Varroa destructor: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability.

    Ewan M Campbell

    Full Text Available Varroa destructor is the major pest of Apis mellifera and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat Varroa will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR. Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA, NADH dehydrogenase (NADH, large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S, heat-shock protein 90 (HSP90, cyclophilin, α-tubulin, actin, were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90 in Varroa gene expression studies. These four genes remain stable in phoretic and reproductive stage Varroa and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH. Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from Varroa up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of Varroa samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in Varroa by proposing a validated sampling protocol to obtain high quality Varroa

  12. A Toolbox for Quantitative Gene Expression in Varroa destructor: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability.

    Campbell, Ewan M; McIntosh, Catriona H; Bowman, Alan S

    2016-01-01

    Varroa destructor is the major pest of Apis mellifera and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat Varroa will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA), NADH dehydrogenase (NADH), large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S), heat-shock protein 90 (HSP90), cyclophilin, α-tubulin, actin), were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90) in Varroa gene expression studies. These four genes remain stable in phoretic and reproductive stage Varroa and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR) for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH). Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from Varroa up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of Varroa samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in Varroa by proposing a validated sampling protocol to obtain high quality Varroa RNA and the

  13. A Toolbox for Quantitative Gene Expression in Varroa destructor: RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability

    Campbell, Ewan M.; McIntosh, Catriona H.; Bowman, Alan S.

    2016-01-01

    Varroa destructor is the major pest of Apis mellifera and contributes to the global honey bee health crisis threatening food security. Developing new control strategies to combat Varroa will require the application of molecular biology, including gene expression studies by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Both high quality RNA samples and suitable stable internal reference genes are required for accurate gene expression studies. In this study, ten candidate genes (succinate dehydrogenase (SDHA), NADH dehydrogenase (NADH), large ribsosmal subunit, TATA-binding protein, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA (18S), heat-shock protein 90 (HSP90), cyclophilin, α-tubulin, actin), were evaluated for their suitability as normalization genes using the geNorm, Normfinder, BestKeeper, and comparative ΔCq algorithims. Our study proposes the use of no more than two of the four most stable reference genes (NADH, 18S, SDHA and HSP90) in Varroa gene expression studies. These four genes remain stable in phoretic and reproductive stage Varroa and are unaffected by Deformed wing virus load. When used for determining changes in vitellogenin gene expression, the signal-to-noise ratio (SNR) for the relatively unstable genes actin and α-tubulin was much lower than for the stable gene combinations (NADH + HSP90 +18S; NADH + HSP90; or NADH). Using both electropherograms and RT-qPCR for short and long amplicons as quality controls, we demonstrate that high quality RNA can be recovered from Varroa up to 10 days later stored at ambient temperature if collected into RNAlater and provided the body is pierced. This protocol allows the exchange of Varroa samples between international collaborators and field sample collectors without requiring frozen collection or shipping. Our results make important contributions to gene expression studies in Varroa by proposing a validated sampling protocol to obtain high quality Varroa RNA and the

  14. A recurrent homozygous nonsense mutation within the LAMA3 gene as a cause of Herlitz junctional epidermolysis bullosa in patients of Pakistani ancestry: evidence for a founder effect.

    McGrath, J A; Kivirikko, S; Ciatti, S; Moss, C; Christiano, A M; Uitto, J

    1996-04-01

    The anchoring filament protein laminin 5 is abnormally expressed in the skin of patients with Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a H-JEB child of first-cousin Pakistani parents, and identified a homozygous C-to-T transition in the LAMA3 gene of laminin 5 resulting in a premature termination codon (CGA-TGA) on both alleles. This mutation, R650X, has been previously reported in two other seemingly unrelated H-JEB individuals of Pakistani ancestry. Although this mutation may represent a mutational hotspot within the LAMA3 gene, haplotype analysis based on a silent intragenic polymorphism (GCC/GCG, alanine 429; GenBank no. L34155), and on three flanking microsatellite polymorphism (D18S45, D18S478, and D18S480), suggests that a common ancestral allele may be present in all three cases. PMID:8618022

  15. Ampelomyces mycoparasites from apple powdery mildew identified as a distinct group based on single-stranded conformation polymorphism analysis of the rDNA ITS region.

    Szentiványi, Orsolya; Kiss, Levente; Russell, John C; Kovács, Gábor M; Varga, Krisztina; Jankovics, Tünde; Lesemann, Silke; Xu, Xiang-Ming; Jeffries, Peter

    2005-04-01

    Pycnidial fungi belonging to the genus Ampelomyces are the most common natural antagonists of powdery mildews worldwide. During a study of the interactions between apple powdery mildew (Podosphaera leucotricha) and Ampelomyces mycoparasites, 52 new Ampelomyces isolates were obtained from P. leucotricha and, in addition, 13 new isolates from other species of the Erysiphaceae in four European countries. Their genetic diversity was screened using single-stranded conformation polymorphism (SSCP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). For comparison, 24 isolates obtained from genetic resource collections or other sources were included in this study. Based on the ITS-SSCP patterns, the isolates were placed in eight groups. The isolates belonged to two types based on their growth in culture. The faster-growing and the slower-growing isolates were included in different SSCP groups. A phylogenetic analysis of the ITS sequences of representatives of these groups confirmed the results obtained with the SSCP method, and showed that the faster-growing isolates do not belong to Ampelomyces as suggested by earlier studies. All the isolates from P. leucotricha fell into a distinct SSCP group of genetically homogeneous isolates. This suggests that Ampelomyces mycoparasites which occur in apple powdery mildew are slightly different from the other Ampelomyces groups which contain mycoparasites from various powdery mildew species. This may be because the main growth period of Ampelomyces mycoparasites in apple powdery mildew is isolated in time from that of Ampelomyces isolates that occur in other species of the Erysiphaceae. P. leucotricha starts its life-cycle early in the season, usually in March-April, while most powdery mildews are active in the same environments only late in the year. PMID:15912930

  16. Molecular identification of Dunaliella viridis Teod. strain MSV-1 utilizing rDNA ITS sequences and its growth responses to salinity and copper toxicity

    Ali Moradshahi

    2012-01-01

    Full Text Available In addition to biochemical, physiological and morphological analysis, molecular studies provide additional information for establishing phylogenetic relationships among different species and strains of the genus Dunaliella. In the present study, based on neighbor- joining analysis of the nuclear rDNA ITS sequence, a novel strain of the green algae Dunaliella viridis was identified from Maharlu salt lake in Shiraz, Iran. The phylogenetic tree shows that the new strain is part of a clade containing several strains of D. viridis. The new strain was designated Dunaliella viridis MSV-1 and submitted to the GenBank under the accession number HQ864830. The optimum salinity for MSV-1 growth is between 1.0 to 1.5 M NaCl and does not turn red up to 4.5 M NaCl, confirming identity of the isolated strain. With respect to growth response to copper toxicity, increase in Cu2+ concentration from 1 to 30 µM, caused progressive increase in cell number ml-1 of culture over time, whereas reduction in cell number occurred at 100 and 200 µM Cu+2. Nano copper (colloidal copper with 40 nm dimensions showed less toxicity compared to the ionic form. Cell number ml-1 of culture did not change up to 200 µM nano copper but decreased at 500 µM. In conclusion, the analysis of the ITS sequence is a reliable basis for establishing evolutionary relationships among species and strains of the genus Dunaliella and due to rapid growth at 1.5 M NaCl and high cell density, D. viridis MSV-1 is a good candidate for biofuel production from microalgae.

  17. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    S.M. Azwai

    2016-03-01

    Full Text Available The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk. Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6% yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS with culture characteristics of Vibrio spp. More than half (n=27 of processed seafood samples (n=46 yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 х104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 х104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9% were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  18. Evaluation of the effects of intrapartum antibiotic prophylaxis on newborn intestinal microbiota using a sequencing approach targeted to multi hypervariable 16S rDNA regions.

    Aloisio, Irene; Quagliariello, Andrea; De Fanti, Sara; Luiselli, Donata; De Filippo, Carlotta; Albanese, Davide; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Di Gioia, Diana

    2016-06-01

    Different factors are known to influence the early gut colonization in newborns, among them the perinatal use of antibiotics. On the other hand, the effect on the baby of the administration of antibiotics to the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has received less attention, although routinely used in group B Streptococcus positive women to prevent the infection in newborns. In this work, the fecal microbiota of neonates born to mothers receiving IAP and of control subjects were compared taking advantage for the first time of high-throughput DNA sequencing technology. Seven different 16S rDNA hypervariable regions (V2, V3, V4, V6 + V7, V8, and V9) were amplified and sequenced using the Ion Torrent Personal Genome Machine. The results obtained showed significant differences in the microbial composition of newborns born to mothers who had received IAP, with a lower abundance of Actinobacteria and Bacteroidetes as well as an overrepresentation of Proteobacteria. Considering that the seven hypervariable regions showed different discriminant ability in the taxonomic identification, further analyses were performed on the V4 region evidencing in IAP infants a reduced microbial richness and biodiversity, as well as a lower number of bacterial families with a predominance of Enterobacteriaceae members. In addition, this analysis pointed out a significant reduction in Bifidobacterium spp. strains. The reduced abundance of these beneficial microorganisms, together with the increased amount of potentially pathogenic bacteria, may suggest that IAP infants are more exposed to gastrointestinal or generally health disorders later in age. PMID:26971496

  19. Use of Single-Enzyme PCR-Restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA to Identify Dermatophyte Species

    L Hosseinpour

    2012-04-01

    Full Text Available Background: Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes.Methods: The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis.Results: The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings.Conclusion: It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.

  20. Bacterial diversity assessment in soil of an active Brazilian copper mine using high-throughput sequencing of 16S rDNA amplicons.

    Rodrigues, Viviane D; Torres, Tatiana T; Ottoboni, Laura M M

    2014-11-01

    Mining activities pose severe environmental risks worldwide, generating extreme pH conditions and high concentrations of heavy metals, which can have major impacts on the survival of organisms. In this work, pyrosequencing of the V3 region of the 16S rDNA was used to analyze the bacterial communities in soil samples from a Brazilian copper mine. For the analysis, soil samples were collected from the slopes (geotechnical structures) and the surrounding drainage of the Sossego mine (comprising the Sossego and Sequeirinho deposits). The results revealed complex bacterial diversity, and there was no influence of deposit geographic location on the composition of the communities. However, the environment type played an important role in bacterial community divergence; the composition and frequency of OTUs in the slope samples were different from those of the surrounding drainage samples, and Acidobacteria, Chloroflexi, Firmicutes, and Gammaproteobacteria were responsible for the observed difference. Chemical analysis indicated that both types of sample presented a high metal content, while the amounts of organic matter and water were higher in the surrounding drainage samples. Non-metric multidimensional scaling (N-MDS) analysis identified organic matter and water as important distinguishing factors between the bacterial communities from the two types of mine environment. Although habitat-specific OTUs were found in both environments, they were more abundant in the surrounding drainage samples (around 50 %), and contributed to the higher bacterial diversity found in this habitat. The slope samples were dominated by a smaller number of phyla, especially Firmicutes. The bacterial communities from the slope and surrounding drainage samples were different in structure and composition, and the organic matter and water present in these environments contributed to the observed differences. PMID:25129578

  1. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

    Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products. PMID:27004169

  2. Dynamic changes in functional gene copy numbers and microbial communities during degradation of pyrene in soils

    This study investigates the dynamics of pyrene degradation rates, microbial communities, and functional gene copy numbers during the incubation of pyrene-spiked soils. Spiking pyrene to the soil was found to have negligible effects on the bacterial community present. Our results demonstrated that there was a significant difference in nidA gene copy numbers between sampling dates in QZ soil. Mycobacterium 16S rDNA clone libraries showed that more than 90% mycobacteria detected were closely related to fast-growing PAH-degrading Mycobacterium in pyrene-spiked soil, while other sequences related to slow-growing Mycobacterium were only detected in the control soil. It is suggested that nidA gene copy number and fast-growing PAH-degrading Mycobacterium could be used as indicators to predict pyrene contamination and its degradation activity in soils. - nidA gene and fast-growing PAH-degrading Mycobacterium can serve as indicators for pyrene contamination.

  3. Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise

    Reverse transcription and real-time PCR have become the method of choice for the detection of low-abundance mRNA transcripts obtained from small human muscle biopsy samples. GAPDH, β-actin, β-2M, and 18S rRNA are widely employed as endogenous control genes, with the assumption that their expression is unregulated and constant for given experimental conditions. The aim of this study was to determine if mRNA transcripts could be performed on isolated human single muscle fibers and to determine reliable housekeeping genes (HKGs) using quantitative gene expression protocols at rest and in response to an acute exercise bout. Muscle biopsies were obtained from the gastrocnemius of three adult males before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO2max. A total of 40 single fibers (MHC I and IIa) were examined for GAPDH, β-actin, β-2M, and 18S rRNA using quantitative RT-PCR and SYBR Green detection. All analyzed single fiber segments showed ribosomal RNA (28S/18S). No degradation or additional bands below ribosomal were detected (rRNA ratio 1.5-1.8). Also, no high or low-molecular weight genomic DNA contamination was observed. For each housekeeping gene the duplicate average SD was ±0.13 with a CV of 0.58%. Stable expression of GAPDH was observed at all time points for each fiber type (MHC I and IIa). Inconsistent expression of β-actin, β-2M, and 18S rRNA was observed during the post-exercise time points for each fiber type. These data indicate that successful extraction of high quality RNA from human single muscle fibers along with quantification of mRNA of selected genes can be performed. Furthermore, exercise does influence the expression of certain HKGs with GAPDH being the most stable

  4. Variabilidade genética na região its do rDNA de isolados de trichoderma spp. (Biocontrolador e Fusarium oxysporum f. sp. Chrysanthemi Genetic variability in rDNA ITS region of Trichoderma spp. (biocontrole agent and Fusarium oxysporum f. sp. chrysanthemi isolates

    Josiane Pacheco Menezes

    2010-02-01

    Full Text Available A análise de características morfológicas e culturais podem não ser suficientes para uma caracterização precisa das espécies de Trichoderma e Fusarium. Objetivou-se, neste trabalho, caracterizar a região do Espaço Interno Transcrito (ITS do rDNA dos isolados UFSMT15.1, UFSMT16 e UFSMT17 de Trichoderma spp. utilizados no biocontrole de Fusarium oxysporum f. sp. chrysanthemi (isolado UFSMF6. A extração de DNA de cada isolado foi realizada a partir de micélio produzido em meio líquido Batata-Dextrose. As amostras de DNA genômico foram submetidas à Reação em Cadeia da Polimerase (PCR com os oligonucleotídeos iniciadores universais ITS1 e ITS4 e o produto gerado foi sequenciado. Os fragmentos gerados pela amplificação da PCR foram tratados com as enzimas de restrição HaeIII, HinfI e MboI. As regiões ITS1, ITS2 e 5.8S do rDNA desses isolados fúngicos foram amplificadas com sucesso. A região ITS dos isolados UFSMT15.1, UFSMT16 e UFSMT17 de Trichoderma e o isolado UFSMF6 de Fusarium apresentaram uma banda simples com um fragmento de aproximadamente 600 pares de base (pb. As enzimas de restrição HaeIII, HinfI e MboI geraram polimorfismo de bandas entre os isolados. Com base nas análises da sequência de DNA, os isolados UFSMT15.1, UFSMT16, UFSMT17 e UFSMF6 apresentaram maior similaridade com as espécies Trichoderma koningiopsis, Hypocrea virens, Hypocrea lixii e Fusarium oxysporum, respectivamente.The analysis of morphological and cultural characteristics may not enough for the characterization of the species of Trichoderma and Fusarium. The aim of this work was to characterize the Internal Transcribed Spacer (ITS region of the rDNA of UFSMT15.1, UFSMT16 and UFSMT17 isolates of Trichoderma spp. used in the biocontrol of Fusarium oxysporum f. sp. chrysanthemi UFSMF6. DNA extraction of each isolate was accomplished starting from hyphae produced in liquid medium Potato-Dextrose-Agar. The samples of genomic DNA were submitted to

  5. Validation of endogenous control genes for gene expression studies on human ocular surface epithelium.

    Bina Kulkarni

    Full Text Available PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG with quantitative reverse transcription PCR (qPCR, for identification of stably expressed endogenous control genes in the ocular surface (OS epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH, beta actin (ACTB, peptidylprolyl isomerase (PPIA, TATA-box binding protein (TBP1, hypoxanthine guanine phosphoribosyl transferase (HPRT1, beta glucuronidase (GUSB, Eucaryotic 18S ribosomal RNA (18S, phosphoglycerate kinase (PGK1, beta-2-microglobulin (B2M, ribosomal protein, large, P0 (RPLP0--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18Sgenes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use

  6. Evidence for Interspecies Gene Transfer in the Evolution of 2,4-Dichlorophenoxyacetic Acid Degraders

    McGowan, Catherine; Fulthorpe, Roberta; Wright, Alice; Tiedje, J M

    1998-01-01

    Small-subunit ribosomal DNA (SSU rDNA) from 20 phenotypically distinct strains of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria was partially sequenced, yielding 18 unique strains belonging to members of the alpha, beta, and gamma subgroups of the class Proteobacteria. To understand the origin of 2,4-D degradation in this diverse collection, the first gene in the 2,4-D pathway, tfdA, was sequenced. The sequences fell into three unique classes found in various members of the beta a...

  7. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

    Xiaoyang Zhu

    Full Text Available Real-time reverse transcription PCR (RT-qPCR is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A, TBP1 (TATA binding protein 1 and TBP2 (TATA binding protein 2 genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2, 18S rRNA (18S ribosomal RNA and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental

  8. Phylogeny of the cuttlefishes (Mollusca:Cephalopoda) based on mitochondrial COI and 16S rRNA gene sequence data

    LIN Xiangzhi; ZHENG Xiaodong; XIAO Shu; WANG Rucai

    2004-01-01

    To clarify cuttlefish phylogeny, mitochondrial cytochrome c oxidase subunit I (COI) gene and partial 16S rRNA gene are sequenced for 13 cephalopod species. Phylogenetic trees are constructed, with the neighbor-joining method.Coleoids are divided into two main lineages, Decabrachia and Octobrachia. The monophyly of the order Sepioidea,which includes the families Sepiidae, Sepiolidae and Idiosepiidae, is not supported. From the two families of Sepioidea examined, the Sepiolidae are polyphyletic and are excluded from the order. On the basis of 16S rRNA and amino acid of COI gene sequences data, the two genera (Sepiella and Sepia) from the Sepiidae can be distinguished, but do not have a visible boundary using COI gene sequences. The reason is explained. This suggests that the 16S rDNA of cephalopods is a precious tool to analyze taxonomic relationships at the genus level, and COI gene is fitter at a higher taxonomic level (i.e., family).

  9. Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

    LI Xin; ZHU Xiao-fei; ZHANG Cheng-fei; Peter Cathro; CJ Seneviratne; SHEN Song

    2013-01-01

    Background Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions,with no information available for the Chinese population.As such,we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing,China using 16S rDNA gene sequencing techniques.Methods Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction.Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected.Purified plasmid DNA with 16S rDNA gene inserts was sequenced,and the sequences were searched against GenBank databases using the BLASTN algorithm.Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping.Results More than 150 taxa were obtained.Primary endodontic infection was mainly associated with Burkholderia cepacia,Actinomyces,Aranicola spp.and Streptococcus sanguinis,whilst Burkholderia cepacia was predominant in the persistent endodontic infections.Conclusion There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beiiing in comoarison to other geographical or ethnic reports.

  10. Molecular phylogenetic lineage of Plagiopogon and Askenasia (Protozoa, Ciliophora) revealed by their gene sequences

    Liu, An; Yi, Zhenzhen; Lin, Xiaofeng; Hu, Xiaozhong; Al-Farraj, Saleh A.; Al-Rasheid, Khaled A. S.

    2015-08-01

    Prostomates and haptorians are two basal groups of ciliates with limited morphological characteristics available for taxonomy. Morphologically, the structures used to identify prostomates and haptorians are similar or even identical, which generate heavy taxonomic and phylogenetic confusion. In present work, phylogenetic positions lineage of two rare genera, Plagiopogon and Askenasia, were investigated. Three genes including small subunit ribosomal RNA gene (hereafter SSU rDNA), internal transcribed spacer region (ITS region), and large subunit ribosomal RNA gene (LSU rDNA) were analyzed, 10 new sequences five species each. Our findings included 1) class Prostomatea and order Haptorida are multiphyletic; 2) it may not be appropriate to place order Cyclotrichiida in subclass Haptoria, and the systematic lineage of order Cyclotrichiida needs to be verified further; 3) genus Plagiopogon branches consistently within a clade covering most prostomes and is basal of clade Colepidae, implying its close lineage to Prostomatea; and 4) Askenasia is phylogenetically distant from the subclass Haptoria but close to classes Prostomatea, Plagiopylea and Oligohymenophorea. We supposed that the toxicyst of Askenasia may be close to taxa of prostomes instead of haptorians, and the dorsal brush is a more typical morphological characteristics of haptorians than toxicysts.

  11. Suitable reference genes for real-time PCR in human HBV-related hepatocellular carcinoma with different clinical prognoses

    Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes. We evaluated 6 commonly used housekeeping genes' expression levels in 108 HBV-related HCCs' matched tumor and non-tomor tissue samples with different clinical outcomes and 26 normal liver specimens by real-time PCR. The expression stability of the 6 genes was compared using the software programs geNorm and NormFinder. To show the impact of reference genes on data analysis, we took PGK1 as a target gene normalized by each reference gene, and performed one-way ANOVA and the equivalence test. With the geNorm and NormFinder software programs, analysis of TBP and HPRT1 showed the best stability in all tissue samples, while 18s and ACTB were less stable. When 18s or ACTB was used for normalization, no significant difference of PGK1 expression (p > 0.05) was found among HCC tissues with and without metastasis, and normal liver specimens; however, dramatically differences (p < 0.001) were observed when either TBP or the combination of TBP and HPRT1 were selected as reference genes. TBP and HPRT1 are the most reliable reference genes for q-PCR normalization in HBV-related HCC specimens. However, the well-used ACTB and 18S are not suitable, which actually lead to the misinterpretation of the results in gene expression analysis

  12. 芸薹属基本种45S rDNA重复序列的染色体定位%Chromosomal localization of 45S rDNA in Brassica dipioids

    张永泰; 孔芳; 李爱民; 王幼平

    2011-01-01

    The position and number of 45S rDNA on mitotic and meiotic chromosomes of the three basic diploid species (Brassica rapa, B.oleracea and B.nigra) were detected by fluorescence in situ hybridization (FISH) analysis using 45S rDNA as probe.Ten out of 20 chromosomes can be easily identified in B.rapa, six out of 16 in B.nigra, and four out of 18 in B.oleracea.Among them, meiotic FISH analysis of 45S rDNA of B.rapa and B.nigra was reported for the first time.These investigations are useful for identifying the chromosomes of genome A, B and C and studying the evolution and phylogeny of the Brassica genomes.%用45S rDNA作探针,对芸薹属3个二倍体种(白菜型油菜、甘蓝和黑芥)进行有丝分裂和减数分裂的原位杂交分析,发现在白菜型油菜的20条染色体中有10条、黑芥16条染色体中有6条、甘蓝18条染色体中有4条染色体上存在杂交信号.其中白菜型油菜和黑芥的减数分裂为首次报道,研究结果对研究芸薹属二倍体基因组A、B或C的亲缘关系以及基因组间的相互进化关系有一定的意义.

  13. The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense.

    Jijón-Moreno, Saúl; Marcos-Jiménez, Cynthia; Pedraza, Raúl O; Ramírez-Mata, Alberto; de Salamone, I García; Fernández-Scavino, Ana; Vásquez-Hernández, Claudia A; Soto-Urzúa, Lucia; Baca, Beatriz E

    2015-06-01

    Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2 μg mg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains. PMID:25842039

  14. Caracterização da região espaçadora 16-23S rDNA para diferenciação de estirpes de rizóbios utilizadas na produção de inoculantes comerciais no Brasil Characterization of the spacer region 16-23S rDNA for differentiation of strains of rhizobia used in the production of commercial inoculants in Brazil

    Andréia Mara Rotta Oliveira

    2012-08-01

    Full Text Available A identificação de estirpes de rizóbio tem sido feita pela especificidade por hospedeiros e ensaios microbiológicos tradicionais. Por constituírem um grupo filogeneticamente heterogêneo, diferentes técnicas moleculares têm sido empregadas para auxiliar na caracterização genética e na identificação de estirpes eficientes e competitivas para a produção de inoculantes. Este trabalho teve por objetivos caracterizar a região espaçadora 16S-23S rDNA das estirpes de rizóbios utilizadas nos inoculantes comercializados no Brasil para espécies leguminosas, utilizando a técnica da PCR em combinação com a de RFLP, e avaliar a possibilidade do uso desse marcador molecular como método auxiliar para identificação das estipes. A amplificação da região espaçadora 16-23 S rDNA das estirpes de rizóbios gerou fragmentos com tamanhos que variaram entre 700pb e 1350pb. Os produtos resultantes da amplificação foram submetidos à digestão com as endonucleases. Mps I, Dde I e Hae III. Os resultados obtidos neste estudo indicam a possibilidade do uso da técnica de PCR-RFLP da região espaçadora 16S-23S rDNA como marcador molecular para a diferenciar as estirpes de rizóbios, em complemento às técnicas microbiológicas tradicionais. Contudo, este marcador não é suficientemente discriminatório para ser usado na identificação das estirpes recomendadas para a produção de inoculantes comerciais.The identification of strains of rhizobia has been made by host specificity and regular microbiological tests. By forming a phylogenetically heterogeneous group, different molecular techniques have been employed to assist in the genetic characterization and identification of efficient and competitive strains for production of inoculants. This study aimed to characterize the spacer region 16S-23S rDNA of the strains of rhizobia used in commercial inoculants in Brazil for legume species, using PCR combined with RFLP, and assess the possibility

  15. Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla.

    Balbutskaya, A; Sammra, O; Nagib, S; Hijazin, M; Alber, J; Lämmler, C; Foster, G; Erhard, M; Wragg, P N; Abdulmawjood, A; Prenger-Berninghoff, E

    2014-01-31

    In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals. PMID:24345409

  16. 基于45S rDNA和雷蒙德氏棉gDNA为探针的草棉FISH核型研究%FISH-based Karyotype of Gossypium herbaceum Generated with 45S Rdna and Gdna of Gossypium raimondii as Probes

    王坤波; 宋国立; 王春英; 刘三宏; 刘方; 李懋学; 黎绍惠; 张香娣; 王玉红

    2008-01-01

    the same numbers, locations and sizes as 45S rDNA NORs. It could be therefore concluded that FISH-based karyotype analyses were more detailed than previous karyotype (non-FISH). Based on this study in conjunction with our other FISH results, there might be great amplifications or pericentric inversions of rDNA in modern A genome species after its contribution to allotetraploid originations, or deamplifications/deletions of tandem repeats like rDNA in extant allotetraploids following their polyploidization. An explanation to D genome specific GISH-NORs is that rDNA contents in D genome species may be much more than those in A genome species. The NORs or GISH-NORs herein may facilitate future locus-specific studies on rRNA gene evolution and function, and also may be useful in developing physical map specific to chromosome order in Gossypium.

  17. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

    Van Zeveren Alex

    2005-12-01

    Full Text Available Abstract Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

  18. Expression of isgylation related genes in regenerating rat liver

    Kuklin A. V.

    2015-10-01

    Full Text Available Our recent studies have revealed the early up-regulated expression of interferon alpha (IFNα in the liver, induced by partial hepatectomy. The role of this cytokine of innate immune response in liver regeneration is still controversial. Aim. To analyze expression of canonical interferon-stimulated genes Ube1l, Ube2l6, Trim25, Usp18 and Isg15 during the liver transition from quiescence to proliferation induced by partial hepatectomy, and acute phase response induced by laparotomy. These genes are responsible for posttranslational modification of proteins by ISGylation. The expression of genes encoding TATA binding protein (TBP and 18S rRNA served as indirect general markers of transcriptional and translational activities. Methods. The abundance of investigated RNAs was assessed in total liver RNA by real time RT–qPCR. Results. Partial hepatecomy induced steady upregulation of the Tbp and 18S rRNA genes expression during 12 hours post-surgery and downregulation or no change in expression of ISGylation-related genes during the first 3 hours followed by slight upregulation at 12 hours. The level of Isg15 transcripts was permanently below that of the control during the prereplicative period. Laparotomy induced a continuous downregulation of Tbp and 18S rRNA expression and early (1–3h upregulation of ISGylation–related transcripts followed by a sharp drop at 6 hours and slight increase/decrease at 12 hours. The changes in the abundance of Ifnα and ISGylation-related mRNAs were oppositely directed at each stage of the response to partial hepatectomy and laparotomy. Conclusion. We suggest that the expression of ISGylation-related genes does not depend on the expression of Ifnα gene after both surgeries. The indirect indices of transcription and translation as well as the expression of ISGylation-relaled genes are principally different in response to partial hepatectomy and laparotomy and argue for the high specificity of innate immune response.

  19. PHYLOGENETIC ANALYSIS AND HOMOLOGY MODELLING OF LACCASE GENE OF FUSARIUM OXYSPORUM Lcc2.

    S.Shanmugam

    2012-12-01

    Full Text Available Morphological identification of fungi is the first and the most difficult step in the identification process. This is especially true for Fusarium species. Fusarium has a cosmopolitan distribution. Fusarium sp was isolated from Anamalai Hills region and Subjected to Morphological identification using standard microbiological methods.Genotypic identification was carried out by PCR amplification and partial sequencing of the 16S rRNA for the confirmation of morphological identity. The rDNA sequence of 16S ribosomal RNA gene was amplified by PCR and the PCR product .The fungus, was bi directionally sequenced using forward and reverse primers. Using BLAST Tool the sequence was searched for the similarity matches .F.oxysporum shows maximum similarity. The Gene of the species was obtained from Uniprot. Since the unavailability of the 3D structure ofFusarium Laccase gene this attempt was carried out and Homology modeling was performed using Modeller Tool.

  20. Insights into the relationships of Palearctic and Nearctic lymnaeids (Mollusca : Gastropoda by rDNA ITS-2 sequencing and phylogeny of stagnicoline intermediate host species of Fasciola hepatica

    Bargues M.D.

    2003-09-01

    Full Text Available Fascioliasis by Fasciola hepatica is the vector-borne disease presenting the widest latitudinal, longitudinal and altitudinal distribution known. F. hepatica shows a great adaptation power to new environmental conditions which is the consequence of its own capacities together with the adaptation and colonization abilities of its specific vector hosts, freshwater snails of the family Lymnaeidae. Several lymnaeid species only considered as secondary contributors to the liver fluke transmission have, however, played a very important role in the geographic expansion of this disease. Many of them belong to the so-called "stagnicoline" type group. Stagnicolines have, therefore, a very important applied interest in the Holarctic region, to which they are geographically restricted. The present knowledge on the genetics of stagnicolines and on their parasite-host interrelationships is, however, far from being sufficient. The present paper analyses the relationships between Palaearctic and Nearctic stagnicoline species on the base of the new light furnished by the results obtained in nuclear rDNA ITS-2 sequencing and corresponding phylogenetic studies of the lymnaeid taxa Lymnaea (Stagnicola occulta, L. (S. palustris palustris (topotype specimens and L.(S. p. turricula from Europe. Natural infections with F. hepatica have been reported in all of them. Surprisingly, ITS-2 length and G C content of L. occulta were similar and perfectly fitted within the respective ranges known in North American stagnicolines. Nucleotide differences and genetic distances were higher between L. occulta and the other European stagnicolines than between L. occulta and the North American ones. The ITS-2 sequence of L. p. turricula from Poland differed from the other genotypes known from turricula in Europe. The phylogenetic trees using the maximum-parsimony, distance and maximum-likelihood methods confirmed (i the inclusion of L. occulta in the branch of North American

  1. Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.

    Galli, Vanessa; Borowski, Joyce Moura; Perin, Ellen Cristina; Messias, Rafael da Silva; Labonde, Julia; Pereira, Ivan dos Santos; Silva, Sérgio Delmar Dos Anjos; Rombaldi, Cesar Valmor

    2015-01-10

    The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts. PMID:25445290

  2. Gene expression

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn2+ or Cd2+. We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  3. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    Ostrup, Olga, E-mail: osvarcova@gmail.com [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway); Hyttel, Poul; Klaerke, Dan A. [Institute of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C (Denmark); Collas, Philippe, E-mail: philc@medisin.uio.no [Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo (Norway); Norwegian Center for Stem Cell Research, Oslo (Norway)

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  4. Differential gene expression in Neurospora crassa cell types: heterogeneity and amplification of rRNA genes. Progress report, July 1980-June 30, 1981

    The significant results obtained during 1980-1981 year of the current research program are as follows: I. Studies on heterogeneity of multiple copies of rDNAs from N. crassa cell types are being continued, such as: (1) Autoradiographs of Southern transfers of EcoR1 restricted fragments of nuclear DNA from conidia, germinated conidia (sprouts) and mycelia of N. crassa were compared after hybridization with 32P-rDNA probe. The nuclear DNA of two hours sprout and of 16 hours mycelia gave similar hybridization patterns with EcoR1 digest, but no such hybridization pattern was evident in conidial DNA digest; (2) Procedure for concentration of rDNAs from Neurospora species and cell types was standardized; restriction analysis of purified rDNAs is being done; (3) 35S total rDNA clone, 17S rDNA clone and 26S rDNA subclone are being used to see gross differences in the precursor rRNAs of different cell types; (4) Comparison of DNA:DNA homologies of rRNA genes with different Neurospora species. II. Post-mitochondrial DNAs of N. crassa are found to be rDNA-like and were further characterized by electron microscopic studies and are found to be approximately twice the size of SV-40 DNAs. These N. crassa post-mitochondrial DNAs hybridized with 32P-labeled N. crassa nuclear DNAs. III. Previous studies on differential RNase sensitive DNA polymerase activity in N. Crassa cell types and on evolution of sexual morphogenesis in the genus Neurospora are completed and published. RNase sensitive DNA polymerase activity is found to be in the post-mitochondrial fraction. Heterothallism in the genus Neurospora is evolved from homothallism

  5. The role of ciliate protozoa in the rumen

    Newbold, Charles J.; de la Fuente, Gabriel; Belanche, Alejandro; Ramos-Morales, Eva; McEwan, Neil R.

    2015-01-01

    First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear. Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult ...

  6. Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus during Vaccination and Infection

    Erlong Wang

    2015-04-01

    Full Text Available qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

  7. The worldwide holoparasitic Apodanthaceae confidently placed in the Cucurbitales by nuclear and mitochondrial gene trees

    Renner Susanne S

    2010-07-01

    Full Text Available Abstract Background Of the c. 450 families of flowering plants, only two are left "unplaced" in the most recent APG classification of angiosperms. One of these is the Apodanthaceae, a clade of c. 19 holoparasitic species in two or three genera occurring in North and South America, Africa, the Near East, and Australia. Because of lateral gene transfer between Apodanthaceae and their hosts it has been difficult to infer the family's true closest relatives. Results Here we report a phylogenetic analysis of 16 accessions representing six species of Apodanthaceae from the United States, Chile, Iran, and Australia, using the mitochondrial matR gene and the nuclear 18S gene. Data matrices include 190 matR sequences from up to 95 families in 39 orders of flowering plants and 197 18S sequences from 101 families representing the 16 orders of rosids. Analyses were performed at the nucleotide and at the amino acid level. Both gene trees agree with angiosperm phylogenies found in other studies using more genes. Apodanthaceae and the seven families of the order Cucurbitales form a clade with 100% bootstrap support from matR and 56% from 18 S. In addition, the Apodanthaceae and Cucurbitales matR gene sequences uniquely share two non-synonymous codon changes and one synonymous change, as well as a codon insertion, already found by Barkman et al. (2007. Conclusions Apodanthaceae belong in the Cucurbitales with which they share inferior ovaries, parietal placentation and a dioecious mating system, traits that are ancestral in Cucurbitales and which can now be interpreted as possible synapomorphies of an enlarged order Cucurbitales. The occurrence of Apodanthaceae in the Americas, Africa, the Near East, and Australia, and their adaptation to distantly related host species in the Fabaceae and Salicaceae suggest a long evolutionary history.

  8. Gene therapy

    2005-01-01

    2005147 CNHK200-hA-a gene-viral therapeutic system and its antitumor effect on lung cancer. WANG Wei-guo(王伟国),et al. Viral & Gene Ther Center, Eastern Hepatobilli Surg Instit 2nd Milit Univ, Shanghai 200438. Chin J Oncol,2005:27(2):69-72. Objective: To develop a novel vector system, which combines the advantages of the gene therapy,

  9. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment

    Callard Gloria V

    2008-11-01

    Full Text Available Abstract Background Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR, normalized to an internal reference ("housekeeping" gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. Results QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1, glyceraldehyde-3-phosphate dehydrogenase (gapdh, glucose-6-phosphate dehydrogenase (g6pd, TATA-box binding protein (tbp, beta-2-microglobulin (b2m, elongation factor 1 alpha (elfa, and 18s ribosomal RNA (18s during development (2 – 120 hr postfertilization, hpf; in different tissue types (brain, eye, liver, heart, muscle, gonads of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using

  10. Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

    Rongying TANG; Andrew DODD; Daniel LAI; Warren C.MCNABB; Donald R.LOVE

    2007-01-01

    The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.

  11. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus using quantitative real-time PCR

    Van Bockstaele Erik

    2010-02-01

    Full Text Available Abstract Background Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported. Results Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD, actin (ACT, β-tubulin (TUB, glyceraldehyde-3-phosphate-dehydrogenase (GADPH, histone H3 (H3, elongation factor 1-alpha (EF and 18S rRNA (rRNA were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants. Conclusions geNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.

  12. Screening of B chromosomes for presence of two genes in yellow-necked mice, Apodemus flavicollis (Mammalia, Rodentia

    Rajičić Marija

    2015-01-01

    Full Text Available B chromosomes (Bs are a very heterogeneous group of extra chromosomes. In various species Bs occur with different nucleotide sequences ranging from repetitive to protein coding. In yellow-necked field mice, Apodemus flavicollis Bs are small euchromatic chromosomes and untill now, only few molecular analyses have been conducted. In this study we examined A. flavicollis individuals with different number of Bs for presence of two genes, C-KIT and 18S rRNA. The C-KIT proto-oncogene was found on Bs in three Canidae species and one Cervidae species. This gene is a coding receptor critical for proliferation and cell differentiation of hematopoietic, melanoblast and primordial germ cells, and is highly conserved within mammals. While using semiquantitative PCR, we did not notice any difference in the C-KIT band intensity among animals with different number of Bs (0-3. The presence of only one copy of C- KIT gene was confirmed using real time-PCR on genomic DNA of A. flavicollis specimens with different number of Bs. rRNA genes in eukaryotes’ genome are organized like units of tandem repeated sequences. The units form distinct clusters on one to several chromosome pairs. rRNA genes were found on Bs in different species including two species of genus Apodemus. One particular sample with 2 Bs showed the number of 18S rRNA gene about three times that of the calibrator 0 B sample. This result can indicate the presence of 18S rRNA gene on Bs, but its confirmation requires the implementation of other methods. Still, we can neither confirm nor deny the existence of pseudogen of tested target genes, or lose of exon 1 of C-KIT protooncogen in Bs of A. flavicollis. Our findings are further discussed. [Projekat Ministarstva nauke Republike Srbije, br. 173003

  13. Gene therapy.

    Mota Biosca, Anna

    1992-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases.

  14. Trichoderma genes

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  15. Screening of B chromosomes for presence of two genes in yellow-necked mice, Apodemus flavicollis (Mammalia, Rodentia)

    Rajičić Marija; Adnađević Tanja; Stamenković Gorana; Blagojević Jelena; Vujošević Mladen

    2015-01-01

    B chromosomes (Bs) are a very heterogeneous group of extra chromosomes. In various species Bs occur with different nucleotide sequences ranging from repetitive to protein coding. In yellow-necked field mice, Apodemus flavicollis Bs are small euchromatic chromosomes and untill now, only few molecular analyses have been conducted. In this study we examined A. flavicollis individuals with different number of Bs for presence of two genes, C-KIT and 18S rRNA. Th...

  16. 基于ITS序列分析对紫花地丁的分子鉴别%Molecular authentication of Viola yedoensis Makino and adulterants by analysis of rDNA ITS sequences

    朱烨; 张春; 庄元春; 税丕先

    2013-01-01

    Objective: The rDNA ITS sequences of Viola yedoensis Makino and the adulterants were analyzed to establish the molecular biological method for authentication of Viola yedoensis Makino and the adulterants. Methods: Total DNA of Viola yedoensis Makino and the adulterants were extracted. The ITS sequence was amplified by universal primer. The PCR products were directly sequenced and both directions were sequenced. Results: 8 specific authenticate sites were found in the ITS1 and ITS2 region. Conclusion: The rDNA ITS sequence can be used as good marker for authenticating Viola yedoensis Makino from the adulterants.%目的:通过研究比较紫花地丁与其易混中药材ITS序列的差异和规律,建立紫花地丁与其易混药材之间的分子鉴别方法.方法:分别从紫花地丁和易混品中提取总DNA,用ITS序列通用引物扩增,扩增产物双向测序.结果:紫花地丁与其他易混品在ITS1和ITS2区段存在特异鉴别位点共8个.结论:根据ITS序列特征,能有效区分紫花地丁和其他易混中药材.ITS序列是紫花地丁鉴定的有效的分子标记.

  17. Amplification and Sequence Analysis of ITS and 5.8S rDNA of Ascarid Nematodes from Xiaocrhua Pig%小耳花猪蛔虫ITS及5.8SrDNA序列扩增与分析

    吴晓斌; 张志宏; 谢东

    2012-01-01

    以分离自小耳花猪消化道内的蛔虫为研究对象,提取虫体的DNA片断,然后用引物对核糖体DNA(rDNA)的内转录间隔区ITS-1、ITS-2及5.8S序列进行PCR扩增。结果显示,目的片段ITS总长为1441bp,2个不同样品之间的ITS序列没有差异。通过BLAST检索,与相关蛔虫ITS序列进行比较,发现与拜林蛔线虫(Baylisascaristransfuga)、猪蛔虫(Ascarissuum)和人蛔虫(hscarislumbricoides)的ITS序列相似性分别为88%、98%和86%,与其他蛔虫的相似性均小于90%。%Our research objective was to identify ascarid samples isolated from small ear floral pig. The ascarid DNA in small ear floral pig was extracted.The internal transcribed spacer (ITS) and 5.8S of ribosomal DNA (rDNA) of two ascarid samples were amplified by PCR using a pair of conserved primers.The lengths of ITS sequence were both 1441 bp long. The two ascarid specimens had identical ITS and 5.8S rDNA sequences.The sequences were blasted in GenBank and the similarities was 88%, 98%, 86% or below 90% with the sequences of Baylisascaris transfuga, Ascaris suum, Ascaris lumbricoides or other ascarid.

  18. Identification of a splice-site mutation in the human growth hormone-variant gene.

    MacLeod, J.N.; Liebhaber, S A; MacGillivray, M H; Cooke, N E

    1991-01-01

    The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analys...

  19. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment.

    Kwasniewska, Jolanta; Jaskowiak, Joanna

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  20. Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR.

    XueYan Li

    Full Text Available Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.

  1. rRNA Gene Expression of Abundant and Rare Activated-Sludge Microorganisms and Growth Rate Induced Micropollutant Removal.

    Vuono, David C; Regnery, Julia; Li, Dong; Jones, Zackary L; Holloway, Ryan W; Drewes, Jörg E

    2016-06-21

    The role of abundant and rare taxa in modulating the performance of wastewater-treatment systems is a critical component of making better predictions for enhanced functions such as micropollutant biotransformation. In this study, we compared 16S rRNA genes (rDNA) and rRNA gene expression of taxa in an activated-sludge-treatment plant (sequencing batch membrane bioreactor) at two solids retention times (SRTs): 20 and 5 days. These two SRTs were used to influence the rates of micropollutant biotransformation and nutrient removal. Our results show that rare taxa (micropollutant biotransformation. An analysis of micropollutant-associated degradation genes via metagenomics and direct measurements of a suite of micropollutants and nutrients further corroborates the loss of enhanced functions at 5-day SRT operation. This work advances our knowledge of the underlying ecosystem properties and dynamics of abundant and rare organisms associated with enhanced functions in engineered systems. PMID:27196630

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

    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...... functions of nuclear group-I intron proteins....

  3. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit.

    Ferradás, Yolanda; Rey, Laura; Martínez, Óscar; Rey, Manuel; González, M Victoria

    2016-05-01

    Identification and validation of reference genes are required for the normalization of qPCR data. We studied the expression stability produced by eight primer pairs amplifying four common genes used as references for normalization. Samples representing different tissues, organs and developmental stages in kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) were used. A total of 117 kiwifruit samples were divided into five sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds). All samples were also analysed as a single set. The expression stability of the candidate primer pairs was tested using three algorithms (geNorm, NormFinder and BestKeeper). The minimum number of reference genes necessary for normalization was also determined. A unique primer pair was selected for amplifying the 18S rRNA gene. The primer pair selected for amplifying the ACTIN gene was different depending on the sample set. 18S 2 and ACT 2 were the candidate primer pairs selected for normalization in the three sample sets (mature leaves, fruit flesh and stigmatic arms). 18S 2 and ACT 3 were the primer pairs selected for normalization in axillary buds. No primer pair could be selected for use as the reference for the seed sample set. The analysis of all samples in a single set did not produce the selection of any stably expressing primer pair. Considering data previously reported in the literature, we validated the selected primer pairs amplifying the FLOWERING LOCUS T gene for use in the normalization of gene expression in kiwifruit. PMID:26897117

  4. [Language gene].

    Takahashi, Hiroshi

    2006-11-01

    The human capacity for acquiring speech and language must derive, at least in part, from the genome. Recent advance in the field of molecular genetics finally discovered 'Language Gene'. Disruption of FOXP2 gene, the firstly identified 'language gene' causes severe speech and language disorder. To elucidate the anatomical basis of language processing in the brain, we examined the expression pattern of FOXP2/Foxp2 genes in the monkey and rat brains through development. We found the preferential expression of FOXP2/Foxp2 in the striosomal compartment of the developing striatum. Thus, we suggest the striatum, particularly striosomal system may participate in neural information processing for language and speech. Our suggestion is consistent with the declarative/ procedural model of language proposed by Ullman (1997, 2001), which the procedural memory-dependent mental grammar is rooted in the basal ganglia and the frontal cortex, and the declarative memory-dependent mental lexicon is rooted in the temporal lobe. PMID:17432197

  5. Gene Silencing

    Kertbundit, Sunee; Juříček, Miloslav; Hall, T.C.

    Dordrecht : Springer, 2010 - (Jain, S.; Brar, D.), s. 631-652 ISBN 978-90-481-2966-9 Institutional research plan: CEZ:AV0Z50380511 Keywords : Gene Silencing * RISC complex Subject RIV: EB - Genetics ; Molecular Biology

  6. Phylogenetic affiliation of the desert truffles Picoa juniperi and Picoa lefebvrei.

    Sbissi, Imed; Neffati, Mohamed; Boudabous, Abdellatif; Murat, Claude; Gtari, Maher

    2010-11-01

    The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation. PMID:20559873

  7. Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer

    In real-time RT quantitative PCR (qPCR) the accuracy of normalized data is highly dependent on the reliability of the reference genes (RGs). Failure to use an appropriate control gene for normalization of qPCR data may result in biased gene expression profiles, as well as low precision, so that only gross changes in expression level are declared statistically significant or patterns of expression are erroneously characterized. Therefore, it is essential to determine whether potential RGs are appropriate for specific experimental purposes. Aim of this study was to identify and validate RGs for use in the differentiation of normal and tumor lung expression profiles. A meta-analysis of lung cancer transcription profiles generated with the GeneChip technology was used to identify five putative RGs. Their consistency and that of seven commonly used RGs was tested by using Taqman probes on 18 paired normal-tumor lung snap-frozen specimens obtained from non-small-cell lung cancer (NSCLC) patients during primary curative resection. The 12 RGs displayed showed a wide range of Ct values: except for rRNA18S (mean 9.8), the mean values of all the commercial RGs and ESD ranged from 19 to 26, whereas those of the microarray-selected RGs (BTF-3, YAP1, HIST1H2BC, RPL30) exceeded 26. RG expression stability within sample populations and under the experimental conditions (tumour versus normal lung specimens) was evaluated by: (1) descriptive statistic; (2) equivalence test; (3) GeNorm applet. All these approaches indicated that the most stable RGs were POLR2A, rRNA18S, YAP1 and ESD. These data suggest that POLR2A, rRNA18S, YAP1 and ESD are the most suitable RGs for gene expression profile studies in NSCLC. Furthermore, they highlight the limitations of commercial RGs and indicate that meta-data analysis of genome-wide transcription profiling studies may identify new RGs

  8. Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer

    Novello Silvia

    2006-07-01

    Full Text Available Abstract Background In real-time RT quantitative PCR (qPCR the accuracy of normalized data is highly dependent on the reliability of the reference genes (RGs. Failure to use an appropriate control gene for normalization of qPCR data may result in biased gene expression profiles, as well as low precision, so that only gross changes in expression level are declared statistically significant or patterns of expression are erroneously characterized. Therefore, it is essential to determine whether potential RGs are appropriate for specific experimental purposes. Aim of this study was to identify and validate RGs for use in the differentiation of normal and tumor lung expression profiles. Methods A meta-analysis of lung cancer transcription profiles generated with the GeneChip technology was used to identify five putative RGs. Their consistency and that of seven commonly used RGs was tested by using Taqman probes on 18 paired normal-tumor lung snap-frozen specimens obtained from non-small-cell lung cancer (NSCLC patients during primary curative resection. Results The 12 RGs displayed showed a wide range of Ct values: except for rRNA18S (mean 9.8, the mean values of all the commercial RGs and ESD ranged from 19 to 26, whereas those of the microarray-selected RGs (BTF-3, YAP1, HIST1H2BC, RPL30 exceeded 26. RG expression stability within sample populations and under the experimental conditions (tumour versus normal lung specimens was evaluated by: (1 descriptive statistic; (2 equivalence test; (3 GeNorm applet. All these approaches indicated that the most stable RGs were POLR2A, rRNA18S, YAP1 and ESD. Conclusion These data suggest that POLR2A, rRNA18S, YAP1 and ESD are the most suitable RGs for gene expression profile studies in NSCLC. Furthermore, they highlight the limitations of commercial RGs and indicate that meta-data analysis of genome-wide transcription profiling studies may identify new RGs.

  9. Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues

    Riond Barbara

    2009-12-01

    Full Text Available Abstract Background Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. Results RNA extraction from tissues was optimised for minimal genomic DNA (gDNA contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL, β-actin (ACTB, β-2-microglobulin (B2M, β-glucuronidase (GUSB, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyltransferase (HPRT, ribosomal protein S7 (RPS7, and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ. The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ. The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA and the least abundant genes (ABL, GUSB, and HMBS. The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the

  10. Evidence for natural horizontal transfer of the pcpB gene in the evolution of polychlorophenol-degrading sphingomonads.

    Tiirola, Marja A; Wang, Hong; Paulin, Lars; Kulomaa, Markku S

    2002-09-01

    The chlorophenol degradation pathway in Sphingobium chlorophenolicum is initiated by the pcpB gene product, pentachlorophenol-4-monooxygenase. The distribution of the gene was studied in a phylogenetically diverse group of polychlorophenol-degrading bacteria isolated from contaminated groundwater in Kärkölä, Finland. All the sphingomonads isolated were shown to share pcpB gene homologs with 98.9 to 100% sequence identity. The gene product was expressed when the strains were induced by 2,3,4,6-tetrachlorophenol. A comparative analysis of the 16S rDNA and pcpB gene trees suggested that a recent horizontal transfer of the pcpB gene was involved in the evolution of the catabolic pathway in the Kärkölä sphingomonads. The full-length Kärkölä pcpB gene allele had approximately 70% identity with the three pcpB genes previously sequenced from sphingomonads. It was very closely related to the environmental clones obtained from chlorophenol-enriched soil samples (M. Beaulieu, V. Becaert, L. Deschenes, and R. Villemur, Microbiol. Ecol. 40:345-355, 2000). The gene was not present in polychlorophenol-degrading nonsphingomonads isolated from the Kärkölä source. PMID:12200305

  11. Utility of internally transcribed spacer region of rDNA (ITS) and β‐tubulin gene sequences to infer genetic diversity and migration patterns of Colletotrichum truncatum infecting Capsicum spp.

    Rampersad, Kandyce; Ramdial, Hema; Rampersad, Sephra N

    2016-01-01

    Abstract Anthracnose is among the most economically important diseases affecting pepper (Capsicum spp.) production in the tropics and subtropics. Of the three species of Colletotrichum implicated as causal agents of pepper anthracnose, C. truncatum is considered to be the most destructive in agro‐ecosystems worldwide. However, the genetic variation and the migration potential of C. truncatum infecting pepper are not known. Five populations were selected for study and a two‐locus (internally t...

  12. Phylogenetic systematics of Barn Owl (Tyto alba (Scopoli, 1769 complex inferred from mitochondrial rDNA (16S rRNA taxonomic implication

    Mansour Aliabadian

    2012-09-01

    Full Text Available The Barn owl, Tyto alba (Scopoli, 1769, occurs worldwide and shows a considerable amount of morphological and geographical variations, leading to the recognition of many subspecies throughout the world. Yet, no comprehensive study has not been done on this species. Data from mitochondrial gene (16S Ribosomal RNA (16S with 569 bp length were analyzed for 41 individuals around the world. Maximum likelihood (ML, maximum parsimony (MP and Bayesian analysis showed two distinct clades including alba clad (old world and furcata clad (new world. The amount of genetic variation within each of these clades ranged from 0.5-1.7 but variation between clades was 3.7. This data may suggest that Barn owls of the Old World may be a separate species from those of the New World.

  13. Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

    Pasquali Giancarlo

    2010-09-01

    Full Text Available Abstract Background Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR; however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. Results By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Conclusion Our study showed that expression stability varied between putative reference genes

  14. Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin▿

    Tremblay, Maxime; Teng, Yumin; Paquette, Michel; Waters, Raymond; Conconi, Antonio

    2008-01-01

    Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER ...

  15. Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury

    Escriou Virginie

    2008-07-01

    Full Text Available Abstract Background Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. Results We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0–48 h post-trauma (PT of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH, β-microtubulin and S100β were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen; GAPDH > 18S rRNA > S100β > β-microtubulin > β-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen > S100β > 18S rRNA > β-actin > β-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen > 18S rRNA; GAPDH > S100β > β-microtubulin > β-actin. Conclusion This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that β-actin and β-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a

  16. Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

    Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

  17. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard

    2009-12-01

    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

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

    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.

  19. Chromosomal evolutionary dynamics of four multigene families in Coreidae and Pentatomidae (Heteroptera) true bugs.

    Bardella, Vanessa Bellini; Fernandes, José Antônio Marin; Cabral-de-Mello, Diogo Cavalcanti

    2016-10-01

    Previous chromosome mapping of multigene families in Pentatomomorpha (Heteroptera) insects, which was restricted to the major rDNA, revealed remarkable conservation of number of clusters and chromosomal positions. Aiming to understand the chromosomal organization and evolutionary patterns of multigene families in karyotypes of Heteroptera, we performed a chromosomal mapping using four distinct multigene families in representatives of Coreidae (ten species) and Pentatomidae (five species). A single pair of the major rDNA cluster (18S rDNA probe) and a single pair of the minor rDNA cluster (5S rDNA probe), both terminally located were primarily observed, being, in most species, located in distinct chromosomes. However, some alternative patterns were also observed. In species in which the U2 snDNA and H4 gene clusters were mapped, they were mainly located in one autosomal pair each, wherein the H4 gene cluster was located in different positions. Our data suggest that the karyotype diversity reported in Coreidae is not reflected in the distribution diversity of multigene families. This contrasts with the data for Pentatomidae, with a conserved gross karyotype but a discrete diversity in the location of the clusters of multigene families, indicating genome dynamics for these markers. The findings are discussed to shed light on the possible causes for the conservation or variation observed and to assist in understanding the chromosomal evolutionary trends in the group. PMID:27380138

  20. Genetic diversity and phylogenetic analysis of Citrus (L from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA

    Marlykynti Hynniewta

    2014-12-01

    Full Text Available The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L. species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply

  1. Simple DNA extraction protocol for a 16S rDNA study of bacterial diversity in tropical landfarm soil used for bioremediation of oil waste.

    Maciel, B M; Santos, A C F; Dias, J C T; Vidal, R O; Dias, R J C; Gross, E; Cascardo, J C M; Rezende, R P

    2009-01-01

    Landfarm soil is used to bioremediate oil wastes from petrochemical industries. We developed a simplified protocol for microbial DNA extraction of tropical landfarm soil using only direct lysis of macerated material. Two samples of tropical landfarm soil from a Brazilian refinery were analyzed by this protocol (one consisted of crude oil-contaminated soil; the other was continuously enriched for nine months with petroleum). The soil samples were lysed by maceration with liquid nitrogen, eliminating the need for detergents, organic solvents and enzymatic cell lysis. Then, the DNA from the lysed soil sample was extracted using phenol-chloroform-isoamyl alcohol or guanidium isothiocyanate, giving high DNA yields (more than 1 micro g DNA/g soil) from both soil types. This protocol compared favorably with an established method of DNA template preparation that included mechanical, chemical and enzymatic treatment for cell lysis. The efficiency of this extraction protocol was confirmed by polymerase chain reaction amplification of the 16S rRNA gene, denaturing gradient gel electrophoresis and cloning assays. Fifty-one different clones were obtained; their sequences were classified into at least seven different phyla of the Eubacteria group (Proteobacteria - alpha, gamma and delta, Chloroflexi, Actinobacteria, Acidobac teria, Planctomycetes, Bacteroidetes, and Firmicutes). Forty percent of the sequences could not be classified into these phyla, demonstrating the genetic diversity of this microbial community. Only eight isolates had sequences similar to known sequences of 16S rRNA of cultivable organisms or of known environmental isolates and therefore could be identified to the genus level. This method of DNA extraction is a useful tool for analysis of the bacteria responsible for petroleum degradation in contaminated environments. PMID:19440973

  2. Fecal microbial diversity in pre-weaned dairy calves as described by pyrosequencing of metagenomic 16S rDNA. Associations of Faecalibacterium species with health and growth.

    Georgios Oikonomou

    Full Text Available In this study, we use barcoded pyrosequencing of the 16S rRNA gene to characterize the fecal microbiota of neonatal calves and identify possible relationships of certain microbiota profiles with health and weight gain. Fecal samples were obtained weekly from 61 calves from birth until weaning (seventh week of the calves' life. Firmicutes was the most prevalent phylum, with a prevalence ranging from 63.84% to 81.90%, followed by Bacteroidetes (8.36% to 23.93%, Proteobacteria (3.72% to 9.75%, Fusobacteria (0.76% to 5.67%, and Actinobacteria (1.02% to 2.35%. Chao1 index gradually increased from the first to the seventh postnatal week. Chao1 index was lower during the third, fourth, and fifth week of life in calves that suffered from pneumonia and were treated with antibiotics. Diarrhea incidence during the first four weeks of the calves' life was also associated with a reduction of microbial diversity during the third week of life. Increased fecal microbial diversity after the second week of life was associated with higher weight gain. Using discriminant analysis we were able to show differences in the microbiota profiles between different weeks of life, between high and low weight gain groups of calves, and between calves affected and not affected with diarrhea during the first four weeks life. The prevalence of Faecalibacterium spp. in the first week of life was associated with weight gain and the incidence of diarrhea, with higher prevalence being associated with higher weight gain and less diarrhea. Representative sequences from Faecalibacterium spp. were closely affiliated to Faecalibacterium prausnitzii. Results presented here provide new information regarding the intestinal microbiota of neonatal calves and its association with health and growth. Fecal microbial diversity was associated with calf age, disease status and growth rates. Results suggesting a possible beneficial effect of Faecalibacterium spp. on health and growth are promising.

  3. SSU rDNA sequence diversity and seasonally differentiated distribution of nanoplanktonic ciliates in neritic Bohai and Yellow Seas as revealed by T-RFLP.

    Jun Dong

    Full Text Available Nanociliates have been frequently found to be important players in the marine microbial loop, however, little is known about their diversity and distribution in coastal ecosystems. We investigated the molecular diversity and distribution patterns of nanoplanktonic oligotrich and choreotrich (OC ciliates in surface water of three neritic basins of northern China, the South Yellow Sea (SYS, North Yellow Sea (NYS, and Bohai Sea (BS in June and November 2011. SSU rRNA gene clone libraries generated from three summertime samples (sites B38, B4 and H8 were analyzed and revealed a large novel ribotype diversity, of which many were low-abundant phylotypes belonging to the subclass Oligotrichia, but divergent from described morphospecies. Based on the data of terminal-restriction fragment length polymorphism (T-RFLP analysis of all 35 samples, we found that the T-RF richness was generally higher in the SYS than in the BS, and negatively correlated with the molar ratio of P to Si. Overall, multidimensional scaling and permutational multivariate analysis of variance of the community turnover demonstrated a distinct seasonal pattern but no basin-to-basin differentiation across all samples. Nevertheless, significant community differences among basins were recognized in the winter dataset. Mantel tests showed that the environmental factors, P:Si ratio, water temperature and concentration of dissolved oxygen (DO, determined the community across all samples. However, both biogeographic distance and environment shaped the community in winter, with DO being the most important physicochemical factor. Our results indicate that the stoichiometric ratio of P:Si is a key factor, through which the phytoplankton community may be shaped, resulting in a cascade effect on the diversity and community composition of OC nanociliates in the N-rich, Si-limited coastal surface waters, and that the Yellow Sea Warm Current drives the nanociliate community, and possibly the

  4. Bipteria formosa (Kovaleva et Gaevskaya, 1979) comb. n. (Myxozoa: Myxosporea) in whiting Merlangius merlangus (Teleostei: Gadidae) from Denmark

    Karlbakk, Egil; Køie, Marianne

    2009-01-01

    spore of is described in detail and compared with other Bipteria spp. The phylogenetic position of B. formosa, based on partial 18S rDNA sequences, is closest to Leptotheca fugu Tun, spp. The phylogenetic position of , based on partial 18S rDNA sequences, is closest to Tun, Yokoyama, Ogawa et...

  5. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.).

    Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

    2016-01-01

    The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family. PMID:27308855

  6. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L..

    Maryam Moazzam Jazi

    Full Text Available The tree species, Pistacia vera (P. vera is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family.

  7. Identification of Reference Genes for Quantitative Gene Expression Studies in a Non-Model Tree Pistachio (Pistacia vera L.)

    Moazzam Jazi, Maryam; Ghadirzadeh Khorzoghi, Effat; Botanga, Christopher; Seyedi, Seyed Mahdi

    2016-01-01

    The tree species, Pistacia vera (P. vera) is an important commercial product that is salt-tolerant and long-lived, with a possible lifespan of over one thousand years. Gene expression analysis is an efficient method to explore the possible regulatory mechanisms underlying these characteristics. Therefore, having the most suitable set of reference genes is required for transcript level normalization under different conditions in P. vera. In the present study, we selected eight widely used reference genes, ACT, EF1α, α-TUB, β-TUB, GAPDH, CYP2, UBQ10, and 18S rRNA. Using qRT-PCR their expression was assessed in 54 different samples of three cultivars of P. vera. The samples were collected from different organs under various abiotic treatments (cold, drought, and salt) across three time points. Several statistical programs (geNorm, NormFinder, and BestKeeper) were applied to estimate the expression stability of candidate reference genes. Results obtained from the statistical analysis were then exposed to Rank aggregation package to generate a consensus gene rank. Based on our results, EF1α was found to be the superior reference gene in all samples under all abiotic treatments. In addition to EF1α, ACT and β-TUB were the second best reference genes for gene expression analysis in leaf and root. We recommended β-TUB as the second most stable gene for samples under the cold and drought treatments, while ACT holds the same position in samples analyzed under salt treatment. This report will benefit future research on the expression profiling of P. vera and other members of the Anacardiaceae family. PMID:27308855

  8. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    Xiao Li Shi

    Full Text Available The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX, which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  9. 不同产地大麻的ITS全序列分析%Identification of cannabis from different growing areas based on rDNA ITS sequencing

    代勇; 姜黎; 皮建华; 孙琴

    2012-01-01

      目的:对分别采自云南、新疆两个地区的大麻植物,通过ITS序列的分析,从分子水平进行鉴定。方法:采用DNA提取试剂盒提取大麻的ITS全序列DNA,经过PCR扩增,胶回收后测序,所得序列与从Genbank中获取3个大麻科植物的ITS全序列比对,以葎草的ITS全序列为外源基因构建了大麻属的系统发育树。结果:对获得的基因序列进行同源性分析,NCBI数据库中登陆的大麻相应序列与新疆大麻(毒品大麻)的ITS全序列亲缘关系性最近,为99%;云南大麻(工业大麻)与新疆大麻(毒品大麻)亲缘关系较远。结论:ITS序列能够较准确的将毒品大麻和工业大麻区分开。%  objective:To identify cannabis sativa plants from Yunnan and Xinjiang areas on molecular level by internal transcribed spaces (ITS) sequence analysis. Methods:The complete ITS sequencing of cannabis sativa was determined through DNA extraction by Plant Genomic DNA Kit, PCR amplification, glue recovery and sequencing. Then the phylogenetic tree of cannabinaceae was constructed by comparing with the complete ITS sequencing of three cannabis plants from GenBank. Results: The gene sequence of cannabis by homology analysis showed that the genetic relationship between NCBI database and Xinjiang cannabis (drugs hemp) was the nearest, the homology reaches 99%; but the relationship between Yunnan cannabis (industrial hemp) and Xinjiang cannabis is farther. Conclusion: The cannabis from different sources could be identified by ITS sequence .

  10. 16s rDNA文库法分析番石榴果实蝇共生菌组成%Composition of symbiotic bacteria associated with Bactrocera correcta analyzed by 16s rDNA libraries

    戴阳; 李志红; 柳丽君; 吴佳教; 邓裕亮

    2012-01-01

    [Objective] The composition of symbiotic bacteria associated with Guava fruit fly, Bacirocera correcta (Bezzi), was investigated to provide the basis for further study on physiological function of the symbionts and co-evolution between bacteria and the host. [Method] 16s rDNA libraries of laboratory population for B. correcta were constructed, and BLAST analysis of bacterial composition and abundance was carried out in this study. [Result] The dominant symbionts in B. correcta male adults were Pseudomonas aeruginosa and Lactococcus lactis, while symbiotic bacteria in female adults were Enterobacter bacteria. [Conclusion] Various bacteria are present in the Guava fruit fly, B. correcta.%[目的]探索分析番石榴果实蝇[ Bactrocera correcta (Bezzi)]的共生菌组成,为进一步探索共生菌的生理功能以及与宿主的协同进化关系奠定基础.[方法]构建番石榴果实蝇室内种群共生菌的16s rDNA文库,BLAST分析其共生菌的组成以及丰度.[结果]室内雄虫共生菌主要为Pseudomonas aeruginosa和Lactococcus lactis,雌虫共生菌主要为Enterobacter属的细菌.[结论]本研究结果证明番石榴果实蝇存在多种共生菌.

  11. Application of 16S rDNA sequencing technique in infantile intestinal microecological research%16SrDNA测序技术在婴儿肠道微生态研究中的应用

    马丽亚; 王辉林; 陈睿; 张敏; 黄艳; 卢光进

    2015-01-01

    目的:探讨16S rDNA测序技术在新生儿、婴儿肠道微生态研究中的应用。方法于生后3天、1月、6月、1岁时收集2例健康婴儿粪便标本共8份,提取细菌总DNA ,以Illumina Hiseq 2000为测序平台,采用新一代高通量16S rDNA宏基因组测序技术对V6可变区测序,并进行生物信息分析(物种分类和丰度分析;多样性分析)。结果8份样品共产生原始测序数据为1027.47 Mbp ,Unique tags序列数量均值为58630,OTU数量63~209;优势菌门为Proteobacteria和Firmicutes ;在科水平,>1%的物种1个月之内2~4种,6月后达7~10种;1号婴儿一直以Enterobacteriaceae占优势,2号婴儿优势菌群包括 Enterobacteriaceae、Lachnospiraceae、Streptococcaceae和Bacteroidaceae;4个时间点的npShannon和Simpson指数分别为1.17、1.29、2.16、2.51和0.43、0.40、0.26、0.14。结论16S rDNA测序技术能满足新生儿、婴儿肠道微生态研究需求;新生儿、婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;从出生到1岁,婴儿肠道菌群结构趋向复杂和多样。%Objective To investigate the application of 16S rDNA sequencing technique in studying intestinal microecology of neonates and infants .Methods Eight fecal samples were collected on 3 d ,1 month ,6 months and 1 year in 2 healthy infants .Total bacterial DNAs were extracted and submitted the high throughout 16S rDNA sequen‐cing on the V6 viable region by using Illumia genome analyzer Hiseq 2000 (101 bp pair‐end sequencing strategy) .The 16S rDNA tags and operational taxonomic units (OTU) were then obtained from the sequences .The bioinformatic a‐nalysis including analysis of taxonomy ,abundance and alpha diversity were performed .Results Total 1 027 .47 Mbp raw data were produced .The mean unique tags number was 58630 .The OTU numbers ranged from 36 to 308 .The bacterial families more than 1% were increased

  12. Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.; Deinhard, G.; Poralla, K.

    1992-01-01

    Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

  13. 16SrDNA测序技术在婴儿肠道微生态研究中的应用%Application of 16S rDNA sequencing technique in infantile intestinal microecological research

    马丽亚; 王辉林; 陈睿; 张敏; 黄艳; 卢光进

    2015-01-01

    目的:探讨16S rDNA测序技术在新生儿、婴儿肠道微生态研究中的应用。方法于生后3天、1月、6月、1岁时收集2例健康婴儿粪便标本共8份,提取细菌总DNA ,以Illumina Hiseq 2000为测序平台,采用新一代高通量16S rDNA宏基因组测序技术对V6可变区测序,并进行生物信息分析(物种分类和丰度分析;多样性分析)。结果8份样品共产生原始测序数据为1027.47 Mbp ,Unique tags序列数量均值为58630,OTU数量63~209;优势菌门为Proteobacteria和Firmicutes ;在科水平,>1%的物种1个月之内2~4种,6月后达7~10种;1号婴儿一直以Enterobacteriaceae占优势,2号婴儿优势菌群包括 Enterobacteriaceae、Lachnospiraceae、Streptococcaceae和Bacteroidaceae;4个时间点的npShannon和Simpson指数分别为1.17、1.29、2.16、2.51和0.43、0.40、0.26、0.14。结论16S rDNA测序技术能满足新生儿、婴儿肠道微生态研究需求;新生儿、婴儿粪便中含丰富细菌基因组;细菌物种丰度及分类存在个体差异;从出生到1岁,婴儿肠道菌群结构趋向复杂和多样。%Objective To investigate the application of 16S rDNA sequencing technique in studying intestinal microecology of neonates and infants .Methods Eight fecal samples were collected on 3 d ,1 month ,6 months and 1 year in 2 healthy infants .Total bacterial DNAs were extracted and submitted the high throughout 16S rDNA sequen‐cing on the V6 viable region by using Illumia genome analyzer Hiseq 2000 (101 bp pair‐end sequencing strategy) .The 16S rDNA tags and operational taxonomic units (OTU) were then obtained from the sequences .The bioinformatic a‐nalysis including analysis of taxonomy ,abundance and alpha diversity were performed .Results Total 1 027 .47 Mbp raw data were produced .The mean unique tags number was 58630 .The OTU numbers ranged from 36 to 308 .The bacterial families more than 1% were increased

  14. Moestrupia oblonga gen. et comb. nov. (syn.: Gyrodinium oblongum), a new marine dinoflagellate genus characterized by light and electron microscopy, photosynthetic pigments and LSU rDNA sequence

    Hansen, Gert; Daugbjerg, Niels

    2011-01-01

    A small-sized, peridinin-containing, athecate dinoflagellate (13–17 µm long) was isolated into clonal culture from a water sample collected at a nearshore location in Tenerife, Spain (October 2004). Based on phenotypic characters (size, shape, pyrenoid and nucleus position), the culture was ident...... the anterior part of the ventral flange to the middorsal side of the episome. Thus, it differs markedly from that in species of Gyrodinium and emphasizes it being distantly related to this genus...... identified as Gyrodinium oblongum. However, a detailed ultrastructural examination revealed a number of features that did not fit into the current delineation of the genus Gyrodinium or any other dinoflagellate. Likewise, the molecular phylogeny and sequence divergence estimates based on partial LSU r......DNA sequences indicated that this isolate has a taxonomically isolated position, and we therefore propose the new genus Moestrupia to accommodate this species within the Dinophyceae. The ultrastructural study uncovered a number of unique features for Moestrupia, for example, a microtubule supported ventral...

  15. Validation of reference genes for real-time quantitative RT-PCR studies in Talaromyces marneffei.

    Dankai, Wiyada; Pongpom, Monsicha; Vanittanakom, Nongnuch

    2015-11-01

    Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions. PMID:26327538

  16. Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes

    Pia Miglietta, Maria; Hourdez, Stephane; Cowart, Dominique A.; Schaeffer, Stephen W.; Fisher, Charles

    2010-11-01

    At least six morphospecies of vestimentiferan tubeworms are associated with cold seeps in the Gulf of Mexico (GOM). The physiology and ecology of the two best-studied species from depths above 1000 m in the upper Louisiana slope (Lamellibrachia luymesi and Seepiophila jonesi) are relatively well understood. The biology of one rare species from the upper slope (escarpiid sp. nov.) and three morphospecies found at greater depths in the GOM (Lamellibrachia sp. 1, L. sp. 2, and Escarpia laminata) are not as well understood. Here we address species distributions and boundaries of cold-seep tubeworms using phylogenetic hypotheses based on two mitochondrial genes. Fragments of the mitochondrial large ribosomal subunit rDNA (16S) and cytochrome oxidase subunit I (COI) genes were sequenced for 167 vestimentiferans collected from the GOM and analyzed in the context of other seep vestimentiferans for which sequence data were available. The analysis supported five monophyletic clades of vestimentiferans in the GOM. Intra-clade variation in both genes was very low, and there was no apparent correlation between the within-clade diversity and collection depth or location. Two of the morphospecies of Lamellibrachia from different depths in the GOM could not be distinguished by either mitochondrial gene. Similarly, E. laminata could not be distinguished from other described species of Escarpia from either the west coast of Africa or the eastern Pacific using COI. We suggest that the mitochondrial COI and 16S genes have little utility as barcoding markers for seep vestimentiferan tubeworms.

  17. Genes and Psoriasis

    ... Diet Tips" to find out more! Email * Zipcode Genes and Psoriasis Genes hold the key to understanding ... is responsible for causing psoriatic disease. How do genes work? Genes control everything from height to eye ...

  18. Genes and Hearing Loss

    ... Meeting Calendar Find an ENT Doctor Near You Genes and Hearing Loss Genes and Hearing Loss Patient ... mutation may only have dystopia canthorum. How Do Genes Work? Genes are a road map for the ...

  19. Preliminary evaluation of the use of soil bacterial 16S rDNA DNA markers in sediment fingerprinting in two small endorheic lagoons in southern Spain

    Gomez, Jose Alfonso; Landa del Castillo, Blanca; Guzman, Gema; Petticrew, Ellen L.; Owens, Phillip N.

    2016-04-01

    bulk community of DNA was extracted from 250 mg of soil samples (three replicates per sample) using the procedure described in Landa et al. (2014). The bacterial 16S rRNA gene V1-V2 hypervariable regions were amplified in polymerase chain reaction (PCR). The sequencing procedure was performed according to the manufacturer's recommendations using MiSeq Reagent Kit v2 for 300 cycles on MiSeq desktop sequencer. The raw dataset for each sample consisted of the number of counts for each of the 6640 operational taxonomic units (OTU) analyzed. All the screening and analysis was performed independently for each lagoon. Given the large number of OTUs, a first screening was made discarding any OTU that did not presented at least five samples with counts >20 for that OTU. This lowered the number of OTUs to 205 in Dulce and 217 in Zoñar. Because of the limited number of samples, we did not perform independent analysis for each soil depth. All the analyses were performed twice; one with the original number of counts and another with the normalized number of counts. We screened the OTU following a 4-step method to determine those with the best ability to discriminate among the three potential source areas. These steps were: 1) eliminate OTUs with no readings or very few, that could be experimental noise; 2) keep only OTUs that are different among source areas; 3) eliminate OTUs that range outside of feasible solutions to explain average values found in sediment; and 4) eliminate OTUs with the largest variability. Afterwards, several over-determined mixing models were solved considering different combinations of OTUs using limSolve (Soetaert et al., 2014) in R. Preliminary results show that 0.2 to 0.6 % of the searched OTUs (i.e. 14 to 42) had the potential for use in the mixing models after the four-step screening process. The results indicate a large variability in the number of counts among the samples from different areas within the subcatchments ranging, on average, from 49 to

  20. Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish

    Tyler Charles R

    2007-02-01

    Full Text Available Abstract Background Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples. Results We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA], ribosomal protein l8 [rpl8], elongation factor 1 alpha [ef1a], glucose-6-phosphate dehydrogenase [g6pd], beta actin [bactin], glyceraldehyde-3-phosphate dehydrogenase [gapdh], hypoxanthine phosphoribosyltransferase 1 [hprt1], and tata box binding protein [tbp] following exposure to the environmental estrogen, 17α-ethinylestradiol (EE2, in the fathead minnow (Pimephales promelas. Exposure to 10 ng/L EE2 for 21 days down-regulated the expression of ef1a, g6pd, bactin and gapdh in the liver, and bactin and gapdh in the gonad. Some of these effects were gender-specific, with bactin in the liver and gapdh in the gonad down-regulated by EE2 in males only. Furthermore, when ef1a, g6pd, bactin or gapdh were used for normalization, the hepatic expression of two genes of interest, vitellogenin (vtg and cytochrome P450 1A (cyp1a following exposure to EE2 was overestimated. Conclusion Based on the data presented, we recommend 18S rRNA, rpl8, hprt1 and/or tbp, but not ef1a, g6pd, bactin and/or gapdh, as likely appropriate internal controls in real-time PCR studies of estrogens effects in fish. Our studies show that pre-validation of control genes considering the scope and nature of the experiments to be performed, including both gender and tissue type, is critical for accurate assessments of the effects of environmental estrogens on gene

  1. Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp. strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes.

    Sugita, M; Sugishita, H; Fujishiro, T; Tsuboi, M; Sugita, C; Endo, T; Sugiura, M

    1997-08-11

    The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp. strain PCC6301 is presented. Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified. The gene order is similar to that of the E. coli S10, spc and alpha operons. Unlike the corresponding E. coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster. The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species. This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium. PMID:9300823

  2. Molecular Phylogeny Analysis of Allium Sativum in Alliaceae%大蒜在葱科的分子分类地位研究

    侯进慧; 李同祥; 蔡文佳

    2014-01-01

    通过扩增获得18S rRNA和叶绿体16S rRNA基因序列,测序并提交GenBank ,登录号分别是JF719285和JF719286.利用大蒜和GenBank相关序列构建系统发育树,进行分子演化分析.结果表明:大蒜18S rRNA 基因与球序韭、韭菜、茖葱等葱科植物序列相似度高;叶绿体16S rRNA基因与龙舌兰科和薯蓣科的物种序列相似度高.大蒜与葱科植物在18S rRNA序列上具有较高的同源性.18S rRNA序列在植物演化方面的区分度比16S rRNA高.%In the paper ,molecular phylogeny of Allium sativum were discussded with the analysis of rRNA gene .18S rRNA gene and chloroplast 16S rRNA gene sequences were amplified .The two rRNA genes were submitted to Genbank and the accession numbers were JF719285 and JF719286 .Gene sequences of Allium sativum was analyzed with related species in GenBank .The results showed that :Allium sativum 18S rRNA gene has a high homology with many species within Alliaceae ,such as Allium thunbergii ,Allium tuberosum and Allium victorialis .Allium sativum and Alliaceae plants has a high similarity in 18S rDNA . The discrimination accusation of 18Sr RNA sequences in plant phylogeny analysis is better than that of 16S rRNA .

  3. Cloning and Sequence Analysis of ITS and 5.8S rDNA of Ascarid Nematodes from Giant Panda%大熊猫蛔虫ITS及5.8SrDNA序列的克隆与分析

    陈绚姣; 唐志玲; 陈武; 金超宇

    2011-01-01

    To identify the species of ascarid samples isolated from a giant panda in Guangzhou Zoo,China,we designed a pair of conserved primers(NC2/NC5) to amplify the first and second internal transcribed spacers(ITS) and 5.8S of ribosomal DNA(5.8S rDNA).The ITS rDNA was amplified by PCR from the ascarid samples.The amplicons were then purified and cloned into pGEM - T Easy vector.After being identified by PCR amplification and restriction digestion,the positive clones were sequenced and the sequences were compared with those of Baylisascaris transfuga,Ascaris sum and Ascaris lumbricoides available in GenBank~(TM).The results revealed that the two ascarid specimens had identical ITS and 5.8S rDNA sequences of 910bp length.The ITS sequences of the ascarid nematodes from the giant panda were significantly different from those of Baylisascaris transfuga,Ascaris sum,and Ascaris lumbricoides,with similarities of 96.6%,82.9%,and 82.7%,respectively. The ascarid nematodes from the giant panda differed from the species listed above and their ITS sequence had not been previously reported.We provisionally identify the species as Baylisascaris transfuga,the species showing greatest similarity.%基于核糖体DNA第一与第二转录间隔序列以及5.8S序列,以分离自广州动物园大熊猫体内的蛔虫为研究对象,用保守引物NC_5和NC_2对核糖体DNA(rDNA)的内转录间隔区ITS-1,ITS-2及5.8S序列进行PCR扩增,扩增后的片段纯化后克隆至pGEM-Teasy载体,重组质粒通过菌液PCR鉴定后,对阳性菌落进行序列测定及分析,鉴定大熊猫蛔虫的种类。结果显示,目的片段总长为910 bp,2个不同样品之间的ITS及5.8S序列没有差异,与GenBank~(TM)中的拜林蛔线虫(Baylisascaris transfuga)、猪蛔虫(Ascaris suum)和人蛔虫(Ascaris lumbricoides)的ITS序列相似性分别为96.6%、82.9%和82.7%。结果表明,此次分离的大熊猫蛔线虫可能为拜林蛔线虫。

  4. RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

    Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

    2016-01-01

    Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

  5. Genetic diversity of bacterial communities and gene transfer agents in northern South China Sea.

    Fu-Lin Sun

    Full Text Available Pyrosequencing of the 16S ribosomal RNA gene (rDNA amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA major capsid gene (g5 was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS, temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments.

  6. Variation in Stability of Endogenous Reference Genes in Fallopian Tubes and Endometrium from Healthy and Ectopic Pregnant Women

    Justin C. Konje

    2012-03-01

    Full Text Available RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes and UBC and ATP5B (endometrium. mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.

  7. Selection of accurate reference genes in mouse trophoblast stem cells for reverse transcription-quantitative polymerase chain reaction.

    Motomura, Kaori; Inoue, Kimiko; Ogura, Atsuo

    2016-06-17

    Mouse trophoblast stem cells (TSCs) form colonies of different sizes and morphologies, which might reflect their degrees of differentiation. Therefore, each colony type can have a characteristic gene expression profile; however, the expression levels of internal reference genes may also change, causing fluctuations in their estimated gene expression levels. In this study, we validated seven housekeeping genes by using a geometric averaging method and identified Gapdh as the most stable gene across different colony types. Indeed, when Gapdh was used as the reference, expression levels of Elf5, a TSC marker gene, stringently classified TSC colonies into two groups: a high expression groups consisting of type 1 and 2 colonies, and a lower expression group consisting of type 3 and 4 colonies. This clustering was consistent with our putative classification of undifferentiated/differentiated colonies based on their time-dependent colony transitions. By contrast, use of an unstable reference gene (Rn18s) allowed no such clear classification. Cdx2, another TSC marker, did not show any significant colony type-specific expression pattern irrespective of the reference gene. Selection of stable reference genes for quantitative gene expression analysis might be critical, especially when cell lines consisting of heterogeneous cell populations are used. PMID:26853688

  8. Genetic Diversity of Daphnia pulex in the Middle and Lower Reaches of the Yangtze River.

    Wenping Wang

    Full Text Available Increased human activities and environmental changes may lead to genetic diversity variations of Cladocerans in water. Daphnia pulex are distributed throughout the world and often regarded as a model organism. The 16S rDNA, cytochrome c oxidase subunit I (COI, and 18S genes were used as molecular marks. The genetic diversity and phylogeny of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River were studied. For 16S rDNA, COI gene, and 18S gene, the A+T content (65.4%, 58.4%, and 54.6% was significantly higher than the G+C content (34.6%, 41.6% and 45.4%. This result was consistent with higher A and T contents among invertebrates. Based on the genetic distances of 16S rDNA and COI genes, the genetic differences of D. pulex from 10 water bodies located in the middle and lower reaches of the Yangtze River in China was minimal (0%-0.8% for 16S rDNA and 0%-1.5% for COI gene. However, D. pulex evolved into two branches in the phylogenetic trees, which coincided with its geographical distribution. Compared with D. pulex from other countries, the average genetic distance of D. pulex obtained from 10 water bodies in the middle and lower reaches of the Yangtze River reached 9.1%-10.5%, thereby indicating that D. pulex may have evolved into different subspecies.

  9. PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

    Decelle, Johan; Romac, Sarah; Stern, Rowena F; Bendif, El Mahdi; Zingone, Adriana; Audic, Stéphane; Guiry, Michael D; Guillou, Laure; Tessier, Désiré; Le Gall, Florence; Gourvil, Priscillia; Dos Santos, Adriana L; Probert, Ian; Vaulot, Daniel; de Vargas, Colomban; Christen, Richard

    2015-11-01

    Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing. PMID:25740460

  10. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and sh...

  11. Cervikobrahijalni sindrom - rad i rdna nesposobnost

    KRAPAC, L.; Krmpotić, A.; Pavićević, L.; Domljan, Z.

    1992-01-01

    Istraživanjem je obuhvaćeno 114 ispitanika koji su odabrani iz skupine invalidskih umirovljenika te bolesnika koji su bili na zdravstvenoj obradi za utvrđivanje invalidnosti. Ispitivanu je skupinu činilo 57 invalidskih umirovljenika s jasno izraženim cervikobrahijalnim sindromom. Poredbena skupina, formirana metodom ekvivalentnih parova s obzirom na spol i dob, bili su bolesnici kojima se ovaj sindrom sa sigurnošću mogao isključiti. Za sve je ispitanike uz klinički pregled ispunjen i standard...

  12. Teriparatide (rDNA origin) Injection

    ... patients). Teriparatide injection contains a synthetic form of natural human hormone called parathyroid hormone (PTH). It works ... container.Teriparatide injection controls osteoporosis but does not cure it. Continue to use teriparatide injection even if ...

  13. Insulin Degludec (rDNA Origin) Injection

    ... cloudy, or contains solid particles, or if the expiration date on the bottle has passed.Do not ... body wheezing difficulty breathing or swallowing shortness of breath sweating swelling of the eyes, face, lips, tongue, ...

  14. Insulin Glargine (rDNA origin) Injection

    ... cloudy, or contains solid particles, or if the expiration date on the bottle has passed.Do not ... body wheezing difficulty breathing or swallowing shortness of breath fast pulse sweating swelling of the eyes, face, ...

  15. Insulin Aspart (rDNA Origin) Injection

    ... not use any type of insulin after the expiration date printed on the bottle has passed.Insulin ... or itching over the whole body shortness of breath wheezing dizziness blurred vision fast heartbeat sweating weakness ...

  16. Insulin Detemir (rDNA Origin) Injection

    ... thickened, or contains solid particles, or if the expiration date on the bottle has passed.You can ... or itching over the whole body shortness of breath wheezing dizziness low blood pressure blurred vision fast ...

  17. Insulin Degludec (rDNA Origin) Injection

    ... azilsartan (Edarbi, in Edarbyclor), candesartan (Atacand, in Atacand HCT), eprosartan (Teveten), irbesartan (Avapro, in Avalide), losartan (Cozaar, in Hyzaar), telmisartan (Micardis, in Micardis HCT, in Twynsta), valsartan (Diovan, in Diovan HCT, in ...

  18. Gene gymnastics

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  19. A multi-gene transcriptional profiling approach to the discovery of cell signature markers.

    Wada, Youichiro; Li, Dan; Merley, Anne; Zukauskas, Andrew; Aird, William C; Dvorak, Harold F; Shih, Shou-Ching

    2011-01-01

    A profile of transcript abundances from multiple genes constitutes a molecular signature if the expression pattern is unique to one cell type. Here we measure mRNA copy numbers per cell by normalizing per million copies of 18S rRNA and identify 6 genes (TIE1, KDR, CDH5, TIE2, EFNA1 and MYO5C) out of 79 genes tested as excellent molecular signature markers for endothelial cells (ECs) in vitro. The selected genes are uniformly expressed in ECs of 4 different origins but weakly or not expressed in 4 non-EC cell lines. A multi-gene transcriptional profile of these 6 genes clearly distinguishes ECs from non-ECs in vitro. We conclude that (i) a profile of mRNA copy numbers per cell from a well-chosen multi-gene panel can act as a sensitive and accurate cell type signature marker, and (ii) the method described here can be applied to in vivo cell fingerprinting and molecular diagnosis. PMID:20972619

  20. The methanol dehydrogenase gene, mxaF, as a functional and phylogenetic marker for proteobacterial methanotrophs in natural environments.

    Evan Lau

    Full Text Available The mxaF gene, coding for the large (α subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene's usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.