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Sample records for 18s internal transcribed

  1. The internal transcribed spacer region of belonolaimus (nemata: belonolaimidae).

    PubMed

    Cherry, T; Szalanski, A L; Todd, T C; Powers, T O

    1997-03-01

    Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates suggest that the California isolate is a relatively recent introduction into the state.

  2. 7th International Workshop on the Identification of Transcribed Sequences. Beyond the Identification of Transcribed Sequences

    SciTech Connect

    Gardner, Kathleen

    1997-11-19

    The Seventh Annual Human Genome Conference: Beyond the Identification of Transcribed Sequences (BITS) was held November 16-19, 1997 at the Asilomar Conference Center in Monterey, California. The format for the meeting was a combination of oral presentations, group discussions and poster sessions. The original workshop was held to discuss methodologies for the identification of transcribed sequences in mammalian genomes. Over the years, the focus of the workshops has gradually shifted towards functional analysis, with the most dramatic change in emphasis at this meeting, as reflected in the modest change in the workshop title. Topics presented and discussed included: (1) large scale expression and mutational analysis in yeast, C. elegans, Drosophila and zebrafish, (2) comparative mapping of zebrafish, chicken and Fugu; (3) functional analysis in mouse using promoter traps, mutational analysis of biochemical pathways, and Cre/lox constructs; (4) construction of 5 foot end and complete cDNA libraries; (5) expression analysis in mammalian organisms by array screening and differential display; (6) genome organization as determined by detailed transcriptional mapping and genomic sequence analysis; (7) analysis of genomic sequence, including gene and regulatory sequence predictions, annotation of genomic sequence, development of expression databases and verification of sequence analysis predictions; and (8) structural/functional relationships as determined by RNA secondary structure analysis and evolutionary conservation of non-coding sequences.

  3. Analysis of Mammalian rDNA Internal Transcribed Spacers

    PubMed Central

    Coleman, Annette W.

    2013-01-01

    Nuclear rDNA Internal Transcribed Spacers, ITS1 and ITS2, are widely used for eukaryote phylogenetic studies from the ordinal level to the species level, and there is even a database for ITS2 sequences. However, ITS regions have been ignored in mammalian phylogenetic studies, and only a few rodent and ape sequences are represented in GenBank. The reasons for this dearth, and the remedies, are described here. We have recovered these sequences, mostly >1 kb in length, for 36 mammalian species. Sequence alignment and transcript folding comparisons reveal the rRNA transcript secondary structure. Mammalian ITS regions, though quite long, still fold into the recognizable secondary structure of other eukaryotes. The ITS2 in particular bears the four standard helix loops, and loops II and III have the hallmark characters universal to eukaryotes. Both sequence and insertions/deletions of transcript secondary structure helices observed here support the four superorder taxonomy of Placentalia. On the family level, major unique indels, neatly excising entire helices, will be useful when additional species are represented, resulting in significant further understanding of the details of mammalian evolutionary history. Furthermore, the identification of a highly conserved element of ITS1 common to warm-blooded vertebrates may aid in deciphering the complex mechanism of RNA transcript processing. This is the last major group of terrestrial vertebrates for which rRNA ITS secondary structure has been resolved. PMID:24260162

  4. Characterization of Dermanyssus gallinae (Acarina: Dermanissydae) by sequence analysis of the ribosomal internal transcribed spacer regions.

    PubMed

    Potenza, L; Cafiero, M A; Camarda, A; La Salandra, G; Cucchiarini, L; Dachà, M

    2009-10-01

    In the present work mites previously identified as Dermanyssus gallinae De Geer (Acari, Mesostigmata) using morphological keys were investigated by molecular tools. The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from mites were amplified and sequenced to examine the level of sequence variations and to explore the feasibility of using this region in the identification of this mite. Conserved primers located at the 3'end of 18S and at the 5'start of 28S rRNA genes were used first, and amplified fragments were sequenced. Sequence analyses showed no variation in 5.8S and ITS2 region while slight intraspecific variations involving substitutions as well as deletions concentrated in the ITS1 region. Based on the sequence analyses a nested PCR of the ITS2 region followed by RFLP analyses has been set up in the attempt to provide a rapid molecular diagnostic tool of D. gallinae.

  5. Identification of Aspergillus Species Using Internal Transcribed Spacer Regions 1 and 2

    PubMed Central

    Henry, Travis; Iwen, Peter C.; Hinrichs, Steven H.

    2000-01-01

    Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1–5.8S–ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA. PMID:10747135

  6. Molecular Systematics of Dictyostelids: 5.8S Ribosomal DNA and Internal Transcribed Spacer Region Analyses▿

    PubMed Central

    Romeralo, María; Escalante, Ricardo; Sastre, Leandro; Lado, Carlos

    2007-01-01

    The variability and adaptability of the amoebae from the class Dictyosteliomycetes greatly complicate their systematics. The nucleotide sequences of the ribosomal internal transcribed spacers and the 5.8S ribosomal DNA gene have been determined for 28 isolates, and their utility to discriminate between different species and genera has been shown. PMID:17056743

  7. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food

    PubMed Central

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2015-01-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411. PMID:26981389

  8. Metagenomic data of fungal internal transcribed spacer from serofluid dish, a traditional Chinese fermented food.

    PubMed

    Chen, Peng; Zhao, Yang; Wu, Zhengrong; Liu, Ronghui; Xu, Ruixiang; Yan, Lei; Li, Hongyu

    2016-03-01

    Serofluid dish (or Jiangshui, in Chinese), a traditional food in the Chinese culture for thousands of years, is made from vegetables by fermentation. In this work, microorganism community of the fermented serofluid dish was investigated by the culture-independent method. The metagenomic data in this article contains the sequences of fungal internal transcribed spacer (ITS) regions of rRNA genes from 12 different serofluid dish samples. The metagenome comprised of 50,865 average raw reads with an average of 8,958,220 bp and G + C content is 45.62%. This is the first report on metagenomic data of fungal ITS from serofluid dish employing Illumina platform to profile the fungal communities of this little known fermented food from Gansu Province, China. The Metagenomic data of fungal internal transcribed spacer can be accessed at NCBI, SRA database accession no. SRP067411.

  9. Molecular Identification of Two Strains of Phellinus sp. by Internal Transcribed Spacer Sequence Analysis

    PubMed Central

    2011-01-01

    Two species of cultivated Phellinus sp. were identified as P. baumii by internal transcribed spacer (ITS) sequence analysis. The fruit bodies of the examined strains were similar to those of naturally occurring strains, having a bracket-like form, yellow-to-orange color, and poroid hymenial surfaces. The DNA sequences of ITS region of both strains showed a homology of 99% with ITS1 to ITS2 sequences of P. (Inonotus) baumii strain PB0806. PMID:22783119

  10. Genetic diversity of Ephedra plants in mongolia inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

    PubMed

    Kitani, Yuki; Zhu, Shu; Batkhuu, Javzan; Sanchir, Chinbat; Komatsu, Katsuko

    2011-01-01

    Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I-V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.

  11. Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout

    USGS Publications Warehouse

    Rasmussen, C.; Purcell, M.K.; Gregg, J.L.; LaPatra, S.E.; Winton, J.R.; Hershberger, P.K.

    2010-01-01

    The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of ~740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

  12. Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA.

    PubMed

    Varela, Eduardo S; Lima, João P M S; Galdino, Alexsandro S; Pinto, Luciano da S; Bezerra, Walderly M; Nunes, Edson P; Alves, Maria A O; Grangeiro, Thalles B

    2004-01-01

    The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.

  13. Imidacloprid and Thiamethoxam Induced Mutations in Internal Transcribed Spacer 2 (ITS2) of Anopheles stephensi.

    PubMed

    Bhinder, Preety; Chaudhry, Asha; Barna, Bhupinder; Kaur, Satvinderjeet

    2012-05-01

    The present article deals with the polymerase chain reaction (PCR)-based genotoxicity evaluation of neonicotinoid pesticides, imidacloprid and thiamethoxam, by using the genome of a mosquito Anopheles stephensi taken as an experimental model. After treatment of the second instar larvae with LC(20) of the pesticides for 24 h, the induced nucleotide sequence variations in the internal transcribed spacer 2 (ITS2) of freshly hatched unfed control and treated individuals was studied from the sequence alignment data and the mutations in the form of insertion, deletion and substitution of bases were recorded. Measurable differences, indicative of the genetic damage due to imidacloprid and thiamethoxam were observed when ITS2 sequences of control and treated individuals were compared. It was found that imidacloprid-treated individual had 8 deletions, 29 insertions, 18 transitions and 33 transversions, whereas thiamethoxam-treated individual had 10 deletions, 8 insertions, 47 transitions and 68 transversions.

  14. Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows.

    PubMed

    Vargas-Bello-Pérez, E; Cancino-Padilla, N; Romero, J

    2016-12-01

    Molecular techniques are important tools for microbiological studies in different habitats, and the internal transcribed spacer (ITS) has been proved to be useful for analyzing fungal diversity. The aim of this study was to use the ITS region to generate ruminal yeast profile and to identify ruminal yeast. DNA from ruminal digesta was extracted to amplify the ribosomal ITS region. The profile from the PCR products was visualized and the excised bands from the profile were identified as the genera Millerozyma, Pichia, Rhizomucor and Hyphopichia. Overall, the ITS resulted to be a simple, fast and sensitive approach that allowed profiling and identification of ruminal yeast that have not been previously described (Millerozyma and Hyphopichia) in the rumen microbial community.

  15. Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.

    PubMed

    Coleman, Annette W

    2015-03-01

    rRNA is one of the few universal features of life, making it uniquely suited to assess phylogenetic relationships. The processing of the initial polycistronic rRNA transcript is also a conserved process, involving numerous cleavage events and the generation of secondary structures. The secondary structure of the internal transcribed spacer (ITS) regions of nuclear rRNA transcripts are well known for a wide variety of eukaryotes and have been used to aid in the alignment of these sequences for phylogenetic comparisons. By contrast, study of the processing of the initial rRNA transcripts has been largely limited to yeast, mice, rats, and humans. Here I examine the known cleavage sites in the two ITS regions and their positions relative to the secondary structure. A better understanding of the conservation of secondary structures and cleavage sites within the ITS regions will improve evolutionary inferences based on these sequences.

  16. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1.

    PubMed

    Perera, Omaththage P; Allen, Kerry C; Jain, Devendra; Purcell, Matthew; Little, Nathan S; Luttrell, Randall G

    2015-01-01

    Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

  17. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

    PubMed

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

    2015-10-22

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

  18. Sequence analysis of the ribosomal internal transcribed spacer DNA of the crayfish parasite Psorospermium haeckeli.

    PubMed

    Bangyeekhun, E; Ryynänen, H J; Henttonen, P; Huner, J V; Cerenius, L; Söderhäll, K

    2001-10-08

    Two morphotypes of the crayfish parasite Psorospermium haeckeli were isolated from 2 crayfish species of different geographical origin. The oval-shaped sporocysts were obtained from the epidermal and connective tissue beneath the carapace of the noble crayfish Astacus astacus from Sweden and Finland. Elongated spores were isolated from the abdominal muscle tissue of the red swamp crayfish Procambarus clarkii from USA. To compare genetic divergence of 2 morphotypes of the parasite, the ribosomal internal transcribed spacer (ITS) DNA (ITS 1 and ITS 2) and the 5.8S rRNA gene were cloned and sequenced. The analysed region is variable in length, with the ribosomal ITS sequence of the European morphotype longer than the North American one. Sequence diversity is found mainly in ITS 1 and ITS 2 regions, and there is 66% and 58% similarity between the 2 morphotypes, respectively. Thus, analysis of the ribosomal ITS DNA suggests that P. haeckeli forms obtained from Europe and North America are genetically diverse, which supports the previously reported morphological characteristics.

  19. Internal transcribed spacer sequence phylogeny of Crambe L. (Brassicaceae): molecular data reveal two Old World disjunctions.

    PubMed

    Francisco-Ortega, J; Fuertes-Aguilar, J; Gómez-Campo, C; Santos-Guerra, A; Jansen, R K

    1999-04-01

    Crambe L. (Brassicaceae) is an Old World genus with a disjunct distribution among four major centers of species diversity. A phylogenetic analysis of nucleotide sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal repeat was conducted with 27 species of Crambe and 18 related genera. Cladistic analyses using weighted and unweighted parsimony support Crambe as a monophyletic genus with three major lineages. The first comprises those taxa endemic to the Macaronesian archipelagos. Taxa with a predominant Mediterranean distribution form the second assemblage, and a disjunction between east Africa (C. abyssinica) and the Mediterranean (C. hispanica) occurs in this clade. The third lineage includes all Eurosiberian-Asian taxa and C. kilimandscharica, a species from the highlands of east Africa. A basal biogeographic split between east Africa and Eurasia is present in the third clade. The patterns of relationships in the ITS tree are concordant with known climatic events in northern Africa and southwestern Asia since the middle Miocene. The ITS trees are congruent with the current sectional classification except for a few members of sections Crambe, Leptocrambe, and Orientecrambe (C. cordifolia, C. endentula, C. kilimandscharica, and C. kotschyana). Low levels of support in the basal branches do not allow resolution of which genera of the subtribes Raphaniae or Brassicinae are sister to Crambe. Both subtribes appear to be highly polyphyletic in the ITS trees.

  20. A Phylogenetic Analysis of Heterorhabditis (Nemata: Rhabditidae) Based on Internal Transcribed Spacer 1 DNA Sequence Data

    PubMed Central

    Adams, B. J.; Burnell, A. M.; Powers, T. O.

    1998-01-01

    Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations. PMID:19274196

  1. Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens.

    PubMed

    Brännström, S; Morrison, D A; Mattsson, J G; Chirico, J

    2008-06-01

    We investigated the presence of the poultry red mite or the chicken mite, Dermanyssus gallinae De Geer, Acari: Dermanyssidae, in wild bird populations in four different geographical regions of Sweden. The mites identified as D. gallinae were compared genetically with D. gallinae from egg-producing poultry farms in the same regions. The small subunit (SSU) gene, the 5.8S ribosomal RNA (rRNA) gene and the two internal transcribed spacers (ITS) of the rRNA genes were used in the genetic analysis. All D. gallinae mites had identical SSU rRNA, 5.8S rRNA and ITS2 sequences independent of their origin. By contrast, we identified significant differences in the ITS1 sequences. Based on the differences in the ITS1 sequences, the mites could be divided into two genotypes, of wild and domesticated origin, with no variation within the groups. These results imply that wild bird populations are of low importance, if any, as natural reservoirs of D. gallinae in these four geographical regions of Sweden.

  2. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

    PubMed

    Robideau, Gregg P; De Cock, Arthur W A M; Coffey, Michael D; Voglmayr, Hermann; Brouwer, Henk; Bala, Kanak; Chitty, David W; Désaulniers, Nicole; Eggertson, Quinn A; Gachon, Claire M M; Hu, Chia-Hui; Küpper, Frithjof C; Rintoul, Tara L; Sarhan, Ehab; Verstappen, Els C P; Zhang, Yonghong; Bonants, Peter J M; Ristaino, Jean B; Lévesque, C André

    2011-11-01

    Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.

  3. Molecular phylogenetic analysis of Indonesia Solanaceae based on DNA sequences of internal transcribed spacer region

    NASA Astrophysics Data System (ADS)

    Hidayat, Topik; Priyandoko, Didik; Islami, Dina Karina; Wardiny, Putri Yunitha

    2016-02-01

    Solanaceae is one of largest family in Angiosperm group with highly diverse in morphological character. In Indonesia, this group of plant is very popular due to its usefulness as food, ornamental and medicinal plants. However, investigation on phylogenetic relationship among the member of this family in Indonesia remains less attention. The purpose of this study was to evaluate the phylogenetics relationship of the family especially distributed in Indonesia. DNA sequences of Internal Transcribed Spacer (ITS) region of 19 species of Solanaceae and three species of outgroup, which belongs to family Convolvulaceae, Apocynaceae, and Plantaginaceae, were isolated, amplified, and sequenced. Phylogenetic tree analysis based on parsimony method was conducted with using data derived from the ITS-1, 5.8S, and ITS-2, separately, and the combination of all. Results indicated that the phylogenetic tree derived from the combined data established better pattern of relationship than separate data. Thus, three major groups were revealed. Group 1 consists of tribe Datureae, Cestreae, and Petunieae, whereas group 2 is member of tribe Physaleae. Group 3 belongs to tribe Solaneae. The use of the ITS region as a molecular markers, in general, support the global Solanaceae relationship that has been previously reported.

  4. Determination of internal transcribed spacer regions (ITS) in Trichomonas vaginalis isolates and differentiation among Trichomonas species.

    PubMed

    Ibáñez-Escribano, Alexandra; Nogal-Ruiz, Juan José; Arán, Vicente J; Escario, José Antonio; Gómez-Barrio, Alicia; Alderete, J F

    2014-04-01

    The nucleotide sequence of the 5.8S rRNA gene and the flanked internal transcribed spacer (ITS) regions of six Trichomonas vaginalis isolates with different metronidazole sensitivity and geographic origin were genotyped. A multiple sequence alignment was performed with different sequences of other isolates available at the GenBank/EMBL/DDBJ databases, which revealed 5 different sequence patterns. Although a stable mutation in position 66 of the ITS1 (C66T) was observed in 26% (9/34) of the T. vaginalis sequences analyzed, there was 99.7% ITS nucleotide sequence identity among isolates for this sequence. The nucleotide sequence variation among other species of the genus Trichomonas ranged from 3.4% to 9.1%. Surprisingly, the % identity between T. vaginalis and Pentatrichomonas hominis was ~83%. There was >40% divergence in the ITS sequence between T. vaginalis and Tritrichomonas spp., including Tritrichomonas augusta, Tritrichomonas muris, and Tritrichomonas nonconforma and with Tetratrichomonas prowazeki. Dendrograms grouped the trichomonadid sequences in robust clades according to their genera. The absence of nucleotide divergence in the hypervariable ITS regions between T. vaginalis isolates suggests the early divergence of the parasite. Importantly, these data show this ITS1-5.8S rRNA-ITS2 region suitable for inter-species differentiation.

  5. Identification of dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence.

    PubMed

    Takamiya, Tomoko; Wongsawad, Pheravut; Tajima, Natsuko; Shioda, Nao; Lu, Jun Feng; Wen, Chi Luan; Wu, Jin Bin; Handa, Takashi; Iijima, Hiroshi; Kitanaka, Susumu; Yukawa, Tomohisa

    2011-01-01

    Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).

  6. Internal Transcribed Spacer 1 (ITS1) based sequence typing reveals phylogenetically distinct Ascaris population.

    PubMed

    Das, Koushik; Chowdhury, Punam; Ganguly, Sandipan

    2015-01-01

    Taxonomic differentiation among morphologically identical Ascaris species is a debatable scientific issue in the context of Ascariasis epidemiology. To explain the disease epidemiology and also the taxonomic position of different Ascaris species, genome information of infecting strains from endemic areas throughout the world is certainly crucial. Ascaris population from human has been genetically characterized based on the widely used genetic marker, internal transcribed spacer1 (ITS1). Along with previously reported and prevalent genotype G1, 8 new sequence variants of ITS1 have been identified. Genotype G1 was significantly present among female patients aged between 10 to 15 years. Intragenic linkage disequilibrium (LD) analysis at target locus within our study population has identified an incomplete LD value with potential recombination events. A separate cluster of Indian isolates with high bootstrap value indicate their distinct phylogenetic position in comparison to the global Ascaris population. Genetic shuffling through recombination could be a possible reason for high population diversity and frequent emergence of new sequence variants, identified in present and other previous studies. This study explores the genetic organization of Indian Ascaris population for the first time which certainly includes some fundamental information on the molecular epidemiology of Ascariasis.

  7. Sequence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species.

    PubMed

    Aktas, Münir; Bendele, Kylie G; Altay, Kürsat; Dumanli, Nazir; Tsuji, Masayoshi; Holman, Patricia J

    2007-07-20

    The genomic region spanning the two ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was cloned and sequenced from sixteen Theileria isolates. Each Theileria species possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among ruminant Theileria species. The spacers were most polymorphic in the agent of tropical theileriosis, Theileria annulata, and were more conserved in two benign species, Theileria buffeli and Theileria sergenti Chitose. Phylogenetic analysis of the rDNA ITS1-5.8S rRNA gene-ITS2 region clearly separated each taxon, placing them in three clusters. One held T. annulata, Theileria parva, and Theileria mutans, with the latter two most closely related. The second held T. sergenti Ikeda, T. sergenti Chitose, and T. buffeli, with the latter two most closely related. The third cluster held the Theileria ovis isolates.

  8. Definition of Eight Mulberry Species in the Genus Morus by Internal Transcribed Spacer-Based Phylogeny.

    PubMed

    Zeng, Qiwei; Chen, Hongyu; Zhang, Chao; Han, Minjing; Li, Tian; Qi, Xiwu; Xiang, Zhonghuai; He, Ningjia

    2015-01-01

    Mulberry, belonging to the order Rosales, family Moraceae, and genus Morus, has received attention because of both its economic and medicinal value, as well as for its important ecological function. The genus Morus has a worldwide distribution, however, its taxonomy remains complex and disputed. Many studies have attempted to classify Morus species, resulting in varied numbers of designated Morus spp. To address this issue, we used information from internal transcribed spacer (ITS) genetic sequences to study the taxonomy of all the members of generally accepted genus Morus. We found that intraspecific 5.8S rRNA sequences were identical but that interspecific 5.8S sequences were diverse. M. alba and M. notabilis showed the shortest (215 bp) and the longest (233 bp) ITS1 sequence length, respectively. With the completion of the mulberry genome, we could identify single nucleotide polymorphisms within the ITS locus in the M. notabilis genome. From reconstruction of a phylogenetic tree based on the complete ITS data, we propose that the Morus genus should be classified into eight species, including M. alba, M. nigra, M. notabilis, M. serrata, M. celtidifolia, M. insignis, M. rubra, and M. mesozygia. Furthermore, the classification of the ITS sequences of known interspecific hybrid clones into both paternal and maternal clades indicated that ITS variation was sufficient to distinguish interspecific hybrids in the genus Morus.

  9. Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas.

    PubMed

    Taylor, D Lee; McCormick, Melissa K

    2008-01-01

    Despite advances owing to molecular approaches, several hurdles still obstruct the identification of fungi forming orchid mycorrhizas. The Tulasnellaceae exhibit accelerated evolution of the nuclear ribosomal operon, causing most standard primers to fail in polymerase chain reaction (PCR) trials. Insufficient sequences are available from well characterized isolates and fruitbodies. Lastly, taxon-specific PCR primers are needed in order to explore the ecology of the fungi outside of the orchid root. Here, progress in overcoming these hurdles is reported. Broad-spectrum basidiomycete internal transcribed spacer (ITS) primers that do not exclude most known Tulasnellaceae are presented. blast searches and empirical PCR tests support their wide utility within the Basidiomycota. Taxon-specific ITS primers are presented targeted to orchid-associated Tulasnella, and a core component of the Thelephora-Tomentella complex. The efficiency and selectivity of these primer sets are again supported by blast searches and empirical tests. Lastly, ITS DNA sequences are presented from several strains of Epulorhiza, Ceratorhiza, Ceratobasidium, Sistotrema, Thanatephorus and Tulasnella that were originally described in the landmark mycorrhizal studies of Currah and Warcup. Detailed phylogenetic analyses reveal some inconsistencies in species concepts in these taxonomically challenging resupinate basidiomycetes, but also help to place several sequences from environmental samples.

  10. Internal transcribed spacer guided multiplex PCR for species identification of Convolvulus prostratus and Evolvulus alsinoides

    PubMed Central

    Sharma, Sonal; Shrivastava, Neeta

    2016-01-01

    Shankhpushpi is a reputed drug from an Indian system of medicine for treating mental disorders and enhancing memory. Two herbs, namely Convolvulus prostratus Forssk. and Evolvulus alsinoides (L.) L., are commonly known as Shankhpushpi. Ambiguous vernacular identity can affect the scientific validity of the Shankpushpi-based herbal drug therapy. In the present investigation, a novel and sensitive multiplex PCR method based on polymorphism in the internal transcribed spacer (ITS) region was developed to establish the molecular identity of C. prostratus and E. alsinoides. DNA was isolated and the ITS region was amplified, sequenced and assembled. Sequences were aligned to identify variable nucleotides in order to develop plant-specific primers. Primers were validated in singleplex reactions and eventually a multiplex assay was developed. This assay was tested for sensitivity and validated by amplifying DNA isolated from the simulated blended powdered plant material. Primers developed for C. prostratus resulted into a 200 bp amplicon and 596 bp for E. alsinoides. The assay was found to be sensitive enough for amplification of low quantities of DNA. The method can detect 10% of the mixing of plants with each other in blended material. This PCR assay can be used for rapid botanical identification of Shankhpushpi plant materials and will improve evidence-based herbal drug therapy. PMID:27175337

  11. Endangered Uyghur Medicinal Plant Ferula Identification through the Second Internal Transcribed Spacer

    PubMed Central

    Fan, Congzhao; Li, Xiaojin; Zhu, Jun; Song, Jingyuan; Yao, Hui

    2015-01-01

    The medicinal plant Ferula has been widely used in Asian medicine, especially in Uyghur medicine in Xinjiang, China. Given that various substitutes and closely related species have similar morphological characteristics, Ferula is difficult to distinguish based on morphology alone, thereby causing confusion and threatening the safe use of Ferula. In this study, internal transcribed spacer 2 (ITS2) sequences were analyzed and assessed for the accurate identification of two salable Ferula species (Ferula sinkiangensis and Ferula fukangensis) and eight substitutes or closely related species. Results showed that the sequence length of ITS2 ranged from 451 bp to 45 bp, whereas guanine and cytosine contents (GC) were from 53.6% to 56.2%. A total of 77 variation sites were detected, including 63 base mutations and 14 insertion/deletion mutations. The ITS2 sequence correctly identified 100% of the samples at the species level using the basic local alignment search tool 1 and nearest-distance method. Furthermore, neighbor-joining tree successfully identified the genuine plants F. sinkiangensis and F. fukangensis from their succedaneum and closely related species. These results indicated that ITS2 sequence could be used as a valuable barcode to distinguish Uyghur medicine Ferula from counterfeits and closely related species. This study may broaden DNA barcoding application in the Uyghur medicinal plant field. PMID:26120347

  12. Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts

    PubMed Central

    Ciardo, D. E.; Schär, G.; Böttger, E. C.; Altwegg, M.; Bosshard, P. P.

    2006-01-01

    In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region. PMID:16390952

  13. Heterogeneity of the internal transcribed spacer region in Leishmania tropica isolates from southern Iran.

    PubMed

    Ghatee, Mohammad Amin; Sharifi, Iraj; Kuhls, Katrin; Kanannejad, Zahra; Harandi, Majid Fasihi; de Almeida, Marcos E; Hatam, Gholamreza; Mirhendi, Hossein

    2014-09-01

    Most of cutaneous leishmaniasis cases occur in only 7 countries, including Iran. Leishmania tropica is the main cause of anthroponotic cutaneous leishmaniasis in Iran. In order to study the heterogeneity and phylogeny of L. tropica in southern Iran, a total of 61 isolates were obtained from Bam district and the cities Kerman and Shiraz. The internal transcribed spacer (ITS) from the ribosomal DNA locus was amplified and then analysed by sequencing. Analysis of the ITS sequences showed four haplotypes in the isolates, including 3 haplotypes among the 58 isolates from the south eastern region, including Bam district and Kerman city, and 2 haplotypes among the 3 isolates from Shiraz city. The results showed a monophyletic structure for the south eastern population. In comparison to GenBank sequences of L. tropica from different countries, most of the southeast Iranian and Indian isolates are comprised in one cluster, while isolates from other countries and few other Iranian isolates group in a different cluster. Analysis of ITS sequences of south eastern L. tropica showed a homogeneous population which could be the basis for other molecular epidemiology studies using more discriminative markers and tracing possible changes in the population structure of L. tropica.

  14. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  15. Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR

    PubMed Central

    Couto, Isabel; Pereira, Sandro; Miragaia, Maria; Sanches, Ilda Santos; de Lencastre, Hermínia

    2001-01-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems. PMID:11526135

  16. Internal transcribed spacer 2 barcode: a good tool for identifying Acanthopanacis cortex

    PubMed Central

    Zhao, Sha; Chen, Xiaochen; Song, Jingyuan; Pang, Xiaohui; Chen, Shilin

    2015-01-01

    Acanthopanacis cortex has been used in clinical applications for a long time. Considering some historical and geographical factors, Acanthopanacis cortex is easily confused with other herbs in medicine markets, thereby causing potential safety issues. In this study, we used the internal transcribed spacer 2 (ITS2) barcode to identify 69 samples belonging to six species, including Acanthopanacis cortex and its adulterants. The nearest distance, single-nucleotide polymorphisms (SNPs), and neighbor-joining (NJ) tree methods were used to evaluate the identification ability of the ITS2 barcode. According to the kimura-2-parameter model, the intraspecific distance of Eleutherococcus nodiflorus ITS2 sequences ranged from 0 to 0.0132. The minimum interspecific distance between E. nodiflorus and E. giraldii was 0.0221, which was larger than the maximum intraspecific distance of E. nodiflorus. Three stable SNPs in ITS2 can be used to distinguish Acanthopanacis cortex and its closely related species. The NJ tree indicated that the Acanthopanacis cortex samples clustered into one clade, which can be distinguished clearly from the adulterants of this herb. A secondary structure of ITS2 provided another dimensionality to identify species. In conclusion, the ITS2 barcode effectively identifies Acanthopanacis cortex, and DNA barcoding is a convenient tool for medicine market supervision. PMID:26500674

  17. Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region.

    PubMed

    Chiu, Hua-Hsien

    2007-04-01

    The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.

  18. Molecular analysis of fungal populations in patients with oral candidiasis using internal transcribed spacer region.

    PubMed

    Ieda, Shinsuke; Moriyama, Masafumi; Takeshita, Toru; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection.

  19. Identification of Medically Important Yeast Species by Sequence Analysis of the Internal Transcribed Spacer Regions

    PubMed Central

    Leaw, Shiang Ning; Chang, Hsien Chang; Sun, Hsiao Fang; Barton, Richard; Bouchara, Jean-Philippe; Chang, Tsung Chain

    2006-01-01

    Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods. PMID:16517841

  20. Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA.

    PubMed

    Goodwin, S B; Dunkle, L D; Zismann, V L

    2001-07-01

    ABSTRACT Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting that the ability to produce this compound had a single evolutionary origin. Intraspecific variation for 25 taxa in the Mycosphaerella clade averaged 1.7 nucleotides (nts) in the ITS region. Thus, isolates with ITS sequences that differ by two or more nucleotides may be distinct species. ITS sequences of groups I and II of the gray leaf spot pathogen Cercospora zeae-maydis differed by 7 nts and clearly represent different species. There were 6.5 nt differences on average between the ITS sequences of the sorghum pathogen Cercospora sorghi and the maize pathogen Cercospora sorghi var. maydis, indicating that the latter is a separate species and not simply a variety of Cercospora sorghi. The large monophyletic Mycosphaerella cluster contained a number of anamorph genera with no known teleomorph associations. Therefore, the number of anamorph genera related to Mycosphaerella may be much larger than suspected previously.

  1. Molecular Analysis of Fungal Populations in Patients with Oral Candidiasis Using Internal Transcribed Spacer Region

    PubMed Central

    Ieda, Shinsuke; Moriyama, Masafumi; Takashita, Toru; Maehara, Takashi; Imabayashi, Yumi; Shinozaki, Shoichi; Tanaka, Akihiko; Hayashida, Jun-Nosuke; Furukawa, Sachiko; Ohta, Miho; Yamashita, Yoshihisa; Nakamura, Seiji

    2014-01-01

    Oral candidiasis is closely associated with changes in the oral fungal flora and is caused primarily by Candida albicans. Conventional methods of fungal culture are time-consuming and not always conclusive. However, molecular genetic analysis of internal transcribed spacer (ITS) regions of fungal rRNA is rapid, reproducible and simple to perform. In this study we examined the fungal flora in patients with oral candidiasis and investigated changes in the flora after antifungal treatment using length heterogeneity-polymerization chain reaction (LH-PCR) analysis of ITS regions. Fifty-two patients with pseudomembranous oral candidiasis (POC) and 30 healthy controls were included in the study. Fungal DNA from oral rinse was examined for fungal species diversity by LH-PCR. Fungal populations were quantified by real-time PCR and previously-unidentified signals were confirmed by nucleotide sequencing. Relationships between the oral fungal flora and treatment-resistant factors were also examined. POC patients showed significantly more fungal species and a greater density of fungi than control individuals. Sixteen fungi were newly identified. The fungal populations from both groups were composed predominantly of C. albicans, though the ratio of C. dubliniensis was significantly higher in POC patients than in controls. The diversity and density of fungi were significantly reduced after treatment. Furthermore, fungal diversity and the proportion of C. dubliniensis were positively correlated with treatment duration. These results suggest that C. dubliniensis and high fungal flora diversity might be involved in the pathogenesis of oral candidiasis. We therefore conclude that LH-PCR is a useful technique for diagnosing and assessing the severity of oral candidal infection. PMID:24979710

  2. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    PubMed Central

    Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

    2012-01-01

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

  3. Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting

    PubMed Central

    2014-01-01

    Background Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. Results We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. Conclusions We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference

  4. Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA.

    PubMed

    Lin, Q; Li, H M; Gao, M; Wang, X Y; Ren, W X; Cong, M M; Tan, X C; Chen, C X; Yu, S K; Zhao, G H

    2012-03-01

    In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3' end of 18S rDNA, complete ITS-1 rDNA, and 5' end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.

  5. Nucleotide sequence of the internal transcribed spacers and 5.8S region of ribosomal DNA in Pinus pinea L.

    PubMed

    Marrocco, R; Gelati, M T; Maggini, F

    1996-01-01

    The nucleotide sequence of the first internal transcribed spacer (ITS1) belonging to different ribosomal RNA genes from Pinus pinea are reported. The analyzed ITS1 can be distinguished on the basis of their length, being one 2631 bp and the other 271 bp long. Nucleotide comparison of these regions did not show appreciable sequence homology. The larger ITS1 contains five tandem arranged subrepeats with size ranging between 219 bp and 237 bp. The nucleotide sequence of the 5.8S and the ITS2 regions belonging to the larger ribosomal RNA gene are also reported.

  6. Identification of fungi based on the nucleotide sequence homology of their internal transcribed spacer 1 (ITS1) region.

    PubMed

    Narutaki, Shoji; Takatori, Kosuke; Nishimura, Hidekatsu; Terashima, Hiroshi; Sasaki, Tsuguo

    2002-01-01

    In this study, we examined the identification of fungi based on the sequence homology of the internal transcribed spacer 1 (ITS1) region. A newly designed primer pair could amplify the target region of all 42 strains tested. The PCR products were sequenced and the sequence homologies were searched by BLAST. It was demonstrated that this method is a reliable identification method at the genus or species level. At present, available databases are still insufficient to identify some fungi, but with the accumulation of further data in the ITS1 database, this method will be available for the identification of fungi.

  7. Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure.

    PubMed

    Campbell, Christopher S; Wright, Wesley A; Cox, Margaret; Vining, Thomas F; Major, C Smoot; Arsenault, Matthew P

    2005-04-01

    The nrDNA ITS1 of Picea is 2747-3271 bp, the longest known of all plants. We obtained 24 cloned ITS1 sequences from six individuals of Picea glehnii, Picea mariana, Picea orientalis, and Picea rubens. Mean sequence divergence within these individuals (0.018+/-0.009) is more than half that between the species (0.031+/-0.011) and may be maintained against concerted evolution by separation of Picea 18S-26S rDNA repeats on multiple chromosomes. Picea ITS1 contains three subrepeats with a motif (5'-GGCCACCCTAGTC) that is conserved across Pinaceae. Two subrepeats are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. Coalescence of ITS1 sequences occurs in P. orientalis but not in the other species.

  8. Molecular Systematics of Genus Atractylodes (Compositae, Cardueae): Evidence from Internal Transcribed Spacer (ITS) and trnL-F Sequences

    PubMed Central

    Peng, Hua-Sheng; Yuan, Qing-Jun; Li, Qian-Quan; Huang, Lu-Qi

    2012-01-01

    To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis. PMID:23203084

  9. Molecular systematics of Genus Atractylodes (Compositae, Cardueae): evidence from Internal Transcribed Spacer (ITS) and trnL-F sequences.

    PubMed

    Peng, Hua-Sheng; Yuan, Qing-Jun; Li, Qian-Quan; Huang, Lu-Qi

    2012-11-09

    To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.

  10. Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences▿ †

    PubMed Central

    Alvarado, Pablo; Manjón, Jose L.

    2009-01-01

    Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs. PMID:19465521

  11. Phylogenetic relationships of the Hamamelidaceae inferred from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA.

    PubMed

    Li, J; Bogle, A L; Klein, A S

    1999-07-01

    Intergeneric relationships in the Hamamelidaceae have long been controversial. In this study, sequences of the internal transcribed spacers of nuclear ribosomal DNA were used to reconstruct the phylogeny for the Hamamelidaceae. Three major clades were recognized in the ITS-based phylogenetic tree: (1) Mytilaria-Exbucklandia-Rhodoleia, (2) Disanthus, and (3) the Hamamelidoideae. Within the Hamamelidoideae there were three well-supported lineages: (1) Corylopsis-Loropetalum-Tetrathyrium-Maingaya-Matudaea, (2) Eustigmateae sensu Endress, plus Molinadendron-Dicoryphinae, and (3) Hamamelis-Fothergilleae sensu Endress, excluding Matudaea and Molinadendron. The Exbucklandioideae sensu Endress were not monophyletic, nor were the tribes in the Hamamelidoideae in their current circumscriptions except for the Corylopsideae. Strap-shaped petals, apetaly, and wind pollination have evolved three times independently in the Hamamelidaceae s.s. (Hamamelidaceae minus Altingioideae), suggesting that homoplasy should be considered in future classifications of the family.

  12. Detection of multiple infections by Monocystis strains in a single earthworm host using ribosomal internal transcribed spacer sequence variation.

    PubMed

    Velavan, T P; Schulenburg, H; Michiels, N K

    2010-01-01

    Monocystis sp. are sporocyst-forming apicomplexan parasites common in seminal vesicles of the earthworm Lumbricus terrestris where they may account for temporary castration. This study describes the internal transcribed spacer (ITS) region of the ribosomal cistron of Monocystis sp. This region, including ITS-1, the 5.8S ribosomal RNA gene, and ITS-2, was PCR amplified, cloned, and sequenced for Monocystis sp. isolated from the seminal vesicles of several wild-caught L. terrestris. Our analysis revealed substantial polymorphisms, also within single host organisms, indicating intra-host diversity of parasites. These genetic markers are the first that allow distinction of Monocystis sp. genotypes, opening new avenues for the study of parasite diversity within and between hosts.

  13. Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA

    PubMed Central

    Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying; Liu, Chang; Lv, Aiping; Deng, Youping; Larson-Rabin, Zachary; Wilkinson, Mike; Chen, Shilin

    2012-01-01

    Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification. PMID:22952830

  14. Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

    PubMed

    Abbott, Cathryn L; Gilmore, Scott R; Lowe, Geoff; Meyer, Gary; Bower, Susan

    2011-02-22

    Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

  15. Internal phylogeny of the Chilopoda (Myriapoda, Arthropoda) using complete 18S rDNA and partial 28S rDNA sequences.

    PubMed Central

    Giribet, G; Carranza, S; Riutort, M; Baguñà, J; Ribera, C

    1999-01-01

    The internal phylogeny of the 'myriapod' class Chilopoda is evaluated for 12 species belonging to the five extant centipede orders, using 18S rDNA complete gene sequence and 28S rDNA partial gene sequence data. Equally and differentially weighted parsimony, neighbour-joining and maximum-likelihood were used for phylogenetic reconstruction, and bootstrapping and branch support analyses were performed to evaluate tree topology stability. The results show that the Chilopoda constitute a monophyletic group that is divided into two lines, Notostigmophora (= Scutigeromorpha) and Pleurostigmophora, as found in previous morphological analyses. The Notostigmophora are markedly modified for their epigenic mode of life. The first offshoot of the Pleurostigmophora are the Lithobiomorpha, followed by the Craterostigmomorpha and by the Epimorpha s. str. (= Scolopendromorpha + Geophilomorpha), although strong support for the monophyly of the Epimorpha s. lat. (= Craterostigmomorpha + Epimorpha s. str.) is only found in the differentially weighted parsimony analysis. PMID:10087567

  16. Genetic diversity and molecular evolution of Naga King Chili inferred from internal transcribed spacer sequence of nuclear ribosomal DNA.

    PubMed

    Kehie, Mechuselie; Kumaria, Suman; Devi, Khumuckcham Sangeeta; Tandon, Pramod

    2016-02-01

    Sequences of the Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNAs were explored to study the genetic diversity and molecular evolution of Naga King Chili. Our study indicated the occurrence of nucleotide polymorphism and haplotypic diversity in the ITS regions. The present study demonstrated that the variability of ITS1 with respect to nucleotide diversity and sequence polymorphism exceeded that of ITS2. Sequence analysis of 5.8S gene revealed a much conserved region in all the accessions of Naga King Chili. However, strong phylogenetic information of this species is the distinct 13 bp deletion in the 5.8S gene which discriminated Naga King Chili from the rest of the Capsicum sp. Neutrality test results implied a neutral variation, and population seems to be evolving at drift-mutation equilibrium and free from directed selection pressure. Furthermore, mismatch analysis showed multimodal curve indicating a demographic equilibrium. Phylogenetic relationships revealed by Median Joining Network (MJN) analysis denoted a clear discrimination of Naga King Chili from its closest sister species (Capsicum chinense and Capsicum frutescens). The absence of star-like network of haplotypes suggested an ancient population expansion of this chili.

  17. Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

    PubMed

    Booton, G C; Kaufman, L; Chandler, M; Oguto-Ohwayo, R; Duan, W; Fuerst, P A

    1999-03-01

    The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

  18. Phylogenetics of Bonamia parasites based on small subunit and internal transcribed spacer region ribosomal DNA sequence data.

    PubMed

    Hill, Kristina M; Stokes, Nancy A; Webb, Stephen C; Hine, P Mike; Kroeck, Marina A; Moore, James D; Morley, Margaret S; Reece, Kimberly S; Burreson, Eugene M; Carnegie, Ryan B

    2014-07-24

    The genus Bonamia (Haplosporidia) includes economically significant oyster parasites. Described species were thought to have fairly circumscribed host and geographic ranges: B. ostreae infecting Ostrea edulis in Europe and North America, B. exitiosa infecting O. chilensis in New Zealand, and B. roughleyi infecting Saccostrea glomerata in Australia. The discovery of B. exitiosa-like parasites in new locations and the observation of a novel species, B. perspora, in non-commercial O. stentina altered this perception and prompted our wider evaluation of the global diversity of Bonamia parasites. Samples of 13 oyster species from 21 locations were screened for Bonamia spp. by PCR, and small subunit and internal transcribed spacer regions of Bonamia sp. ribosomal DNA were sequenced from PCR-positive individuals. Infections were confirmed histologically. Phylogenetic analyses using parsimony and Bayesian methods revealed one species, B. exitiosa, to be widely distributed, infecting 7 oyster species from Australia, New Zealand, Argentina, eastern and western USA, and Tunisia. More limited host and geographic distributions of B. ostreae and B. perspora were confirmed, but nothing genetically identifiable as B. roughleyi was found in Australia or elsewhere. Newly discovered diversity included a Bonamia sp. in Dendostrea sandvicensis from Hawaii, USA, that is basal to the other Bonamia species and a Bonamia sp. in O. edulis from Tomales Bay, California, USA, that is closely related to both B. exitiosa and the previously observed Bonamia sp. from O. chilensis in Chile.

  19. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer

    PubMed Central

    Ruegger, Paul M.; Clark, Robin T.; Weger, John R.; Braun, Jonathan; Borneman, James

    2014-01-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses. PMID:25034229

  20. Application of Partial Internal Transcribed Spacer Sequences for the Discrimination of Artemisia capillaris from Other Artemisia Species

    PubMed Central

    Doh, Eui Jeong; Paek, Seung-Ho; Lee, Guemsan; Lee, Mi-Young; Oh, Seung-Eun

    2016-01-01

    Several Artemisia species are used as herbal medicines including the dried aerial parts of Artemisia capillaris, which are used as Artemisiae Capillaris Herba (known as “Injinho” in Korean medicinal terminology and “Yin Chen Hao” in Chinese). In this study, we developed tools for distinguishing between A. capillaris and 11 other Artemisia species that grow and/or are cultured in China, Japan, and Korea. Based on partial nucleotide sequences in the internal transcribed spacer (ITS) that differ between the species, we designed primers to amplify a DNA marker for A. capillaris. In addition, to detect other Artemisia species that are contaminants of A. capillaris, we designed primers to amplify DNA markers of A. japonica, A. annua, A. apiacea, and A. anomala. Moreover, based on random amplified polymorphic DNA analysis, we confirmed that primers developed in a previous study could be used to identify Artemisia species that are sources of Artemisiae Argyi Folium and Artemisiae Iwayomogii Herba. By using these primers, we found that multiplex polymerase chain reaction (PCR) was a reliable tool to distinguish between A. capillaris and other Artemisia species and to identify other Artemisia species as contaminants of A. capillaris in a single PCR. PMID:27313651

  1. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.

    PubMed

    Ruegger, Paul M; Clark, Robin T; Weger, John R; Braun, Jonathan; Borneman, James

    2014-10-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses.

  2. Preliminary analysis of length and GC content variation in the ribosomal first internal transcribed spacer (ITS1) of marine animals.

    PubMed

    Chow, S; Ueno, Y; Toyokawa, M; Oohara, I; Takeyama, H

    2009-01-01

    Length and guanine-cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.

  3. Variation of the internal transcribed spacer 1 sequence within individual strains and among different strains of Neospora caninum.

    PubMed

    Gondim, Luis F P; Laski, Paul; Gao, Liying; McAllister, Milton M

    2004-02-01

    Small differences have been reported in the internal transcribed spacer 1 (ITS1) region among strains of Neospora caninum. We compared ITS1 sequences among 6 N. caninum strains analyzed in our laboratory, including 2 strains that have not been examined previously (NC-Illinois and NC-Bahia). Five sequences showed 100% similarity and also were identical to 7 of 11 sequences that were previously reported by others. In contrast, initial attempts to sequence the ITS1 of NC-Bahia generated 12 nucleotide differences compared with the other 5 strains, and several ambiguous bases. However, the single band containing the ITS1 region, as observed after electrophoresis on a 2% agarose gel, became divided into 2 distinct bands when reanalyzed using 5 or 10% polyacrylamide gel electrophoresis (PAGE), and the ITS1 within these separate bands were sequenced without ambiguity. The other 5 N. caninum strains were also reexamined using PAGE, and in each strain 2 distinct bands were discovered. In comparison, 2 strains of Toxoplasma gondii continued to show only 1 band when examined using PAGE. The ITS1 sequence of NC-Bahia, from Brazil, differs in several base pairs from those of North American and European strains of N. caninum. Intrastrain variation of the ITS1 region appears to be common in N. caninum, in contrast to T. gondii.

  4. Conservation in the first internal transcribed spacer (ITS1) region of Hematodinium perezi (genotype III) from Callinectes sapidus .

    PubMed

    Pagenkopp Lohan, Katrina M; Small, Hamish J; Shields, Jeffrey D; Place, Allen R; Reece, Kimberly S

    2013-03-13

    Hematodinium spp. infections have been reported from blue crabs Callinectes sapidus in high-salinity waters of the USA from New Jersey to Texas. Recently, H. perezi (genotype III) has been proposed as the parasite species and genotype infecting blue crabs from Virginia; however, it is unknown whether this same genotype is present in blue crabs from other locations. To address this question, we collected 317 blue crabs from Massachusetts, Virginia, Georgia, Florida, Louisiana, and Texas to test for the presence of H. perezi (III) using a specific PCR assay targeting the first internal transcribed spacer (ITS1) region of the ribosomal RNA gene complex. To examine the genetic variation within H. perezi (III), ITS1 region sequences from the parasite in blue crabs from multiple locations were compared to each other and to those of H. perezi (III) found in alternate hosts from Virginia. In total, 34 distinct ITS1 sequence variants of the parasite were identified from blue crabs alone, and 38 distinct variants were identified when alternate hosts were included. However, a single ITS1 sequence variant appeared in all geographic regions and hosts, and also in blue crabs sampled from a previous study. The high similarity among all the ITS1 region sequences examined (>98%) and the observation of a single variant found throughout a large geographic range, strongly suggests that a single species and genotype of Hematodinium, specifically H. perezi (III), infects blue crabs from Virginia to Texas and multiple alternate host species in Virginia.

  5. Genetic variability in Melipona quinquefasciata (Hymenoptera, Apidae, Meliponini) from northeastern Brazil determined using the first internal transcribed spacer (ITS1).

    PubMed

    Pereira, J O P; Freitas, B M; Jorge, D M M; Torres, D C; Soares, C E A; Grangeiro, T B

    2009-01-01

    Melipona quinquefasciata is a ground-nesting South American stingless bee whose geographic distribution was believed to comprise only the central and southern states of Brazil. We obtained partial sequences (about 500-570 bp) of first internal transcribed spacer (ITS1) nuclear ribosomal DNA from Melipona specimens putatively identified as M. quinquefasciata collected from different localities in northeastern Brazil. To confirm the taxonomic identity of the northeastern samples, specimens from the state of Goiás (Central region of Brazil) were included for comparison. All sequences were deposited in GenBank (accession numbers EU073751-EU073759). The mean nucleotide divergence (excluding sites with insertions/deletions) in the ITS1 sequences was only 1.4%, ranging from 0 to 4.1%. When the sites with insertions/deletions were also taken into account, sequence divergences varied from 0 to 5.3%. In all pairwise comparisons, the ITS1 sequence from the specimens collected in Goiás was most divergent compared to the ITS1 sequences of the bees from the other locations. However, neighbor-joining phylogenetic analysis showed that all ITS1 sequences from northeastern specimens along with the sample of Goiás were resolved in a single clade with a bootstrap support of 100%. The ITS1 sequencing data thus support the occurrence of M. quinquefasciata in northeast Brazil.

  6. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

    PubMed

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-06-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

  7. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2014-11-26

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. In this study, co-dominant ITS PCR-RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR-RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR-RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area.

  8. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction–restriction fragment length polymorphism

    PubMed Central

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2015-01-01

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) techniques. In this study, co-dominant ITS PCR–RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR–RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR–RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area. PMID:25429047

  9. Multiple origins and nrDNA internal transcribed spacer homeologue evolution in the Glycine tomentella (Leguminosae) allopolyploid complex.

    PubMed Central

    Rauscher, Jason T; Doyle, Jeff J; Brown, A H D

    2004-01-01

    Despite the importance of polyploidy in the evolution of plants, patterns of molecular evolution and genomic interactions following polyploidy are not well understood. Nuclear ribosomal DNA is particularly complex with respect to these genomic interactions. The composition of nrDNA tandem arrays is influenced by intra- and interlocus concerted evolution and their expression is characterized by patterns such as nucleolar dominance. To understand these complex interactions it is important to study them in diverse natural polyploid systems. In this study we use direct sequencing to isolate and characterize nrDNA internal transcribed spacer (ITS) homeologues from multiple accessions of six different races in the Glycine tomentella allopolyploid complex. The results indicate that in most allopolyploid accessions both homeologous nrDNA repeats are present, but that there are significant biases in copy number toward one homeologue, possibly resulting from interlocus concerted evolution. The predominant homeologue often differs between races and between accessions within a race. A phylogenetic analysis of ITS sequences provides evidence for multiple origins in several of the polyploid races. This evidence for diverse patterns of nrDNA molecular evolution and multiple origins of polyploid races will provide a useful system for future studies of natural variation in patterns of nrDNA expression. PMID:15020482

  10. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    PubMed Central

    Lindner, Daniel L; Carlsen, Tor; Henrik Nilsson, R; Davey, Marie; Schumacher, Trond; Kauserud, Håvard

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions. PMID:23789083

  11. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The IT...

  12. DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

    PubMed

    Wipf, D; Munch, J C; Botton, B; Buscot, F

    1996-09-01

    The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.

  13. High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

    PubMed Central

    Janse, Ingmar; Meima, Marion; Kardinaal, W. Edwin A.; Zwart, Gabriel

    2003-01-01

    For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed. PMID:14602623

  14. Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio.

    PubMed

    Minamoto, Toshifumi; Uchii, Kimiko; Takahara, Teruhiko; Kitayoshi, Takumi; Tsuji, Satsuki; Yamanaka, Hiroki; Doi, Hideyuki

    2017-03-01

    The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.

  15. Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications for molecular diagnosis in clinical microbiology laboratories.

    PubMed

    Woo, Patrick C Y; Leung, Shui-Yee; To, Kelvin K W; Chan, Jasper F W; Ngan, Antonio H Y; Cheng, Vincent C C; Lau, Susanna K P; Yuen, Kwok-Yung

    2010-01-01

    Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping.

  16. RAPD and internal transcribed spacer sequence analyses reveal Zea nicaraguensis as a section Luxuriantes species close to Zea luxurians.

    PubMed

    Wang, Pei; Lu, Yanli; Zheng, Mingmin; Rong, Tingzhao; Tang, Qilin

    2011-04-15

    Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.

  17. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens.

    PubMed

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated.

  18. Genetic Analyses of the Internal Transcribed Spacer Sequences Suggest Introgression and Duplication in the Medicinal Mushroom Agaricus subrufescens

    PubMed Central

    Chen, Jie; Moinard, Magalie; Xu, Jianping; Wang, Shouxian; Foulongne-Oriol, Marie; Zhao, Ruilin; Hyde, Kevin D.; Callac, Philippe

    2016-01-01

    The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is widely used in fungal taxonomy and phylogeographic studies. The medicinal and edible mushroom Agaricus subrufescens has a worldwide distribution with a high level of polymorphism in the ITS region. A previous analysis suggested notable ITS sequence heterogeneity within the wild French isolate CA487. The objective of this study was to investigate the pattern and potential mechanism of ITS sequence heterogeneity within this strain. Using PCR, cloning, and sequencing, we identified three types of ITS sequences, A, B, and C with a balanced distribution, which differed from each other at 13 polymorphic positions. The phylogenetic comparisons with samples from different continents revealed that the type C sequence was similar to those found in Oceanian and Asian specimens of A. subrufescens while types A and B sequences were close to those found in the Americas or in Europe. We further investigated the inheritance of these three ITS sequence types by analyzing their distribution among single-spore isolates from CA487. In this analysis, three co-dominant markers were used firstly to distinguish the homokaryotic offspring from the heterokaryotic offspring. The homokaryotic offspring were then analyzed for their ITS types. Our genetic analyses revealed that types A and B were two alleles segregating at one locus ITSI, while type C was not allelic with types A and B but was located at another unlinked locus ITSII. Furthermore, type C was present in only one of the two constitutive haploid nuclei (n) of the heterokaryotic (n+n) parent CA487. These data suggest that there was a relatively recent introduction of the type C sequence and a duplication of the ITS locus in this strain. Whether other genes were also transferred and duplicated and their impacts on genome structure and stability remain to be investigated. PMID:27228131

  19. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using internal transcribed spacer and cytochrome oxidase subunit l DNA sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genetic diversity of Tamarixia radiata laboratory colonies derived from collections in China, northern Vietnam, Pakistan, and a mixed colony from Taiwan and southern Vietnam was evaluated using the internal transcribed spacer region 1 (ITS-1), internal transcribed spacer region 2 (ITS-2) and the...

  20. Comparative evolution of S7 Intron 1 and ribosomal internal transcribed spacer in Coilia nasus (Clupeiformes: Engraulidae).

    PubMed

    Liu, Dong; Guo, Hong-Yi; Tang, Wen-Qiao; Yang, Jin-Quan

    2012-01-01

    Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.

  1. Assessment of phylogenetic relationship of rare plant species collected from Saudi Arabia using internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Al-Qurainy, F; Khan, S; Nadeem, M; Tarroum, M; Alaklabi, A

    2013-03-11

    The rare and endangered plants of any country are important genetic resources that often require urgent conservation measures. Assessment of phylogenetic relationships and evaluation of genetic diversity is very important prior to implementation of conservation strategies for saving rare and endangered plant species. We used internal transcribed spacer sequences of nuclear ribosomal DNA for the evaluation of sequence identity from the available taxa in the GenBank database by using the Basic Local Alignment Search Tool (BLAST). Two rare plant species viz, Heliotropium strigosum claded with H. pilosum (98% branch support) and Pancratium tortuosum claded with P. tenuifolium (61% branch support) clearly. However, some species, viz Scadoxus multiflorus, Commiphora myrrha and Senecio hadiensis showed close relationships with more than one species. We conclude that nuclear ribosomal internal transcribed spacer sequences are useful markers for phylogenetic study of these rare plant species in Saudi Arabia.

  2. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  3. Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

    PubMed

    Areekit, Supatra; Khuchareontaworn, Sintawee; Kanjanavas, Pornpimon; Sriyapai, Thayat; Pakpitchareon, Arda; Khawsak, Paisarn; Chansiri, Kosum

    2009-01-01

    This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

  4. Development of a PCR assay based on the 16S-23S rDNA internal transcribed spacer for identification of strictly anaerobic bacterium Zymophilus.

    PubMed

    Felsberg, Jurgen; Jelínková, Markéta; Kubizniaková, Petra; Matoulková, Dagmar

    2015-06-01

    PCR-primers were designed for identification of strictly anaerobic bacteria of the genus Zymophilus based on genus-specific sequences of the 16S-23S rDNA internal transcribed spacer region. The specificity of the primers was tested against 37 brewery-related non-target microorganisms that could potentially occur in the same brewery specimens. None DNA was amplified from any of the non-Zymophilus strains tested including genera from the same family (Pectinatus, Megasphaera, Selenomonas), showing thus 100% specificity. PCR assay developed in this study allows an extension of the spectra of detected beer spoilage microorganisms in brewery laboratories.

  5. Molecular Identification of Ptychodera flava (Hemichordata: Enteropneusta): Reconsideration in Light of Nucleotide Polymorphism in the 18S Ribosomal RNA Gene.

    PubMed

    Urata, Makoto

    2015-06-01

    Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

  6. Molecular phylogenetic relationships among members of the family Phytolaccaceae sensu lato inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Lee, J; Kim, S Y; Park, S H; Ali, M A

    2013-02-28

    The phylogeny of a phylogenetically poorly known family, Phytolaccaceae sensu lato (s.l.), was constructed for resolving conflicts concerning taxonomic delimitations. Cladistic analyses were made based on 44 sequences of the internal transcribed spacer of nuclear ribosomal DNA from 11 families (Aizoaceae, Basellaceae, Didiereaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae s.l., Polygonaceae, Portulacaceae, Sarcobataceae, Tamaricaceae, and Nepenthaceae) of the order Caryophyllales. The maximum parsimony tree from the analysis resolved a monophyletic group of the order Caryophyllales; however, the members, Agdestis, Anisomeria, Gallesia, Gisekia, Hilleria, Ledenbergia, Microtea, Monococcus, Petiveria, Phytolacca, Rivinia, Schindleria, Seguieria, Stegnosperma, and Trichostigma, which belong to the family Phytolaccaceae s.l., did not cluster under a single clade, demonstrating that Phytolaccaceae is polyphyletic.

  7. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region

    PubMed Central

    Sutton, Bruce D.; Steck, Gary J.; Norrbom, Allen L.; Rodriguez, Erick J.; Srivastava, Pratibha; Alvarado, Norma Nolazco; Colque, Fredy; Landa, Erick Yábar; Sánchez, Juan José Lagrava; Quisberth, Elizabeth; Peñaranda, Emilio Arévalo; Clavijo, P. A. Rodriguez; Alvarez-Baca, Jeniffer K.; Zapata, Tito Guevara; Ponce, Patricio

    2015-01-01

    Abstract The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included. PMID:26798259

  8. A new PCR primer for the identification of Paracoccidioides brasiliensis based on rRNA sequences coding the internal transcribed spacers (ITS) and 5 x 8S regions.

    PubMed

    Imai, T; Sano, A; Mikami, Y; Watanabe, K; Aoki, F H; Branchini, M L; Negroni, R; Nishimura, K; Miyaji, M

    2000-08-01

    Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.

  9. Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA.

    PubMed

    Marrelli, M T; Souza, L F; Marques, R C; Labruna, M B; Matioli, S R; Tonon, A P; Ribolla, P E M; Marinotti, O; Schumaker, T T S

    2007-03-01

    The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.

  10. Sequence variation of the ribosomal DNA second internal transcribed spacer region in two spatially-distinct populations of Amblyomma americanum (L.) (Acari: Ixodidae).

    PubMed

    Reichard, M V; Kocan, A A; Van Den Bussche, R A; Barker, R W; Wyckoff, J H; Ewing, S A

    2005-04-01

    Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

  11. Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

    PubMed

    Elbadri, Gamal A A; De Ley, Paul; Waeyenberge, Lieven; Vierstraete, Andy; Moens, Maurice; Vanfleteren, Jacques

    2002-02-01

    Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.

  12. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions.

    PubMed

    Scorzetti, Gloria; Fell, J W; Fonseca, A; Statzell-Tallman, Adele

    2002-12-01

    Basidiomycetous yeasts in the Urediniomycetes and Hymenomycetes were examined by sequence analysis in two ribosomal DNA regions: the D1/D2 variable domains at the 5' end of the large subunit rRNA gene (D1/D2) and the internal transcribed spacers (ITS) 1 and 2. Four major lineages were recognized in each class: Microbotryum, Sporidiobolus, Erythrobasidium and Agaricostilbum in the Urediniomycetes; Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales in the Hymenomycetes. Bootstrap support for many of the clades within those lineages is weak; however, phylogenetic analysis provides a focal point for in-depth study of biological relationships. Combined sequence analysis of the D1/D2 and ITS regions is recommended for species identification, while species definition requires classical biological information such as life cycles and phenotypic characterization.

  13. Molecular identification of the light brown apple moth (Lepidoptera: Tortricidae) in California using a polymerase chain reaction assay of the internal transcribed spacer 2 locus.

    PubMed

    Barr, N B; Ledezma, L A; Vasquez, J D; Epstein, M; Kerr, P H; Kinnee, S; Sage, O; Gilligan, T M

    2009-12-01

    A molecular protocol using a hemi-nested polymerase chain reaction (PCR) of the internal transcribed spacer region 2 (ITS2) is reported for the diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in California. This protocol distinguishes the light brown apple moth from other moths in California based on size differences of PCR amplicons that are visualized on agarose gels. The molecular diagnostic tool generated no false negatives based on analysis of 337 light brown apple moths collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 424 moths representing other tortricid species generated correct identification for >95% of the samples and only two false positives. Of the 761 moths tested only fourteen produced no PCR amplicons and five generated inconclusive data.

  14. Sequence differences in the internal transcribed spacer 1 and 5.8S ribosomal RNA among three Moniezia species isolated from ruminants in Japan.

    PubMed

    Ohtori, Maiko; Aoki, Mikiko; Itagaki, Tadashi

    2015-01-01

    This study was designed to clarify the differences in the internal transcribed spacer (ITS) 1 and 5.8S nucleotide sequences of Moniezia expansa, M. benedeni and M. monardi isolated from ruminants in Japan and to determine their phylogenetic relationships. A 98% similarity in the 5.8S sequences was observed among the 3 Moniezia species, whereas many nucleotide indels and substitutions were observed in the ITS1 sequences among the three Moniezia species. These results suggest that the ITS1 region could serve as a potential marker for discriminating the 3 Moniezia species. In the phylogenetic tree based on the ITS1 sequences, M. monardi and M. benedeni showed genetically closer relationship to each other than to M. expansa.

  15. [The phylogeny of Schistidium (Bryophyta, Grimmiaceae) based on primary and secondary structure study of nuclear rDNA internal transcribed spacers].

    PubMed

    Miliutina, I A; Goriunov, D V; Ignatov, M S; Ignatova, E A; Troitskiĭ, A V

    2010-01-01

    The phylogeny of Schistidium (Bryophyta, Grimmiaceae) was studied on the basis of nucleotide sequences of internal transcribed spacers ITS1-2 of nuclear DNA and trnT-trnD region of chloroplast DNA. The consistency of phylogenetic trees constructed from nuclear and chloroplast sequences was shown. A basal grade and two large clades were resolved on the phylogenetic trees. Morphological characteristics specific for these clades were described. ITS1 and ITS2 secondary structures of Schistidium species were modeled using thermodynamic criteria. Four different structures of the longest ITS1 hairpin were identified. Possible paths of Schistidium evolution were considered based on the four types of ITS1 secondary structure and phylogenetic trees.

  16. Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

    PubMed Central

    Gupta, Aditya K.; Boekhout, Teun; Theelen, Bart; Summerbell, Richard; Batra, Roma

    2004-01-01

    Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular techniques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal transcribed spacer, and sequencing of the D1 and D2 domains of the large-subunit ribosomal DNA region, was applied for the identification of Malassezia species. All species could be correctly identified by means of these methods. The results of AFLP analysis and sequencing were in complete agreement with each other. However, some discrepancies were noted when the molecular methods were compared with the phenotypic method of identification. Specific genotypes were distinguished within a collection of Malassezia furfur isolates from Canadian sources. AFLP analysis revealed significant geographical differences between the North American and European M. furfur strains. PMID:15365020

  17. Nuclear ribosomal internal transcribed spacer 1 (ITS1) variation in the Anastrepha fraterculus cryptic species complex (Diptera, Tephritidae) of the Andean region.

    PubMed

    Sutton, Bruce D; Steck, Gary J; Norrbom, Allen L; Rodriguez, Erick J; Srivastava, Pratibha; Alvarado, Norma Nolazco; Colque, Fredy; Landa, Erick Yábar; Sánchez, Juan José Lagrava; Quisberth, Elizabeth; Peñaranda, Emilio Arévalo; Clavijo, P A Rodriguez; Alvarez-Baca, Jeniffer K; Zapata, Tito Guevara; Ponce, Patricio

    2015-01-01

    The nuclear ribosomal internal transcribed spacer 1 (ITS1) was sequenced for Anastrepha fraterculus (Wiedemann, 1830) originating from 85 collections from the northern and central Andean countries of South America including Argentina (Tucumán), Bolivia, Perú, Ecuador, Colombia, and Venezuela. The ITS1 regions of additional specimens (17 collections) from Central America (México, Guatemala, Costa Rica, and Panamá), Brazil, Caribbean Colombia, and coastal Venezuela were sequenced and together with published sequences (Paraguay) provided context for interpretation. A total of six ITS1 sequence variants were recognized in the Andean region comprising four groups. Type I predominates in the southernmost range of Anastrepha fraterculus. Type II predominates in its northernmost range. In the central and northern Andes, the geographic distributions overlap and interdigitate with a strong elevational effect. A discussion of relationships between observed ITS1 types and morphometric types is included.

  18. Internal transcribed spacer (ITS) evolution in populations of the hyperparasitic European mistletoe pathogen fungus, Sphaeropsis visci (Botryosphaeriaceae): The utility of ITS2 secondary structures.

    PubMed

    Poczai, Péter; Varga, Ildikó; Hyvönen, Jaakko

    2015-03-01

    We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Sphaeropsis visci, a hyperparasitic fungus that causes the leaf spot disease of the hemiparasite European mistletoe (Viscum album). Samples of S. visci were obtained from Hungary covering all major infected forest areas. For obtaining PCR products we used a fast and efficient direct PCR approach based on a high fidelity DNA polymerase. A total of 140 ITS sequences were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, nucleotide polymorphism patterns, GC content, and presence of conserved motifs. Analysed sequences exhibited features of functional rRNAs. Overall, polymorphism was observed within less conserved motifs, such as loops and bulges, or, alternatively, as non-canonical G-U pairs within conserved regions of double stranded helices. The secondary structure of ITS2 provides new opportunities for obtaining further valuable information, which could be used in phylogenetic analyses, or at population level as demonstrated in our study. This is due to additional information provided by secondary structures and their models. The combined score matrix was used with the methods implemented in the programme 4SALE. Besides the pseudoprotein coding method of 4SALE, the molecular morphometric character coding also has potential for gaining further information for phylogenetic analyses based on the geometric features of the sub-structural elements of the ITS2 RNA transcript.

  19. Systematics of marine brown alga Sargassum from Thailand: A preliminary study based on morphological data and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences

    NASA Astrophysics Data System (ADS)

    Kantachumpoo, Attachai; Uwai, Shinya; Noiraksar, Thidarat; Komatsu, Teruhisa

    2015-06-01

    The marine brown algal genus Sargassum has been investigated extensively based on genetic information. In this report, we performed the first comparative study of morphological and molecular data among common species of Sargassum found in Thailand and explored the phylogenetic diversity within the genus. Our results revealed an incongruent pattern for species classification in Thai Sargassum. Morphologically, our Sargassum specimens were distinguishable and represented 8 species, namely, S. aquifolium (Turner) C.Agardh, Sargassum baccularia (Mertens) C. Agardh, S. cinereum J. Agardh, S. ilicifolium (Turner) C.Agardh, S. oligocystum Montagne, S. plagiophyllum C. Agardh, S. polycystum C. Agardh and S. swartzii (Turuner) C. Agardh. In contrast, using three different methods, phylogenetic analysis of nuclear ribosomal internal transcribed spacer 2 (ITS2) revealed six distinct clades, including S. baccularia/ S. oligosyntum clade, S. aquifolium/ S. swartzii clade, S. cinereum clade, S. aquifolium/ S. ilicifolium clade, S. polycystum clade, and S. plagiophyllum clade, which was suggestive of a phenotypic plasticity species complex. Our molecular data also confirmed the paraphyletic relationship in the section Binderianae and suggested that this section requires reassessment. Overall, further studies are required to increase our understanding of the taxonomy, phylogenetic relationships and species boundaries among Sargassum species in Thailand.

  20. Secondary structure analyses of the nuclear rRNA internal transcribed spacers and assessment of its phylogenetic utility across the Brassicaceae (mustards).

    PubMed

    Edger, Patrick P; Tang, Michelle; Bird, Kevin A; Mayfield, Dustin R; Conant, Gavin; Mummenhoff, Klaus; Koch, Marcus A; Pires, J Chris

    2014-01-01

    The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.

  1. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

  2. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

    PubMed Central

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-01-01

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

  3. Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

    PubMed Central

    Daffonchio, Daniele; Cherif, Ameur; Borin, Sara

    2000-01-01

    Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus. PMID:11097928

  4. Evaluation of Medicinal Categorization of Atractylodes japonica Koidz. by Using Internal Transcribed Spacer Sequencing Analysis and HPLC Fingerprinting Combined with Statistical Tools

    PubMed Central

    Kim, Jung-Hoon; Doh, Eui-Jeong; Lee, Guemsan

    2016-01-01

    Atractylodes rhizomes have been used as the herbal medicine “Changchul” or “Baekchul,” according to their clinical purpose, in Korea, China, and Japan. Among the Atractylodes species, the medicinal use of Atractylodes japonica has been controversial, as it is categorized as both Changchul and Baekchul in those countries, and, moreover, parts of the rhizome have been differently used, depending on age of the plant, in Korea. Chromatographic fingerprinting by using HPLC combined with chemometric analyses and internal transcribed spacer (ITS) sequencing analysis were conducted to classify and identify 34 crude drugs derived from Atractylodes rhizomes. The identification of the samples, authenticated by their morphological features as A. japonica Koidz. (Changchul and Baekchul), A. chinensis Koidz., and A. macrocephala Koidz., was confirmed as A. japonica, A. chinensis, and A. macrocephala by ITS sequencing. The results from chemometric analyses showed that the chemical components of the crude drugs from A. japonica were significantly different from those from A. macrocephala but were similar to those from A. chinensis. The analyses also suggested that the categorization by age of A. japonica as Changchul or Baekchul is not recommended. The results indicate that A. japonica should be categorized as “Changchul” and should not be further categorized by age. PMID:27190530

  5. Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions.

    PubMed

    Harpke, Doerte; Peterson, Angela

    2008-05-01

    The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems.

  6. Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

    PubMed Central

    Sang, T; Crawford, D J; Stuessy, T F

    1995-01-01

    The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species. Images Fig. 1 PMID:7624325

  7. Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    ZARRIN, MAJID; GANJ, FARZANEH; FARAMARZI, SAMA

    2016-01-01

    The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species. PMID:27073635

  8. The value of the D1/D2 and internal transcribed spacers (ITS) domains for the identification of yeast species belonging to the genus Yamadazyma.

    PubMed

    Groenewald, M; Robert, V; Smith, M Th

    2011-06-01

    In a taxonomic study of yeasts that have been isolated in French Guiana and Thailand, five yeast strains isolated from plants were found to belong to the Yamadazyma clade of Saccharomycotina. On the basis of morphology, physiology and the nucleotide divergence in the D1/D2 domain of the 26S nuclear ribosomal RNA (nrRNA) gene, as well as the internal transcribed spacers (ITS) domain of the nrRNA gene operon, these strains were identified to represent three novel species in this teleomorphic clade. An additional isolate, that is publicly available from the CBS yeast collection and isolated from Taiwan, was found to be similar to one of the novel species described from Thailand. Yeast species belonging to the Yamadazyma clade have previously been described as members of the Candida membranifaciens clade. These species are widely distributed and were isolated from diverse habitats, including water, plants, animals and guts of insects and termites. In the present study the ITS region is shown to be a valuable region for species identification within this clade, and the novel species proposed are Candida vaughaniae (ex-type strain CBS 8583), Candida khao-thaluensis (ex-type strain CBS 8535) and Candida tallmaniae (ex-type strain CBS 8575).

  9. Characterization of Penicillium species isolated from grape berries by their internal transcribed spacer (ITS1) sequences and by gas chromatography-mass spectrometry analysis of geosmin production.

    PubMed

    La Guerche, Stéphane; Garcia, Carole; Darriet, Philippe; Dubourdieu, Denis; Labarère, Jacques

    2004-06-01

    Geosmin (trans-1,10-dimethyl-trans-9-decalol), an earthy-musty compound, has been identified in wines and in grape juice, in which its presence is highly detrimental to the aromatic quality. Geosmin has a biological origin, and the analysis of rotten grape microflora has been done on two grape varieties (Semillon, Cabernet Sauvignon) from six parcels of the Bordeaux region over 3 years (1999, 2000, 2001). Forty-three Penicillium-related species have been analyzed by gas chromatography-mass spectrometry (GC-MS) for their geosmin production. GC-MS analysis has demonstrated that the earthy odor was always correlated with the presence of geosmin. Phenotypic characterization of Penicillium spp. being ambiguous, a molecular characterization by rDNA internal transcribed spacer (ITS1) sequencing was performed for all strains. The results evidenced that all strains producing geosmin belonged to only one species, P. expansum, and that the other strains, not producing geosmin, belonged to three species: P. purpurogenum, P. thomii, and Talaromyces wortmanii.

  10. Genetic diversity of Histoplasma capsulatum strains isolated from Argentina based on nucleotide sequence variations in the internal transcribed spacer regions of rDNA.

    PubMed

    Landaburu, Fernanda; Cuestas, María Luján; Rubio, Andrea; Elías, Nahuel Alejandro; Daneri, Gabriela Lopez; Veciño, Cecilia; Iovannitti, Cristina A; Mujica, María Teresa

    2014-05-01

    The internal transcribed spacer (ITS) regions of rDNA genes of 49 Histoplasma capsulatum (48 from clinical samples and one from soil) isolates were examined. Nucleotide sequence heterogeneity within this region was useful for phylogenetic classification of H. capsulatum and species identification. Thus, in 45 of 49 isolates we observed higher percentages of identity in the nucleotide sequences of ITS regions when the isolates studied herein were compared with those reported in our country in the South America B clade. Phylogenetic analyses of rDNA sequences corresponding to the 537 bp of the ITS region obtained from H. capsulatum isolates assigned South America type B clade (45 isolates), North America type 1 and Asia clade (2 isolates each one). H. capsulatum strains isolated from soil and from patients living in Argentina (45 of 49) clustered together with the H. capsulatum isolates of the South America B clade. The high level of genetic similarity among our isolates suggests that almost one genetic population is present in the microenvironment. Isolates described as H. capsulatum var. capsulatum or var. farciminosum (2 isolates) did not form a monophyletic group and were found in the Asia clade. Subsequent studies are needed to properly identify these isolates.

  11. Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification.

    PubMed

    Yeh, W B; Tseng, M J; Chang, N T; Wu, S Y; Tsai, Y S

    2015-02-01

    Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

  12. Conservation in the first internal transcribed spacer region (ITS1) in Hematodinium species infecting crustacean hosts found in the UK and Newfoundland.

    PubMed

    Small, H J; Shields, J D; Moss, J A; Reece, K S

    2007-05-09

    Parasitic dinoflagellates in the genus Hematodinium infect a number of decapod crustaceans in waters off the UK, including the Norway lobster Nephrops norvegicus and the edible crab Cancer pagurus. This study investigated sequence variability in the first internal transcribed spacer (ITS1) region of the ribosomal RNA complex of Hematodinium spp. infecting N. norvegicus, C. pagurus, and Pagurus bernhardus from 4 locations in the UK and from the Hematodinium sp. infecting Chionoecetes opilio from the province of Newfoundland and Labrador, Canada. Phylogenetic analysis of the Hematodinium ITS1 sequences from N. norvegicus, C. pagurus, P. bernhardus and C. opilio suggest that these crustaceans are infected with the same species of Hematodinium. Length variability of the ITS1 region was observed (324 to 345 bp) and attributed to 4 variable microsatellite regions (CATG)n' (GCC)nTCCGC(TG)n' (TA)n' and (GAA)n(GGA)n within the sequenced ITS1 fragment. The observed variation may be due to co-infection of the host crustacean with several different strains of Hematodinium or differences among copies of ITS1 region within the genome of a single parasite cell. The Hematodinium ITS1 sequence from N. norvegicus, C. pagurus, P. bernhardus and C. opilio isolates was sufficiently conserved in primer binding regions targeted by previous molecular diagnostic assays; therefore, we suggest that this assay could be used to screen for Hematodinium infections in these crustacean hosts.

  13. Genotyping of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes.

    PubMed

    Li, Kai; He, Ai; Cai, Wei Ping; Tang, Xiao Ping; Zheng, Xiao Ying; Li, Zhuo Ya; Zhan, Xi Mei

    2013-01-01

    Genetic diversity of Pneumocystis jirovecii isolates based on internal transcribed spacer (ITS) of the nuclear rRNA locus has previously been reported. The information about ITS genotype and epidemiology of this organism in Chinese human immunodeficiency virus-infected patients has not been available. In this study, 12 bronchoalveolar lavage fluid specimens obtained from HIV-infected patients were analyzed by PCR followed by cloning, sequencing and typing. Three ITS1 genotypes (E, B and 'H') and four ITS2 genotypes (b, g, i and r) as previously reported were identified, the most common of which were E, b and i. Five ITS haplotypes (Eg, Eb, Bi, Er and 'H'r) and 19 new combination types were also identified with the most common types being Eg (four of 12 patients, 10 of 60 clones), Eb (three of 12 patients, 11 of 60 clones) and Bi (three of 12 patients, 10 of 60 clones). Nine patients were found to be co-infected with more than one ITS genotype of P. jirovecii. The prevalence of ITS genotypes in HIV patients from one Chinese hospital did not seem to be significantly different when compared to reports from other countries.

  14. Characterization of internal transcribed spacer (ITS1)-ITS2 region of ribosomal RNA gene from 25 species of Culicoides biting midges (Diptera: Ceratopogonidae) in Japan.

    PubMed

    Matsumoto, Y; Yanase, T; Tsuda, T; Noda, H

    2009-09-01

    We determined nucleotide sequences of the nuclear rDNA internal transcribed spacer (ITS)1-5.8S-ITS2a-2S-ITS2 region in 103 individuals of 25 Culicoides species (Diptera: Ceratopogonidae) from 11 locations in Japan. Ribosomal RNA genes, 5.8S and 2S rDNA, were highly conserved among the species with few variations. The ITS2a region showed length variation among species. Both ITS1 and ITS2 showed highly varied sequences among species. The noticeable indel regions among ITS1 sequences are present in some Culicoides species, separating species into two types having long or short ITS1 region. However, Culicoides cylindratus Kitaoka possesses both types of ITS1 in each individual; these results seem to indicate that the ITS1-long type was the prototype and the short type was produced through deletion in many Culicoides species. One species, belonging to subgenus Avaritia, possessed an Avaritia-specific sequence in ITS1 and phylogenetically formed a monophyletic group. Geographical genotypes in a species were not clear. Species-specific sequence features were observed, enabling molecular identification of Culicoides species.

  15. Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers.

    PubMed

    Cacciò, Simone M; Beck, Relja; Almeida, Andre; Bajer, Anna; Pozio, Edoardo

    2010-05-01

    PCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3' end of the small subunit and in the 5' end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

  16. Molecular characterization of the Indian poultry nodular tapeworm, Raillietina echinobothrida (Cestoda: Cyclophyllidea: Davaineidae) based on rDNA internal transcribed spacer 2 region.

    PubMed

    Ramnath; Jyrwa, D B; Dutta, A K; Das, B; Tandon, V

    2014-03-01

    The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

  17. Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

    PubMed Central

    Zarrin, Majid; Erfaninejad, Maryam

    2016-01-01

    Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085

  18. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

    PubMed

    Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

    2016-05-05

    Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided.

  19. ITS1 sequence variabilities correlate with 18S rDNA sequence types in the genus Acanthamoeba (Protozoa: Amoebozoa).

    PubMed

    Köhsler, Martina; Leitner, Brigitte; Blaschitz, Marion; Michel, Rolf; Aspöck, Horst; Walochnik, Julia

    2006-01-01

    The subgenus classification of the ubiquitously spread and potentially pathogenic acanthamoebae still poses a great challenge. Fifteen 18S rDNA sequence types (T1-T15) have been established, but the vast majority of isolates fall into sequence type T4, and so far, there is no means to reliably differentiate within T4. In this study, the first internal transcribed spacer (ITS1), a more variable region than the 18S rRNA gene, was sequenced, and the sequences of 15 different Acanthamoeba isolates were compared to reveal if ITS1 sequence variability correlates with 18S rDNA sequence typing and if the ITS1 sequencing allows a differentiation within T4. It was shown that the variability in ITS1 is tenfold higher than in the 18S rDNA, and that ITS1 clusters correlate with the 18S rDNA clusters and thus corroborate the Acanthamoeba sequence type system. Moreover, high sequence dissimilarities and distinctive microsatellite patterns could enable a more detailed differentiation within T4.

  20. Double trouble for grasshopper molecular systematics: intra-individual heterogeneity of both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer ribosomal DNA sequences in Hesperotettix viridis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hesperotettix viridis grasshoppers (Orthoptera: Acrididae:Melanoplinae) exhibit intra-individual variation in both mitochondrial 12S-valine-16S and nuclear internal transcribed spacer (ITS) ribosomal DNA sequences. These findings violate core assumptions underlying DNA sequence data obtained via pol...

  1. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.

    PubMed

    Li, De-Zhu; Gao, Lian-Ming; Li, Hong-Tao; Wang, Hong; Ge, Xue-Jun; Liu, Jian-Quan; Chen, Zhi-Duan; Zhou, Shi-Liang; Chen, Shi-Lin; Yang, Jun-Bo; Fu, Cheng-Xin; Zeng, Chun-Xia; Yan, Hai-Fei; Zhu, Ying-Jie; Sun, Yong-Shuai; Chen, Si-Yun; Zhao, Lei; Wang, Kun; Yang, Tuo; Duan, Guang-Wen

    2011-12-06

    A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

  2. A Real-Time PCR Method for Quantifying Viable Ascaris Eggs Using the First Internally Transcribed Spacer Region of Ribosomal DNA▿

    PubMed Central

    Pecson, Brian M.; Barrios, José Antonio; Johnson, David R.; Nelson, Kara L.

    2006-01-01

    Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications. PMID:17056687

  3. Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China

    PubMed Central

    Li, Qin-Qin; Zhou, Song-Dong; He, Xing-Jin; Yu, Yan; Zhang, Yu-Cheng; Wei, Xian-Qin

    2010-01-01

    Background and Aims The genus Allium comprises more than 800 species, placing it among the largest monocotyledonous genera. It is a variable group that is spread widely across the Holarctic region. Previous studies of Allium have been useful in identifying and assessing its evolutionary lineages. However, there are still many gaps in our knowledge of infrageneric taxonomy and evolution of Allium. Further understanding of its phylogeny and biogeography will be achieved only through continued phylogenetic studies, especially of those species endemic to China that have often been excluded from previous analyses. Earlier molecular studies have shown that Chinese Allium is not monophyletic, so the goal of the present study was to infer the phylogeny and biogeography of Allium and to provide a classification of Chinese Allium by placement of Chinese species in the context of the entire phylogeny. Methods Phylogenetic studies were based on sequence data of the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast rps16 intron, analysed using parsimony and Bayesian approaches. Biogeographical patterns were conducted using statistical dispersal–vicariance analysis (S-DIVA). Key Results Phylogenetic analyses indicate that Allium is monophyletic and consists of three major clades. Optimal reconstructions have favoured the ancestors of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum as originating in eastern Asia. Conclusions Phylogenetic analyses reveal that Allium is monophyletic but that some subgenera are not. The large genetic distances imply that Allium is of ancient origin. Molecular data suggest that its evolution proceeded along three separate evolutionary lines. S-DIVA indicates that the ancestor of Amerallium, Anguinum, Vvedenskya, Porphyroprason and Melanocrommyum originated from eastern Asia and underwent different biogeographical pathways. A taxonomic synopsis of Chinese Allium at sectional level is given, which divides Chinese

  4. Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

    PubMed Central

    Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

    2014-01-01

    Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

  5. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  6. Internal Transcribed Spacer 2 (nu ITS2 rRNA) Sequence-Structure Phylogenetics: Towards an Automated Reconstruction of the Green Algal Tree of Life

    PubMed Central

    Buchheim, Mark A.; Keller, Alexander; Koetschan, Christian; Förster, Frank; Merget, Benjamin; Wolf, Matthias

    2011-01-01

    Background Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta. Methodology/Principal Findings Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses. Conclusions/Significance Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated

  7. Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

    PubMed

    Tashakori, Mahnaz; Mahnaz, Tashakori; Kuhls, Katrin; Katrin, Kuhls; Al-Jawabreh, Amer; Amer, Al-Jawabreh; Mauricio, Isabel L; Isabel, Mauricio; Schönian, Gabriele; Gabriele, Schönian; Farajnia, Safar; Safar, Farajnia; Alimohammadian, Mohammad Hossein; Hossein, Alimohammadian Mohammad

    2006-04-01

    Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

  8. Molecular approaches to differentiate three species of Nematodirus in sheep and goats from China based on internal transcribed spacer rDNA sequences.

    PubMed

    Zhao, G H; Jia, Y Q; Bian, Q Q; Nisbet, A J; Cheng, W Y; Liu, Y; Fang, Y Q; Ma, X T; Yu, S K

    2015-05-01

    Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.

  9. Sequence variation in two mitochondrial DNA regions and internal transcribed spacer among isolates of the nematode Oesophagostomum asperum originating from goats in Hunan Province, China.

    PubMed

    Li, F; Hu, T; Duan, N C; Li, W Y; Teng, Q; Li, H; Liu, W; Liu, Y; Cheng, T Y

    2016-01-01

    The present study examined sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1), and internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) among Oesophagostomum asperum isolates from goats in Hunan Province, China. A portion of the cox1 (pcox1), nad1 (pnad1) genes and the ITS (ITS1+5.8S rDNA+ITS2) rDNA were amplified by polymerase chain reaction (PCR) separately from adult O. asperum individuals and the representative amplicons were subjected to sequencing from both directions. The lengths of pcox1, pnad1 and ITS rDNA were 366 bp, 681 bp and 785 bp, respectively. The A+T contents of gene sequences were 71.5-72% for pcox1, 73.7-74.2% for pnad1 and 58-58.8% for ITS rDNA. Intra-specific sequence variations within O. asperum were 0-1.6% for pcox1, 0-1.9% for pnad1 and 0-1.7% for ITS rDNA, while inter-specific sequence differences among members of the genus Oesophagostomum were significantly higher, being 11.1-12.5%, 13.3-17.7% and 8.5-18.6% for pcox1, pnad1 and ITS rDNA, respectively. Phylogenetic analyses using combined sequences of pcox1 and pnad1, with three different computational algorithms (Bayesian inference, maximum likelihood and maximum parsimony), revealed distinct groups with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA and rDNA sequences among O. asperum isolates from goats in Hunan Province, China, and have implications for studying molecular epidemiology and population genetics of O. asperum.

  10. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys.

    PubMed

    Walters, William; Hyde, Embriette R; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A; Jansson, Janet K; Caporaso, J Gregory; Fuhrman, Jed A; Apprill, Amy; Knight, Rob

    2016-01-01

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for

  11. Myxobolus cerebralis internal transcribed spacer 1 (ITS-1) sequences support recent spread of the parasite to North America and within Europe

    USGS Publications Warehouse

    Whipps, C.M.; El-Matbouli, M.; Hedrick, R.P.; Blazer, V.; Kent, M.L.

    2004-01-01

    Molecular approaches for resolving relationships among the Myxozoa have relied mainly on small subunit (SSU) ribosomal DNA (rDNA) sequence analysis. This region of the gene is generally used for higher phylogenetic studies, and the conservative nature of this gene may make it inadequate for intraspecific comparisons. Previous intraspecific studies of Myxobolus cerebralis based on molecular analyses reported that the sequence of SSU rDNA and the internal transcribed spacer (ITS) were highly conserved in representatives of the parasite from North America and Europe. Considering that the ITS is usually a more variable region than the SSU, we reanalyzed available sequences on GenBank and obtained sequences from other M. cerebralis representatives from the states of California and West Virginia in the USA and from Germany and Russia. With the exception of 7 base pairs, most of the sequence designated as ITS-1 in GenBank was a highly conserved portion of the rDNA near the 3-prime end of the SSU region. Nonetheless, the additional ITS-1 sequences obtained from the available geographic representatives were well conserved. It is unlikely that we would have observed virtually identical ITS-1 sequences between European and American M. cerebralis samples had it spread naturally over time, particularly when compared to the variation seen between isolates of another myxozoan (Kudoa thyrsites) that has most likely spread naturally. These data further support the hypothesis that the current distribution of M. cerebralis in North America is a result of recent introductions followed by dispersal via anthropogenic means, largely through the stocking of infected trout for sport fishing.

  12. Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

    PubMed Central

    Chen, Sharon C.-A.; Wang, He; Zhang, Li; Fan, Xin; Xu, Zhi-Peng; Cheng, Jing-Wei; Kong, Fanrong; Zhao, Yu-Pei; Xu, Ying-Chun

    2016-01-01

    Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species. PMID:27105313

  13. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  14. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    PubMed Central

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy

    2015-01-01

    ABSTRACT Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers

  15. Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata.

    PubMed

    Sumida, Masayuki; Kato, Yoji; Kurabayashi, Atsushi

    2004-04-01

    The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.

  16. DNA Methylation within Transcribed Regions

    PubMed Central

    To, Taiko K.; Saze, Hidetoshi; Kakutani, Tetsuji

    2015-01-01

    DNA methylation within transcribed genes is commonly found in diverse animals and plants. Here, we provide an overview of recent advances and the remaining mystery regarding intragenic DNA methylation. PMID:26143255

  17. Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbc L and 18S rDNA sequence data

    NASA Astrophysics Data System (ADS)

    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 rbc L genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-1 407 bp long rbc L 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 rbc L genes. The sequencing results showed that the three Ulva prolifera (or U. pertusa) gene sequences from Qingdao and Nan'ao Island were identical. The ITS, 18S rDNA and rbc L 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.

  18. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    PubMed

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05).

  19. Gradual processing of the ITS1 from the nucleolus to the cytoplasm during synthesis of the human 18S rRNA.

    PubMed

    Preti, Milena; O'Donohue, Marie-Françoise; Montel-Lehry, Nathalie; Bortolin-Cavaillé, Marie-Line; Choesmel, Valérie; Gleizes, Pierre-Emmanuel

    2013-04-01

    Defects in ribosome biogenesis trigger stress response pathways, which perturb cell proliferation and differentiation in several genetic diseases. In Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia, mutations in ribosomal protein genes often interfere with the processing of the internal transcribed spacer 1 (ITS1), the mechanism of which remains elusive in human cells. Using loss-of-function experiments and extensive RNA analysis, we have defined the precise position of the endonucleolytic cleavage E in the ITS1, which generates the 18S-E intermediate, the last precursor to the 18S rRNA. Unexpectedly, this cleavage is followed by 3'-5' exonucleolytic trimming of the 18S-E precursor during nuclear export of the pre-40S particle, which sets a new mechanism for 18S rRNA formation clearly different from that established in yeast. In addition, cleavage at site E is also followed by 5'-3' exonucleolytic trimming of the ITS1 by exonuclease XRN2. Perturbation of this step on knockdown of the large subunit ribosomal protein RPL26, which was recently associated to DBA, reveals the putative role of a highly conserved cis-acting sequence in ITS1 processing. These data cast new light on the original mechanism of ITS1 elimination in human cells and provide a mechanistic framework to further study the interplay of DBA-linked ribosomal proteins in this process.

  20. Primers ITS1, ITS2 and ITS4 detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi.

    PubMed

    Korabecná, M; Liska, V; Fajfrlík, K

    2003-01-01

    Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.

  1. Sequence variation in the cytochrome oxidase I, internal transcribed spacer 1, and Ts14 diagnostic antigen sequences of Taenia solium isolates from South and Central America, India, and Asia.

    PubMed

    Hancock, K; Broughel, D E; Moura, I N; Khan, A; Pieniazek, N J; Gonzalez, A E; Garcia, H H; Gilman, R H; Tsang, V C

    2001-12-01

    We examined the genetic variability in the pig-human tapeworm, Taenia solium, by sequencing the genes for cytochrome oxidase I, internal transcribed spacer 1, and a diagnostic antigen, Ts14, from individual cysts isolated from Peru, Colombia, Mexico, India, China, and the Philippines. For these genes, the rate of nucleotide variation was minimal. Isolates from these countries can be distinguished based on one to eight nucleotide differences in the 396 nucleotide cytochrome oxidase I (COI) sequence. However, all of the 15 isolates from within Peru had identical COI sequences. The Ts14 sequences from India and China were identical and differed from the Peru sequence by three nucleotides in 333. These data indicate that there is minimal genetic variability within the species T. solium. Minimal variability was also seen in the ITS1 sequence, but this variation was observed within the individual. Twenty-two cloned sequences from six isolates sorted into 13 unique sequences. The variability observed within the sequences from individual cysts was as great as the variability between the isolates.

  2. Development of SCAR markers for the discrimination of three species of medicinal plants, Angelica decursiva (Peucedanum decursivum), Peucedanum praeruptorum and Anthricus sylvestris, based on the internal transcribed spacer (ITS) sequence and random amplified polymorphic DNA (RAPD).

    PubMed

    Choo, Byung Kil; Moon, Byeong Cheol; Ji, Yunui; Kim, Bo Bae; Choi, Goya; Yoon, Taesook; Kim, Ho Kyoung

    2009-01-01

    Angelicae decursivae radix ('Jeonho' in Korean) is prescribed as the root of Angelica decursiva (= Peucedanum decursivum) and Peucedanum praeruptorum in Korean pharmacopoeia. However, Anthricus sylvestris has been usually distributed on the market because it is identical to the Korean plant name 'Jeonho'. Furthermore, due to the morphological similarity of the aerial parts and herbal medicines, the correct identification of these roots is difficult. Therefore, to develop a reliable method for discriminating among A. decursiva (= P. decursivum), P. praeruptorum and A. sylvestris, we applied the tools of molecular genetics, such as the analysis of ribosomal DNA internal transcribed spacer (rDNA-ITS) and random amplified polymorphic DNA (RAPD). In the comparison of rDNA-ITS sequences, we found a specific primer region for the identification of A. sylvestris among three varieties of the herb that produced a 273 bp strand of DNA specific to A. sylvestris. As the result of RAPD analysis, we developed one sequence characterized amplified region (SCAR) marker for A. decursiva and P. praeruptorum that amplified a 363 bp DNA fragment specific to both A. decursiva and P. praeruptorum and two markers for P. praeruptorum that amplified 145 bp and 305 bp DNA fragments specific to P. praeruptorum. Furthermore, we established the SCAR markers for the simultaneous discrimination of the three species by applying a multiplex-polymerase chain reaction (PCR) with a combination of primers. This method of discrimination would be useful in preventing the distribution of adulterates because it can identify each herb and distinguish it from inauthentic substitutions.

  3. Intra-individual internal transcribed spacer 1 (ITS1) and ITS2 ribosomal sequence variation linked with multiple rDNA loci: a case of triploid Atractolytocestus huronensis, the monozoic cestode of common carp.

    PubMed

    Králová-Hromadová, Ivica; Stefka, Jan; Spakulová, Marta; Orosová, Martina; Bombarová, Marta; Hanzelová, Vladimíra; Bazsalovicsová, Eva; Scholz, Tomás

    2010-02-01

    Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological characters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and triploidy in all analysed specimens. Analysis of 20 sequences of each ITS1 and ITS2 spacer yielded eight and 10 different sequence types, respectively. In individual tapeworms, two to four ITS1 and three to four ITS2 sequence types were found. Divergent intra-genomic ITS copies were mostly induced by nucleotide substitutions and different numbers of short repetitive motifs within the sequence. In addition, triploidy was found to be a common feature of A. huronensis. The karyotype of Slovakian A. huronensis possesses three sets of chromosomes (3n=24, n=4m+3st+1minute chromosome), similar to the previously described triploidy in conspecific tapeworms from North America. Fluorescent in situ hybridisation (FISH) with a ssrDNA probe revealed two distinct rDNA clusters for each homologue of the triplet number 2. To date, A. huronensis is the only cestode species in which intra-individual ITS sequence variants were found in parallel with its triploid nature and multiple rDNA loci. Some of these molecular and genetic features were observed in several other species of basal or nearly basal tapeworms of the orders Caryophyllidea and Diphyllobothriidea, which indicates that the phenomena may be characteristic for evolutionarily lower tapeworms and deserve more attention in future studies.

  4. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    PubMed Central

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  5. Unusual features of the sequences of copies of the 16S-23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events.

    PubMed

    Maslunka, Christopher; Gürtler, Volker; Seviour, Robert

    2015-02-01

    The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

  6. Novel genetic diversity within Anopheles punctimacula s.l.: phylogenetic discrepancy between the Barcode cytochrome c oxidase I (COI) gene and the rDNA second internal transcribed spacer (ITS2).

    PubMed

    Loaiza, Jose R; Scott, Marilyn E; Bermingham, Eldredge; Sanjur, Oris I; Rovira, Jose R; Dutari, Larissa C; Linton, Yvonne-Marie; Bickersmith, Sara; Conn, Jan E

    2013-10-01

    Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni, have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3' COI), the Barcode region in the five prime end of the COI (5' COI), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3' COI depicted six highly supported molecular lineages (A-F), none of which was An. malefactor. In contrast, phylogenetic inference with the 5' COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3' COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama.

  7. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria

    PubMed Central

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J.; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC. PMID:27749897

  8. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

    PubMed Central

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-01-01

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

  9. Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: characterization by internal transcribed spacer, β-Tubulin, and calmodulin gene sequencing, metabolic fingerprinting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Tam, Emily W T; Chen, Jonathan H K; Lau, Eunice C L; Ngan, Antonio H Y; Fung, Kitty S C; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2014-04-01

    Aspergillus nomius and Aspergillus tamarii are Aspergillus species that phenotypically resemble Aspergillus flavus. In the last decade, a number of case reports have identified A. nomius and A. tamarii as causes of human infections. In this study, using an internal transcribed spacer, β-tubulin, and calmodulin gene sequencing, only 8 of 11 clinical isolates reported as A. flavus in our clinical microbiology laboratory by phenotypic methods were identified as A. flavus. The other three isolates were A. nomius (n = 2) or A. tamarii (n = 1). The results corresponded with those of metabolic fingerprinting, in which the A. flavus, A. nomius, and A. tamarii strains were separated into three clusters based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC MS) analysis. The first two patients with A. nomius infections had invasive aspergillosis and chronic cavitary and fibrosing pulmonary and pleural aspergillosis, respectively, whereas the third patient had A. tamarii colonization of the airway. Identification of the 11 clinical isolates and three reference strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed that only six of the nine strains of A. flavus were identified correctly. None of the strains of A. nomius and A. tamarii was correctly identified. β-Tubulin or the calmodulin gene should be the gene target of choice for identifying A. flavus, A. nomius, and A. tamarii. To improve the usefulness of MALDI-TOF MS, the number of strains for each species in MALDI-TOF MS databases should be expanded to cover intraspecies variability.

  10. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  11. Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences.

    PubMed

    Zhao, Guang-Hui; Li, Hong-Mei; Ryan, Una M; Cong, Mei-Mei; Hu, Bing; Gao, Man; Ren, Wan-Xin; Wang, Xing-Ye; Zhang, Shui-Ping; Lin, Qing; Zhu, Xing-Quan; Yu, San-Ke

    2012-09-01

    The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.

  12. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

    PubMed

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  15. Distanced Data: Transcribing Other People's Research Tapes

    ERIC Educational Resources Information Center

    Tilley, Susan A.; Powick, Kelly D.

    2004-01-01

    In this article, we report on our qualitative study involving eight individuals hired to transcribe research tapes in university contexts. We consider issues of data analysis and data trustworthiness and the implications for both when transcription is assigned to someone other than the researcher. We explore the challenges transcribers faced…

  16. A quick transcribing technique for oral data

    USGS Publications Warehouse

    Schleicher, David

    1972-01-01

    Stenographic techniques offer a means for transcribing oral data accurately and efficiently. In one such application, during five Appolo lunar missions, a rough but helpful transcript was produced within minutes. Similarly, lectures, conferences, and audio tapes can be accurately transcribed as promptly as necessary. Computer programs for translating shorthand notes are being developed; they will increase both speed and accuracy of translation.

  17. 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

    PubMed Central

    Meli, Marina L.; Novacco, Marilisa; Borel, Nicole

    2016-01-01

    The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects. PMID:27672657

  18. Two F-18s in Autonomous Formation Flight

    NASA Technical Reports Server (NTRS)

    2001-01-01

    This 32 second video clip shows two F-18s in NASA's Autonomous Formation Flight (AFF) program. The aircraft use smoke contrails to gather data on wingtip vortices. Flight research attempts to utilize the energy in the vortices for more efficient flight.

  19. Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA.

    PubMed

    Zhao, Ya-E; Wu, Li-Ping; Hu, Li; Xu, Yang; Wang, Zheng-Hang; Liu, Wen-Yan

    2012-11-01

    Due to the difficulty of DNA extraction for Demodex, few studies dealt with the identification and the phyletic evolution of Demodex at molecular level. In this study, we amplified, sequenced, and analyzed a complete (Demodex folliculorum) and an almost complete (D12 missing) (Demodex brevis) ribosomal DNA (rDNA) sequence and also analyzed the primary sequences of divergent domains in small-subunit ribosomal RNA (rRNA) of 51 species and in large-subunit rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea, and Ixodoidea). The results revealed that 18S rDNA sequence was relatively conserved in rDNA-coding regions and was not evolving as rapidly as 28S rDNA sequence. The evolutionary rates of transcribed spacer regions were much higher than those of the coding regions. The maximum parsimony trees of 18S and 28S rDNA appeared to be almost identical, consistent with their morphological classification. Based on the fact that the resolution capability of sequence length and the divergence of the 13 segments (D1-D6, D7a, D7b, and D8-D12) of 28S rDNA were stronger than that of the nine variable regions (V1-V9) of 18S rDNA, we were able to identify Demodex (Cheyletoidea) by the indels occurring in D2, D6, and D8.

  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

    PubMed Central

    Matsuda, Tomoko; Morishita, Maiko; Hinomoto, Norihide; Gotoh, Tetsuo

    2014-01-01

    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. PMID:25289639

  1. Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

    PubMed

    Guo, Liliang; Sui, Zhenghong; Zhang, Shu; Ren, Yuanyuan; Liu, Yuan

    2015-04-01

    Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

  2. 5. international workshop on the identification of transcribed sequences

    SciTech Connect

    1995-12-31

    This workshop was held November 5--8, 1995 in Les Embiez, France. The purpose of this conference was to provide a multidisciplinary forum for exchange of state-of-the-art information on mapping the human genome. Attention is focused on the following topics: transcriptional maps; functional analysis; techniques; model organisms; and tissue specific libraries and genes. Abstracts are included of the papers that were presented.

  3. Transcribed dark matter: meaning or myth?

    PubMed Central

    Ponting, Chris P.; Belgard, T. Grant

    2010-01-01

    Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic ‘dark matter’ transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted. PMID:20798109

  4. Seeing What They Meant: Transcribing as a Route To Noticing.

    ERIC Educational Resources Information Center

    Lynch, Tony

    2001-01-01

    Describes a reflective noticing activity in which pairs of adult learners of English for academic purposes transcribe their own performances of a routine classroom speaking task. Analysis of the process and product of these cycles of work suggest that collaborative transcribing and editing can encourage learners to focus on their form in output in…

  5. Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

    PubMed Central

    Nyaku, Seloame T.; Sripathi, Venkateswara R.; Kantety, Ramesh V.; Gu, Yong Q.; Lawrence, Kathy; Sharma, Govind C.

    2013-01-01

    The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. PMID:23593343

  6. Chromosomal localization of 5S and 18S-5.8S-25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization.

    PubMed

    Liu, Z-L; Zhang, D; Hong, D-Y; Wang, X-R

    2003-01-01

    Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.

  7. Training and Availability of Braille Transcribers in the United States.

    ERIC Educational Resources Information Center

    Corn, Anne L.; Wall, Robert S.

    2002-01-01

    A survey of Braille transcribers in 40 states found Braille production systems rely on volunteers and hence Braille transcription is not considered a bona fide career. The majority cited low funding and resources as obstacles to change. Issues of certification, training, availability, and definition are discussed, along with recommendations.…

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

    PubMed

    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.

  9. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    PubMed

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  10. Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

    PubMed Central

    Gillespie, J J; Johnston, J S; Cannone, J J; Gutell, R R

    2006-01-01

    As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome

  11. Compositional properties and thermal adaptation of 18S rRNA in vertebrates

    PubMed Central

    Varriale, Annalisa; Torelli, Giuseppe; Bernardi, Giorgio

    2008-01-01

    In order to investigate the influence of temperature on the GC level of the paired sequences of ribosomal 18S RNAs in vertebrates, we have studied their base composition in cold- and warm-blooded vertebrates using a stem-by-stem comparison. We observed that a number of stems of 18S ribosomal RNAs (rRNAs) are variable among species and that the majority of such stems are GC richer in warm-blooded than in cold-blooded vertebrates. We also constructed the secondary structures of the 18S rRNAs of a polar fish, a marsupial, and a monotreme to compare them with those of temperate/tropical fishes and of eutherians, respectively. In these cases, differences similar to those already mentioned were found. We conclude that there is a correlation between stem stability and body temperature even within the relatively limited temperature range of vertebrates. PMID:18567811

  12. Chemical probing of adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA

    SciTech Connect

    Rairkar, A.; Rubino, H.M.; Lockard, R.E.

    1988-01-26

    The location of unpaired adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA was determined by chemical probing. Naked /sup 18/S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked /sup 18/S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of /sup 32/P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in /sup 18/S rRNA determined by comparative sequence analysis. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit /sup 18/S rRNA than for E. coli /sup 16/S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian /sup 18/S rRNA, as compared with prokaryotic /sup 16/S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

  13. Taxonomic resolutions based on 18S rRNA genes: a case study of subclass copepoda.

    PubMed

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

    Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1-9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy.

  14. Project to transcribe old ship logs provides important weather data

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-11-01

    Kathy Wendolkowski is a citizen scientist. It's a term that Wendolkowski considers far too lofty for what she claims is simply a happy addiction that she and others have for transcribing old logs from naval ship and other vessels. They perform this task to glean the regularly recorded weather data from those logs for the benefit of science. For Wendolkowski, though, greater satisfaction comes from reading what the logs also reveal about the daily lives of the sailors as well as any accompanying historical drama.

  15. 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.

    PubMed

    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.

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

    PubMed

    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.

  17. PCR Primers for Metazoan Nuclear 18S and 28S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Knowlton, Nancy

    2012-01-01

    Background Metagenetic analyses, which amplify and sequence target marker DNA regions from environmental samples, are increasingly employed to assess the biodiversity of communities of small organisms. Using this approach, our understanding of microbial diversity has expanded greatly. In contrast, only a few studies using this approach to characterize metazoan diversity have been reported, despite the fact that many metazoan species are small and difficult to identify or are undescribed. One of the reasons for this discrepancy is the availability of universal primers for the target taxa. In microbial studies, analysis of the 16S ribosomal DNA is standard. In contrast, the best gene for metazoan metagenetics is less clear. In the present study, we have designed primers that amplify the nuclear 18S and 28S ribosomal DNA sequences of most metazoan species with the goal of providing effective approaches for metagenetic analyses of metazoan diversity in environmental samples, with a particular emphasis on marine biodiversity. Methodology/Principal Findings Conserved regions suitable for designing PCR primers were identified using 14,503 and 1,072 metazoan sequences of the nuclear 18S and 28S rDNA regions, respectively. The sequence similarity of both these newly designed and the previously reported primers to the target regions of these primers were compared for each phylum to determine the expected amplification efficacy. The nucleotide diversity of the flanking regions of the primers was also estimated for genera or higher taxonomic groups of 11 phyla to determine the variable regions within the genes. Conclusions/Significance The identified nuclear ribosomal DNA primers (five primer pairs for 18S and eleven for 28S) and the results of the nucleotide diversity analyses provide options for primer combinations for metazoan metagenetic analyses. Additionally, advantages and disadvantages of not only the 18S and 28S ribosomal DNA, but also other marker regions as targets

  18. Secondary structure of rabbit 18S ribosomal RNA determined from biochemical and phylogenetic data

    SciTech Connect

    Rairkar, A.; Rubino, H.; Lockard, R.E.

    1986-05-01

    To understand the functional role of 18S rRNA in the eukaryotic 40S subunit, its higher order structure must first be determined. Native deproteinized 18S rRNA was isolated from purified rabbit 40S subunits, fractionated on SDS-sucrose density gradients and concentrated using centricon-30 microconcentrators. The structure of native 18S rRNA was probed chemically with both diethylpyrocarbonate (DEPC) and dimethyl sulfate (DMS) which react with unpaired adenosine and guanosine residues, respectively. After /sup 32/P-end-labeling of intact and fragmented RNA, the modified nucleotides were identified by polyacrylamide sequencing gel electrophoresis upon aniline induced strand scission. On the basis of both the biochemical and phylogenetic data, a secondary structure model is proposed which includes the two major G + C rich insertion elements. A comparison of the structure data with previously published phylogenetic models suggests an instability of certain predicted helices. These unstable helices may normally be stabilized by ribosomal proteins and could represent the flexible elements involved in biologically significant conformational switches within 40S subunit.

  19. Taenia spp.: 18S rDNA microsatellites for molecular systematic diagnosis.

    PubMed

    Foronda, P; Casanova, J C; Martinez, E; Valladares, B; Feliu, C

    2005-06-01

    The 18S rDNA gene of adult worms of Taenia parva found in Genetta genetta in the Iberian Peninsula and larval stages of T. pisiformis from the wild rabbit (Oryctolagus cuniculus) in Tenerife (Canary Islands) were amplified and sequenced. The sequences of the 18S rDNA gene of T. parva (1768 bp) and T. pisiformis (1760 bp) are reported for the first time (GenBank accession nos. AJ555167-AJ555168 and AJ555169-AJ555170, respectively). In 168 alignment positions microsatellites in the 18S rDNA of both taxa were detected for the first time (TGC in T. parva and TGCT in T. pisiformis) and differences in their sequences with different repetition numbers were observed. The use of nucleotide sequences of this gene in the resolution of systematic problems in cestodes is discussed with reference to the systematic status of Taenia spp. and mainly in human taeniids such as T. solium, T. saginata, and Asian human isolates of Taenia.

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

    PubMed

    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.

  1. Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

    PubMed Central

    Lim, K Y; Skalicka, K; Koukalova, B; Volkov, R A; Matyasek, R; Hemleben, V; Leitch, A R; Kovarik, A

    2004-01-01

    An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions. PMID:15126410

  2. Rate accelerations in nuclear 18S rDNA of mycoheterotrophic and parasitic angiosperms.

    PubMed

    Lemaire, Benny; Huysmans, Suzy; Smets, Erik; Merckx, Vincent

    2011-09-01

    Rate variation in genes from all three genomes has been observed frequently in plant lineages with a parasitic and mycoheterotrophic mode of life. While the loss of photosynthetic ability leads to a relaxation of evolutionary constraints in genes involved in the photosynthetic apparatus, it remains to be determined how prevalent increased substitution rates are in nuclear DNA of non-photosynthetic angiosperms. In this study we infer rates of molecular evolution of 18S rDNA of all parasitic and mycoheterotorphic plant families (except Lauraceae and Polygalaceae) using relative rate tests. In several holoparasitic and mycoheterotrophic plant lineages extremely high substitution rates are observed compared to other photosynthetic angiosperms. The position and frequency of these substitutions have been identified to understand the mutation dynamics of 18S rRNA in achlorophyllous plants. Despite the presence of significantly elevated substitution rates, very few mutations occur in major functional and structural regions of the small ribosomal molecule, providing evidence that the efficiency of the translational apparatus in non-photosynthetic plants has not been affected.

  3. Karyotypes, heterochromatin, and physical mapping of 18S-26S rDNA in Cactaceae.

    PubMed

    Las Peñas, M L; Urdampilleta, J D; Bernardello, G; Forni-Martins, E R

    2009-01-01

    Karyotype analyses in members of the four Cactaceae subfamilies were performed. Numbers and karyotype formula obtained were: Pereskioideae = Pereskiaaculeata(2n = 22; 10 m + 1 sm), Maihuenioideae = Maihuenia patagonica (2n = 22, 9 m + 2 sm; 2n = 44, 18 m + 4 sm), Opuntioideae = Cumulopuntia recurvata(2n = 44; 20 m + 2 sm), Cactoideae = Acanthocalycium spiniflorum (2n = 22; 10 m + 1 sm),Echinopsis tubiflora (2n = 22; 10 m + 1 sm), Trichocereus candicans (2n = 22, 22 m). Chromosomes were small, the average chromosome length was 2.3 mum. Diploid species and the tetraploid C. recurvata had one terminal satellite, whereas the remaining tetraploid species showed four satellited chromosomes. Karyotypes were symmetrical. No CMA(-)/DAPI(+) bands were detected, but CMA(+)/DAPI(-) bands associated with NOR were always found. Pericentromeric heterochromatin was found in C. recurvata, A. spiniflorum, and the tetraploid cytotype of M. patagonica. The locations of the 18S-26S rDNA sites in all species coincided with CMA(+)/DAPI(-) bands; the same occurred with the sizes and numbers of signals for each species. This technique was applied for the first time in metaphase chromosomes in cacti. NOR-bearing pair no.1 may be homeologous in all species examined. In Cactaceae, the 18S-26S loci seem to be highly conserved.

  4. Optical and electrical stability of viral-templated copper sulfide (Cu1.8S) films

    NASA Astrophysics Data System (ADS)

    Shahriar Zaman, Mohammed; Bernard Grajeda, Gabriel; Haberer, Elaine D.

    2014-04-01

    The optical and electrical stabilities of viral-templated non-stoichiometric copper sulfide, digenite (Cu1.8S) films were investigated. The films were composed of large agglomerates of randomly aligned Cu1.8S-coated M13 filamentous phage. Free carrier optical absorption associated with localized surface plasmon resonance (LSPR) was observed in the near infrared spectral region, and the films were electrically active, displaying a linear current-voltage relationship. Under ambient conditions, the magnitude of the LSPR absorption increased, following a power law relationship with time, and the electrical resistance of viral-templated films decreased significantly. In contrast, the resistance of films stored under low oxygen, low humidity conditions experienced a smaller reduction in electrical resistance. Changes in optical and electrical film properties under ambient conditions were associated with an increase in free carrier concentration within the copper chalcogenide material due to oxygen exposure. X-ray photoelectron spectroscopy was used to relate this increase in free carrier concentration to compositional changes on the viral-templated material surface.

  5. Loop-mediated isothermal amplification targeting 18S ribosomal DNA for rapid detection of Acanthamoeba.

    PubMed

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-06-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

  6. Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.

    PubMed

    Berbee, M L; Taylor, J W

    1992-01-01

    For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.

  7. Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

    PubMed Central

    Yang, Hye-Won; Lee, Yu-Ran; Inoue, Noboru; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Kim, Hong-Kyun; Lee, Junhun; Goo, Youn-Kyoung; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner. PMID:23864737

  8. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  9. Medical Terminology of the Musculoskeletal System. Medical Records. Instructional Unit for the Medical Transcriber.

    ERIC Educational Resources Information Center

    Gosman, Minna L.

    Following an analysis of the task of transcribing as practiced in a health facility, this study guide was developed to teach the knowledge and skills required of a medical transcriber. The medical record department was identified as a major occupational area, and a task inventory for medical records was developed and used as a basis for a…

  10. 18S rRNA suggests that Entoprocta are protostomes, unrelated to Ectoprocta.

    PubMed

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

    1996-05-01

    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, two annelids, and a platyhelminth. Phylogenetic analyses of these data show that (1) entoprocts and lophophorates have spiralian, protostomous affinities, (2) Ento- and Ectoprocta are not sister taxa, (3) phoronids and brachiopods form a monophyletic clade, and (4) neither Ectoprocta or Annelida appear to be monophyletic. Both deuterostomous and pseudocoelomate features may have arisen at least two times in evolutionary history. These results advocate a Spiralia-Radialia-based classification rather than one based on the Protostomia-Deuterostomia concept.

  11. Phylogenetic relationships of Spiruromorpha from birds of prey based on 18S rDNA.

    PubMed

    Honisch, M; Krone, O

    2008-06-01

    A total of 153 free-ranging birds from Germany belonging to 15 species were examined for nematodes in their digestive and respiratory tracts. In 51.7% of the birds 14 different nematode species were found: the intestinal ascarids Porrocaecum depressum and P. angusticolle, the strongylid Hovorkonema variegatum, which inhabits the trachea and bronchi, the hairworms Eucoleus dispar and Capillaria tenuissima isolated from the digestive system, the spirurid nematodes Cyrnea leptoptera, C. mansioni, C. seurati, Microtetrameres cloacitectus, Physaloptera alata, P. apivori, Synhimantus hamatus and S. laticeps, which inhabit the proventriculus and gizzard of the raptors, and the spirurid nematode Serratospiculum tendo, which lives in the air sacs. To revise their systematic positions the ribosomal 18S gene regions of the nematode species were analysed and a phylogenetic tree was constructed. The molecular data confirmed the morphological systematics, except the spirurid family Physalopteridae, which grouped together with the Acuariidae.

  12. 18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines.

    PubMed

    Rivera, Windell L; Adao, Davin Edric V

    2009-10-01

    This study was carried out to document the genotypes of Acanthamoeba present in contact lens cases from 50 randomly selected contact lens wearers living in Quezon City, Metro Manila, Philippines. Acanthamoeba species were isolated from eight (16%) in 50 contact lens cases examined. We analyzed partial 18S ribosomal DNA (Rns) sequences of the eight isolates and found that the sequence differences were sufficient to distinguish the genotypes. After the isolates were genotyped, using the Basic Local Alignment Search Tool program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. The model-based (GTR+Gamma+Iota) neighbor-joining, maximum likelihood, and Bayesian inference analyses, as well as the non-model-based maximum parsimony analysis were used. Results showed that of the eight isolates, six were Rns genotype T5 while two were Rns genotype T4. This present study indicates that genotype T5 is also a common contaminant in contact lens storage cases.

  13. Novelty in phylogeny of gastrotricha: evidence from 18S rRNA gene.

    PubMed

    Wirz, A; Pucciarelli, S; Miceli, C; Tongiorgi, P; Balsamo, M

    1999-11-01

    Gastrotricha form a phylum which is crucial for defining the origin of pseudocoelomates, in that they share a number of characters with Rotifera and Nematoda but also with acoelomates, and even the evolutionary relationships within the phylum are anything but defined. For this reason the first extensive molecular data on Gastrotricha from the 18S rRNA sequences of both orders have been obtained and analyzed. Sequence analyses show that the phylum Gastrotricha is strictly monophyletic along an evolutionary line quite distinct from that of both Rotifera and Nematoda. A new view of the evolutionary history of the phylum Gastrotricha is put forward, in which Chaetonotida, and not Macrodasyida, are the most primitive forms of the group, contrary to the commonly held view. A polyphyletic origin of aschelminthes is supported, and the misleading term pseudocoelomates should be discarded.

  14. The phylogenetic status of arthropods, as inferred from 18S rRNA sequences.

    PubMed

    Turbeville, J M; Pfeifer, D M; Field, K G; Raff, R A

    1991-09-01

    Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.

  15. Chromosome Mapping of 18S Ribosomal RNA Genes in Eleven Hypostomus Species (Siluriformes, Loricariidae): Diversity Analysis of the Sites.

    PubMed

    Rubert, Marceléia; da Rosa, Renata; Zawadzki, Claudio H; Mariotto, Sandra; Moreira-Filho, Orlando; Giuliano-Caetano, Lucia

    2016-08-01

    We investigated the chromosomal distribution of 18S ribosomal DNA (rDNA) in different populations of 11 species of Hypostomus collected in important Brazilian basins, namely South Atlantic, Upper Paraná, and Paraguay applying the fluorescence in situ hybridization (FISH). Hypostomus cochliodon, Hypostomus commersoni, Hypostomus hermanni, Hypostomus regani, Hypostomus albopunctatus, Hypostomus paulinus, Hypostomus aff. paulinus, Hypostomus iheringii, and Hypostomus mutucae presented multiple 18S rDNA sites while Hypostomus strigaticeps and Hypostomus nigromaculatus exhibited a single pair of chromosomes with 18S rDNA sites. The studied species presented variations in the number and position of these sites. The results accomplished were similar to those obtained by the analysis of AgNORs, revealing the same interspecific variability. Each species exhibited distinctive patterns of AgNOR and 18S rDNA distribution, which can be considered cytogenetic markers in each species of the genus and help improve the discussions on the phylogeny of the group.

  16. Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification.

    PubMed

    El-Sherry, Shiem; Ogedengbe, Mosun E; Hafeez, Mian A; Barta, John R

    2013-07-01

    Multiple 18S rDNA sequences were obtained from two single-oocyst-derived lines of each of Eimeria meleagrimitis and Eimeria adenoeides. After analysing the 15 new 18S rDNA sequences from two lines of E. meleagrimitis and 17 new sequences from two lines of E. adenoeides, there were clear indications that divergent, paralogous 18S rDNA copies existed within the nuclear genome of E. meleagrimitis. In contrast, mitochondrial cytochrome c oxidase subunit I (COI) partial sequences from all lines of a particular Eimeria sp. were identical and, in phylogenetic analyses, COI sequences clustered unambiguously in monophyletic and highly-supported clades specific to individual Eimeria sp. Phylogenetic analysis of the new 18S rDNA sequences from E. meleagrimitis showed that they formed two distinct clades: Type A with four new sequences; and Type B with nine new sequences; both Types A and B sequences were obtained from each of the single-oocyst-derived lines of E. meleagrimitis. Together these rDNA types formed a well-supported E. meleagrimitis clade. Types A and B 18S rDNA sequences from E. meleagrimitis had a mean sequence identity of only 97.4% whereas mean sequence identity within types was 99.1-99.3%. The observed intraspecific sequence divergence among E. meleagrimitis 18S rDNA sequence types was even higher (approximately 2.6%) than the interspecific sequence divergence present between some well-recognized species such as Eimeria tenella and Eimeria necatrix (1.1%). Our observations suggest that, unlike COI sequences, 18S rDNA sequences are not reliable molecular markers to be used alone for species identification with coccidia, although 18S rDNA sequences have clear utility for phylogenetic reconstruction of apicomplexan parasites at the genus and higher taxonomic ranks.

  17. Sequence variation of ribosomal internal transcribed spacers (ITS) in commercially important Phytoseiidae mites.

    PubMed

    Navajas, M; Lagnel, J; Fauvel, G; de Moraes, G

    1999-11-01

    Preliminary work is needed to assess the usefulness of different markers at different taxonomic scales when a new group is analyzed, such as the commercially important Phytoseiidae mites. We investigate here the level of sequence variation of the nuclear ribosomal spacers ITS 1 and 2 and the 5.8S gene in six species of Phytoseiidae: Neoseiulus culifornicus, N. fallacis, Euseius concordis, Metaseiulus occidentalis, Typhlodromus pyri and Phytoseiulus persimilis. As expected, the 5.8S gene (148 base pairs) is markedly conserved and displays little variation in between genera comparisons. ITS1 and ITS2 show contrasting patterns: while the ITS2 is short (80-89 bp) and shows little variation, the ITS1 is longer (303-404 bp) and is very variable in sequence. This fact compromises reliable nucleotide homologies when comparing the genera. The comparison of ITS1 sequence similarity at the species level might be useful for species identification, however, the value of ITS in taxonomic studies does not extend to the level of the family. The intraspecific variations of ITS were investigated in three species: N. californicus, N. fallacis and E. concordis. The first species has identical ITS1 sequences and the last two display low polymorphism (2 nucleotide substitutions). The ITS2 and 5.8S sequences were identical in all three subspecies comparisons.

  18. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six DNA regions were evaluated in a multi-national, multi-laboratory consortium as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it...

  19. Confirmation of hybrid origin of Cyrtanthus based on the sequence analysis of internal transcribed spacer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to create interspecific hybrids between Cyrtanthus elatus and C. sanguineus and to confirm the hybrid origin of the progeny based on morphological characters and using molecular markers. The tip of the leaves, the shape and size of cells, and stomata distribution i...

  20. Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region.

    PubMed

    GokulRaj, Kathamuthu; Sundaresan, Natesan; Ganeshan, Enthai Jagan; Rajapriya, Pandi; Muthumary, Johnpaul; Sridhar, Jayavel; Pandi, Mohan

    2014-01-01

    Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence - secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.

  1. Testing three pipelines for 18S rDNA-based metabarcoding of soil faunal diversity.

    PubMed

    Yang, ChenXue; Ji, YingQiu; Wang, XiaoYang; Yang, ChunYang; Yu, Douglas W

    2013-01-01

    A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.

  2. 18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa.

    PubMed

    Kudryavtsev, Alexander; Bernhard, Detlef; Schlegel, Martin; Chao, Ema E Y; Cavalier-Smith, Thomas

    2005-08-01

    Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.

  3. 18S rDNA phylogeny of lamproderma and allied genera (Stemonitales, Myxomycetes, Amoebozoa).

    PubMed

    Fiore-Donno, Anna Maria; Kamono, Akiko; Meyer, Marianne; Schnittler, Martin; Fukui, Manabu; Cavalier-Smith, Thomas

    2012-01-01

    The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (~600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species.

  4. 18S rDNA Phylogeny of Lamproderma and Allied Genera (Stemonitales, Myxomycetes, Amoebozoa)

    PubMed Central

    Fiore-Donno, Anna Maria; Kamono, Akiko; Meyer, Marianne; Schnittler, Martin; Fukui, Manabu; Cavalier-Smith, Thomas

    2012-01-01

    The phylogenetic position of the slime-mould genus Lamproderma (Myxomycetes, Amoebozoa) challenges traditional taxonomy: although it displays the typical characters of the order Stemonitales, it appears to be sister to Physarales. This study provides a small subunit (18S or SSU) ribosomal RNA gene-based phylogeny of Lamproderma and its allies, with new sequences from 49 specimens in 12 genera. We found that the order Stemonitales and Lamproderma were both ancestral to Physarales and that Lamproderma constitutes several clades intermingled with species of Diacheopsis, Colloderma and Elaeomyxa. We suggest that these genera may have evolved from Lamproderma by multiple losses of fruiting body stalks and that many taxonomic revisions are needed. We found such high genetic diversity within three Lamproderma species that they probably consist of clusters of sibling species. We discuss the contrasts between genetic and morphological divergence and implications for the morphospecies concept, highlighting the phylogenetically most reliable morphological characters and pointing to others that have been overestimated. In addition, we showed that the first part (∼600 bases) of the SSU rDNA gene is a valuable tool for phylogeny in Myxomycetes, since it displayed sufficient variability to distinguish closely related taxa and never failed to cluster together specimens considered of the same species. PMID:22530009

  5. Transcribing and digitizing eighteenth- and nineteenth-century letters for a historical digital repository.

    PubMed

    Dunster, Emily S; Kipnis, Daniel G; Angelo, F Michael

    2014-01-01

    In fall 2011, the Scott Memorial Library purchased 53 letters belonging to an 1841 graduate of Jefferson Medical College, John Plimpton Green. The library staff transcribed and digitized the letters, creating an online collection in the university's institutional repository, Jefferson Digital Commons. This article will detail the process of transcribing and digitizing the collection along with sharing statistics and the benefits of this project to global researchers.

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

    PubMed Central

    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 sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89–95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73–5.04 μm) × 3.94 μm (3.50–4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships. PMID:25772204

  7. Identification of transcribed sequences in the human genome

    SciTech Connect

    Gardiner, K.

    1992-12-01

    The workshop was held at the National Institutes of Mental Health, Bethesda, Maryland, on October 4 and 5, 1991. Twenty-four investigators attended from England, Germany and the United States. The topics discussed included: Genome sequence analysis using computer assisted detection of open reading frames, splice sites and hexamer patterns, direct exon identification using trapping of internal and 3' exons, and a recombination based system, cDNA library construction and screening, including the use of normalization and subtraction procedures, Alu and splice donor site PCR from hybrid cell lines, and microdissection clones as probes, use of labeled CDNAS as probes to screen lambda and cosmid libraries, and sequencing of random cDNAs.

  8. Radiolaria Divided into Polycystina and Spasmaria in Combined 18S and 28S rDNA Phylogeny

    PubMed Central

    Dolven, Jane K.; Ose, Randi F.; Klaveness, Dag; Kristensen, Tom; Bjørklund, Kjell R.; Shalchian-Tabrizi, Kamran

    2011-01-01

    Radiolarians are marine planktonic protists that belong to the eukaryote supergroup Rhizaria together with Foraminifera and Cercozoa. Radiolaria has traditionally been divided into four main groups based on morphological characters; i.e. Polycystina, Acantharia, Nassellaria and Phaeodaria. But recent 18S rDNA phylogenies have shown that Phaeodaria belongs within Cerocozoa, and that the previously heliozoan group Taxopodida should be included in Radiolaria. 18S rDNA phylogenies have not yet resolved the sister relationship between the main Radiolaria groups, but nevertheless suggests that Spumellaria, and thereby also Polycystina, are polyphyletic. Very few sequences other than 18S rDNA have so far been generated from radiolarian cells, mostly due to the fact that Radiolaria has been impossible to cultivate and single cell PCR has been hampered by low success rate. Here we have therefore investigated the mutual evolutionary relationship of the main radiolarian groups by using the novel approach of combining single cell whole genome amplification with targeted PCR amplification of the 18S and 28S rDNA genes. Combined 18S and 28S phylogeny of sequences obtained from single cells shows that Radiolaria is divided into two main lineages: Polycystina (Spumellaria+Nassellaria) and Spasmaria (Acantharia+Taxopodida). Further we show with high support that Foraminifera groups within Radiolaria supporting the Retaria hypothesis. PMID:21853146

  9. The Voice Transcription Technique: Use of Voice Recognition Software to Transcribe Digital Interview Data in Qualitative Research

    ERIC Educational Resources Information Center

    Matheson, Jennifer L.

    2007-01-01

    Transcribing interview data is a time-consuming task that most qualitative researchers dislike. Transcribing is even more difficult for people with physical limitations because traditional transcribing requires manual dexterity and the ability to sit at a computer for long stretches of time. Researchers have begun to explore using an automated…

  10. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis

    PubMed Central

    Michael, E. S.; Kuliopulos, A.; Covic, L.; Steer, M. L.

    2013-01-01

    Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2−/− mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca2+ concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP. PMID:23275617

  11. Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis.

    PubMed

    Michael, E S; Kuliopulos, A; Covic, L; Steer, M L; Perides, G

    2013-03-01

    Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2(-/-) mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca(2+) concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

  12. Metabolism of 18S rRNA in rat liver cells in different functional states of protein-synthesizing apparatus

    SciTech Connect

    Chirkov, G.P.; Druzhinina, M.K.; Todorov, I.N.

    1986-04-10

    The ratio of the absolute radioactivities of 28S and 18S RNAs in the fractions of membrane-bound and free polysomes and the fraction of free rat liver ribosomes was studied under conditions of inhibition of translation by cycloheximide, insulin, and cAMP. It was found that insulin and cAMP, in contrast to cycloheximide, do not induce selective degradation of 18S rRNA. The results are discussed from the standpoint of the possible role of the phosphorylation of protein S6 in the degradation of the 40S ribosomal subunit.

  13. Transcribed sequences in the human genome to be held in San Francisco, November 7 and 8, 1992. Final report, September 1, 1992--August 31, 1993

    SciTech Connect

    Gardiner, K.

    1993-11-01

    The Second International Workshop on the Identification of Transcribed Sequences was held in San Francisco on November 7--8, 1992. The purpose of the workshop was to discuss and evaluate techniques for developing a complete transcriptional map of the human genome. Such a map requires the positions, sequences, and expression patterns of all genes. This goal is being approached from two different directions, each with strengths and weaknesses. One method is to identify the transcribed sequences from genomic DNA of a given region; the other is to systematically sequence and map cDNAs. The cDNA approach yields sequence information rapidly, but mapping each cDNA is a technical challenge. In the first approach, the map locations of genomic sequences are known at the outset, and the challenge is to identify exons. The efficient construction of a transcriptional map will require a diverse array of techniques.

  14. Sequencing and characterization of full-length sequence of 18S rRNA gene from the reniform nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Variation within this gene is rare but it has been observed in few metazoan species. For the first time, we h...

  15. Eukaryotic diversity in premise drinking water using 18S rDNA sequencing: implications for health risks

    EPA Science Inventory

    The goal of this study was to characterize microbial eukaryotes over a 12 month period, so as to provide insight into the occurrence of potentially important predators and bacterial hosts in hot and cold premise plumbing. Nearly 6,300 partial (600 bp) 18S rRNA gene sequences from...

  16. Nucleosomes are depleted at the VSG expression site transcribed by RNA polymerase I in African trypanosomes.

    PubMed

    Figueiredo, Luisa M; Cross, George A M

    2010-01-01

    In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.

  17. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  18. TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed

    PubMed Central

    Papantonis, Argyris; Kohro, Takahide; Baboo, Sabyasachi; Larkin, Joshua D; Deng, Binwei; Short, Patrick; Tsutsumi, Shuichi; Taylor, Stephen; Kanki, Yasuharu; Kobayashi, Mika; Li, Guoliang; Poh, Huay-Mei; Ruan, Xiaoan; Aburatani, Hiroyuki; Ruan, Yijun; Kodama, Tatsuhiko; Wada, Youichiro; Cook, Peter R

    2012-01-01

    Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories. PMID:23103767

  19. The nuclear 18S ribosomal RNA gene as a source of phylogenetic information in the genus Taenia.

    PubMed

    Yan, Hongbin; Lou, Zhongzi; Li, Li; Ni, Xingwei; Guo, Aijiang; Li, Hongmin; Zheng, Yadong; Dyachenko, Viktor; Jia, Wanzhong

    2013-03-01

    Most species of the genus Taenia are of considerable medical and veterinary significance. In this study, complete nuclear 18S rRNA gene sequences were obtained from seven members of genus Taenia [Taenia multiceps, Taenia saginata, Taenia asiatica, Taenia solium, Taenia pisiformis, Taenia hydatigena, and Taenia taeniaeformis] and a phylogeny inferred using these sequences. Most of the variable sites fall within the variable regions, V1-V5. We show that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.

  20. Cystoisospora spp. from dogs in China and phylogenetic analysis of its 18S and ITS1 gene.

    PubMed

    He, Pengfei; Li, Jianhua; Gong, Pengtao; Huang, Jingui; Zhang, Xichen

    2012-11-23

    Cystoisospora spp. oocysts isolated from dog feces in Changchun, China were morphologically similar to those of Cystoisospora ohioensis and Cystoisospora sp. 1-MM recently isolated from dogs in Japanese. Sequencing results of the 18S subunit RNA gene from isolates in the present study were compared to other Cystoisospora spp. and the results suggested that Cystoisospora spp. from dogs in Changchun was homologous to C. ohioensis and Cystoisospora sp. 1-MM. Phylogenetic analysis of the 18S rRNA sequences showed that the Cystoisospora sp. ChangChun 1 and Cystoisospora sp. ChangChun 2 were nested in a clade with other Cystoisospora spp., including C. ohioensis, Cystoisospora belli, Cystoisospora suis, Isospora sp. Harbin/01/08 and C. orlovi,. Cystoisospora sp. ChangChun 2 was confirmed as C. ohioensis, and the other isolate was in a separate clade but the genetic relationship was relatively close to C. suis after analysis of the ITS-1gene.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

    PubMed

    Thompson, C; Baravalle, M E; Valentini, B; Mangold, A; Torioni de Echaide, S; Ruybal, P; Farber, M; Echaide, I

    2014-06-01

    The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

  3. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    PubMed

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

  4. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

    PubMed Central

    de Souza, Sandro J.; Camargo, Anamaria A.; Briones, Marcelo R. S.; Costa, Fernando F.; Nagai, Maria Aparecida; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; de Fátima Sonati, Maria; Tajara, Eloiza H.; Valentini, Sandro R.; Acencio, Marcio; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Bengtson, Mário Henrique; Carraro, Dirce M.; Carvalho, Alex F.; Carvalho, Lúcia Helena; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Costa, Maria Cristina R.; Curcio, Cyntia; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Leite, Luciana C. C.; Maia, Gustavo; Majumder, Paromita; Marins, Mozart; Matsukuma, Adriana; Melo, Analy S. A.; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana Gilbert; Rahal, Paula; Rainho, Claudia A.; da Ro's, Nancy; de Sá, Renata G.; Sales, Magaly M.; da Silva, Neusa P.; Silva, Tereza C.; da Silva, Wilson; Simão, Daniel F.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Zalcberg, Heloisa; Brentani, Ricardo R.; Reis, Luis F. L.; Dias-Neto, Emmanuel; Simpson, Andrew J. G.

    2000-01-01

    Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html). PMID:11070084

  5. Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells.

    PubMed

    Hagenbüchle, O; Santer, M; Steitz, J A; Mans, R J

    1978-03-01

    DNA sequencing methods have been used to determine a sequence of about 20 nucleotides at the 3' termini of various 18S (small ribosomal subunit) RNA molecules. Polyadenylated rRNA was first synthesized using the enzyme ATP:polynucleotidyl transferase from mainze. Then in the presence of an oligonucleotide primer uniquely complementary to the end of each adenylated rRNA, a cDNA copy was produced using AMV reverse transcriptase. In every case, the cDNA transcript was of finite size, which we ascribe to the appearance of an oligonucleotide containing m62A near the 3' end of the 18S rRNAs. Sequences at the 3' termini of 18S rRNA molecules from the four eucaryotic species examined here (mouse, silk worm, wheat embryo and slime mold) are highly conserved. They also exhibit strong homology to the 3' end of E. coli 16S rRNA. Two important differences, however, are apparent. First, the 16S sequence CCUCC, implicated in mRNA binding by E. coli ribosomes, is absent from each eucaryotic rRNA sequence. Second, a purine-rich region which exhibits extensive complementarity to the 5' noncoding regions of many eucaryotic mRNAs appears consistently.

  6. Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

    PubMed Central

    1976-01-01

    4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders. PMID:944187

  7. Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp.

    PubMed

    Walochnik, J; Obwaller, A; Aspöck, H

    2001-02-01

    Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.

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

    PubMed Central

    Tsagkogeorga, Georgia; Turon, Xavier; Hopcroft, Russell R; Tilak, Marie-Ka; Feldstein, Tamar; Shenkar, Noa; Loya, Yossi; Huchon, Dorothée; Douzery, Emmanuel JP; Delsuc, Frédéric

    2009-01-01

    Background Tunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea. Results Thirty new complete 18S rRNA sequences were acquired from previously unsampled tunicate species, with special focus on groups presenting high evolutionary rate. The updated 18S rRNA dataset has been aligned with respect to the constraint on homology imposed by the rRNA secondary structure. A probabilistic framework of phylogenetic reconstruction was adopted to accommodate the particular evolutionary dynamics of this ribosomal marker. Detailed Bayesian analyses were conducted under the non-parametric CAT mixture model accounting for site-specific heterogeneity of the evolutionary process, and under RNA-specific doublet models accommodating the occurrence of compensatory substitutions in stem regions. Our results support the division of tunicates into three major clades: 1) Phlebobranchia + Thaliacea + Aplousobranchia, 2) Appendicularia, and 3) Stolidobranchia, but the position of Appendicularia could not be firmly resolved. Our study additionally reveals that most Aplousobranchia evolve at extremely high rates involving changes in secondary structure of their 18S rRNA, with the exception of the family Clavelinidae, which appears to be slowly evolving. This extreme rate heterogeneity precluded resolving with certainty the exact phylogenetic placement of Aplousobranchia. Finally, the best fitting secondary-structure and CAT-mixture models suggest a sister

  9. Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA.

    PubMed

    Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M; Villegas, Leopoldo; Escalante, Ananias A; Kachur, S Patrick; Barnwell, John W; Peterson, David S; Udhayakumar, Venkatachalam; Kissinger, Jessica C

    2011-07-01

    Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.

  10. Molecular phylogenetics of subclass Peritrichia (Ciliophora: Oligohymenophorea) based on expanded analyses of 18S rRNA sequences.

    PubMed

    Utz, Laura R P; Eizirik, Eduardo

    2007-01-01

    Phylogenetic relationships among peritrich ciliates remain unclear in spite of recent progress. To expand the analyses performed in previous studies, and to statistically test hypotheses of monophyly, we analyzed a broad sample of 18s rRNA sequences (including 15 peritrich genera), applying a conservative alignment strategy and several phylogenetic approaches. The main results are that: (i) the monophyly of Peritrichia cannot be rejected; (ii) the two main clades of Sessilida do not correspond to formally recognized taxa; (iii) the monophyly of genera Vorticella and Epistylis is significantly rejected; and (iv) morphological structures commonly used in peritrich taxonomy may be evolutionarily labile.

  11. The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes

    PubMed Central

    Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

    2011-01-01

    Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression. PMID:22081017

  12. A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases

    PubMed Central

    Heberling, Tamra; Davis, Lisa; Gedeon, Jakub; Morgan, Charles; Gedeon, Tomáš

    2016-01-01

    In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs) transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases. PMID:27517607

  13. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    NASA Astrophysics Data System (ADS)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  14. Replication-dependent histone genes are actively transcribed in differentiating and aging retinal neurons.

    PubMed

    Banday, Abdul Rouf; Baumgartner, Marybeth; Al Seesi, Sahar; Karunakaran, Devi Krishna Priya; Venkatesh, Aditya; Congdon, Sean; Lemoine, Christopher; Kilcollins, Ashley M; Mandoiu, Ion; Punzo, Claudio; Kanadia, Rahul N

    2014-01-01

    In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.

  15. Conservation and Purifying Selection of Transcribed Genes Located in a Rice Centromere[W

    PubMed Central

    Fan, Chuanzhu; Walling, Jason G.; Zhang, Jianwei; Hirsch, Cory D.; Jiang, Jiming; Wing, Rod A.

    2011-01-01

    Recombination is strongly suppressed in centromeric regions. In chromosomal regions with suppressed recombination, deleterious mutations can easily accumulate and cause degeneration of genes and genomes. Surprisingly, the centromere of chromosome8 (Cen8) of rice (Oryza sativa) contains several transcribed genes. However, it remains unclear as to what selective forces drive the evolution and existence of transcribed genes in Cen8. Sequencing of orthologous Cen8 regions from two additional Oryza species, Oryza glaberrima and Oryza brachyantha, which diverged from O. sativa 1 and 10 million years ago, respectively, revealed a set of seven transcribed Cen8 genes conserved across all three species. Chromatin immunoprecipitation analysis with the centromere-specific histone CENH3 confirmed that the sequenced orthologous regions are part of the functional centromere. All seven Cen8 genes have undergone purifying selection, representing a striking phenomenon of active gene survival within a recombination-free zone over a long evolutionary time. The coding sequences of the Cen8 genes showed sequence divergence and mutation rates that were significantly reduced from those of genes located on the chromosome arms. This suggests that Oryza has a mechanism to maintain the fidelity and functionality of Cen8 genes, even when embedded in a sea of repetitive sequences and transposable elements. PMID:21856794

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Phylogenetic Relationships Among Xiphinema and Xiphidorus Nematode Species from Brazil Inferred from 18S rDNA Sequences

    PubMed Central

    Oliveira, Claudio M. G.; Hübschen, Judith; Brown, Derek J. F.; Ferraz, Luiz C. C. B.; Wright, Frank; Neilson, Roy

    2004-01-01

    Maximum likelihood trees produced from 18S rDNA sequences separated 14 Xiphinema and five Xiphidorus nematode species from Brazil into distinct groups that concurred with their current morphological taxonomic status. Species belonging to the X. americanum group (X. brevicolle, X. diffusum, X. oxycaudatum, and X. peruvianum) formed a single group that was clearly separated from the other Xiphinema species. As with previous taxonomic studies that noted only minor morphological differences between putative X. americanum group species, separation of these species based upon 18S rDNA sequences was inconclusive. Thus it is probable that instead of comprising distinct species, the X. americanum group may in fact represent numerous morphotypes with large inter- and intra- population morphological variability that may be environmentally driven. Within the cluster representing non X. americanum group species, there was little statistical support to clearly separate species. However, three subgroups, comprising (i) the X. setariae/vulgare complex, (ii) X. ifacolum and X. paritaliae, and (iii) X. brasiliense and X. ensiculiferum were well resolved. PMID:19262801

  18. Optical and electrical stability of viral-templated copper sulfide (Cu{sub 1.8}S) films

    SciTech Connect

    Shahriar Zaman, Mohammed; Bernard Grajeda, Gabriel; Haberer, Elaine D.

    2014-04-14

    The optical and electrical stabilities of viral-templated non-stoichiometric copper sulfide, digenite (Cu{sub 1.8}S) films were investigated. The films were composed of large agglomerates of randomly aligned Cu{sub 1.8}S-coated M13 filamentous phage. Free carrier optical absorption associated with localized surface plasmon resonance (LSPR) was observed in the near infrared spectral region, and the films were electrically active, displaying a linear current-voltage relationship. Under ambient conditions, the magnitude of the LSPR absorption increased, following a power law relationship with time, and the electrical resistance of viral-templated films decreased significantly. In contrast, the resistance of films stored under low oxygen, low humidity conditions experienced a smaller reduction in electrical resistance. Changes in optical and electrical film properties under ambient conditions were associated with an increase in free carrier concentration within the copper chalcogenide material due to oxygen exposure. X-ray photoelectron spectroscopy was used to relate this increase in free carrier concentration to compositional changes on the viral-templated material surface.

  19. Early diagnosis of Exophiala CAPD peritonitis by 18S ribosomal RNA gene sequencing and its clinical significance.

    PubMed

    Lau, Susanna K P; Woo, Patrick C Y; Chiu, Siu-kau; Leung, Kit-wah; Yung, Raymond W H; Yuen, Kwok-yung

    2003-06-01

    Phenotypic identification of fungi in clinical microbiology laboratories is often difficult and late, especially for slow growing and rarely encountered fungi. We describe the application of 18S ribosomal RNA (rRNA) gene sequencing in the early diagnosis of a case of Exophiala peritonitis. A yeast-like fungus was isolated from the dialysate fluid of a 66-year-old man undergoing continuous ambulatory peritoneal dialysis. It grew slowly after 12 days of incubation to yield mature cultures to permit recognition of microscopic features resembling those of Exophiala, a dematiacerous mold. 18S rRNA gene sequencing provided results 12 days earlier than phenotypic identification and revealed 15 base difference (0.9%) between the isolate and Exophiala sp. strain GHP 1205 (GenBank Accession no. AJ232954), indicating that the isolate most closely resembles a strain of Exophiala species. The patient responded to 4 weeks of intravenous amphotericin B therapy. Early identification of the fungus was important for the choice of anti-fungal regimen. As opportunistic fungal infections in immunocompromised patients are globally emerging problems, the development of molecular techniques for fungal identification is crucial for early diagnosis and appropriate treatment.

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

    PubMed Central

    Gomez-Rodriguez, Victor Manuel; Rodriguez-Garay, Benjamin; Palomino, Guadalupe; Martínez, Javier; Barba-Gonzalez, Rodrigo

    2013-01-01

    Abstract 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 Agave tequilana Weber, 1902 ‘Azul’, Agave cupreata Trelease et Berger, 1915 and Agave 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. PMID:24260700

  1. Distribution of 18S rDNA sites and absence of the canonical TTAGG insect telomeric repeat in parasitoid Hymenoptera.

    PubMed

    Gokhman, Vladimir E; Anokhin, Boris A; Kuznetsova, Valentina G

    2014-08-01

    Karyotypes of six species belonging to three main clades of parasitoid Hymenoptera, the superfamilies Ichneumonoidea (Ichneumonidae: Ichneumon amphibolus), Cynipoidea (Cynipidae: Diplolepis rosae) and Chalcidoidea (Eurytomidae: Eurytoma robusta, Eu. serratulae and Eu. compressa, and Torymidae: Torymus bedeguaris) were studied using FISH with 18S rDNA and telomeric (TTAGG)n probes. Haploid karyotypes of D. rosae, Eu. robusta and Eu. serratulae carried the only 18S rDNA hybridization signal, whereas those of I. amphibolus and Eu. compressa carried three and two rDNA clusters respectively. In addition, three rDNA sites were visualized in the aneuploid female of T. bedeguaris. The number of rDNA clusters in parasitoid Hymenoptera generally correlates to the chromosome number. Apart from the overwhelming majority of the studied species of aculeate Hymenoptera, no hybridization signals were obtained from FISH with the telomeric (TTAGG)n probe in the examined parasitoid species. These data suggest absence of the canonical (TTAGG)n insect telomeric motif in the Ichneumonoidea, Cynipoidea and Chalcidoidea, and perhaps in parasitoid Hymenoptera in general.

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

    PubMed

    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.

  3. The ATPase hCINAP regulates 18S rRNA processing and is essential for embryogenesis and tumour growth

    PubMed Central

    Bai, Dongmei; Zhang, Jinfang; Li, Tingting; Hang, Runlai; Liu, Yong; Tian, Yonglu; Huang, Dadu; Qu, Linglong; Cao, Xiaofeng; Ji, Jiafu; Zheng, Xiaofeng

    2016-01-01

    Dysfunctions in ribosome biogenesis cause developmental defects and increased cancer susceptibility; however, the connection between ribosome assembly and tumorigenesis remains unestablished. Here we show that hCINAP (also named AK6) is required for human 18S rRNA processing and 40S subunit assembly. Homozygous CINAP−/− mice show embryonic lethality. The heterozygotes are viable and show defects in 18S rRNA processing, whereas no delayed cell growth is observed. However, during rapid growth, CINAP haploinsufficiency impairs protein synthesis. Consistently, hCINAP depletion in fast-growing cancer cells inhibits ribosome assembly and abolishes tumorigenesis. These data demonstrate that hCINAP reduction is a specific rate-limiting controller during rapid growth. Notably, hCINAP is highly expressed in cancers and correlated with a worse prognosis. Genome-wide polysome profiling shows that hCINAP selectively modulates cancer-associated translatome to promote malignancy. Our results connect the role of hCINAP in ribosome assembly with tumorigenesis. Modulation of hCINAP expression may be a promising target for cancer therapy. PMID:27477389

  4. Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region.

    PubMed

    Payvar, F; DeFranco, D; Firestone, G L; Edgar, B; Wrange, O; Okret, S; Gustafsson, J A; Yamamoto, K R

    1983-12-01

    Glucocorticoid receptor protein stimulates transcription initiation within murine mammary tumor virus (MTV) DNA sequences in vivo, and interacts selectively with MTV DNA in vitro. We mapped and compared five regions of MTV DNA that are bound specifically by purified receptor; one resides upstream of the transcription start site, and the others are distributed within transcribed sequences between 4 and 8 kb from the initiation site. Each region contains at least two strong binding sites for receptor, which itself appears to be a tetramer of 94,000 dalton hormone-binding subunits. Three of the five binding regions contain nine nuclease footprints that lack extensive homology, although a family of related octanucleotides can be discerned. Receptor interacts with the different regions with similar efficiencies, suggesting that receptor affinity for upstream and internal regions may differ by less than one order of magnitude. Moreover, each region appears to be bound independent of the others. A restriction fragment containing four footprint sequences from one of the regions has previously been shown to act in vivo as a receptor-dependent transcriptional enhancer element, implying that the binding sites detected in vitro may be biologically functional.

  5. Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data.

    PubMed

    Conti, Anastasia; Carnevali, Davide; Bollati, Valentina; Fustinoni, Silvia; Pellegrini, Matteo; Dieci, Giorgio

    2015-01-01

    Of the ∼ 1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼ 1300 Alu loci corresponding to detectable transcripts, with ∼ 120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5'-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions.

  6. Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data

    PubMed Central

    Conti, Anastasia; Carnevali, Davide; Bollati, Valentina; Fustinoni, Silvia; Pellegrini, Matteo; Dieci, Giorgio

    2015-01-01

    Of the ∼1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼1300 Alu loci corresponding to detectable transcripts, with ∼120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5′-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions. PMID:25550429

  7. The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-04-01

    The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

  8. Further evidence for the variability of the 18S rDNA loci in the family Tingidae (Hemiptera, Heteroptera)

    PubMed Central

    Golub, Natalia V.; Golub, Viktor B.; Kuznetsova, Valentina G.

    2016-01-01

    Abstract As of now, within the lace bug family Tingidae (Cimicomorpha), only 1.5% of the species described have been cytogenetically studied. In this paper, male karyotypes of Stephanitis caucasica, Stephanitis pyri, Physatocheila confinis, Lasiacantha capucina, Dictyla rotundata and Dictyla echii were studied using FISH mapping with an 18S rDNA marker. The results show variability: the major rDNA sites are predominantly located on a pair of autosomes but occasionally on the X and Y chromosomes. All currently available data on the distribution of the major rDNA in the Tingidae karyotypes are summarized and shortly discussed. Our main concern is to clarify whether the chromosomal position of rDNA loci can contribute to resolving the phylogenetic relationships among the Tingidae taxa. PMID:28123675

  9. Granulomatous prostatitis due to Cryptococcus neoformans: diagnostic usefulness of special stains and molecular analysis of 18S rDNA.

    PubMed

    Wada, R; Nakano, N; Yajima, N; Yoneyama, T; Wakasaya, Y; Murakami, C; Yamato, K; Yagihashi, S

    2008-01-01

    A 57-year-old Japanese man complained of pain on micturition. The prostate was of normal size but hard. Transrectal needle biopsy demonstrated granulomatous prostatitis with small focal abscesses. Staining with periodic acid-Schiff, Grocott's methenamine silver and Fontana-Masson revealed yeast-form fungus in the granulomas. The mucoid capsule of the fungus stained with mucicarmine. PCR specific for cryptococcal 18S rDNA using DNA extracted from the pathological specimen was positive, and the sequence was homologous to Cryptococcus neoformans. A diagnosis of cryptococcal granulomatous prostatitis was made. The patient was then found to suffer from meningitis and lung abscess, and was treated with amphotericin B and flucytosine. Careful histological and molecular studies are beneficial to reach the correct diagnosis and to prevent an unfavorable outcome of disseminated cryptococcosis.

  10. Phylogenetic relationships of the Culicomorpha inferred from 18S and 5.8S ribosomal DNA sequences. (Diptera:Nematocera).

    PubMed

    Miller, B R; Crabtree, M B; Savage, H M

    1997-05-01

    We investigated the evolutionary origins of the mosquito family Culicidae by examination of 18S and 5.8S ribosomal gene sequence divergence. Phylogenetic analyses demonstrated that within the infraorder Culicomorpha, taxa in the families Corethrellidae, Chaoboridae and Culicidae formed a monophyletic group; there was support for a sister relationship between this lineage and a representative of the Chironomidae. A chaoborid midge was the closest relative of the mosquitoes. Taxa from four genera of mosquitoes formed a monophyletic group; lack of a spacer in the 5.8S gene was unique to members of the Culicidae. A member of the genus Anopheles formed the most basal lineage among the mosquitoes analysed. Phylogenetic relationships were unresolved for representatives in the families Dixidae, Simuliidae and Ceratopogonidae.

  11. Molecular Diversity of Eukaryotes in Municipal Wastewater Treatment Processes as Revealed by 18S rRNA Gene Analysis

    PubMed Central

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4–8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes. PMID:25491751

  12. Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha).

    PubMed

    Manylov, Oleg G; Vladychenskaya, Natalia S; Milyutina, Irina A; Kedrova, Olga S; Korokhov, Nikolai P; Dvoryanchikov, Gennady A; Aleshin, Vladimir V; Petrov, Nikolai B

    2004-03-01

    Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data.

  13. Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis.

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

    Eukaryotic communities involved in sewage treatment processes have been investigated by morphological identification, but have not yet been well-characterized using molecular approaches. In the present study, eukaryotic communities were characterized by constructing 18S rRNA gene clone libraries. The phylogenetic affiliations of a total of 843 clones were Alveolata, Fungi, Rhizaria, Euglenozoa, Stramenopiles, Amoebozoa, and Viridiplantae as protozoans and Rotifera, Gastrotricha, and Nematoda as metazoans. Sixty percent of the clones had <97% sequence identity to described eukaryotes, indicating the greater diversity of eukaryotes than previously recognized. A core OTU closely related to Epistylis chrysemydis was identified, and several OTUs were shared by 4-8 libraries. Members of the uncultured lineage LKM11 in Cryptomycota were predominant fungi in sewage treatment processes. This comparative study represents an initial step in furthering understanding of the diversity and role of eukaryotes in sewage treatment processes.

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

    NASA Astrophysics Data System (ADS)

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

    2004-08-01

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

  15. Molecular phylogeny of extant gymnosperms and seed plant evolution: analysis of nuclear 18S rRNA sequences.

    PubMed

    Chaw, S M; Zharkikh, A; Sung, H M; Lau, T C; Li, W H

    1997-01-01

    To study the evolutionary relationships among the four living gymnosperm orders and the interfamilial relationships in each order, a set of 65 nuclear 18S rRNA sequences from ferns, gymnosperms, and angiosperms was analyzed using the neighbor-joining and maximum-parsimony methods. With Selaginella as the outgroup, the analysis strongly indicates that the seed plants form a monophyletic group with the ferns as a sister group. Within the seed plants the angiosperms are clearly a monophyletic group. Although the bootstrap support for the monophyly of the gymnosperm clade is moderate, the monophyly is further supported by its lack of angiosperm-specific indels. Within the gymnosperms there appear to be three monophyletic clades: Cycadales-Ginkgoales, Gnetales, and Coniferales. The cycad-ginkgo clade is the earliest gymnosperm lineage. Given the strong support for the sister group relationship between Gnetales and Coniferales, it is unlikely that Gnetales is a sister group of the angiosperms, contrary to the view of many plant taxonomists. Within Coniferales, Pinaceae is monophyletic and basal to the remaining conifer families, among which there are three monophyletic clades: Phyllocladaceae-Podocarpaceae, Araucariaceae, and Sciadopityaceae-Taxaceae-Cephalotaxaceae-Taxodiacea e-Cupressaceae. Within the latter clade, Sciadopityaceae may be an outgroup to the other four families. Among the angiosperms, no significant cluster at the level of subclass was found, but there was evidence that Nymphaeaceae branched off first. Within the remaining angiosperms, the monocots included in this study are nested and form a monophyletic group. This study attests to the utility of nuclear 18S rRNA sequences in addressing relationships among living gymnosperms. Considerable variation in substitution rates was observed among the ferns and seed plants.

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

    PubMed

    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.

  17. Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

    PubMed Central

    Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

    2016-01-01

    The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

  18. Transcriptome map of plant mitochondria reveals islands of unexpected transcribed regions

    PubMed Central

    2011-01-01

    Background Plant mitochondria contain a relatively large amount of genetic information, suggesting that their functional regulation may not be as straightforward as that of metazoans. We used a genomic tiling array to draw a transcriptomic atlas of Oryza sativa japonica (rice) mitochondria, which was predicted to be approximately 490-kb long. Results Whereas statistical analysis verified the transcription of all previously known functional genes such as the ones related to oxidative phosphorylation, a similar extent of RNA expression was frequently observed in the inter-genic regions where none of the previously annotated genes are located. The newly identified open reading frames (ORFs) predicted in these transcribed inter-genic regions were generally not conserved among flowering plant species, suggesting that these ORFs did not play a role in mitochondrial principal functions. We also identified two partial fragments of retrotransposon sequences as being transcribed in rice mitochondria. Conclusion The present study indicated the previously unexpected complexity of plant mitochondrial RNA metabolism. Our transcriptomic data (Oryza sativa Mitochondrial rna Expression Server: OsMES) is publicly accessible at [http://bioinf.mind.meiji.ac.jp/cgi-bin/gbrowse/OsMes/#search]. PMID:21627843

  19. Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

    PubMed Central

    Conconi, Antonio; Bespalov, Vyacheslav A.; Smerdon, Michael J.

    2002-01-01

    Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. PMID:11782531

  20. 11p15-subband specific search for transcribed sequences using exon trapping

    SciTech Connect

    Loebbert, R.; Prawitt, D.; Monroe, D.

    1994-09-01

    Evidence from cytogenetic and molecular data suggest that the region 11p15 contains genes involved in different disorders, like Beckwith-Wiedemann syndrome (BWS), long QT syndrome (LQT), Usher syndrome type I and tumor development. Focusing on the subregion 11p15.1, we are isolating and characterizing new transcribed sequences. The applied strategy includes exon amplification and subsequent PCR screening of cDNA libraries. So far 100 YACs and 38 cosmid clones from 11p15.1-15.3 have been collected and are currently arrayed. 16 cosmids have been analyzed for transcribed sequences using the exon amplification scheme developed by Buckler et al. (1991). We were able to identify 18 exons that contain correct open reading frames and map back to the cosmid clones. A data base search revealed that two exons represent parts of known genes from this region (ST5 and AMPD3). Moreover, we identified one exon that represents an EGF-like repeat with homologies to various proteins. Using PCR and primers from the exon sequences, a fetal brain library, which has been arranged in the form of hierarchic arrayed phage pools, was screened. Up to now, two cDNA clones corresponding to different exons were isolated and are currently sequenced.

  1. Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

    SciTech Connect

    Niedojadlo, Janusz; Perret-Vivancos, Cecile; Kalland, Karl-Henning; Cmarko, Dusan; Cremer, Thomas; Driel, Roel van; Fakan, Stanislav

    2011-02-15

    The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

  2. Infrequently transcribed long genes depend on the Set2/Rpd3S pathway for accurate transcription

    PubMed Central

    Li, Bing; Gogol, Madelaine; Carey, Mike; Pattenden, Samantha G.; Seidel, Chris; Workman, Jerry L.

    2007-01-01

    The presence of Set2-mediated methylation of H3K36 (K36me) correlates with transcription frequency throughout the yeast genome. K36me targets the Rpd3S complex to deacetylate transcribed regions and suppress cryptic transcription initiation at certain genes. Here, using a genome-wide approach, we report that the Set2–Rpd3S pathway is generally required for controlling acetylation at coding regions. When using acetylation as a functional readout for this pathway, we discovered that longer genes and, surprisingly, genes transcribed at lower frequency exhibit a stronger dependency. Moreover, a systematic screen using high-resolution tiling microarrays allowed us to identify a group of genes that rely on Set2–Rpd3S to suppress spurious transcripts. Interestingly, most of these genes are within the group that depend on the same pathway to maintain a hypoacetylated state at coding regions. These data highlight the importance of using the functional readout of histone codes to define the roles of specific pathways. PMID:17545470

  3. Monitoring the mycobiota during Greco di Tufo and Aglianico wine fermentation by 18S rRNA gene sequencing.

    PubMed

    De Filippis, Francesca; La Storia, Antonietta; Blaiotta, Giuseppe

    2017-05-01

    Spontaneous alcoholic fermentation of grape must is a complex process, carried out by indigenous yeast populations arising from the vineyard or the winery environment and therefore representing an autochthonous microbial terroir of the production area. Microbial diversity at species and biotype level is extremely important in order to develop the composite and typical flavour profile of DOCG (Appellation of Controlled and Guaranteed Origin) wines. In this study, we monitored fungal populations involved in spontaneous fermentations of Aglianico and Greco di Tufo grape must by high-throughput sequencing (HTS) of 18S rRNA gene amplicons. We firstly proposed an alternative/addition to ITS as target gene in HTS studies and highlighted consistency between the culture-dependent and -independent approaches. A complex mycobiota was found at the beginning of the fermentation, mainly characterized by non-Saccharomyces yeasts and several moulds, with differences between the two types of grapes. Moreover, Interdelta patterns revealed a succession of several Saccharomyces cerevisiae biotypes and a high genetic diversity within this species.

  4. Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba.

    PubMed

    Fuerst, Paul A; Booton, Gregory C; Crary, Monica

    2015-01-01

    Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.

  5. [Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes].

    PubMed

    Petrov, N B; Pegova, A N; Manylov, O G; Vladychenskaia, N S; Miuge, N S; Aleshin, V V

    2007-01-01

    Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.

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

    PubMed

    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.

  7. Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

    PubMed Central

    Devente, Savannah R.; Kirk, Michelle R.; Seedorf, Henning; Dehority, Burk A.

    2015-01-01

    The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. PMID:25616800

  8. The structure of nucleosomal core particles within transcribed and repressed gene regions.

    PubMed Central

    Studitsky, V M; Belyavsky, A V; Melnikova, A F; Mirzabekov, A D

    1988-01-01

    The arrangement of histones along DNA in nucleosomal core particles within transcribed heat shock gene (hsp 70) region and repressed insertion within ribosomal genes of Drosophila was analysed by using protein-DNA crosslinking methods combined with hybridization tests. In addition, two-dimensional gel electrophoresis was employed to compare the overall nucleosomal shape and the nucleosomal DNA size. The arrangement of histones along DNA and general compactness of nucleosomes were shown to be rather similar in transcriptionally active and inactive genomic regions. On the other hand, nucleosomes within transcriptionally active chromatin are characterized by a larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility. Images PMID:3144704

  9. T7 RNA polymerase cannot transcribe through a highly knotted DNA template.

    PubMed Central

    Portugal, J; Rodríguez-Campos, A

    1996-01-01

    The ability of T7 RNA polymerase to transcribe a plasmid DNA in vitro in its linear, supercoiled, relaxed and knotted forms was analysed. Similar levels of transcription were found on each template with the exception of plasmids showing varying degrees of knotting (obtained using stoichiometric amounts of yeast topoisomerase II). A purified fraction of knotted DNA with a high number of nodes (crosses) was found to be refractory to transcription. The unknotting of the knotted plasmids, using catalytic amounts of topoisomerase II, restored their capacity as templates for transcription to levels similar to those obtained for the other topological forms. These results demonstrate that highly knotted DNA is the only topological form of DNA that is not a template for transcription. We suggest that the regulation of transcription, which depends on the topological state of the template, might be related to the presence of knotted DNA with different number of nodes. PMID:9016657

  10. Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells

    SciTech Connect

    Fromaget, Maud; Cook, Peter R. . E-mail: peter.cook@path.ox.ac.uk

    2007-08-15

    RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors - some of which are used therapeutically - affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.

  11. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells

    PubMed Central

    Arner, Erik; Daub, Carsten O.; Vitting-Seerup, Kristoffer; Andersson, Robin; Lilje, Berit; Drabløs, Finn; Lennartsson, Andreas; Rönnerblad, Michelle; Hrydziuszko, Olga; Vitezic, Morana; Freeman, Tom C.; Alhendi, Ahmad M. N.; Arner, Peter; Axton, Richard; Baillie, J. Kenneth; Beckhouse, Anthony; Bodega, Beatrice; Briggs, James; Brombacher, Frank; Davis, Margaret; Detmar, Michael; Ehrlund, Anna; Endoh, Mitsuhiro; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley; Goldowitz, Daniel; Guler, Reto; Ha, Thomas; Hara, Mitsuko; Herlyn, Meenhard; Ikawa, Tomokatsu; Kai, Chieko; Kawamoto, Hiroshi; Khachigian, Levon M.; Klinken, S. Peter; Kojima, Soichi; Koseki, Haruhiko; Klein, Sarah; Mejhert, Niklas; Miyaguchi, Ken; Mizuno, Yosuke; Morimoto, Mitsuru; Morris, Kelly J.; Mummery, Christine; Nakachi, Yutaka; Ogishima, Soichi; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Qin, Xian-Yang; Roy, Sugata; Sato, Hiroki; Savvi, Suzana; Saxena, Alka; Schwegmann, Anita; Sugiyama, Daisuke; Swoboda, Rolf; Tanaka, Hiroshi; Tomoiu, Andru; Winteringham, Louise N.; Wolvetang, Ernst; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Zabierowski, Susan; Zhang, Peter; Abugessaisa, Imad; Bertin, Nicolas; Diehl, Alexander D.; Fukuda, Shiro; Furuno, Masaaki; Harshbarger, Jayson; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Ishizu, Yuri; Itoh, Masayoshi; Kawashima, Tsugumi; Kojima, Miki; Kondo, Naoto; Lizio, Marina; Meehan, Terrence F.; Mungall, Christopher J.; Murata, Mitsuyoshi; Nishiyori-Sueki, Hiromi; Sahin, Serkan; Nagao-Sato, Sayaka; Severin, Jessica; de Hoon, Michiel J. L.; Kawai, Jun; Kasukawa, Takeya; Lassmann, Timo; Suzuki, Harukazu; Kawaji, Hideya; Summers, Kim M.; Wells, Christine; Hume, David A.; Forrest, Alistair R. R.; Sandelin, Albin; Carninci, Piero; Hayashizaki, Yoshihide

    2015-01-01

    Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation. PMID:25678556

  12. Rapid identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) using ribosomal RNA internal transcribed spacer 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in some countries of South America has increased the risk of this species invading North America. Differentiat...

  13. IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA-LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER-SPECIFIC PCR ASSAYS. (R826791)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  14. Sequence variation of the rDNA internal transcribed spacer (ITS) region among isolates of Rhizoctonia solani

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizoctonia solani is a common and highly heterogeneous fungal species. Sub-specific groups have been created based on hyphal anastomosis (AGs). One of the newer AGs described is AG-11 from soybean and rice seedlings or soil in Arkansas and lupine in Australia (Carling et al. Phytopathology 84:1378-...

  15. Fine‐scale Geographical Structure, Intra‐individual Polymorphism and Recombination in Nuclear Ribosomal Internal Transcribed Spacers in Armeria (Plumbaginaceae)

    PubMed Central

    NIETO FELINER, GONZALO; GUTIÉRREZ LARENA, BELÉN; FUERTES AGUILAR, JAVIER

    2004-01-01

    • Background and Aims Isolation and drift are the main causes for geographic structure of molecular variation. In contrast, the one found in a previous survey in Armeria (Plumbaginaceae) for nuclear ribosomal ITS multicopy regions was species‐independent and has been hypothesized to be due to extensive gene‐flow and biased concerted evolution. Since this was inferred from a genus‐level phylogenetic analysis, the aim of this study was to check for the occurrence of such structure and the validity of the proposed model at a local scale, in a southern Spanish massif (Sierra Nevada), as well as to examine the evolutionary implications at the organism level. • Methods In addition to 117 sequences of direct PCR products from genomic DNA, 50 sequences of PCR products from cloned DNA were obtained to analyse cases of intragenomic polymorphisms for the ITS regions. • Key Results Sequence data confirm the occurrence of a species‐independent structure at a local scale and reveal insights through the analysis of contact areas between different ITS copies (ribotypes). A comparison between cloned and direct sequences (a) confirms that, within these contact areas, ITS copies co‐occur both in different individuals and within single genomes; and (b) reveals recombination between different copies. • Conclusions This study supports the utility of direct sequences for detecting intra‐individual polymorphism and for partially inferring the ITS copies involved, given previous knowledge of the variability. The main evolutionary implication at the organism level is that gene‐flow and concerted evolution shape the geographic structure of ITS variation. PMID:14707002

  16. Total DNA transcription in vitro: a procedure to detect highly repetitive and transcribable sequences with tRNA-like structures

    SciTech Connect

    Endoh, H.; Okada, N.

    1986-01-01

    Total DNAs from various animals were transcribed in vitro in a HeLa cell extract, and it was found that one to several discrete RNAs were transcribed by RNA polymerase III. With tortoise (Geoclemys reevessi) and newt (Cynops pyrrhogaster), distinct 6.5S and 8S RNAs were transcribed from these respective DNAs. Representative phage clones carrying the 6.5S and 8S RNA genes were isolated from genomic libraries of these animals, and the sequences of these genes were determined. The 5' parts of highly repetitive and transcribable sequences of tortoise and newt were found to have close resemblance to tRNA/sub 1//sup Lys/ (rabbit) gene (78% homology) and a tRNA/sup Glu/ (Drosophila) gene (74% homology, not counting the aminoacyl stem region), respectively. The homologies extended to secondary structures, homologous nucleotides being located on similar secondary structures. It is proposed that many, if not all, highly repetitive and transcribable sequences detected by total DNA transcription have specific tRNA genes as their progenitors.

  17. Characterization of the vaginal fungal flora in pregnant diabetic women by 18S rRNA sequencing.

    PubMed

    Zheng, N-N; Guo, X-C; Lv, W; Chen, X-X; Feng, G-F

    2013-08-01

    Pregnancy and diabetes are regarded as individual risk factors for vaginal candidiasis. The high prevalence of vaginal candidiasis in pregnant diabetic women can be explained by disruption of the balance of the vaginal normal flora. However, little is known about the overall structure and composition of the vaginal fungal flora in pregnant diabetic women. In the present study, the diversity and richness of the vaginal fungal flora in healthy non-pregnant women (group HN), healthy pregnant women (group HP), women with gestational diabetes mellitus (group GDM), and pregnant women with diabetes mellitus type I (group T1DM) were investigated using an 18S rRNA gene clone library method. Our data demonstrated that the composition of the vaginal fungal flora in the four groups could be divided into two phyla (Ascomycetes, 20/26, and Basidiomycetes, 6/26). The most predominant vaginal fungal species belonged to the Candida and Saccharomyces genera, uncultured fungi, and a large number of low-abundance taxa that were unrecorded or underrepresented in previous studies using cultivation-dependent methods. Variation in operational taxonomic units (OTUs) between the study cohorts was generally high in the clone libraries, as 9, 13, 17, and 20 phylotypes were identified in groups HN, HP, GDM, and T1DM, respectively. The Shannon indices of groups GDM and T1DM (with poorer glycemic control) were significantly higher compared to groups HN and HP (p < 0.05). The data presented here revealed an increased diversity and varied composition of the vaginal fungal flora in pregnant diabetic women and demonstrated that poor glycemic control might be associated with disturbances in the vaginal fungal flora.

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

    USGS Publications Warehouse

    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.

  19. 17(R),18(S)-Epoxyeicosatetraenoic Acid, A Potent Eicosapentaenoic Acid (EPA)-Derived Regulator of Cardiomyocyte Contraction: Structure-Activity Relationships and Stable Analogs

    PubMed Central

    Falck, John R.; Wallukat, Gerd; Puli, Narender; Goli, Mohan; Arnold, Cosima; Konkel, Anne; Rothe, Michael; Fischer, Robert; Müller, Dominik N.; Schunck, Wolf-Hagen

    2011-01-01

    17(R),18(S)-Epoxyeicosatetraenoic acid [17(R),18(S)-EETeTr], a cytochrome P450 epoxygenase metabolite of eicosapentaenoic acid (EPA), exerts negative chronotropic effects and protects neonatal rat cardiomyocytes against Ca2+-overload with an EC50 ~1–2 nM. Structure-activity studies revealed a cis-Δ11,12- or Δ14,15-olefin and a 17(R),18(S)-epoxide are minimal structural elements for anti-arrhythmic activity whereas antagonist activity was often associated with the combination of a Δ14,15-olefin and a 17(S),18(R)-epoxide. Compared with natural material, the agonist and antagonist analogs are chemically and metabolically more robust and several show promise as templates for future development of clinical candidates. PMID:21591683

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

    PubMed

    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.

  1. The widely used small subunit 18S rDNA molecule greatly underestimates true diversity in biodiversity surveys of the meiofauna.

    PubMed

    Tang, Cuong Q; Leasi, Francesca; Obertegger, Ulrike; Kieneke, Alexander; Barraclough, Timothy G; Fontaneto, Diego

    2012-10-02

    Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding small-subunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.

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

    PubMed

    Abouheif, E; Zardoya, R; Meyer, A

    1998-10-01

    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 among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion.

  3. Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

    PubMed Central

    Kowalchuk, G A; Gerards, S; Woldendorp, J W

    1997-01-01

    Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys. PMID:9327549

  4. Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    The relationships among transcription, recombination, DNA damage, and repair in mammalian cells were investigated. We monitored the effects of transcription on UV-induced intrachromosomal recombination between neomycin repeats including a promoterless allele and an inducible heteroallele regulated by the mouse mammary tumor virus promoter. Although transcription and UV light separately stimulated recombination, increasing transcription levels reduced UV-induced recombination. Preferential repair of UV damage in transcribed strands was shown in highly transcribed DNA, suggesting that recombination is stimulated by unrepaired UV damage and that increased DNA repair in highly transcribed alleles removes recombinogenic lesions. This study indicates that the genetic consequences of DNA damage depend on transcriptional states and provides a basis for understanding tissue- and gene-specific responses to DNA-damaging agents. Images PMID:8264606

  5. Physical mapping of 18S-25S rDNA and 5S rDNA in Lupinus via fluorescent in situ hybridization.

    PubMed

    Naganowska, Barbara; Zielińska, Anna

    2002-01-01

    Double-target fluorescent in situ hybridization (FISH) was used to determine the genomic distribution of ribosomal RNA genes in five Lupinus species: L. cosentinii (2n=32), L. pilosus (2n=42), L. angustifolius (2n=40), L. luteus (2n=52) and L. mutabilis (2n=48). 18S-25S rDNA and 5S rDNA were used as probes. Some interspecific variation was observed in the number and size of the 18S-25S rDNA loci. All the studied species had one chromosome pair carrying 5S rDNA.

  6. Direct chemical probing of the conformation of the 3' functional domain of rabbit 18S rRNA in 40S subunits, 80S ribosomes and polyribosomes

    SciTech Connect

    Rubino, H.M.; Rairkar, A.; Lockard, R.E.

    1987-05-01

    Recent evidence suggests that the 3' minor domain of eukaryotic 18S rRNA, as in prokaryotes, is directly involved in protein biosynthesis. To determine regions of possible functional importance, they have probed the higher order structure of rabbit 18S rRNA in both 40S subunits and 80S ribosomes, as well as polyribosomes using the single-strand specific chemical probes dimethyl sulfate (DMS) and diethyl pyrocarbonate (DEPC) which react with unpaired guanosine and adenosine residues, respectively. The modified 18S rRNA was isolated from these particles and the resultant modified nucleotides identified on polyacrylamide sequencing gels upon either aniline-induced strand scission of /sup 32/P-end-labeled intact rRNA or by DNA primer extension using sequence specific deoxyoligomers with reverse transcriptase. Their results indicate a decreased reactivity of residue C-1692 in rabbit 18S rRNA (corresponding to C-1400 E. coli) within the putative tRNA contact site in polyribosomes as compared with 40S subunits and 80S ribosomes. They have also determined varying reactivities of a number of other residues within specific regions of the 3' functional domain when 40S, 80S, and polyribosomes are compared, which may be important for both subunit association as well as mRNA binding.

  7. Quantitative analysis of dinoflagellates and diatoms community via Miseq sequencing of actin gene and v9 region of 18S rDNA

    PubMed Central

    Guo, Liliang; Sui, Zhenghong; Liu, Yuan

    2016-01-01

    Miseq sequencing and data analysis for the actin gene and v9 region of 18S rDNA of 7 simulated samples consisting of different mixture of dinoflagellates and diatoms were carried out. Not all the species were detectable in all the 18S v9 samples, and sequence percent in all the v9 samples were not consistent with the corresponding cell percent which may suggest that 18S rDNA copy number in different cells of these species differed greatly which result in the large deviation of the amplification. And 18S rDNA amplification of the microalgae was prone to be contaminated by fungus. The amplification of actin gene all was from the dinoflagellates because of its targeted degenerate primers. All the actin sequences of dinoflagellates were detected in the act samples except act4, and sequence percentage of the dinoflagellates in the act samples was not completely consistent with the dinoflagellates percentage of cell samples, but with certain amplification deviations. Indexes of alpha diversity of actin gene sequencing may be better reflection of community structure, and beta diversity analysis could cluster the dinoflagellates samples with identical or similar composition together and was distinguishable with blooming simulating samples at the generic level. Hence, actin gene was more proper than rDNA as the molecular marker for the community analysis of the dinoflagellates. PMID:27721499

  8. Physical mapping of 5S and 18S-5.8S-26S RNA gene families in polyploid series of Cenchrus ciliaris Linnaeus, 1771 (Poaceae)

    PubMed Central

    Kharrat-Souissi, Amina; Siljak-Yakovlev, Sonja; Pustahija, Fatima; Chaieb, Mohamed

    2012-01-01

    Abstract The Buffelgrass (Cenchrus ciliaris L., Poaceae) is one of the most important pasturage grasses due to its high productivity and good forage qualities. This species possess a high adaptability to bioclimatic constraints of arid zones and may be used for the restoration of degraded arid ecosystems. Tunisian populations present three ploidy levels (4x, 5x and 6x) with a basic chromosome number x=9. This study reported for the first time the distribution of the ribosomal genes (rRNA) for pentaploid and hexaploid cytotypes of Cenchrus ciliaris. Molecular cytogenetic study using double fluorescence in situ hybridization has shown that the two rDNA families, 5S and 18S-5.8S-26S (18S), displayed intraspecific variation in number of loci among different ploidy levels. Each ploidy level was characterized by specific number of both 5S and 18S rDNA loci (two loci in tetraploid, five in pentaploid and six in hexaploid level). For three studied cytotypes (4x, 5x and 6x) all 5S rDNA loci were localized on the subcentromeric region of chromosomes, while 18S loci were situated on the telomeric region of short chromosome arms. Data of the FISH experiments show proportional increase of ribosomal loci number during polyploidization processes. PMID:24260668

  9. Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis.

    PubMed

    Takeo, Toshinori; Tanaka, Tetsuya; Matsubayashi, Makoto; Maeda, Hiroki; Kusakisako, Kodai; Matsui, Toshihiro; Mochizuki, Masami; Matsuo, Tomohide

    2014-08-01

    Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata.

  10. Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga.

    PubMed

    Estrada-Gómez, Sebastian; Vargas-Muñoz, Leidy Johana; Saldarriaga-Córdoba, Mónica; Cifuentes, Yeimy; Perafan, Carlos

    2017-04-01

    Theraphosidae spider venoms are well known for possess a complex mixture of protein and non-protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF-hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity.

  11. Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human CellsD⃞

    PubMed Central

    Valgardsdottir, Rut; Chiodi, Ilaria; Giordano, Manuela; Cobianchi, Fabio; Riva, Silvano; Biamonti, Giuseppe

    2005-01-01

    Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs. PMID:15788562

  12. Energy efficiency trade-offs drive nucleotide usage in transcribed regions.

    PubMed

    Chen, Wei-Hua; Lu, Guanting; Bork, Peer; Hu, Songnian; Lercher, Martin J

    2016-04-21

    Efficient nutrient usage is a trait under universal selection. A substantial part of cellular resources is spent on making nucleotides. We thus expect preferential use of cheaper nucleotides especially in transcribed sequences, which are often amplified thousand-fold compared with genomic sequences. To test this hypothesis, we derive a mutation-selection-drift equilibrium model for nucleotide skews (strand-specific usage of 'A' versus 'T' and 'G' versus 'C'), which explains nucleotide skews across 1,550 prokaryotic genomes as a consequence of selection on efficient resource usage. Transcription-related selection generally favours the cheaper nucleotides 'U' and 'C' at synonymous sites. However, the information encoded in mRNA is further amplified through translation. Due to unexpected trade-offs in the codon table, cheaper nucleotides encode on average energetically more expensive amino acids. These trade-offs apply to both strand-specific nucleotide usage and GC content, causing a universal bias towards the more expensive nucleotides 'A' and 'G' at non-synonymous coding sites.

  13. CpSAT-1, a transcribed satellite sequence from the codling moth, Cydia pomonella.

    PubMed

    Věchtová, Pavlína; Dalíková, Martina; Sýkorová, Miroslava; Žurovcová, Martina; Füssy, Zoltán; Zrzavá, Magda

    2016-08-01

    Satellite DNA (satDNA) is a non-coding component of eukaryotic genomes, located mainly in heterochromatic regions. Relevance of satDNA began to emerge with accumulating evidence of its potential yet hardly comprehensible role that it can play in the genome of many organisms. We isolated the first satDNA of the codling moth (Cydia pomonella, Tortricidae, Lepidoptera), a species with holokinetic chromosomes and a single large heterochromatic element, the W chromosome in females. The satDNA, called CpSAT-1, is located on all chromosomes of the complement, although in different amounts. Surprisingly, the satellite is almost missing in the heterochromatic W chromosome. Additionally, we isolated mRNA from all developmental stages (1st-5th instar larva, pupa, adult), both sexes (adult male and female) and several tissues (Malpighian tubules, gut, heart, testes, and ovaries) of the codling moth and showed the CpSAT-1 sequence was transcribed in all tested samples. Using CpSAT-1 specific primers we amplified, cloned and sequenced 40 monomers from cDNA and gDNA, respectively. The sequence analysis revealed a high mutation rate and the presence of potentially functional motifs, mainly in non-conserved regions of the monomers. Both the chromosomal distribution and the sequence analysis suggest that CPSAT-1 has no function in the C. pomonella genome.

  14. High-resolution profiling of novel transcribed regions during rat spermatogenesis.

    PubMed

    Chalmel, Frédéric; Lardenois, Aurélie; Evrard, Bertrand; Rolland, Antoine D; Sallou, Olivier; Dumargne, Marie-Charlotte; Coiffec, Isabelle; Collin, Olivier; Primig, Michael; Jégou, Bernard

    2014-07-01

    Mammalian spermatogenesis is a complex and highly orchestrated combination of processes in which male germline proliferation and differentiation result in the production of mature spermatozoa. If recent genome-wide studies have contributed to the in-depth analysis of the male germline protein-encoding transcriptome, little effort has yet been devoted to the systematic identification of novel unannotated transcribed regions expressed during mammalian spermatogenesis. We report high-resolution expression profiling of male germ cells in rat, using next-generation sequencing technology and highly enriched testicular cell populations. Among 20 424 high-confidence transcripts reconstructed, we defined a stringent set of 1419 long multi-exonic unannotated transcripts expressed in the testis (testis-expressed unannotated transcripts [TUTs]). TUTs were divided into 7 groups with different expression patterns. Most TUTs share many of the characteristics of vertebrate long noncoding RNAs (lncRNAs). We also markedly reinforced the finding that TUTs and known lncRNAs accumulate during the meiotic and postmeiotic stages of spermatogenesis in mammals and that X-linked meiotic TUTs do not escape the silencing effects of meiotic sex chromosome inactivation. Importantly, we discovered that TUTs and known lncRNAs with a peak expression during meiosis define a distinct class of noncoding transcripts that exhibit exons twice as long as those of other transcripts. Our study provides new insights in transcriptional profiling of the male germline and represents a high-quality resource for novel loci expressed during spermatogenesis that significantly contributes to rat genome annotation.

  15. Genetic structure of wild emmer wheat populations as reflected by transcribed versus anonymous SSR markers.

    PubMed

    Peleg, Zvi; Fahima, Tzion; Abbo, Shahal; Krugman, Tamar; Saranga, Yehoshua

    2008-03-01

    Simple sequence repeat (SSR) markers have become a major tool in population genetic analyses. The anonymous genomic SSRs (gSSRs) have been recently supplemented with expressed sequence tag (EST) derived SSRs (eSSRs), which represent the transcribed regions of the genome. In the present study, we used 8 populations of wild emmer wheat (Triticum turgidum subsp. dicoccoides) to compare the usefulness of the two types of SSR markers in assessing allelic diversity and population structure. gSSRs revealed significantly higher diversity than eSSRs in terms of average number of alleles (14.92 vs. 7.4, respectively), polymorphic information content (0.87 vs. 0.68, respectively), and gene diversity (He; 0.55 vs. 0.38, respectively). Despite the overall differences in the level of diversity, Mantel tests for correlations between eSSR and gSSR pairwise genetic distances were found to be significant for each population as well as for all accessions jointly (RM=0.54, p=0.01). Various genetic structure analyses (AMOVA, PCoA, STRUCTURE, unrooted UPGMA tree) revealed a better capacity of eSSRs to distinguish between populations, while gSSRs showed a higher proportion of intrapopulation (among accessions) diversity. We conclude that eSSR and gSSR markers should be employed in conjunction to obtain a high inter- and intra-specific (or inter- and intra-varietal) distinctness.

  16. Electrolyte level and blood pH in dogs infected by various 18S RNA strains of Babaesia canis canis on the early stage of babesiosis.

    PubMed

    Adaszek, Łukasz; Górna, Marta; Winiarczyk, Stanisław

    2012-01-01

    The purpose of the studies was to determine electrolyte disturbances and blood pH changes in dogs with babesiosis and possibly show a connection between the Babesia (B.) canis strain causing the infection and the intensity of these irregularities. 40 animals (group 1) with early babesiosis and 40 healthy dogs (group 2) were studied and their blood pH and blood levels of potassium, chlorides; calcium and sodium were determined. At the same time, molecular typing of parasites was carried out to detect which B.canis strain (18S RNA-A or 185 RNA-B) had caused the disease in dogs of group 1. In group 1, four dogs were acidaemic, twelve had normal blood pH, and 24 were alkalaemic. Potassium concentration was below normal in 16 out of 40 dogs (40%) and normal in 24 dogs. Hypochloremia was present in 36 out of 40 dogs; chloride was normal in the remaining four animals. Serum sodium concentration was low in 16 of 40 dogs, normal in 20 of 40 dogs and high in four dogs. Calcium concentration was normal in all 40 dogs. In dogs of group 2 no abnormalities of haematological or blood biochemical parameters were observed. 29 out of the 40 dogs of group 1 were infected with the 18S RNA-A strain and eleven with the 18S RNA-B strain of Babesia canis canis. We did not observe any correlation between the type of strain causing the infection and the electrolyte disturbances in the serum of sick dogs. Hypocalaemia was observed in ten specimen infected with 18S RNA-A and six infected with 18S RNA-B. Additionally, in dogs infected with 18S RNA-A, hypochloraemia (28), hyponatraemia (10), hypernatraemia (2) were observed, as well as blood pH drop (4) or increase (14). The 18S RNA-B-infected dogs suffered from hypochloraemia (8), hyponatraemia (6), hypernatraemia (2) and increase in blood pH (10).The studies conducted did not answer the question of whether the type of electrolyte disturbances in dogs with babesiosis can be connected with the strain of the parasite that induced the disease, as

  17. Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella.

    PubMed

    Hejazi, Mohammad A; Barzegari, Abolfazl; Gharajeh, Nahid Hosseinzadeh; Hejazi, Mohammad S

    2010-04-08

    Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 mumol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days.

  18. Unified English Braille in the United Kingdom: Part 2--Examination by Literary Braille Users, Braille Teachers, and Transcribers

    ERIC Educational Resources Information Center

    Cryer, Heather; Home, Sarah; Morley Wilkins, Sarah

    2013-01-01

    To inform decision-making around the adoption of the Unified English Braille (UEB) code in the United Kingdom, a suite of research was carried out. This study involved a variety of braille stakeholders--student braille readers (in full time education), adult braille readers, braille teachers, and braille transcribers. Participants were sent…

  19. Genetic characterization and phylogenetic relationships based on 18S rRNA and ITS1 region of small form of canine Babesia spp. from India.

    PubMed

    Mandal, M; Banerjee, P S; Garg, Rajat; Ram, Hira; Kundu, K; Kumar, Saroj; Kumar, G V P P S Ravi

    2014-10-01

    Canine babesiosis is a vector borne disease caused by intra-erythrocytic apicomplexan parasites Babesia canis (large form) and Babesia gibsoni (small form), throughout the globe. Apart from few sporadic reports on the occurrence of B. gibsoni infection in dogs, no attempt has been made to characterize Babesia spp. of dogs in India. Fifteen canine blood samples, positive for small form of Babesia, collected from northern to eastern parts of India, were used for amplification of 18S rRNA gene (∼1665bp) of Babesia sp. and partial ITS1 region (∼254bp) of B. gibsoni Asian genotype. Cloning and sequencing of the amplified products of each sample was performed separately. Based on sequences and phylogenetic analysis of 18S rRNA and ITS1 sequences, 13 were considered to be B. gibsoni. These thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype. The other two isolates could not be assigned to any particular species because of the difference(s) in 18S rRNA sequence with B. gibsoni and closer identity with Babesiaoccultans and Babesiaorientalis. In the phylogenetic tree, all the isolates of B. gibsoni Asian genotype formed a separate major clade named as Babesia spp. sensu stricto clade with high bootstrap support. The two unnamed Babesia sp. (Malbazar and Ludhiana isolates) clustered close together with B. orientalis, Babesia sp. (Kashi 1 isolate) and B. occultans of bovines. It can be inferred from this study that 18S rRNA gene and ITS1 region are highly conserved among 13 B. gibsoni isolates from India. It is the maiden attempt of genetic characterization by sequencing of 18S rRNA gene and ITS1 region of B. gibsoni from India and is also the first record on the occurrence of an unknown Babesia sp. of dogs from south and south-east Asia.

  20. Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer).

    PubMed

    Chaisi, Mamohale E; Collins, Nicola E; Potgieter, Fred T; Oosthuizen, Marinda C

    2013-01-16

    The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.

  1. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

    PubMed Central

    McDonald, James E.; Larsen, Niels; Pennington, Andrea; Connolly, John; Wallis, Corrin; Rooks, David J.; Hall, Neil; McCarthy, Alan J.; Allison, Heather E.

    2016-01-01

    PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by ‘universal’ primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology. PMID:27276347

  2. Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome.

    PubMed

    She, Richard; Chakravarty, Anupam K; Layton, Curtis J; Chircus, Lauren M; Andreasson, Johan O L; Damaraju, Nandita; McMahon, Peter L; Buenrostro, Jason D; Jarosz, Daniel F; Greenleaf, William J

    2017-04-04

    RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

  3. Reverse transcription-polymerase chain reaction detection of transcribed sequences on human chromosome 21

    SciTech Connect

    Cheng, J.F.; Zhu, Y. )

    1994-03-15

    Seventy-four pairs of oligonucleotides derived from sequence-tagged sites (STSs) on the long arm of human chromosome 21, specifically from bands 21q22.1 to 21q22.3, were used in reverse transcription-polymerase chain reactions (RT-PCR) to detect the presence of expressed sequences in a fetal brain. These STSs included 69 that had not been related to transcribed sequences and 5 that had detected two known genes and three previously isolated cDNA clones. Of the 69 STSs analyzed in RT-PCR, 25 allowed amplification of specific cDNA fragments. The sizes of amplified cDNA fragments match those amplified from either human genomic DNA or somatic hybrid cells containing human chromosome 21. Of the 11 cDNA analyzed in Northern blot hybridizations, 6 hybridized to specific RNA species. The rapid screening for cDNA using previously mapped STSs has provided insight into the distribution of expressed sequences in this region of chromosome 21. Northern blot analysis of the amplified cDNA fragments has revealed interesting candidate genes in two disease loci. The marker D21S267 was previously mapped in the Down syndrome region of chromosome 21, and the marker D21S113 is closely linked to progressive myoclonus epilepsy. The cDNA fragments amplified using the primer sequences derived from D21S267 and D21S113 hybridized to 7- and 6.5-kb transcripts, respectively, which seems to express predominantly in brain. 37 refs., 3 figs., 1 tab.

  4. A molecular phylogenetic study of the Palmae (Arecaceae) based on atpB, rbcL, and 18S nrDNA sequences.

    PubMed

    Hahn, William J

    2002-02-01

    Notoriously slow rates of molecular evolution and convergent evolution among some morphological characters have limited phylogenetic resolution for the palm family (Arecaceae). This study adds nuclear DNA (18S SSU rRNA) and chloroplast DNA (cpDNA; atpB and rbcL) sequence data for 65 genera of palms and characterizes molecular variation for each molecule. Phylogenetic relationships were estimated with maximum likelihood and maximum parsimony techniques for the new data and for previously published molecular data for 45 palm genera. Maximum parsimony analysis was also used to compare molecular and morphological data for 33 palm genera. Incongruence among datasets was detected between cpDNA and 18S data and between molecular and morphological data. Most conflict between nuclear and cpDNA data was associated with the genus Nypa. Several taxa showed relatively long branches with 18S data, but phylogenetic resolution of these taxa was essentially the same for 18S and cpDNA data. Base composition bias for 18S that contributed to erroneous phylogenetic resolution in other taxa did not seem to be present in Palmae. Morphological data were incongruent with all molecular data due to apparent morphological homoplasy for Caryoteae, Ceroxyloideae, Iriarteae, and Thrinacinae. Both cpDNA and nuclear 18S data firmly resolved Caryoteae with Borasseae of Coryphoideae, suggesting that at least some morphological characters used to place Caryoteae in Arecoideae are homoplastic. In this study, increased character sampling seems to be more important than increased taxon sampling; a comparison of the full (65-taxon) and reduced (45- and 33-taxon) datasets suggests little difference in core topology but considerably more nodal support with the increased character sample sizes. These results indicate a general trend toward a stable estimate of phylogenetic relationships for the Palmae. Although the 33-taxon topologies are even better resolved, they lack several critical taxa and are

  5. Multiple origins of the ascidian-Prochloron symbiosis: molecular phylogeny of photosymbiotic and non-symbiotic colonial ascidians inferred from 18S rDNA sequences.

    PubMed

    Yokobori, Shin-Ichi; Kurabayashi, Atsushi; Neilan, Brett A; Maruyama, Tadashi; Hirose, Euichi

    2006-07-01

    In the tropics, certain didemnid ascidians harbor the prokaryotic photosymbiont Prochloron. To date, this photosymbiosis has been found in four didemnid genera that include non-symbiotic species. Here, we report the molecular phylogeny of symbiotic and non-symbiotic didemnids based on their 18S rDNA sequences. The data cover all four genera containing symbiotic species and one other genus comprised of only non-symbiotic species. Near-complete nucleotide sequences of 18S rDNAs were determined for four non-didemnid species and 52 didemnid samples (five genera), including 48 photosymbiotic samples collected from the Ryukyu Archipelago, the Great Barrier Reef, Hawaii, and Bali. Our phylogenetic trees indicated a monophyletic origin of the family Didemnidae, as well as each of the didemnid genera. The results strongly support the hypothesis that establishment of the ascidian-Prochloron symbiosis occurred independently in the Didemnidae lineage at least once in each of the genera that possess symbiotic species.

  6. 18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

    PubMed Central

    Wu, Zhihong; Tsumura, Yoshihiko; Blomquist, Göran; Wang, Xiao-Ru

    2003-01-01

    In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring. PMID:12957927

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

    PubMed Central

    Alves, Anderson Luis; de Borba, Rafael Splendore; Pozzobon, Allan Pierre Bonetti; Oliveira, Claudio; Nirchio, Mauro; Granado, Angel; Foresti, Fausto

    2012-01-01

    Abstract 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. PMID:24260671

  8. Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa.

    PubMed

    Chaisi, Mamohale E; Sibeko, Kgomotso P; Collins, Nicola E; Potgieter, Fred T; Oosthuizen, Marinda C

    2011-12-15

    Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the

  9. Distribution of Mosquitoes in the South East of Argentina and First Report on the Analysis Based on 18S rDNA and COI Sequences

    PubMed Central

    Díaz-Nieto, Leonardo M.; Maciá, Arnaldo; Parisi, Gustavo; Farina, Juan L.; Vidal-Domínguez, María E.; Perotti, M. Alejandra; Berón, Corina M.

    2013-01-01

    Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors. PMID:24098700

  10. Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity

    PubMed Central

    2010-01-01

    Background The 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties. Results We report here the first authentic 18S genes of three Solenogastres species (Mollusca), each possessing a unique sequence composition with regions conspicuously rich in guanine and cytosine. For these GC-rich regions we calculated strong secondary structures. The observed high intra-molecular forces hamper standard amplification and appear to increase formation of chimerical sequences caused by contaminating foreign DNAs from potential prey organisms. In our analyses, contamination was avoided by using RNA as a template. Indication for contamination of previously published Solenogastres sequences is presented. Detailed phylogenetic analyses were conducted using RNA specific models that account for compensatory substitutions in stem regions. Conclusions The extreme morphological diversity of mollusks is mirrored in the molecular 18S data and shows elevated substitution rates mainly in three higher taxa: true limpets (Patellogastropoda), Cephalopoda and Solenogastres. Our phylogenetic tree based on 123 species, including representatives of all mollusk classes, shows limited resolution at the class level but illustrates the pitfalls of artificial groupings formed due to shared biased sequence composition. PMID:20214780

  11. Karyotypes, male meiosis and comparative FISH mapping of 18S ribosomal DNA and telomeric (TTAGG) n repeat in eight species of true bugs (Hemiptera, Heteroptera)

    PubMed Central

    Grozeva, S.; Kuznetsova, V.G.; Anokhin, B.A.

    2011-01-01

    Abstract Eight species belonging to five true bug families were analyzed using DAPI/CMA3-staining and fluorescence in situ hybridization (FISH) with telomeric (TTAGG)n and 18S rDNA probes. Standard chromosomal complements are reported for the first time for Deraeocoris rutilus (Herrich-Schäffer, 1838) (2n=30+2m+XY) and Deraeocoris ruber(Linnaeus, 1758) (2n=30+2m+XY) from the family Miridae. Using FISH, the location of a 18S rDNA cluster was detected in these species and in five more species: Megaloceroea recticornis (Geoffroy, 1785) (2n=30+XY) from the Miridae; Oxycarenus lavaterae (Fabricius, 1787) (2n=14+2m+XY) from the Lygaeidae s.l.; Pyrrhocoris apterus (Linnaeus, 1758) (2n=22+X) from the Pyrrhocoridae; Eurydema oleracea (Linnaeus, 1758) (2n=12+XY) and Graphosoma lineatum (Linnaeus, 1758) (2n=12+XY) from the Pentatomidae. The species were found to differ with respect to location of a 18S rRNA gene cluster which resides on autosomes in Oxycarenus lavaterae and Pyrrhocoris apterus, whereas it locates on sex chromosomes in other five species. The 18S rDNA location provides the first physical landmark of the genomes of the species studied. The insect consensus telomeric pentanucleotide (TTAGG)n was demonstrated to be absent in all the species studied in this respect, Deraeocoris rutilus, Megaloceroea recticornis, Cimex lectularius Linnaeus, 1758 (Cimicidae), Eurydema oleracea, and Graphosoma lineatum, supporting the hypothesis that this motif was lost in early evolution of the Heteroptera and secondarily replaced with another motif (yet unknown) or the alternative telomerase-independent mechanisms of telomere maintenance. Dot-blot hybridization analysis of the genomic DNA from Cimex lectularius, Nabis sp. and Oxycarenus lavaterae with (TTAGG)n and six other telomeric probes likewise provided a negative result. PMID:24260641

  12. Reconstructing the Phylogeny of Capsosiphon fulvescens (Ulotrichales, Chlorophyta) from Korea Based on rbcL and 18S rDNA Sequences.

    PubMed

    Sun, Sang-Mi; Yang, Seung Hwan; Golokhvast, Kirill S; Le, Bao; Chung, Gyuhwa

    2016-01-01

    Capsosiphon fulvescens is a filamentous green algae in the class Ulvophyceae. It has been consumed as food with unique flavor and soft texture to treat stomach disorders and hangovers, and its economic value justifies studying its nutritional and potential therapeutic effects. In contrast to these applications, only a few taxonomic studies have been conducted on C. fulvescens. In particular, classification and phylogenetic relationships of the C. fulvescens below the order level are controversial. To determine its phylogenetic position in the class, we used rbcL and 18S rDNA sequences as molecular markers to construct phylogenetic trees. The amplified rbcL and 18S rDNA sequences from 4 C. fulvescens isolates (Jindo, Jangheung, Wando, and Koheung, Korea) were used for phylogenetic analysis by employing three different phylogenetic methods: neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The rbcL phylogenetic tree showed that all taxa in the order Ulvales were clustered as a monophyletic group and resolved the phylogenetic position of C. fulvescens in the order Ulotrichales. The significance of our study is that the 18S rDNA phylogenetic tree shows the detailed taxonomic position of C. fulvescens. In our result, C. fulvescens is inferred as a member of Ulotrichaceae, along with Urospora and Acrosiphonia.

  13. Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences

    PubMed Central

    Patra, Avijit; Mondal, Anjan; Banerjee, Sayani; Adikesavalu, Harresh; Joardar, Siddhartha Narayan; Abraham, Thangapalam Jawahar

    2016-01-01

    The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87–100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species. PMID:28097169

  14. Co-located 18S/5S rDNA arrays: an ancient and unusual chromosomal trait in Julidini species (Labridae, Perciformes)

    PubMed Central

    Amorim, Karlla Danielle Jorge; Cioffi, Marcelo de Bello; Bertollo, Luiz Antonio Carlos; Soares, Rodrigo Xavier; de Souza, Allyson Santos; da Costa, Gideão Wagner Werneck Felix; Molina, Wagner Franco

    2016-01-01

    Abstract Wrasses (Labridae) are extremely diversified marine fishes, whose species exhibit complex interactions with the reef environment. They are widely distributed in the Indian, Pacific and Atlantic oceans. Their species have displayed a number of karyotypic divergent processes, including chromosomal regions with complex structural organization. Current cytogenetic information for this family is phylogenetically and geographically limited and mainly based on conventional cytogenetic techniques. Here, the distribution patterns of heterochromatin, GC-specific chromosome regions and Ag-NORs, and the organization of 18S and 5S rDNA sites of the Atlantic species Thalassoma noronhanum (Boulenger, 1890), Halichoeres poeyi (Steindachner, 1867), Halichoeres radiatus (Linnaeus, 1758), Halichoeres brasiliensis (Bloch, 1791) and Halichoeres penrosei Starks, 1913, belonging to the tribe Julidini were analyzed. All the species exhibited 2n=48 chromosomes with variation in the number of chromosome arms among genera. Thalassoma noronhanum has 2m+46a, while species of the genus Halichoeres Rüppell, 1835 share karyotypes with 48 acrocentric chromosomes. The Halichoeres species exhibit differences in the heterochromatin distribution patterns and in the number and distribution of 18S and 5S rDNA sites. The occurrence of 18S/5S rDNA syntenic arrangements in all the species indicates a functionally stable and adaptive genomic organization. The phylogenetic sharing of this rDNA organization highlights a marked and unusual chromosomal singularity inside the family Labridae. PMID:28123678

  15. Transcription promotes guanine to thymine mutations in the non-transcribed strand of an Escherichia coli gene.

    PubMed

    Klapacz, Joanna; Bhagwat, Ashok S

    2005-07-12

    Transcription of DNA opens the chromatin, causes topological changes in DNA and transiently exposes the two strands to different biochemical environments. Consequently, it has long been argued that transcription may promote damage to DNA and there are data in Escherichia coli and yeast supporting a correlation between high transcription and mutations. We examined the transcription-dependence of the reversion of a nonsense codon (TGA) in E. coli and found that there was a strong dependence of mutations on transcription in strains defective in the repair of 8-oxoguanine in DNA. Under conditions of high transcription there was a three to five-fold increase in mutations that changed TGA in the non-transcribed strand to a sense codon. Furthermore, in both mutY and mutM mutY backgrounds the mutations were overwhelmingly G:C to T:A. In contrast, when the TGA was in the transcribed strand in relation with the inducible promoter, high transcription decreased the rate of reversion. Similar results were obtained in a strain defective in the transcription-repair coupling factor, Mfd, suggesting that transcription dependent increase in base substitutions does not require transcription-dependent DNA repair. However, Mfd does modulate the magnitude of the mutagenic effect of transcription. These data are consistent with a model in which the non-transcribed strand is more susceptible to oxidative damage during transcription than the transcribed strand. These results suggest that the magnitudes of individual base substitutions and their relative numbers in other studies of mutational spectra may also be affected by transcription.

  16. Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

    PubMed Central

    Smit, Eric; Leeflang, Paula; Glandorf, Boet; Dirk van Elsas, Jan; Wernars, Karel

    1999-01-01

    Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic

  17. Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment.

    PubMed

    Zhang, Huan; Lin, Senjie

    2005-11-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and 18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5'-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells x ml(-1)), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).

  18. Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

    PubMed

    Kazi, Mudassar Anisoddin; Reddy, C R K; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

  19. Molecular Phylogeny and Barcoding of Caulerpa (Bryopsidales) Based on the tufA, rbcL, 18S rDNA and ITS rDNA Genes

    PubMed Central

    Kazi, Mudassar Anisoddin; Reddy, C. R. K.; Jha, Bhavanath

    2013-01-01

    The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters. PMID:24340028

  20. Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

    PubMed Central

    Schroeder, Jill M.; Booton, Gregory C.; Hay, John; Niszl, Ingrid A.; Seal, David V.; Markus, Miles B.; Fuerst, Paul A.; Byers, Thomas J.

    2001-01-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK. PMID:11326011

  1. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

    PubMed

    Schroeder, J M; Booton, G C; Hay, J; Niszl, I A; Seal, D V; Markus, M B; Fuerst, P A; Byers, T J

    2001-05-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

  2. Development of a cob-18S rRNA Gene Real-Time PCR Assay for Quantifying Pfiesteria shumwayae in the Natural Environment†

    PubMed Central

    Zhang, Huan; Lin, Senjie

    2005-01-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5′-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml−1), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay). PMID:16269741

  3. Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas.

    PubMed

    Itoh, Naoki; Oda, Tadashi; Yoshinaga, Tomoyoshi; Ogawa, Kazuo

    2003-03-31

    To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20 degrees C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.

  4. Determination of phylogenetic relationships among Eimeria species, which parasitize cattle, on the basis of nuclear 18S rDNA sequence.

    PubMed

    Kokuzawa, Takuya; Ichikawa-Seki, Madoka; Itagaki, Tadashi

    2013-11-01

    We analyzed almost complete 18S rDNA sequences of 10 bovine Eimeria species, namely Eimeria alabamensis, E. auburnensis, E. bovis, E. bukidnonensis, E. canadensis, E. cylindrica, E. ellipsoidalis, E. subspherica, E. wyomingensis and E. zuernii. Although these sequences showed intraspecific variation in 8 species, the sequences of each species were clustered in monophyletic groups in all species, except E. auburnensis. The sequences constituted 3 distinct clusters in a phylogenetic tree with relatively high bootstrap values; however, the members including each cluster shared no similarities in oocyst morphology.

  5. Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

    PubMed

    Paparini, Andrea; Gofton, Alexander; Yang, Rongchang; White, Nicole; Bunce, Michael; Ryan, Una M

    2015-01-01

    Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are

  6. [Mg2+ ions affect the structure of the central domain of the 18S rRNA in the vicinity of the ribosomal protein S13 binding site].

    PubMed

    Ivanov, A V; Malygin, A A; Karpova, G G

    2013-01-01

    It is known that Mg2+ ions at high concentrations stabilize the structure of the 16S rRNA in a conformation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. Effect of Mg2+ on the formation of the 18S rRNA structure at the 40S subunit assembly remains poorly explored. In this paper, we show that the sequentional increase of the Mg2+ concentration from 0.5 mM to 20 mM leads to a significant decrease of the affinity of recombinant human ribosomal protein S13 (rpS13e) to a RNA transcript corresponding to the central domain fragment of the 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD (including the nucleotides of helices H20 and H22), whose availabilities to hydroxyl radicals were dependent on the Mg2+ concentration, were determined. It was found that increase of the concentrations of Mg2+ results in the enhanced accessibilities of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduces those of nucleotides A1023, A1024, and A1028-S1026 in the helix H20. Comparison of the results obtained with the crystallographic data on the structure of the central domain of 18S rRNA in the 40S ribosomal subunit led to conclusion that increase of Mg2+ concentrations results in the reorientation of helices H20 and H24 relatively helices H22 and H23 to form a structure, in which these helices are positioned the same way as in 40S subunits. Hence, saturation of the central domain of 18S rRNA with coordinated Mg2+ ions causes the same changes in its structure as rpS13e binding does, and leads to decreasing of this domain affinity to the protein.

  7. TSH stimulates 32P-labeling of thyroid nuclear HMG 14, a protein associated with actively transcribed chromatin

    SciTech Connect

    Cooper, E.; Palmer, R.J.; Spaulding, S.W.

    1982-04-01

    Thyroid slices were incubated with 32P with or without TSH. 32P-labeling of acid-soluble nuclear proteins was then examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. We found that TSH enhanced the labeling of the high mobility group protein HMG 14, a protein that is preferentially associated with actively transcribed chromatin. This observation suggests that changes in HMG 14 phosphorylation may be involved in mediating TSH-induced effects on the structure and function of active chromatin.

  8. Isolation and characterization of recombinant DNAs containing repeated elements of barley genome: identification of individual actively transcribed families of repeats

    SciTech Connect

    Prosnyak, M.I.; Kartel', N.A.; Ryskov, A.P.

    1986-05-01

    A bank of Escherichia coli clones containing fragments of barley nuclear DNA was obtained using plasmid pBR 322. Clones carrying repeated sequences of the plant genome were selected by means of colony and blot hybridization. Clones with actively transcribed sequences were selected by hybridization to complementary DNA synthesized by means of reverse transcription on a template of total barley poly(A)-containing RNA. Individual families of repeats, two of which contained transcriptionally active sequences of the barley genome, were identified by blot hybridization of recombinant plasmids containing labeled DNA fragments of the inserts of three different clones.

  9. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

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

    PubMed

    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.

  11. Molecular phylogenetic analysis of the coccidian cephalopod parasites Aggregata octopiana and Aggregata eberthi (Apicomplexa: Aggregatidae) from the NE Atlantic coast using 18S rRNA sequences.

    PubMed

    Castellanos-Martínez, Sheila; Pérez-Losada, Marcos; Gestal, Camino

    2013-08-01

    The coccidia genus Aggregata is responsible for intestinal coccidiosis in wild and cultivated cephalopods. Two coccidia species, Aggregata octopiana, (infecting the common octopus Octopus vulgaris), and A. eberthi, (infecting the cuttlefish Sepia officinalis), are identified in European waters. Extensive investigation of their morphology resulted in a redescription of A. octopiana in octopuses from the NE Atlantic Coast (NW Spain) thus clarifying confusing descriptions recorded in the past. The present study sequenced the 18S rRNA gene in A. octopiana and A. eberthi from the NE Atlantic coast in order to assess their taxonomic and phylogenetic status. Phylogenetic analyses revealed conspecific genetic differences (2.5%) in 18S rRNA sequences between A. eberthi from the Ria of Vigo (NW Spain) and the Adriatic Sea. Larger congeneric differences (15.9%) were observed between A. octopiana samples from the same two areas, which suggest the existence of two species. Based on previous morphological evidence, host specificity data, and new molecular phylogenetic analyses, we suggest that A. octopiana from the Ria of Vigo is the valid type species.

  12. Identification of transcribed sequences in the human genome. Final report, September 15, 1991--September 14, 1992

    SciTech Connect

    Gardiner, K.

    1992-12-01

    The workshop was held at the National Institutes of Mental Health, Bethesda, Maryland, on October 4 and 5, 1991. Twenty-four investigators attended from England, Germany and the United States. The topics discussed included: Genome sequence analysis using computer assisted detection of open reading frames, splice sites and hexamer patterns, direct exon identification using trapping of internal and 3` exons, and a recombination based system, cDNA library construction and screening, including the use of normalization and subtraction procedures, Alu and splice donor site PCR from hybrid cell lines, and microdissection clones as probes, use of labeled CDNAS as probes to screen lambda and cosmid libraries, and sequencing of random cDNAs.

  13. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

    SciTech Connect

    Venema, J.; van Hoffen, A.; Karcagi, V.; Natarajan, A.T.; van Zeeland, A.A.; Mullenders, L.H. )

    1991-08-01

    The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5{prime} part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3{prime} part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

  14. FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

    PubMed Central

    Muñoz-Centeno, Mari Cruz; Singh, Rakesh Kumar; Oreal, Vincent; Reddy, Gajjalaiahvari Ugander; Liang, Dun; Géli, Vincent; Gunjan, Akash; Chávez, Sebastián

    2010-01-01

    The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication. PMID:20502685

  15. The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

    PubMed Central

    Bonnet, Jacques; Wang, Chen-Yi; Baptista, Tiago; Vincent, Stéphane D.; Hsiao, Wei-Chun; Stierle, Matthieu; Kao, Cheng-Fu; Tora, László

    2014-01-01

    The SAGA (Spt–Ada–Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription. PMID:25228644

  16. 18S rDNA analysis of alkenone-producing haptophyte(s) preserved in surface sediments of Lake Toyoni, Japan

    NASA Astrophysics Data System (ADS)

    McColl, J. L.; Couto, J.; Bendle, J. A.; Henderson, A. C.; Seki, O.; Phoenix, V. R.; Toney, J. L.

    2013-12-01

    Alkenones (long chain ketones) are readily preserved in sedimentary archives and have the potential to provide quantitative reconstructions of past water temperature. Alkenones are produced by a limited number of haptophyte algae in the marine and also some lacustrine systems. However, lakes are heterogeneous: an individual lake will have a unique combination of ecological conditions, haptophyte species and seasonal alkenone production that contributes to the sedimentary record. Haptophyte algae species have different sensitivities to temperature; therefore identifying the alkenone producer(s) prior to down-core temperature reconstructions is critical before selecting the most appropriate temperature calibration. We present a study from Lake Toyoni, a freshwater lake in Hokkaido, Japan that has alkenones preserved in surface sediments. The aim of this study is to identify the alkenone producer(s) within the lake using 18S rDNA analyses. Preserved rDNA of planktonic phototrophic algae was extracted from surface sediments of Lake Toyoni and phylogenetic analyses of the rDNA sequences suggest alkenones are produced by a single haptophyte within the class Prymnesiophyceae (order Isochrysidales). The Lake Toyoni alkenone-producer shares a distinct phylotype with a haptophyte reported from water filter samples collected in Lake BrayaSø, Greenland (D'Andrea et al., 2006). Similarity between the 18S rDNA sequences from Lake Toyoni and Lake BrayaSø provides a basis for applying (and updating) the Greenland lake temperature calibration. Applying this temperature calibration (T°C = 40.8 [UK37] + 31.8, R2=0.96; n=34) to the surface sediment alkenone unsaturation index from Lake Toyoni gives an estimated lake surface temperature (LST) of 8°C. This is in line with observed LST at Lake Toyoni, which ranges between 7 - 22°C (Apr 2011 to Nov 2011). The occurrence and identification of a single alkenone producer in Lake Toyoni means problems posed by a mixture of haptophytes in

  17. Phylogeny of freshwater parasitic copepods in the Ergasilidae (Copepoda: Poecilostomatoida) based on 18S and 28S rDNA sequences.

    PubMed

    Song, Y; Wang, G T; Yao, W J; Gao, Q; Nie, P

    2008-01-01

    The phylogenetic relationships among the Ergasilidae genera are poorly understood. In this study, 14 species from four genera in the Ergasilidae including Sinergasilus, Ergasilus, Pseudergasilus, and Paraergasilus were collected in China, and their phylogenetic relationships were examined using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods based on partial sequences of 18S and 28S ribosomal deoxyribonucleic acid, respectively. All the analyses suggest that the Sinergasilus and Paraergasilus are both monophyletic, but the Ergasilus is polyphyletic rather than monophyletic. Considering the relationships among the four genera, the phylogenetic analyses and subsequent hypothesis tests all suggest that Pseudergasilus clustered with some Ergasilus species may have a closer relationship with Sinergasilus rather than with Paraergasilus. It is proposed that the Sinergasilus and the Pseudergasilus species might have evolved from Ergasilus species.

  18. Slow molecular evolution in 18S rDNA, rbcL and nad5 genes of mosses compared with higher plants.

    PubMed

    Stenøien, H K

    2008-03-01

    The evolutionary potential of bryophytes (mosses, liverworts and hornworts) has been debated for decades. Fossil record and biogeographical distribution patterns suggest very slow morphological evolution and the retainment of several ancient traits since the split with vascular plants some 450 million years ago. Many have argued that bryophytes may evolve as rapidly as higher plants on the molecular level, but this hypothesis has not been tested so far. Here, it is shown that mosses have experienced significantly lower rates of molecular evolution than higher plants within 18S rDNA (nuclear), rbcL (chloroplast) and nad5 (mitochondrial) genes. Mosses are on an average evolving 2-3 times slower than ferns, gymnosperms and angiosperms; and also green algae seem to be evolving faster than nonvascular plants. These results support the observation of a general correlation between morphological and molecular evolutionary rates in plants and also show that mosses are 'evolutionary sphinxes' regarding both morphological and molecular evolutionary potential.

  19. Molecular characterization and phylogeny of Linguatula serrata (Pentastomida: Linguatulidae) based on the nuclear 18S rDNA and mitochondrial cytochrome c oxidase I gene

    PubMed Central

    MOHANTA, Uday Kumar; ITAGAKI, Tadashi

    2016-01-01

    Linguatula serrata, a cosmopolitan parasite, is commonly known as tongue worm belonging to the subclass Pentastomida.We collected the nymphal stage of the worm from mesenteric lymph nodes of cattle and identified these as L. serrata based on morphology and morphometry. The 18S rDNA sequences showed no intraspecific variation, although cox1 sequences showed 99.7–99.9% homology. In the phylogenies inferred from both gene loci, members of the genus Linguatula (order Porocephalida) were closer to those of the order Cephalobaenida than to those of Porocephalida, reflecting a mismatch with the corresponding morphology-based taxonomy. Accordingly, analyses of additional gene loci using a larger number of taxa across the Pentastomida should be undertaken to determine an accurate phylogenetic position within the Arthropoda. PMID:27941305

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

    SciTech Connect

    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.

  1. 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.

    PubMed

    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

  2. Genetic diversity among Babesia rossi detected in naturally infected dogs in Abeokuta, Nigeria, based on 18S rRNA gene sequences.

    PubMed

    Takeet, Michael I; Oyewusi, Adeoye J; Abakpa, Simon A V; Daramola, Olukayode O; Peters, Sunday O

    2017-03-01

    Adequate knowledge of the genetic diversity among Babesia species infecting dogs is necessary for a better understanding of the epidemiology and control of canine babesiosis. Hence, this study determined the genetic diversity among the Babesia rossi detected in dogs presented for routine examination in Veterinary Hospitals in Abeokuta, Nigeria. Blood were randomly collected from 209 dogs. Field-stained thin smears were made and DNA extracted from the blood. Partial region of the 18S small subunit ribosomal RNA (rRNA) gene was amplified, sequenced and analysed. Babesia species was detected in 16 (7.7%) of the dogs by microscopy. Electrophoresed PCR products from 39 (18.66%) dogs revealed band size of 450 bp and 2 (0.95%) dogs had band size of 430 bp. The sequences obtained from 450 bp amplicon displayed homology of 99.74% (387/388) with partial sequences of 18S rRNA gene of Babesia rossi in the GeneBank. Of the two sequences that had 430 bp amplicon, one was identified as T. annulata and second as T. ovis. A significantly (p<0.05) higher prevalence of B. rossi was detected by PCR compared to microscopy. The mean PCV of Babesia infected dogs was significantly (p<0.05) lower than non-infected dogs. Phylogenetic analysis revealed minimal diversity among B. rossi with the exception of one sequence that was greatly divergent from the others. This study suggests that more than one genotype of B. rossi may be in circulation among the dog population in the study area and this may have potential implication on clinical outcome of canine babesiosis.

  3. Electrophoretic separation of a class of nucleosomes enriched in HMG 14 and 17 and actively transcribed globin genes.

    PubMed Central

    Albanese, I; Weintraub, H

    1980-01-01

    Monomer nucleosomes from chick erythrocytes can be fractionated according to their electrophoretic mobility in (comparatively) high salt acrylamide gels. We show that the fractionation is based predominantly on differences in charge. The monomer heterogeneity persists even when the nucleosomes are trimmed down to 145 bp with Exo III or when H1 and H5 are removed. The slowest migrating monomers are associated with HMG 14 and 17; however, we do not believe that these proteins are entirely responsible for the altered mobility since the nucleosome heterogeneity persists even after removal of HMG 14 and 17. The DNA associated with the HMG 14 and 17 containing nucleosomes is shown to be enriched in actively transcribed globin sequences. Images PMID:6448987

  4. Improvement of in vitro-transcribed amber suppressor tRNAs toward higher suppression efficiency in wheat germ extract.

    PubMed

    Ogawa, Atsushi; Doi, Yasunori; Matsushita, Nobuto

    2011-12-21

    In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

  5. Transcribed pseudogene ψPPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma

    PubMed Central

    Chan, Wen-Ling; Yuo, Chung-Yee; Yang, Wen-Kuang; Hung, Shih-Ya; Chang, Ya-Sian; Chiu, Chien-Chih; Yeh, Kun-Tu; Huang, Hsien-Da; Chang, Jan-Gowth

    2013-01-01

    Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene ψPPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted ψPPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected ψPPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of ψPPM1K. Furthermore, expression of NEK8 in ψPPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of ψPPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth. PMID:23376929

  6. Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers

    PubMed Central

    2012-01-01

    Background Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported. Results We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker. Conclusions This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research. PMID:22929540

  7. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

  8. Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

    PubMed Central

    2016-01-01

    ABSTRACT The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCE The quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential

  9. Phylogeny of coral-inhabiting barnacles (Cirripedia; Thoracica; Pyrgomatidae) based on 12S, 16S and 18S rDNA analysis.

    PubMed

    Simon-Blecher, N; Huchon, D; Achituv, Y

    2007-09-01

    The traditional phylogeny of the coral-inhabiting barnacles, the Pyrgomatidae, is based on morphological characteristics, mainly of the hard parts. It has been difficult to establish the phylogenetic relationships among Pyrgomatidae because of the apparent convergence of morphological characteristics, and due to the use of non-cladistic systematics, which emphasize ancestor-descendant relationships rather than sister-clade relationships. We used partial sequences of two mithochondrial genes, 12S rDNA and 16S rDNA, and a nuclear gene, 18S rDNA, to infer the molecular phylogeny of the pyrgomatids. Our phylogenetic results allowed us to reject previous classifications of Pyrgomatidae based on morphological characteristics. Our results also suggested the possibility of paraphyly of the Pyrgomatidae. The hydrocoral barnacle Wanella is not found on the same clade as the other pyrgomatids, but rather, with the free-living balanids. The basal position of Megatrema and Ceratoconcha is supported. The archeaobalanid Armatobalanus is grouped with Cantellius at the base of the Indo-Pacific pyrgomatines. Fusion of the shell plate and modification of the opercular valves are homoplasious features that occurred more than three times on different clades. The monophyly of the "Savignium" group, comprising four nominal genera, is also not supported, and the different taxa are placed on different clades.

  10. Prevalence of infection and 18S rRNA gene sequences of Cytauxzoon species in Iberian lynx (Lynx pardinus) in Spain.

    PubMed

    Millán, J; Naranjo, V; Rodríguez, A; de la Lastra, J M Pérez; Mangold, A J; de la Fuente, J

    2007-07-01

    The Iberian lynx (Lynx pardinus) is the most endangered felid in the world. Only about 160 individuals remain in 2 separate metapopulations in Southern Spain (Sierra Morena and Doñana). We obtained blood samples of 20 lynxes captured from 2004 to 2006, and determined the prevalence of infection and genetic diversity of Cytauxzoon spp. using 18S rRNA PCR and sequence analysis. Prevalence of infection was 15% (3 of 20). Cytauxzoon sp. was only detected in Sierra Morena. For phylogenetic analysis, we used the sequences reported in the present study and those characterized in different domestic and wild felids and ticks from North and South America, Asia and Europe. Three different Cytauxzoon sp. sequences were obtained. They were closely related to that obtained from a Spanish cat, but diverged in up to 1.0% with respect to the only previously reported sequence from an Iberian lynx. Conversely, the latter sequence clustered together with C. manul sequences obtained from Pallas cats (Otocolobus manul) in Mongolia. Our analysis yields a separate cluster of C. felis sequences from cats, wild felids and ticks in the United States and Brazil. These results suggest that at least 2 different Cytauxzoon spp. may be present in Iberian lynx. The apparent absence in one of the areas, together with the possibility of fatal cytauxzoonosis in lynxes makes necessary disease risks to be taken into account in management conservation strategies, such as translocations and re-introductions.

  11. Morphology and 18S rDNA sequencing identifies Henneguya visibilis n. sp., a parasite of Leporinus obtusidens from Mogi Guaçu River, Brazil.

    PubMed

    Moreira, Gabriel S A; Adriano, Edson A; Silva, Marcia R M; Ceccarelli, Paulo S; Maia, Antônio A M

    2014-01-01

    During a survey of myxozoan parasites of freshwater fish from the Mogi Guaçu River in São Paulo State, Brazil, plasmodia of Henneguya visibilis n. sp. were found on the fins of Leporinus obtusidens (Characiformes: Anostomidae). The plasmodia, which were observed on five out of eight (62.5%) L. obtusidens examined, were 400-1,000 μm long. Mature spores were elongated with a spore body 10.8 ± 0.6 μm long and 3.9 ± 0.2 μm wide, a caudal process 18 ± 1.2 μm long, and a total spore length of 26.8 ± 1.1 μm. Polar capsules were elongated 4.9 ± 0.3 μm long and 1.4 ± 0.1 μm wide. Histological examination indicated that the plasmodia developed in the connective tissue, and no inflammatory infiltrate was observed at the infection site. Ultrastructural analysis showed a plasmodium wall with a single membrane and several pinocytotic canals. Sporogenesis occurred from the periphery to the center of the plasmodia. Phylogenetic analysis of the 18S rDNA sequence using maximum likelihood and maximum parsimony methods showed H. visibilis n. sp. positioned in a sub-clade composed of Henneguya/Myxobolus parasites of several freshwater fish families.

  12. High protists diversity in the plankton of sulfurous lakes and lagoons examined by 18s rRNA gene sequence analyses.

    PubMed

    Triadó-Margarit, Xavier; Casamayor, Emilio O

    2015-12-01

    Diversity of small protists was studied in sulfidic and anoxic (euxinic) stratified karstic lakes and coastal lagoons by 18S rRNA gene analyses. We hypothesized a major sulfide effect, reducing protist diversity and richness with only a few specialized populations adapted to deal with low-redox conditions and high-sulfide concentrations. However, genetic fingerprinting suggested similar ecological diversity in anoxic and sulfurous than in upper oxygen rich water compartments with specific populations inhabiting euxinic waters. Many of them agreed with genera previously identified by microscopic observations, but also new and unexpected groups were detected. Most of the sequences matched a rich assemblage of Ciliophora (i.e., Coleps, Prorodon, Plagiopyla, Strombidium, Metopus, Vorticella and Caenomorpha, among others) and algae (mainly Cryptomonadales). Unidentified Cercozoa, Fungi, Stramenopiles and Discoba were recurrently found. The lack of GenBank counterparts was higher in deep hypolimnetic waters and appeared differentially allocated in the different taxa, being higher within Discoba and lower in Cryptophyceae. A larger number of populations than expected were specifically detected in the deep sulfurous waters, with unknown ecological interactions and metabolic capabilities.

  13. The phylogenetic position of Myxobolus carnaticus (Myxozoa, Myxosporea, Bivalvulida) infecting gill lamellae of Cirrhinus mrigala (Hamilton, 1822) based on 18S rRNA sequence analysis

    PubMed Central

    Banerjee, Sayani; Patra, Avijit; Adikesavalu, Harresh; Mondal, Anjan; Jawahar Abraham, Thangapalam

    2015-01-01

    Myxozoans are an economically important group of microscopic parasites best known for the diseases they cause in commercially important fish hosts. The classification of myxosporeans is generally based on the morphology of their myxospores. Without molecular data, it is very difficult to identify new or existing species. DNA sequence information is therefore, a prerequisite to taxonomic and phylogenic studies of myxosporeans. In the present study, a myxozoan parasite, Myxobolus carnaticus, infecting the gill lamellae of mrigal carp, Cirrhinus mrigala, was characterized by the 18S rRNA gene sequence. The DNA sequence of M. carnaticus clustered phylogenetically with other gill infecting Myxobolus spp. of freshwater clades, forming a dichotomy with closely related M. pavlovskii (HM991164) that infects the gill lamellae epithelium of silver carp, Hypophthalmichthys molitrix with 95% similarity. Evolutionary pair-wise distances among M. carnaticus and other species of myxosporeans indicated high genetic diversity among myxosporeans. The present study demonstrated that tissue tropism, host specificity and habitat play important roles in phylogenetic relationships among myxozoan species. PMID:27844004

  14. Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa.

    PubMed

    Cavalier-Smith, Thomas

    2014-10-01

    Gregarine 18S ribosomal DNA trees are hard to resolve because they exhibit the most disparate rates of rDNA evolution of any eukaryote group. As site-heterogeneous tree-reconstruction algorithms can give more accurate trees, especially for technically unusually challenging groups, I present the first site-heterogeneous rDNA trees for 122 gregarines and an extensive set of 452 appropriate outgroups. While some features remain poorly resolved, these trees fit morphological diversity better than most previous, evolutionarily less realistic, maximum likelihood trees. Gregarines are probably polyphyletic, with some 'eugregarines' and all 'neogregarines' (both abandoned as taxa) being more closely related to Cryptosporidium and Rhytidocystidae than to archigregarines. I establish a new subclass Orthogregarinia (new orders Vermigregarida, Arthrogregarida) for gregarines most closely related to Cryptosporidium and group Orthogregarinia, Cryptosporidiidae, and Rhytidocystidae as revised class Gregarinomorphea. Archigregarines are excluded from Gregarinomorphea and grouped with new orders Velocida (Urosporoidea superfam. n. and Veloxidium) and Stenophorida as a new sporozoan class Paragregarea. Platyproteum and Filipodium never group with Orthogregarinia or Paragregarea and are sufficiently different morphologically to merit a new order Squirmida. I revise gregarine higher-level classification generally in the light of site-heterogeneous-model trees, discuss their evolution, and also sporozoan cell structure and life-history evolution, correcting widespread misinterpretations.

  15. Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

    PubMed Central

    Zhang, Jun; McCann, Kathleen L.; Qiu, Chen; Gonzalez, Lauren E.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2016-01-01

    Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C'-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease, Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins. PMID:27725644

  16. Molecular Characterization of Cryptosporidium spp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

    PubMed Central

    Saki, Jasem; Asadpouri, Reza

    2016-01-01

    Background. Rodents could act as reservoir for Cryptosporidium spp. specially C. parvum, a zoonotic agent responsible for human infections. Since there is no information about Cryptosporidium infection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization of Cryptosporidium spp. in this region. Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method using SspI and VspI restriction enzymes was carried out to genotype the species and then obtained results were sequenced. Results. Three out of 100 samples were diagnosed as positive and overall prevalence of Cryptosporidium spp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples and C. parvum pattern was identified. Finally PCR-RFLP findings were sequenced and presence of C. parvum was confirmed again. Conclusions. Our study showed rodents could be potential reservoir for C. parvum. So an integrated program for control and combat with them should be adopted and continued. PMID:27956905

  17. Nanoscale copper sulfide hollow spheres with phase-engineered composition: covellite (CuS), digenite (Cu1.8S), chalcocite (Cu2S).

    PubMed

    Leidinger, Peter; Popescu, Radian; Gerthsen, Dagmar; Lünsdorf, Heinrich; Feldmann, Claus

    2011-06-01

    Covellite (CuS), digenite (Cu(1.8)S) and chalcocite (Cu(2)S) are prepared as nanoscaled hollow spheres by reaction at the liquid-to-liquid phase boundary of a w/o-microemulsion. According to electron microscopy (SEM, STEM, TEM, HRTEM) the hollow spheres exhibit an outer diameter of 32-36 nm, a wall thickness of 8-12 nm and an inner cavity of 8-16 nm in diameter. The phase composition is determined based on HRTEM, electron-energy loss spectroscopy, X-ray powder diffraction and thermal analysis. In face of the advanced morphology of the hollow spheres, precise control of its phase composition is nevertheless possible by adjusting the experimental conditions (i.e. type and concentration of the copper precursor, concentration of ammonia inside of the micelle). Such phase-engineering of nanoscale hollow spheres is firstly observed and might allow adjusting even further compositions/structures as well as tailoring of phase-specific properties in the future.

  18. Phylogenetic position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology.

    PubMed

    Turbeville, J M; Field, K G; Raff, R A

    1992-03-01

    Partial 18S rRNA sequence of the nemertine Cerebratulus lacteus was obtained and compared with those of coelomate metazoans and acoelomate platyhelminths to test whether nemertines share a most recent common ancestor with the platyhelminths, as traditionally has been implied, or whether nemertines lie within a protostome coelomate clade, as suggested by more recent morphological analyses. Maximum-parsimony analysis supports the inclusion of the nemertine within a protostome-coelomate clade that falls within a more inclusive coelomate clade. Bootstrap analysis indicates strong support for a monophyletic Coelomata composed of a deuterostome and protostome-coelomate clade. Support for a monophyletic protostome Coelomata is weak. Inference by distance analysis is consistent with that of maximum parsimony. Analysis of down-weighted paired sites by maximum parsimony reveals variation in topology only within the protostome-coelomate clade. The relationships among the protostome coelomates cannot be reliably inferred from the partial sequences, suggesting that coelomate protostomes diversified rapidly. Results with evolutionary parsimony are consistent with the inclusion of the nemertine in a coelomate clade. The molecular inference corroborates recent morphological character analyses that reveal no synapomorphies of nemertines and flatworms but instead suggest that the circulatory system and rhynchocoel of nemertines are homologous to coelomic cavities of protostome coelomates, thus supporting the corresponding hypothesis that nemertines belong within a protostome-coelomate clade. The sequence data provide an independent test of morphological character homology.

  19. Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus).

    PubMed

    Harada, Tetsuya; Kawai, Takao; Jinnai, Michio; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko; Kumeda, Yuko

    2012-09-01

    Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10(1) spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations.

  20. Seasonal and geographical distribution of near-surface small photosynthetic eukaryotes in the western North Pacific determined by pyrosequencing of 18S rDNA.

    PubMed

    Kataoka, Takafumi; Yamaguchi, Haruyo; Sato, Mayumi; Watanabe, Tsuyoshi; Taniuchi, Yukiko; Kuwata, Akira; Kawachi, Masanobu

    2017-02-01

    In this study, we investigated the distribution of small photosynthetic eukaryotes in the near-surface layer of the western North Pacific at four stations, including two oceanic stations where the subarctic Oyashio and subtropical Kuroshio currents influence a transition region and the bay mouth and head of the Sendai Bay, from April 2012 to May 2013. Flow cytometry was applied to sort small photosynthetic eukaryotes (<5 μm), and high-throughput sequencing of 18S rDNA was performed. Our taxonomic analysis showed that 19/195 operational taxonomic units (OTUs) were frequently distributed among all sites. Composition analysis showed that the OTUs had characteristic patterns and were divided into four main groups. Two groups reflected the low-saline water and winter season, with the characteristic OTUs belonging to diatoms; Chaetoceros and Leptocylindrus were characteristic of low saline water, and two diatom genera (Minidiscus and Minutocellus) and Cryptomonadales-related OTUs were prevalent in the winter. Our results indicate that the community composition of small photosynthetic eukaryotes seasonally changes in a dynamic manner according to variations in water properties.

  1. Design and validation of four new primers for next-generation sequencing to target the 18S rRNA genes of gastrointestinal ciliate protozoa.

    PubMed

    Ishaq, Suzanne L; Wright, André-Denis G

    2014-09-01

    Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.

  2. Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

    PubMed Central

    Larburu, Natacha; Montellese, Christian; O'Donohue, Marie-Françoise; Kutay, Ulrike; Gleizes, Pierre-Emmanuel; Plisson-Chastang, Célia

    2016-01-01

    Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation. PMID:27530427

  3. Nop9 is a PUF-like protein that prevents premature cleavage to correctly process pre-18S rRNA

    SciTech Connect

    Zhang, Jun; McCann, Kathleen L.; Qiu, Chen; Gonzalez, Lauren E.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2016-10-11

    Numerous factors direct eukaryotic ribosome biogenesis, and defects in a single ribosome assembly factor may be lethal or produce tissue-specific human ribosomopathies. Pre-ribosomal RNAs (pre-rRNAs) must be processed stepwise and at the correct subcellular locations to produce the mature rRNAs. Nop9 is a conserved small ribosomal subunit biogenesis factor, essential in yeast. Here we report a 2.1-Å crystal structure of Nop9 and a small-angle X-ray-scattering model of a Nop9:RNA complex that reveals a ‘C’-shaped fold formed from 11 Pumilio repeats. We show that Nop9 recognizes sequence and structural features of the 20S pre-rRNA near the cleavage site of the nuclease, Nob1. We further demonstrate that Nop9 inhibits Nob1 cleavage, the final processing step to produce mature small ribosomal subunit 18S rRNA. Together, our results suggest that Nop9 is critical for timely cleavage of the 20S pre-rRNA. Moreover, the Nop9 structure exemplifies a new class of Pumilio repeat proteins.

  4. Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

    PubMed Central

    Wright, André-Denis G.

    2014-01-01

    Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen. PMID:24973070

  5. The phylogenetic position of eriophyoid mites (superfamily Eriophyoidea) in Acariformes inferred from the sequences of mitochondrial genomes and nuclear small subunit (18S) rRNA gene.

    PubMed

    Xue, Xiao-Feng; Dong, Yan; Deng, Wei; Hong, Xiao-Yue; Shao, Renfu

    2017-04-01

    Eriophyoid mites (superfamily Eriophyoidea) comprise >4400 species worldwide. Despite over a century of study, the phylogenetic position of these mites within Acariformes is still poorly resolved. Currently, Eriophyoidea is placed in the order Trombidiformes. We inferred the high-level phylogeny of Acari with the mitochondrial (mt) genome sequences of 110 species including four eriophyoid species, and the nuclear small subunit (18S) rRNA gene sequences of 226 species including 25 eriophyoid species. Maximum likelihood (ML), Bayesian inference (BI) and Maximum parsimony (MP) methods were used to analyze the sequence data. Divergence times were estimated for major lineages of Acari using Bayesian approaches. Our analyses consistently recovered the monophyly of Eriophyoidea but rejected the monophyly of Trombidiformes. The eriophyoid mites were grouped with the sarcoptiform mites, or were the sister group of sarcoptiform mites+non-eriophyoid trombidiform mites, depending on data partition strategies. Eriophyoid mites diverged from other mites in the Devonian (384Mya, 95% HPD, 352-410Mya). The origin of eriophyoid mites was dated to the Permian (262Mya, 95% HPD 230-307Mya), mostly prior to the radiation of gymnosperms (Triassic-Jurassic) and angiosperms (early Cretaceous). We propose that the placement of Eriophyoidea in the order Trombidiformes under the current classification system should be reviewed.

  6. Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans

    PubMed Central

    Meyer, Britta; Wurm, Jan Philip; Sharma, Sunny; Immer, Carina; Pogoryelov, Denys; Kötter, Peter; Lafontaine, Denis L. J.; Wöhnert, Jens; Entian, Karl-Dieter

    2016-01-01

    The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. PMID:27084949

  7. When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

    PubMed

    Budaeva, Nataliya; Schepetov, Dmitry; Zanol, Joana; Neretina, Tatiana; Willassen, Endre

    2016-01-01

    Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected.

  8. Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability.

    PubMed

    Harrison, Paul M; Zheng, Deyou; Zhang, Zhaolei; Carriero, Nicholas; Gerstein, Mark

    2005-01-01

    Pseudogenes, in the case of protein-coding genes, are gene copies that have lost the ability to code for a protein; they are typically identified through annotation of disabled, decayed or incomplete protein-coding sequences. Processed pseudogenes (PPsigs) are made through mRNA retrotransposition. There is overwhelming genomic evidence for thousands of human PPsigs and also dozens of human processed genes that comprise complete retrotransposed copies of other genes. Here, we survey for an intermediate entity, the transcribed processed pseudogene (TPPsig), which is disabled but nonetheless transcribed. TPPsigs may affect expression of paralogous genes, as observed in the case of the mouse makorin1-p1 TPPsig. To elucidate their role, we identified human TPPsigs by mapping expressed sequences onto PPsigs and, reciprocally, extracting TPPsigs from known mRNAs. We consider only those PPsigs that are homologous to either non-mammalian eukaryotic proteins or protein domains of known structure, and require detection of identical coding-sequence disablements in both the expressed and genomic sequences. Oligonucleotide microarray data provide further expression verification. Overall, we find 166-233 TPPsigs ( approximately 4-6% of PPsigs). Proteins/transcripts with the highest numbers of homologous TPPsigs generally have many homologous PPsigs and are abundantly expressed. TPPsigs are significantly over-represented near both the 5' and 3' ends of genes; this suggests that TPPsigs can be formed through gene-promoter co-option, or intrusion into untranslated regions. However, roughly half of the TPPsigs are located away from genes in the intergenic DNA and thus may be co-opting cryptic promoters of undesignated origin. Furthermore, TPPsigs are unlike other PPsigs and processed genes in the following ways: (i) they do not show a significant tendency to either deposit on or originate from the X chromosome; (ii) only 5% of human TPPsigs have potential orthologs in mouse. This

  9. OPTICAL TRANSCRIBING OSCILLOSCOPE

    DOEpatents

    Kerns, Q.A.

    1961-09-26

    A device is designed for producing accurate graphed waveforms of very fast electronic pulses. The fast pulse is slowly tracked on a cathode ray tube and a pair of photomultiplier tubes, exposed to the pulse trace, view separate vertical portions thereof at each side of a fixed horizontal reference. Each phototube produces an output signal indicative of vertical movement of the exposed trace, which simultaneous signals are compared in a difference amplifier. The amplifier produces a difference signal which, when applied to the cathode ray tube, maintains the trace on the reference. A graphic recorder receives the amplified difference signal at an x-axis input, while a y-axis input is synchronized with the tracking time of the cathode ray tube and therefore graphs the enlarged waveshape.

  10. Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

    PubMed

    Chattopadhyay, M; Gangadharan, S; Kapur, V; Azfer, M A; Prakash, B; Ali, S

    2001-09-01

    We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.

  11. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells.

    PubMed

    Minsky, Neri; Shema, Efrat; Field, Yair; Schuster, Meromit; Segal, Eran; Oren, Moshe

    2008-04-01

    Histone modifications have emerged as important regulators of transcription. Histone H2B monoubiquitination has also been implicated in transcription; however, better understanding of the biological significance of this modification in mammalian cells has been hindered by the lack of suitable reagents, particularly antibodies capable of specifically recognizing ubiquitinated H2B (ubH2B). Here, we report the generation of anti-ubH2B monoclonal antibodies using a branched peptide as immunogen. These antibodies provide a powerful tool for exploring the biochemical functions of H2B monoubiquitination at both a genome-wide and gene-specific level. Application of these antibodies in high resolution chromatin immunoprecipitation (ChIP)-chip experiments in human cells, using tiling arrays, revealed preferential association of ubiquitinated H2B with the transcribed regions of highly expressed genes. Unlike dimethylated H3K4, ubH2B was not associated with distal promoter regions. Furthermore, experimental modulation of the transcriptional activity of the tumour suppressor p53 was accompanied by rapid changes in the H2B ubiquitination status of its p21 target gene, attesting to the dynamic nature of this process. It has recently been demonstrated that the apparent extent of gene expression often reflects elongation rather than initiation rates; thus, our findings suggest that H2B ubiquitination is intimately linked with global transcriptional elongation in mammalian cells.

  12. Bd oxidase homologue of photosynthetic purple sulfur bacterium Allochromatium vinosum is co-transcribed with a nitrogen fixation related gene.

    PubMed

    Dincturk, H Benan; Demir, Volkan; Aykanat, Tutku

    2011-02-01

    Purple sulfur bacteria, which are known to be the most ancient among anoxygenic phototrophs, play an important role in the global sulfur cycle. Allochromatium vinosum oxidizes reduced sulfur compounds such as hydrogen sulfide, elemental sulfur and thiosulfide. At low oxygen concentrations, A. vinosum can grow chemotrophically using oxygen as the terminal electron acceptor. Being also a nitrogen fixer, A. vinosum is faced with the paradox of co-existence of aerobic metabolism and nitrogen fixation. Due to growth difficulties, only a few studies have dealt with the aerobic metabolism of the organism and, until now, there has been no information about the genes involved in the respiratory metabolism of purple sulfur bacteria. In this article we show the first terminal oxidase gene for A. vinosum. The presence of a Bd type of quinol oxidase is necessary to protect nitrogenases against the inhibitory effects of oxygen. In this case, a nitrogen fixation related gene is part of the cyd operon and this gene is co-transcribed with cydAB genes. Bd oxidase of A. vinosum may be the earliest form of oxidase where the function of the enzyme is to scavenge the contaminant oxygen during nitrogen fixation. This may be an important clue about the early evolution of oxygenic photosynthesis, perhaps as a protective mechanism for nitrogen fixation.

  13. Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

    PubMed Central

    Standring, D N; Venegas, A; Rutter, W J

    1981-01-01

    A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process. Images PMID:6796956

  14. Time-series of water column alkenones and 18S rRNA confirm that Uk'37 is a viable SST proxy in Narragansett Bay, RI

    NASA Astrophysics Data System (ADS)

    Salacup, J.; Theroux, S.; Herbert, T.; Prell, W. L.

    2011-12-01

    Alkenones, produced in the sunlit mixed layer by specific Haptophyte algae, are a well-established and widely-applied proxy for sea surface temperature (SST) in the world's open-oceans. However, the proxy's utility in estuarine environments remains largely untested. A reliable SST proxy is needed to identify the estuary's sensitivity and response to past and present global change because SST can exert strong control on stratification and circulation patterns, and thus oxygenation and ecosystem health, in these shallow basins. Knowing the estuaries response should help local managers and policy-makers plan mitigation and adaptation strategies. Additionally, the rapid deposition of both marine and terrestrial organic and inorganic material in estuarine systems makes them potential archives of high-resolution paleo-environmental information. A previous investigation of estuarine alkenones suggested that the Uk'37 proxy may be sensitive to the composition of the alkenone-producing Haptophyte population, which may be affected by local nutrient and fresh water fluxes. In particular, low-salinity coastal Haptophytes such as Isochrysis galbana may have a different relationship to SST than higher-salinity open-ocean Haptophytes and their presence may complicate interpretations of the Uk'37 proxy in estuaries. To better understand how the alkenone-based Uk'37 SST proxy is produced in estuarine systems, we present a two-year time-series (monthly-to-thrice-weekly resolution) of alkenone concentrations in particulate organic matter from Narragansett Bay. Alkenone concentrations are coupled with 18S ribosomal RNA (rRNA) measurements to identify the alkenone-producing population. Highest concentrations of alkenones are detected at different times in the upper and lower Bay such that the highest alkenone concentrations occur in the winter-spring (upper Bay) and summer/fall (lower Bay). This result is consistent with the established seasonal blooms and seasonal changes in nutrient

  15. Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences▿ †

    PubMed Central

    Valster, Rinske M.; Wullings, Bart A.; Bakker, Geo; Smidt, Hauke; van der Kooij, Dick

    2009-01-01

    Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa. PMID:19465529

  16. Sequence alignment of 18S ribosomal RNA and the basal relationships of Adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae.

    PubMed

    Shull, V L; Vogler, A P; Baker, M D; Maddison, D R; Hammond, P M

    2001-01-01

    Current hypotheses regarding family relationships in the suborder Adephaga (Coleoptera) are conflicting. Here we report full-length 18S ribosomal RNA sequences of 39 adephagans and 13 outgroup taxa. Data analysis focused on the impact of sequence alignment on tree topology, using two principally different approaches. Tree alignments, which seek to minimize indels and substitutions on the tree in a single step, as implemented in an approximate procedure by the computer program POY, were contrasted with a more traditional procedure based on alignments followed by phylogenetic inference based on parsimony, likelihood, and distance analyses. Despite substantial differences between the procedures, phylogenetic conclusions regarding basal relationships within Adephaga and relationships between the four suborders of Coleoptera were broadly similar. The analysis weakly supports monophyly of Adephaga, with Polyphaga usually as its sister, and the two small suborders Myxophaga and Archostemata basal to them. In some analyses, however, Polyphaga was reconstructed as having arisen from within Hydradephaga. Adephaga generally split into two monophyletic groups, corresponding to the terrestrial Geadephaga and the aquatic Hydradephaga, as initially proposed by Crowson in 1955, consistent with a single colonization of the aquatic environment by adephagan ancestors and contradicting the recent proposition of three independent invasions. A monophyletic Hydradephaga is consistently, though not strongly, supported under most analyses, and a parametric bootstrapping test significantly rejects an hypothesis of nonmonophyly. The enigmatic Trachypachidae, which exhibit many similarities to aquatic forms but whose species are entirely terrestrial, were usually recovered as a basal lineage within Geadephaga. Strong evidence opposes the view that terrestrial trachypachids are related to the dytiscoid water beetles.

  17. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data.

    PubMed

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  18. Use of 18S rRNA gene-based PCR assay for diagnosis of acanthamoeba keratitis in non-contact lens wearers in India.

    PubMed

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K

    2003-07-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the "gold standard," the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.

  19. Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis in Non-Contact Lens Wearers in India

    PubMed Central

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K.

    2003-01-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the “gold standard,” the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting. PMID:12843065

  20. Identification of Habitat-Specific Biomes of Aquatic Fungal Communities Using a Comprehensive Nearly Full-Length 18S rRNA Dataset Enriched with Contextual Data

    PubMed Central

    Panzer, Katrin; Yilmaz, Pelin; Weiß, Michael; Reich, Lothar; Richter, Michael; Wiese, Jutta; Schmaljohann, Rolf; Labes, Antje; Imhoff, Johannes F.; Glöckner, Frank Oliver; Reich, Marlis

    2015-01-01

    Molecular diversity surveys have demonstrated that aquatic fungi are highly diverse, and that they play fundamental ecological roles in aquatic systems. Unfortunately, comparative studies of aquatic fungal communities are few and far between, due to the scarcity of adequate datasets. We combined all publicly available fungal 18S ribosomal RNA (rRNA) gene sequences with new sequence data from a marine fungi culture collection. We further enriched this dataset by adding validated contextual data. Specifically, we included data on the habitat type of the samples assigning fungal taxa to ten different habitat categories. This dataset has been created with the intention to serve as a valuable reference dataset for aquatic fungi including a phylogenetic reference tree. The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of sequences suggesting that environmental factors were the main drivers of fungal community structure, rather than species competition. Thus, the diversification process of aquatic fungi must be highly clade specific in some cases.The combined data enabled us to infer fungal community patterns in aquatic systems. Pairwise habitat comparisons showed significant phylogenetic differences, indicating that habitat strongly affects fungal community structure. Fungal taxonomic composition differed considerably even on phylum and class level. Freshwater fungal assemblage was most different from all other habitat types and was dominated by basal fungal lineages. For most communities, phylogenetic signals indicated clustering of

  1. Structural and functional studies of Bud23-Trm112 reveal 18S rRNA N7-G1575 methylation occurs on late 40S precursor ribosomes.

    PubMed

    Létoquart, Juliette; Huvelle, Emmeline; Wacheul, Ludivine; Bourgeois, Gabrielle; Zorbas, Christiane; Graille, Marc; Heurgué-Hamard, Valérie; Lafontaine, Denis L J

    2014-12-23

    The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N(7)-methylguanosine (m(7)G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P- and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a β-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m(7)G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.

  2. Enhanced DNA repair of bleomycin-induced 3'-phosphoglycolate termini at the transcription start sites of actively transcribed genes in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Galea, Anne M

    2014-11-01

    The anti-tumour agent, bleomycin, cleaves DNA to give 3'-phosphoglycolate and 5'-phosphate termini. The removal of 3'-phosphoglycolate to give 3'-OH ends is a very important step in the DNA repair of these lesions. In this study, next-generation DNA sequencing was utilised to investigate the repair of these 3'-phosphoglycolate termini at the transcription start sites (TSSs) of genes in HeLa cells. The 143,600 identified human TSSs in HeLa cells comprised 82,596 non-transcribed genes and 61,004 transcribed genes; and the transcribed genes were divided into quintiles of 12,201 genes comprising the top 20%, 20-40%, 40-60%, 60-80%, 80-100% of expressed genes. Repair of bleomycin-induced 3'-phosphoglycolate termini was enhanced at actively transcribed genes. The top 20% and 20-40% quintiles had a very similar level of enhanced repair, the 40-60% quintile was intermediate, while the 60-80% and 80-100% quintiles were close to the low level of enhancement found in non-transcribed genes. There were also interesting differences regarding bleomycin repair on the sense and antisense strands of DNA at TSSs. The sense strand had highly enhanced repair between 0 and 250bp relative to the TSS, while for the antisense strand highly enhanced repair was between 150 and 450bp. Repair of DNA damage is a major mechanism of resistance to anti-tumour drugs and this study provides an insight into this process in human tumour cells.

  3. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome

    SciTech Connect

    Leadon, S.A. ); Copper, P.K. )

    1993-11-15

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribe genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, the authors examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. They found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. The results extend the generality of preferential repair in active genes to include damage other than bulky lesions.

  4. A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

    PubMed Central

    Diesch, Jeannine; Lesmana, Analia; Poortinga, Gretchen; Hein, Nadine; Lidgerwood, Grace; Cameron, Donald P.; Ellul, Jason; Goodall, Gregory J.; Wong, Lee H.; Dhillon, Amardeep S.; Hamdane, Nourdine; Rothblum, Lawrence I.; Pearson, Richard B.; Haviv, Izhak; Moss, Tom

    2015-01-01

    Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). PMID:25452314

  5. Molecular detection, identification and phylogenetic inference of Leishmania spp. in some desert lizards from Northwest China by using internal transcribed spacer 1 (ITS1) sequences.

    PubMed

    Zhang, Jun-Rong; Guo, Xian-Guang; Liu, Jin-Long; Zhou, Tian-He; Gong, Xiong; Chen, Da-Li; Chen, Jian-Ping

    2016-10-01

    Leishmaniasis caused by Leishmania is still endemic in Northwest China. It has been thought that reptiles could be a reservoir for mammalian leishmaniasis. However, data are still scarce on natural infection of lizards with Leishmania spp. in China. The present study deals with detection, identification and phylogenetic inference of Leishmania parasites at species and intraspecies levels isolated from six desert lizard species from 10 geographical locations in Northwest China using amplification and sequencing of ITS-rDNA. In total, 83 haplotypes were found among 137 ITS1 sequences obtained from up to 64.6% of all captured lizards. Representative sequences of Leishmania available in GenBank were compiled for comparison with the obtained haplotypes. Tree-based species delimitation was achieved by using Bayesian phylogenitc analyses and maximum parsimony approach. Phylogenetic trees congruently supported that the haplotypes were found to belong to three Leishmania species including L. (sauroleishmania) sp., Leishmania tropica and Leishmania donovani complex. A network approach revealed paraphyletic populations of L. (sauroleishmania) sp. and L. tropica at intraspecies level regarding geographical origin and low host specificity. Chinese L. tropica from lizards showed significant heterogeneity as the obtained haplotypes were distributed in different clusters from other countries. Common ancestry was observed between some sequences of L. tropica from lizards and other sequence types from clinical samples from other countries. This may lend support to the potential reservoir role of lizards for human leishmaniasis. Our results appear to be the first molecular evidence for natural infection of lizards in Northwest China with reptilian Leishmania and mammalian Leishmania species. Desert lizards may be considered as putative reservoir hosts for Leishmania in China. Further studies on persistence of the Leishmania parasites in lizards and sandflies are recommended for the better understanding of their epidemiological involvement.

  6. Molecular Analysis of the Lance Nematode, Hoplolaimus spp., Using the First Internal Transcribed Spacer and the D1-D3 Expansion Segments of 28S Ribosomal DNA1

    PubMed Central

    Bae, CH; Szalanski, AL; Robbins, RT

    2008-01-01

    DNA sequence analyses of the nuclear ribosomal ITS1 region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene were conducted to characterize the genetic variation of six amphimictic Hoplolaimus species, including H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus and H. seinhorsti. PCR amplifications of the combined D1-D3 expansion segments and the ITS1 region each yielded one distinct amplicon. In the D1-D3 region, there was no nucleotide sequence variation between populations of H. columbus, H. magnistylus, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, whereas the ITS1 sequences had nucleotide variation among species. We detected conserved ITS1 regions located at the 3’ and 5’ end of ITS1 and also in the middle of the ITS1 among Hoplolaimus species. These regions were compared with sequences of distantly related Heterodera and Globedera. PCR-RFLP and sequence analysis of ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus and Hoplolaimus sp. 1. Maximum likelihood and maximum parsimony analysis using the combined ITS1 and D1-D3 expansion segment sequences always produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species (H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution. PMID:19440260

  7. A case of Beauveria bassiana keratitis confirmed by internal transcribed spacer and LSU rDNA D1-D2 sequencing.

    PubMed

    Ligozzi, M; Maccacaro, L; Passilongo, M; Pedrotti, E; Marchini, G; Koncan, R; Cornaglia, G; Centonze, A R; Lo Cascio, G

    2014-05-01

    We describe a case of fungal keratitis due to Beauveria bassiana in a farmer with Fuchs' dystrophy, treated with amphotericin B. Surgery with penetrating keratoplasty was necessary to resolve the lesions. Susceptibility testing and molecular sequencing permitted the identification and treatment of this rare aetiological agent of invasive fungal disease.

  8. Phylogenetic reconstruction using secondary structures of Internal Transcribed Spacer 2 (ITS2, rDNA): finding the molecular and morphological gap in Caribbean gorgonian corals

    PubMed Central

    Grajales, Alejandro; Aguilar, Catalina; Sánchez, Juan A

    2007-01-01

    Background Most phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics, not found in the primary sequence, that give "morphological" information. Despite the number of recent molecular studies on octocorals, there is no consensus opinion about a region that carries enough phylogenetic resolution to solve intrageneric or close species relationships. Moreover, intrageneric morphological information by itself does not always produce accurate phylogenies; intra-species comparisons can reveal greater differences than intra-generic ones. The search for new phylogenetic approaches, such as by RNA secondary structure analysis, is therefore a priority in octocoral research. Results Initially, twelve predicted RNA secondary structures were reconstructed to provide the basic information for phylogenetic analyses; they accorded with the 6 helicoidal ring model, also present in other groups of corals and eukaryotes. We obtained three similar topologies for nine species of the Caribbean gorgonian genus Eunicea (candelabrum corals) with two sister taxa as outgroups (genera Plexaura and Pseudoplexaura) on the basis of molecular morphometrics of ITS2 RNA secondary structures only, traditional primary sequence analyses and maximum likelihood, and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics; each data partition was allowed to have a different evolution rate. In addition, each helix was partitioned as if it had evolved at a distinct rate. Plexaura flexuosa was found to group within Eunicea; this was best supported by both the molecular morphometrics and combined analyses. We suggest Eunicea flexuosa (Lamouroux, 1821) comb. nov., and we present a new species description including Scanning Electron Microscopy (SEM) images of morphological characteristics (sclerites). Eunicea flexuosa, E. pallida, E. laxispica and E. mammosa formed a separate clade in the molecular phylogenies, and were reciprocally monophyletic with respect to other Eunicea (Euniceopsis subgenus, e.g. E. tourneforti and E. laciniata) in the molecular morphometrics tree, with the exception of E. fusca. Moreover, we suggest a new diagnostic character for Eunicea, also present in E. flexuosa: middle layer sclerites > 1 mm in length. Conclusion ITS2 was a reliable sequence for intrageneric studies in gorgonian octocorals because of the amount of phylogenetic signal, and was corroborated against morphological characters separating Eunicea from Plexaura. The ITS2 RNA secondary structure approach to phylogeny presented here did not rely on alignment methods such as INDELS, but provided clearly homologous characters for partition analysis and RNA molecular morphometrics. These approaches support the divergence of Eunicea flexuosa comb. nov. from the outgroup Plexaura, although it has been considered part of this outgroup for nearly two centuries because of morphological resemblance. PMID:17562014

  9. Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences.

    PubMed

    Martínez-Ibeas, A M; Martínez-Valladares, M; González-Lanza, C; Miñambres, B; Manga-González, M Y

    2011-12-01

    The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.

  10. Improved Molecular Detection of Angiostrongylus cantonensis in Mollusks and Other Environmental Samples with a Species-Specific Internal Transcribed Spacer 1-Based TaqMan Assay ▿

    PubMed Central

    Qvarnstrom, Yvonne; da Silva, Ana Cristina Aramburu; Teem, John L.; Hollingsworth, Robert; Bishop, Henry; Graeff-Teixeira, Carlos; da Silva, Alexandre J.

    2010-01-01

    Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue. PMID:20543049

  11. Population Structure of the Primary Malaria Vector in South America, Anopheles darlingi, Using Isozyme, Random Amplified Polymorphic DNA, Internal Transcribed Spacer 2, and Morphologic Markers

    DTIC Science & Technology

    1999-01-01

    derscoring some congruence, in this case, between two dif- ferent molecular markers. Intraspecific variation in the ITS2 region of 21 members of...morphism in the salivary gland chromosomes of Anopheles darlingi. Mosq News 32: 555-565. 29. Tadei WP, Santos JMN, Rabbani MG, 1982. Biologia de ano...Contel EPB, dos Santos JMM, Tadei Wp, 1984. Biologia de Anophelinos Amazonicos. VI. Enzimatica em Anopheles dar- lingi Root (Dipt.: Culicidae). Acta

  12. PCR-RFLP of ribosomal internal transcribed spacers highlights inter and intra-species variation among Leishmania strains native to La Paz, Bolivia.

    PubMed

    Buitrago, Rosio; Cupolillo, Elisa; Bastrenta, Brigitte; Le Pont, Francois; Martinez, Eddy; Barnabé, Christian; Brenière, Simone Frédérique

    2011-04-01

    Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis, 21 to L. (L.) amazonensis, 1 to Leishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question.

  13. [Analysis of the sequences of internal transcribed spacers ITS1, ITS2 and the 5.8S ribosomal gene of species of the Amaranthus genus].

    PubMed

    Slugina, M A; Torres Minho, K; Filiushin, M A

    2014-01-01

    Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5'-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.

  14. A case of Beauveria bassiana keratitis confirmed by internal transcribed spacer and LSU rDNA D1–D2 sequencing

    PubMed Central

    Ligozzi, M; Maccacaro, L; Passilongo, M; Pedrotti, E; Marchini, G; Koncan, R; Cornaglia, G; Centonze, A R; Lo Cascio, G

    2014-01-01

    We describe a case of fungal keratitis due to Beauveria bassiana in a farmer with Fuchs' dystrophy, treated with amphotericin B. Surgery with penetrating keratoplasty was necessary to resolve the lesions. Susceptibility testing and molecular sequencing permitted the identification and treatment of this rare aetiological agent of invasive fungal disease. PMID:25356350

  15. Construction and expression in vivo of an internally deleted mouse alpha-fetoprotein gene: presence of a transcribed Alu-like repeat within the first intervening sequence.

    PubMed Central

    Young, P R; Scott, R W; Hamer, D H; Tilghman, S M

    1982-01-01

    An alpha-fetoprotein (AFP) 'minigene' was constructed by joining the first two exons along with 0.9 kilobases of 5' flanking sequence of the mouse AFP gene to its last exon and 0.4 kilobases of 3' flanking sequence. This 'minigene' was tested for activity by inserting it into an SV40 vector and infecting African green monkey kidney cells. Correct initiation, termination, polyadenylation and splicing were observed. Furthermore a 220 nucleotide transcript was detected and mapped to a mouse Alu-like or B1 repeat on the opposite strand to that encoding the AFP gene in the first intervening sequence. Images PMID:6179043

  16. Identification of Anopheles (Nyssorhynchus) albitarsis complex species (Diptera: Culicidae) using rDNA internal transcribed spacer 2-based polymerase chain reaction primes

    DTIC Science & Technology

    2005-08-01

    Brazil Para Maraj6 Is. 10 00’S 31 AY828339· (type locality) 49°30’W AY828340 marajoara COI9 Venezuela Cojedes Finca " Rosa 9° 36’ N 9 AY828344- Blanca ...34 68° 54’W AY828345 marajoara COIIO Venezuela Cojedes Finca " Rosa 9° 36’ N 4 AY828346- Blanca " 68° 54’W AY828341 marajoara BR4 Brazil Roraima Boa Vista...domes/icus Galvao & Damesceno with An. mal’ajoara and declared An. allopha Peryassu to be a nomen dubium. Subsequently, Rosa -Freitas (1989) described a

  17. A transcribed ultraconserved noncoding RNA, Uc.173, is a key molecule for the inhibition of lead-induced neuronal apoptosis.

    PubMed

    Nan, Aruo; Zhou, Xinke; Chen, Lijian; Liu, Meiling; Zhang, Nan; Zhang, Li; Luo, Yuanwei; Liu, Zhenzhong; Dai, Lijun; Jiang, Yiguo

    2016-01-05

    As a common toxic metal, lead has significant neurotoxicity to brain development. Long non-coding RNAs (lncRNAs) function in multiple biological processes. However, whether lncRNAs are involved in lead-induced neurotoxicity remains unclear. Uc.173 is a lncRNA from a transcribed ultra-conservative region (T-UCR) of human, mouse and rat genomes. We established a lead-induced nerve injury mouse model. It showed the levels of Uc.173 decreased significantly in hippocampus tissue and serum of the model. We further tested the expression of Uc.173 in serum of lead-exposed children, which also showed a tendency to decrease. To explore the effects of Uc.173 on lead-induced nerve injury, we overexpressed Uc.173 in an N2a mouse nerve cell line and found Uc.173 had an inhibitory effect on lead-induced apoptosis of N2a. To investigate the molecular mechanisms of Uc.173 in apoptosis associated with lead-induced nerve injury, we predicted the target microRNAs of Uc.173 by using miRanda, TargetScan and RegRNA. After performing quantitative real-time PCR and bioinformatics analysis, we showed Uc.173 might inter-regulate with miR-291a-3p in lead-induced apoptosis and regulate apoptosis-associated genes. Our study suggests Uc.173 significantly inhibits the apoptosis of nerve cells, which may be mediated by inter-regulation with miRNAs in lead-induced nerve injury.

  18. Further use of nearly complete 28S and 18S rRNA genes to classify Ecdysozoa: 37 more arthropods and a kinorhynch.

    PubMed

    Mallatt, Jon; Giribet, Gonzalo

    2006-09-01

    This work expands on a study from 2004 by Mallatt, Garey, and Shultz [Mallatt, J.M., Garey, J.R., Shultz, J.W., 2004. Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin. Mol. Phylogenet. Evol. 31, 178-191] that evaluated the phylogenetic relationships in Ecdysozoa (molting animals), especially arthropods. Here, the number of rRNA gene-sequences was effectively doubled for each major group of arthropods, and sequences from the phylum Kinorhyncha (mud dragons) were also included, bringing the number of ecdysozoan taxa to over 80. The methods emphasized maximum likelihood, Bayesian inference and statistical testing with parametric bootstrapping, but also included parsimony and minimum evolution. Prominent findings from our combined analysis of both genes are as follows. The fundamental subdivisions of Hexapoda (insects and relatives) are Insecta and Entognatha, with the latter consisting of collembolans (springtails) and a clade of proturans plus diplurans. Our rRNA-gene data provide the strongest evidence to date that the sister group of Hexapoda is Branchiopoda (fairy shrimps, tadpole shrimps, etc.), not Malacostraca. The large, Pancrustacea clade (hexapods within a paraphyletic Crustacea) divided into a few basic subclades: hexapods plus branchiopods; cirripedes (barnacles) plus malacostracans (lobsters, crabs, true shrimps, isopods, etc.); and the basally located clades of (a) ostracods (seed shrimps) and (b) branchiurans (fish lice) plus the bizarre pentastomids (tongue worms). These findings about Pancrustacea agree with a recent study by Regier, Shultz, and Kambic that used entirely different genes [Regier, J.C., Shultz, J.W., Kambic, R.E., 2005a. Pancrustacean phylogeny: hexapods are terrestrial crustaceans and maxillopods are not monophyletic. Proc. R. Soc. B 272, 395-401]. In Malacostraca, the stomatopod (mantis shrimp) was not at the base of the eumalacostracans

  19. Physical Mapping of the 5S and 18S rDNA in Ten Species of Hypostomus Lacépède 1803 (Siluriformes: Loricariidae): Evolutionary Tendencies in the Genus

    PubMed Central

    César Venere, Paulo; Thums Konerat, Jocicléia; Henrique Zawadzki, Cláudio; Ricardo Vicari, Marcelo; Margarido, Vladimir Pavan

    2014-01-01

    Hypostomus is a diverse group with unclear aspects regarding its biology, including the mechanisms that led to chromosome diversification within the group. Fluorescence in situ hybridization (FISH) with 5S and 18S rDNA probes was performed on ten Hypostomini species. Hypostomus faveolus, H. cochliodon, H. albopunctatus, H. aff. paulinus, and H. topavae had only one chromosome pair with 18S rDNA sites, while H. ancistroides, H. commersoni, H. hermanni, H. regani, and H. strigaticeps had multiple 18S rDNA sites. Regarding the 5S rDNA genes, H. ancistroides, H. regani, H. albopunctatus, H. aff. paulinus, and H. topavae had 5S rDNA sites on only one chromosome pair and H. faveolus, H. cochliodon, H. commersoni, H. hermanni, and H. strigaticeps had multiple 5S rDNA sites. Most species had 18S rDNA sites in the telomeric region of the chromosomes. All species but H. cochliodon had 5S rDNA in the centromeric/pericentromeric region of one metacentric pair. Obtained results are discussed based on existent phylogenies for the genus, with comments on possible dispersion mechanisms to justify the variability of the rDNA sites in Hypostomus. PMID:25405240

  20. Physical mapping of the 5S and 18S rDNA in ten species of Hypostomus Lacépède 1803 (Siluriformes: Loricariidae): evolutionary tendencies in the genus.

    PubMed

    Bueno, Vanessa; Venere, Paulo César; Thums Konerat, Jocicléia; Zawadzki, Cláudio Henrique; Vicari, Marcelo Ricardo; Margarido, Vladimir Pavan

    2014-01-01

    Hypostomus is a diverse group with unclear aspects regarding its biology, including the mechanisms that led to chromosome diversification within the group. Fluorescence in situ hybridization (FISH) with 5S and 18S rDNA probes was performed on ten Hypostomini species. Hypostomus faveolus, H. cochliodon, H. albopunctatus, H. aff. paulinus, and H. topavae had only one chromosome pair with 18S rDNA sites, while H. ancistroides, H. commersoni, H. hermanni, H. regani, and H. strigaticeps had multiple 18S rDNA sites. Regarding the 5S rDNA genes, H. ancistroides, H. regani, H. albopunctatus, H. aff. paulinus, and H. topavae had 5S rDNA sites on only one chromosome pair and H. faveolus, H. cochliodon, H. commersoni, H. hermanni, and H. strigaticeps had multiple 5S rDNA sites. Most species had 18S rDNA sites in the telomeric region of the chromosomes. All species but H. cochliodon had 5S rDNA in the centromeric/pericentromeric region of one metacentric pair. Obtained results are discussed based on existent phylogenies for the genus, with comments on possible dispersion mechanisms to justify the variability of the rDNA sites in Hypostomus.

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

    PubMed

    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.

  2. 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

    PubMed Central

    Sage, E; Drobetsky, E A; Moustacchi, E

    1993-01-01

    We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand. Images PMID:8440233

  3. Oral Academic Discourse Socialisation: Challenges Faced by International Undergraduate Students in a Malaysian Public University

    ERIC Educational Resources Information Center

    Mahfoodh, Omer Hassan Ali

    2014-01-01

    This paper reports a qualitative study which examines the challenges faced by six international undergraduate students in their socialisation of oral academic discourse in a Malaysian public university. Data were collected employing interviews. Students' presentations were also collected. Semi-structured interviews were transcribed verbatim and…

  4. Seasonal Diversity of Planktonic Protists in Southwestern Alberta Rivers over a 1-Year Period as Revealed by Terminal Restriction Fragment Length Polymorphism and 18S rRNA Gene Library Analyses

    PubMed Central

    Thomas, Matthew C.; Selinger, L. Brent

    2012-01-01

    The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure. PMID:22685143

  5. TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification.

    PubMed

    Pinto, C Miguel; Kalko, Elisabeth K V; Cottontail, Iain; Wellinghausen, Nele; Cottontail, Veronika M

    2012-08-01

    We report TcBat, a recently described genetic lineage of Trypanosoma cruzi, in fruit-eating bats Artibeus from Panama. Infections were common (11.6% prevalence), but no other T. cruzi cruzi genotypes were detected. Phylogenetic analyses show an unambiguous association with Brazilian TcBat, but raise questions about the phylogenetic placement of this genotype using the 18S rRNA gene alone. However, analyses with three concatenated genes (18S rRNA, cytb, and H2B) moderately support TcBat as sister to the discrete typing unit (DTU) TcI. We demonstrate that short fragments (>500 bp) of the 18S rRNA gene are useful for identification of DTUs of T. cruzi, and provide reliable phylogenetic signal as long as they are analyzed within a matrix with reference taxa containing additional informative genes. TcBat forms a very distinctive monophyletic group that may be recognized as an additional DTU within T. cruzi cruzi.

  6. Phylogenetic position of the enigmatic clawless eutardigrade genus Apodibius Dastych, 1983 (Tardigrada), based on 18S and 28S rRNA sequence data from its type species A. confusus.

    PubMed

    Dabert, Miroslawa; Dastych, Hieronymus; Hohberg, Karin; Dabert, Jacek

    2014-01-01

    The systematics of Eutardigrada, the largest lineage among the three classes of the phylum Tardigrada, is based mainly on the morphology of the leg claws and of the buccal apparatus. However, three members of the rarely recorded and poorly known limno-terrestrial eutardigrade genus Apodibius have no claws on their strongly reduced legs, a unique character among all tardigrades. This absence of all claws makes the systematic position of Apodibius one of the most enigmatic among the whole class. Until now all known associates of the genus Apodibius have been located in the incertae sedis species group or, quite recently, included into the Necopinatidae family. In the present study, phylogenetic analyses of 18S and 28S rRNA sequence data from 31 tardigrade species representing four parachelan superfamilies (Isohypsibioidea, Hypsibioidea, Macrobiotoidea, Eohypsibioidea), the apochelan Milnesium tardigradum, and the type species of the genus Apodibius, A. confusus, indicated close relationship of the Apodibius with tardigrade species recently included in the superfamily Isohypsibioidea. This result was well-supported and consistent across all markers (separate 18S rRNA, 28S rRNA, and combined 18S rRNA+28S rRNA datasets) and methods (MP, ML) applied.

  7. DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase.

    PubMed

    Sabouri, Nasim; McDonald, Karin R; Webb, Christopher J; Cristea, Ileana M; Zakian, Virginia A

    2012-03-15

    Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

  8. Topoisomerase 1-dependent deletions initiated by incision at ribonucleotides are biased to the non-transcribed strand of a highly activated reporter

    PubMed Central

    Cho, Jang-Eun; Kim, Nayun; Jinks-Robertson, Sue

    2015-01-01

    DNA polymerases incorporate ribonucleoside monophosphates (rNMPs) into genomic DNA at a low level and such rNMPs are efficiently removed in an error-free manner by ribonuclease (RNase) H2. In the absence of RNase H2 in budding yeast, persistent rNMPs give rise to short deletions via a mutagenic process initiated by Topoisomerase 1 (Top1). We examined the activity of a 2-bp, rNMP-dependent deletion hotspot [the (TG)2 hotspot] when on the transcribed or non-transcribed strand (TS or NTS, respectively) of a reporter placed in both orientations near a strong origin of replication. Under low-transcription conditions, hotspot activity depended on whether the (TG)2 sequence was part of the newly synthesized leading or lagging strand of replication. In agreement with an earlier study, deletions occurred at a much higher rate when (TG)2 was on the nascent leading strand. Under high-transcription conditions, however, hotspot activity was not dependent on replication direction, but rather on whether the (TG)2 sequence was on the TS or NTS of the reporter. Deletion rates were several orders of magnitude higher when (TG)2 was on the NTS. These results highlight the complex interplay between replication and transcription in regulating Top1-dependent genetic instability. PMID:26271994

  9. A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways.

    PubMed Central

    van Tol, H; Stange, N; Gross, H J; Beier, H

    1987-01-01

    tRNA splicing enzymes had been identified in mammalian and plant cells long before homologous intron-containing tRNA genes were detected. The tRNATyr gene presented here is the first intron-containing, human tRNA gene for which transcription and pre-tRNA maturation has been studied in a homologous system. This gene is disrupted by a 20-bp long intron and encodes one of the two major human tRNAsTyr which have been purified and sequenced. A tRNATyr gene recently isolated from Nicotiana also contains an intron and codes for a functional, major cytoplasmic tRNATyr. Both tRNA genes are efficiently transcribed in a HeLa cell nuclear extract. Each of them produces two independent primary transcripts because of two initiation and termination sites, respectively. The maturation of the tRNATyr precursors proceeds along different pathways. The intervening sequence of the human pre-tRNATyr is excised first, followed by ligation of the tRNA halves and maturation of the flanks, as has been shown for all intron-containing tRNA genes transcribed in HeLa extract. The order of maturation steps is reversed for the plant pre-tRNATyr: processing of the flanking sequences precedes intron excision. This maturation pathway corresponds to that observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. PMID:3502708

  10. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation.

    PubMed

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2016-01-29

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation.

  11. Basic cytogenetics and physical mapping of 5S and 18S ribosomal genes in Hoplias malabaricus (Osteichthyes, Characiformes, Erythrinidae) from isolated natural lagoons: a conserved karyomorph along the Iguaçu river basin

    PubMed Central

    Gemi, Gisele; Lui, Roberto Laridondo; Treco, Fernando Rodrigo; Paiz, Leonardo Marcel; Moresco, Rafaela Maria; Margarido, Vladimir Pavan

    2014-01-01

    Abstract Erythrinidae include Neotropical teleost fish that are widely distributed in South America. Hoplias Gill, 1903 include two large groups: H. malabaricus Bloch, 1794 and H. lacerdae Miranda Ribeiro, 1908. Hoplias malabaricus is characterized by remarkable karyotype diversity, with some karyomorphs widely distributed geographically while others are more restricted to certain river basins. Cytogenetic analyzes were performed in a population of Hoplias malabaricus from the Wildlife Refuge of Campos de Palmas, the Iguaçu River basin. The specimens showed diploid number of 42 chromosomes (24m+18sm) without differentiated sex chromosomes system. The impregnation by silver nitrate showed multiple AgNORs. Seven pairs (4, 7, 10, 13, 16, 20 and 21) carrying 18S rDNA were detected by FISH. Heterochromatin was verified in the centromeric and pericentromeric region of most chromosomes and the terminal region of some pairs. FISH with 5S rDNA probes showed two chromosome pairs carrying these sites in the interstitial region (8 and 14). The data obtained in this study are similar to those found for two other populations of H. malabaricus already studied in the basin of the Iguaçu River, confirming the hypothesis that this species is natural, not having been introduced, as well as having an intrinsic characteristic, such as the largest number of sites of 18S rDNA. PMID:25349672

  12. Secondary structure prediction for complete rDNA sequences (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari.

    PubMed

    Zhao, Ya-E; Wang, Zheng-Hang; Xu, Yang; Wu, Li-Ping; Hu, Li

    2013-10-01

    According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.

  13. 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.

    PubMed

    Oldach, D W; Delwiche, C F; Jakobsen, K S; Tengs, T; Brown, E G; Kempton, J W; Schaefer, E F; Bowers, H A; Glasgow, H B; Burkholder, J M; Steidinger, K A; Rublee, P A

    2000-04-11

    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 strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

  14. Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene

    SciTech Connect

    Lommel, L.; Hanawalt, P.C. )

    1993-02-01

    People that suffer from xeroderma pigmentosum (XP) are sun sensitive and experience elevated incidences of cancer, particularly skin cancers on sun-light exposed parts of their bodies. Cultured cells from XP patients are found to be subtantially more sensitive to lethal and mutagenic effects of ultraviolet (UV) radiation than are cells from unaffected individuals. Using the cells from XP individuals, researchers study the roles that cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts play in UV resistance. The results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs form the transcribed strands of expressed genes is essential for UV resistance. 36 refs., 5 figs., 1 tab.

  15. The Nine Genes of the Nocardia lactamdurans Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region

    PubMed Central

    Enguita, Francisco J.; Coque, Juan Jose R.; Liras, Paloma; Martin, Juan F.

    1998-01-01

    The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp. PMID:9765587

  16. Both open reading frames of the linear plasmid pMC3-2 from the ascomycete Morchella conica are transcribed in vivo.

    PubMed

    Rohe, M; Meinhardt, F

    1992-12-01

    Mitochondrial RNA was isolated from the morel strain Morchella conica 3 harbouring the linear plasmid pMC3-2 and subjected to gel electrophoresis followed by a Northern analysis using cloned fragments of the plasmid pMC3-2 as probes. Hybridization was obtained only with central parts of pMC3-2 and specific bands of mtRNA. The hybridization bands (2.8 kb and 1.0 kb) correspond in size to the length of the two ORFs of pMC3-2 which were deduced from nucleotide-sequence data. Thus, both ORFs, one encoding a DNA polymerase and the other a yet unknown protein, are transcribed in the mitochondria of the plasmid-bearing Morchella conica strain.

  17. Verification of false-positive blood culture results generated by the BACTEC 9000 series by eubacterial 16S rDNA and panfungal 18S rDNA directed polymerase chain reaction (PCR).

    PubMed

    Daxboeck, Florian; Dornbusch, Hans Jürgen; Krause, Robert; Assadian, Ojan; Wenisch, Christoph

    2004-01-01

    A small but significant proportion of blood cultures processed by the BACTEC 9000 series systems is signaled positive, while subsequent Gram's stain and culture on solid media yield no pathogens. In this study, 15 "false-positive" vials (7 aerobes, 8 anaerobes) from 15 patients were investigated for the presence of bacteria and fungi by eubacterial 16S rDNA and panfungal 18S rDNA amplification, respectively. All samples turned out negative by both methods. Most patients (7) had neutropenia, which does not support the theory that high leukocyte counts enhance the generation of false-positive results. In conclusion, the results of this study indicate that false-negative results generated by the BACTEC 9000 series are inherent to the automated detection and not due to the growth of fastidious organisms.

  18. Population studies for STR loci (D3S1358, D5S818, D7S820, D18S51 and FGA) in NWFP and Sindhi populations of Pakistan for forensic use.

    PubMed

    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.

  19. Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells

    PubMed Central

    Abdullah, Rasedee; Kassim, Nur Kartinee Bt; Rosli, Rozita; Yeap, Swee Keong; Waziri, Peter; Etti, Imaobong Christopher; Bello, Muhammad Bashir

    2017-01-01

    Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer. PMID:28103302

  20. Cytogenetic comparison between two allopatric populations of Astyanax altiparanae Garutti et Britski, 2000 (Teleostei, Characidae), with emphasis on the localization of 18S and 5S rDNA.

    PubMed

    Pacheco, Rosiley Berton; da Rosa, Renata; Giuliano-Caetano, Lucia; Júlio, Horácio Ferreira; Dias, Ana Lúcia

    2011-01-01

    Two populations of Astyanax altiparanae (Garutti & Britski, 2000) of the Água dos Patos stream/SP and lake Igapó/PR were analyzed. All individuals showed 2n = 50, however, different karyotypic formulae were observed. The population of the Água dos Patos stream showed 8m +24sm+6st+12a (NF=88) and the population of lake Igapó, 8m+28sm+4st+10a (NF=90). Nucleolus organizing regions (AgNORs) were observed in the terminal position on the short and long arm of different chromosomes of both populations, showing a variation from 3 to 4 chromosomes. Fluorescent in situ hybridization (FISH) using 18S rDNA probes revealed only one pair of chromosomes with fluorescent signals in the terminal site on the short arm in the Igapó lake population, while the population of Água dos Patos stream showed 4 fluorescence terminal signals, characterizing a system of simple and multiple NORs, respectively. 5S rDNA fluorescent signals were detected in the interstitial position of a pair of chromosomes in the two studied populations. Some AgNOR sites revealed to be GC-rich when stained with Chromomycin A3 (CMA3), however, AT positive regions were not observed. The data obtained show that, despite the conservation of the diploid number and location of 5S DNAr, differences in both the distribution of 18S rDNA and karyotypic formula among the populations were found, thus corroborating the existing data on chromosome variability in Astyanax altiparanae that can be significant for cytotaxonomy in this group.

  1. 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)

    USGS Publications Warehouse

    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.                   

  2. Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

    PubMed

    Malami, Ibrahim; Abdul, Ahmad Bustamam; Abdullah, Rasedee; Kassim, Nur Kartinee Bt; Rosli, Rozita; Yeap, Swee Keong; Waziri, Peter; Etti, Imaobong Christopher; Bello, Muhammad Bashir

    2017-01-01

    Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

  3. International Health

    MedlinePlus

    ... create refugee populations with immediate and long-term health problems. Some of the major diseases currently affecting ... also an international problem which can affect people's health. Many countries and health organizations are working together ...

  4. International Geology

    ERIC Educational Resources Information Center

    Hoover, Linn

    1977-01-01

    Briefly discusses recent international programs in various areas of geology, including land-use problems, coping with geological hazards, and conserving the environment while searching for energy and mineral resources. (MLH)

  5. Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

    PubMed Central

    Chang, K S; Stass, S A; Chu, D T; Deaven, L L; Trujillo, J M; Freireich, E J

    1992-01-01

    A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the

  6. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene.

    PubMed Central

    Linton, J P; Yen, J Y; Selby, E; Chen, Z; Chinsky, J M; Liu, K; Kellems, R E; Crouse, G F

    1989-01-01

    The mouse dihydrofolate reductase gene (dhfr) is a housekeeping gene expressed under the control of a promoter region embedded in a CpG island--a region rich in unmethylated CpG dinucleotides. A divergent transcription unit exists immediately upstream of the dhfr gene which is coamplified with dhfr in some but not all methotrexate-resistant cell lines. We show that the promoter region for this gene pair consists of two bidirectional promoters, a major and minor promoter, which are situated within a 660-base-pair region upstream of the dhfr ATG translation initiation codon. The major promoter controls over 90% of dhfr transcription, while the minor promoter directs the transcription of the remaining dhfr mRNAs. The major promoter functions bidirectionally, transcribing a divergent 4.0-kilobase poly(A) mRNA (class A) in the direction opposite that of dhfr transcription. The predicted protein product of this mRNA is 105 kilodaltons. The minor promoter also functions bidirectionally, directing the transcription of at least two divergent RNAs (class B). These RNAs, present in quantities approximately 1/10 to 1/50 that of the class A mRNAs, are 4.4- and 1.6-kilobase poly(A) mRNAs. cDNAs representing both class A and class B mRNAs have been cloned from a mouse fibroblast cell line which has amplified the dhfr locus (3T3R500). DNA sequence analysis of these cDNAs reveals that the class A and class B mRNAs share, for the most part, the same exons. On the basis of S1 nuclease protection analysis of RNA preparations from several mouse tissues, both dhfr and divergent genes showed similar levels of expression but did show some specificity in start site utilization. Computer homology searches have revealed sequence similarity of the divergent transcripts with bacterial genes involved in DNA mismatch repair, and we therefore have named the divergently transcribed gene Rep-1. Images PMID:2674679

  7. How eukaryotic genes are transcribed

    PubMed Central

    Venters, Bryan J.; Pugh, B. Franklin

    2009-01-01

    Summary Regulation of eukaryotic gene expression is far more complex than one might have imagined thirty years ago. However, progress towards understanding gene regulatory mechanisms has been rapid and comprehensive, which has made the integration of detailed observations into broadly connected concepts a challenge. This review attempts to integrate the following concepts: 1) a well-defined organization of nucleosomes and modification states at most genes, 2) regulatory networks of sequence-specific transcription factors, 3) chromatin remodeling coupled to promoter assembly of the general transcription factors and RNA polymerase II, and 4) phosphorylation states of RNA polymerase II coupled to chromatin modification states during transcription. The wealth of new insights arising from the tools of biochemistry, genomics, cell biology, and genetics is providing a remarkable view into the mechanics of gene regulation. PMID:19514890

  8. Heat shock factor 1 binds to and transcribes satellite II and III sequences at several pericentromeric regions in heat-shocked cells

    SciTech Connect

    Eymery, Angeline; Souchier, Catherine; Vourc'h, Claire; Jolly, Caroline

    2010-07-01

    Cells respond to stress by activating the synthesis of heat shock proteins (HSPs) which protect the cells against the deleterious effects of stress. This mechanism is controlled by the heat shock factor 1 (HSF1). In parallel to HSP gene transcription, in human cells, HSF1 also binds to and transcribes satellite III repeated sequences present in numerous copies in the 9q12 pericentromeric region of chromosome 9. These HSF1 accumulation sites are termed nuclear stress bodies (nSBs). In tumor cells, however, the number of nSBs is higher than the number of 9q12 copies, suggesting the existence of other HSF1 targets. In this paper, we were interested in characterizing these other HSF1 binding sites. We show that HSF1 indeed binds to the pericentromeric region of 14 chromosomes, thereby directing the formation of 'secondary nSBs'. The appearance of secondary nSBs depends on the number of satellite sequences present in the target locus, and on the cellular amount of HSF1 protein. Moreover, secondary nSBs also correspond to transcription sites, thus demonstrating that heat shock induces a genome-wide transcription of satellite sequences. Finally, by analyzing published transcriptomic data, we show that the derepression of these large heterochromatic blocks does not significantly affect the transcription of neighboring genes.

  9. Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation

    SciTech Connect

    Beckmann, R.P.; Agostino, M.J.; McHugh, M.M.; Sigmund, R.D.; Beerman, T.A.

    1987-08-25

    Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene previously described served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the threshold LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.

  10. Intraspecific variation and population structure of the German cockroach, Blattella germanica, revealed with RFLP analysis of the non-transcribed spacer region of ribosomal DNA.

    PubMed

    Mukha, D V; Kagramanova, A S; Lazebnaya, I V; Lazebnyi, O E; Vargo, E L; Schal, C

    2007-06-01

    Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.

  11. Regulation of spblimp1/krox1a, an alternatively transcribed isoform expressed in midgut and hindgut of the sea urchin gastrula.

    PubMed

    Livi, Carolina B; Davidson, Eric H

    2007-01-01

    The sea urchin regulatory gene Spblimp1/krox produces alternatively transcribed and spliced isoforms, 1a and 1b, which have different temporal and spatial patterns of expression. Here we describe a cis-regulatory module that controls the expression of the 1a splice form in the midgut and hindgut at the beginning of gastrulation. Conserved sequence patches revealed by a comparison of the blimp1/krox locus in Strongylocentrotus purpuratus and Lytechinus variegatus genomes were tested by gene transfer, in association with GFP or CAT reporter genes. An expression construct containing a conserved sequence patch immediately 5' of exon 1a included the transcription initiation site for blimp1/krox1a. This construct displays specific mid and hindgut expression, indicating that these are the locations of endogenous blimp1/krox1a transcription during the gastrula stage. Its sequence contains binding sites for Brn1/2/4, Otx, and Blimp1/Krox itself, as predicted in a prior regulatory network analysis.

  12. cis-acting sequences required for expression of the divergently transcribed Drosophila melanogaster Sgs-7 and Sgs-8 glue protein genes.

    PubMed Central

    Hofmann, A; Garfinkel, M D; Meyerowitz, E M

    1991-01-01

    The Sgs-7 and Sgs-8 glue genes at 68C are divergently transcribed and are separated by 475 bp. Fusion genes with Adh or lacZ coding sequences were constructed, and the expression of these genes, with different amounts of upstream sequences present, was tested by a transient expression procedure and by germ line transformation. A cis-acting element for both genes is located asymmetrically in the intergenic region between -211 and -43 bp relative to Sgs-7. It is required for correct expression of both genes. This element can confer the stage- and tissue-specific expression pattern of glue genes on a heterologous promoter. An 86-bp portion of the element, from -133 to -48 bp relative to Sgs-7, is shown to be capable of enhancing the expression of a truncated and therefore weakly expressed Sgs-3 fusion gene. Recently described common sequence motifs of glue gene regulatory elements (T. Todo, M. Roark, K. Vijay Raghavan, C. A. Mayeda, and E.M. Meyerowitz, Mol. Cell. Biol. 10:5991-6002, 1990) are located within this 86-bp region. PMID:1903838

  13. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  14. Internal shim

    DOEpatents

    Barth, Clyde H.; Blizinski, Theodore W.

    2003-05-13

    An internal shim used to accurately measure spaces in conjunction with a standard small probe has a shim top and a chassis. The internal shim is adjustably fixed within the space to be measured using grippers that emerge from the chassis and which are controlled by an arm pivotably attached to the shim top. A standard small probe passes through the shim along guides on the chassis and measures the distance between the exterior of the chassis and the boundary. By summing the measurements on each side of the chassis and the width of the chassis, the dimension of the space can be determined to within 0.001 inches.

  15. International Reports.

    ERIC Educational Resources Information Center

    Anderson, Nancy D.; And Others

    1994-01-01

    Three reports discuss the International Federation of Library Associations and Institutions; the Frankfurt Book Fair, focusing on electronics; and Canadian library trends, including resource sharing, technology projects, information policy, censorship, services for persons with disabilities, construction projects, and library education and…

  16. Internet International.

    ERIC Educational Resources Information Center

    Woodard, Colin

    1995-01-01

    The unexpectedly rapid expansion of the Internet in Eastern and Central Europe is having a significant effect on institutions of higher education, still suffering from decades of isolation. The benefits include global access to information and cost-effective communications. A number of international efforts are under way to expand Internet access,…

  17. International Systems.

    ERIC Educational Resources Information Center

    Saba, Farhad, Ed.

    1999-01-01

    Completes a discussion of a systems model of distance education (in articles since May 1999) focusing on the most complex level, international. Discussion includes transfer of technology from United States universities to developing nations, the free market, and the age of the global economy. Presents a list of "early indicators" of changes in…

  18. International Entomology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pests and diseases of plants in agriculture are a shared international problem. Yet some of the very places that pest invaders come from often lack the institutional structure and organization necessary to help in understanding the biology of the pest or disease. Strengthening entomology by stimulat...

  19. International Reports.

    ERIC Educational Resources Information Center

    Tabb, Winston; Bender, David R.; Haycock, Ken; Horodyski, John

    2001-01-01

    Includes three annual reports: one from the International Federation of Library Associations and Institutions, the Special Libraries Association, and a report on innovations in Canadian libraries that discusses electronic initiatives, partnerships, books and publishing, school libraries, national issues, local challenges, and funding. (LRW)

  20. (001–002) Proposals to amend Art. 32.2, 23.5, and 24.2 to clarify the treatment of transcribed Greek terminations of epithets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The International Code of Nomenclature for algae, fungi and plants is revised every six years to incorporate decisions of the Nomenclature Section of successive International Botanical Congresses (IBC) on proposals to amend the Code. The proposals in this paper will be considered at the IBC in Shenz...

  1. Detection of Cryptosporidium species in feces or gastric contents from snakes and lizards as determined by polymerase chain reaction analysis and partial sequencing of the 18S ribosomal RNA gene.

    PubMed

    Richter, Barbara; Nedorost, Nora; Maderner, Anton; Weissenböck, Herbert

    2011-05-01

    Cryptosporidiosis is a well-known gastrointestinal disease of snakes and lizards. In the current study, 672 samples (feces and/or gastric contents or regurgitated food items) of various snakes and lizards were examined for the presence of cryptosporidia by polymerase chain reaction (PCR) assay targeting a part of the 18S ribosomal RNA gene. A consecutive sequencing reaction was used to identify the cryptosporidian species present in PCR-positive samples. Cryptosporidium varanii (saurophilum) was detected in 17 out of 106 (16%) samples from corn snakes (Pantherophis guttatus) and in 32 out of 462 (7%) samples from leopard geckos (Eublepharis macularius). Cryptosporidium serpentis was found in 8 out of 462 (2%) leopard gecko samples, but in no other reptile. The Cryptosporidium sp. "lizard genotype" was present in 1 leopard gecko sample, and 1 sample from a corn snake showed a single nucleotide mismatch to this genotype. Pseudoparasitic cryptosporidian species were identified in 5 out of 174 (3%) ophidian samples, but not in lizards. Other sequences did not show complete similarity to previously published Cryptosporidium sequences. The results stress the importance for diagnostic methods to be specific for Cryptosporidium species especially in snakes and show a relatively high prevalence of C. varanii in leopard geckos and corn snakes.

  2. Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples

    PubMed Central

    Balzano, Sergio; Marie, Dominique; Gourvil, Priscillia; Vaulot, Daniel

    2012-01-01

    The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture. PMID:22278671

  3. Cytogenetic analysis of the tamaraw (Bubalus mindorensis): a comparison of R-banded karyotype and chromosomal distribution of centromeric satellite DNAs, telomeric sequence, and 18S-28S rRNA genes with domestic water buffaloes.

    PubMed

    Tanaka, K; Matsuda, Y; Masangkay, J S; Solis, C D; Anunciado, R V; Kuro-o, M; Namikawa, T

    2000-01-01

    The karyotype of the tamaraw (Bubalus mindorensis, 2n = 46) was investigated by RBG-banding technique and compared with those of the river and the swamp cytotypes of domestic water buffalo (B. bubalis). The tamaraw karyotype consisted of 6 submetacentric and 16 acrocentric autosome pairs (NAA = 56), and X and Y chromosomes. The RBG-banded karyotype of the three taxa had a high degree of homology, and the tamaraw karyotype could be explained by a Robertsonian translocation between chromosomes 7 and 15 and by a telomere-centromere tandem fusion between chromosomes 4p and 12 of the standardized river buffalo cytotype (2n = 50, NAA = 58). The buffalo satellite I and II DNAs were localized to the centromeric regions of all the tamaraw chromosomes. The biarmed chromosome 2 of the tamaraw resulting from the fusion between chromosomes 7 and 15 of the standard contained much larger amounts of the satellite I DNA than the other biarmed chromosomes, suggesting that this chromosome was formed by a relatively recent Robertsonian fusion. The (TTAGGG)n telomeric sequence was specifically localized to the telomeric region of all the buffalo chromosomes. The 18S + 28S rDNA was localized to the telomeric regions of the chromosomes 5p, 7, 19, 21, and 22 of the tamaraw and of their homologous chromosomes in the river and swamp buffalo cytotypes.

  4. Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).

    PubMed

    Kishida, Naohiro; Miyata, Ryo; Furuta, Atsushi; Izumiyama, Shinji; Tsuneda, Satoshi; Sekiguchi, Yuji; Noda, Naohiro; Akiba, Michihiro

    2012-01-01

    We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.

  5. Chromosome studies of Astyanax jacuhiensis Cope, 1894 (Characidae) from the Tramandai River Basin, Brazil, using in situ hybridization with the 18S rDNA probe, DAPI and CMA3 staining.

    PubMed

    da Silva, Laura Lahr Lourenço; Giuliano-Caetano, Lucia; Dias, Ana Lúcia

    2012-01-01

    The genus Astyanax comprises 86 species of fish distributed in Brazilian river basins and is considered of the Incertae sedis group within the family Characidae. This study presents an analysis of 12 specimens of Astyanax jacuhiensis from the Tramandai River Basin, RS Brazil: 6 from the Maquiné River and 6 from the Quadros Lagoon. All specimens showed a diploid number equal to 50 chromosomes with different karyotypic formula between the two localities. The population from the Maquiné River showed 10m+26sm+6st+8a (FN=92). Fish from the Quadros Lagoon showed 12m+20sm+6st+12a (FN=88). AgNORs were evidenced in the short arm of one acrocentric chromosome pair in both populations, confirmed by FISH with the 18S rDNA probe. CMA3 fluorochrome corresponded with the AgNOR sites, while DAPI staining was negative in these regions. C banding revealed that heterochromatin was weakly distributed, mainly in the pericentromeric and terminal regions in most chromosomes. Analyses of male gonadal tissue were conducted with the objective of characterizing the meiotic chromosome behavior in A. jacuhiensis. The following stages were evidenced: spermatogonial with 50 chromosomes, pachytene and metaphase I with 25 bivalents, and metaphase II with 25 chromosomes, thus confirming the diploid number of the species. Chromosomal abnormalities were not observed. This study shows preliminary data on A. jacuhiensis from the Tramandai River Basin, contributing with more chromosomal information for this group of fish.

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

    PubMed

    Buse, Helen Y; Lu, Jingrang; Lu, Xinxin; Mou, Xiaozhen; Ashbolt, Nicholas J

    2014-05-01

    Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, yet knowledge about the microbial composition of DW biofilms developed on common in-premise plumbing material is limited. Utilizing 16S and 18S rRNA gene pyrosequencing, this study characterized the microbial community structure within DW biofilms established on unplasticized polyvinyl chloride (uPVC) and copper (Cu) surfaces and the impact of introducing Legionella pneumophila (Lp) and Acanthamoeba polyphaga. Mature (> 1 year old) biofilms were developed before inoculation with sterilized DW (control, Con), Lp, or Lp and A. polyphaga (LpAp). Comparison of uPVC and Cu biofilms indicated significant differences between bacterial (P = 0.001) and eukaryotic (P < 0.01) members attributable to the unique presence of several family taxa: Burkholderiaceae, Characeae, Epistylidae, Goniomonadaceae, Paramoebidae, Plasmodiophoridae, Plectidae, Sphenomonadidae, and Toxariaceae within uPVC biofilms; and Enterobacteriaceae, Erythrobacteraceae, Methylophilaceae, Acanthamoebidae, and Chlamydomonadaceae within Cu biofilms. Introduction of Lp alone or with A. polyphaga had no effect on bacterial community profiles (P > 0.05) but did affect eukaryotic members (uPVC, P < 0.01; Cu, P = 0.001). Thus, established DW biofilms host complex communities that may vary based on substratum matrix and maintain consistent bacterial communities despite introduction of Lp, an environmental pathogen.

  7. Conformation of yeast 18S rRNA. Direct chemical probing of the 5' domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible.

    PubMed Central

    Lempereur, L; Nicoloso, M; Riehl, N; Ehresmann, C; Ehresmann, B; Bachellerie, J P

    1985-01-01

    The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized. Images PMID:2417197

  8. Transcription factor Reb1 is required for proper transcriptional start site usage at the divergently transcribed TFC6-ESC2 locus in Saccharomyces cerevisiae.

    PubMed

    Wang, Qing; Donze, David

    2016-12-05

    Eukaryotic promoters generally contain nucleosome depleted regions near their transcription start sites. In the model organism Saccharomyces cerevisiae, these regions are adjacent to binding sites for general regulatory transcription factors, and the Reb1 protein is commonly bound to promoter DNA near such regions. The yeast TFC6 promoter is a unique RNA polymerase II promoter in that it is autoregulated by its own gene product Tfc6p, which is part of the RNA polymerase III transcription factor complex TFIIIC. We previously demonstrated that mutation of a potential Reb1 binding site adjacent to the TFIIIC binding site in the TFC6 promoter modestly reduces transcript levels, but leads to a severe decrease in Tfc6 protein levels due to an upstream shift in the TFC6 transcription start site. Here we confirm that Reb1p indeed binds to the TFC6 promoter, and is important for proper transcription start site selection and protein expression. Interestingly, loss of Reb1p association at this site has a similar effect on the adjacent divergently transcribed ESC2 promoter, resulting in a significant increase of 5'-extended ESC2 transcripts and reduction of Esc2 protein levels. This altered divergent transcription may be the result of changes in nucleosome positioning at this locus in the absence of Reb1p binding. We speculate that an important function of general regulatory factors such as Reb1p is to establish and maintain proper transcription start sites at promoters, and that when binding of such factors is compromised, resulting effects on mRNA translation may be an underappreciated aspect of gene regulation studies.

  9. Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

    PubMed Central

    Toth, I. K.; Avrova, A. O.; Hyman, L. J.

    2001-01-01

    Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica. PMID:11526007

  10. Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA.

    PubMed

    Chan, G C; Hess, P; Meenakshi, T; Carlstedt-Duke, J; Gustafsson, J A; Payvar, F

    1991-11-25

    Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

  11. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

    PubMed

    Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl

    2015-06-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

  12. MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

    PubMed

    Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A M

    2013-01-01

    Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

  13. DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions.

    PubMed

    Dingman, Douglas W

    2009-01-01

    Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

  14. dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes.

    PubMed

    Lloret-Llinares, Marta; Pérez-Lluch, Sílvia; Rossell, David; Morán, Tomás; Ponsa-Cobas, Joan; Auer, Herbert; Corominas, Montserrat; Azorín, Fernando

    2012-10-01

    H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol llo(ser5) form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

  15. Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein.

    PubMed Central

    Fennewald, S; van Santen, V; Kieff, E

    1984-01-01

    The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, established by in vitro growth transformation with virus, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome. The RNA was transcribed from right to left, and two introns were spliced out. This region of the genome was sequenced, and the exons of the RNA were identified by S1 analysis of DNA-RNA hybrids and primer extension. The first start codon in the RNA was 40 nucleotides from its 5' end. Beginning with the start codon, there was a 1,158-nucleotide open reading frame which crossed both introns. The important characteristics of the translated protein were as follows. (i) The amino terminus was highly charged and not suggestive of a leader sequence. (ii) There were six markedly hydrophobic alpha-helical domains, each having 21 amino acids and connected by 5 to 7 amino acid segments predicted to be reverse turns. (iii) The carboxy-terminal 200 amino acids were markedly acidic, containing 6 glutamic and 37 aspartic acids. The hydrophobic region is predicted to form six membrane-spanning regions, leaving the short charged amino terminus and long acidic carboxy terminus on the inside of the membrane. This protein could be responsible for the new antigen detected in the plasma membrane of Epstein-Barr virus-transformed cells, lymphocyte-determined membrane antigen. There were two other open reading frames in the RNA. Images PMID:6086953

  16. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  17. Teaching International Law: Concepts in International Relations

    ERIC Educational Resources Information Center

    Starbird, Caroline; Pettit, Jenny; Singleton, Laurel

    2004-01-01

    This book is designed to introduce students to public international law. Topics covered include international public organizations, such as the United Nations and World Trade Organization, international courts, international human rights law, international trade law, and international environmental law. The goal of each study is to examine how…

  18. 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

    PubMed Central

    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

  19. 18S Ribosomal DNA Typing and Tracking of Acanthamoeba Species Isolates from Corneal Scrape Specimens, Contact Lenses, Lens Cases, and Home Water Supplies of Acanthamoeba Keratitis Patients in Hong Kong

    PubMed Central

    Booton, G. C.; Kelly, D. J.; Chu, Y.-W.; Seal, D. V.; Houang, E.; Lam, D. S. C.; Byers, T. J.; Fuerst, P. A.

    2002-01-01

    We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of ∼113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections. PMID:11980931

  20. 18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

    PubMed

    Booton, G C; Kelly, D J; Chu, Y-W; Seal, D V; Houang, E; Lam, D S C; Byers, T J; Fuerst, P A

    2002-05-01

    We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

  1. Operating internationally

    SciTech Connect

    Seeley, R.S.

    1994-02-01

    When Enron Power Corp. took over a 28 MW power facility at the former US Naval base in Subic Bay, the Philippines, the company was required to employ 139 people to run the plant. This large labor force was necessary not because of the plant's operational needs, but because of local labor practices and unemployment pressures. Independent power companies have become all too familiar with the high cost and complexity of developing projects in emerging international markets. Some of the most significant issues involve taxation, unfamiliar legal systems, changing regulations, and foreign investment restrictions. In addition, questions about currency exchange, national credit worthiness, and political stability add to the difficulty of international development. However, one of the most daunting challenges centers not on development, but on long-term operations and maintenance (O M). A key concern is finding qualified labor. Most developers and O M companies agree that local people should run the plant, with the top person, or persons, thoroughly trained in the developer's company philosophy.

  2. Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

    PubMed Central

    Maslunka, Christopher; Gifford, Bianca; Tucci, Joseph; Gürtler, Volker; Seviour, Robert J.

    2014-01-01

    To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species. PMID:25141005

  3. 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.

    PubMed

    Hynniewta, Marlykynti; Malik, Surendra Kumar; Rao, Satyawada Rama

    2014-12-01

    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 clear that C. reticulata also has close relation to C. ichagensis, as these species have clustered together, denoting their close genetic relationship. On the other hand, our studies did not demonstrate a clear differentiation between subgenera Citrus and Papeda at the rDNA level. The combined approach of cytogenetical and molecular analysis did complement our early karyological findings and helped in resolving many a taxonomic riddles.

  4. The ectomycorrhizal symbiosis between Lactarius deliciosus and Pinus sylvestris in forest soil samples: symbiotic efficiency and development on roots of a rDNA internal transcribed spacer-selected isolate of L. deliciosus.

    PubMed

    Guerin-Laguette, Alexis; Conventi, Serge; Ruiz, Guy; Plassard, Claude; Mousain, Daniel

    2003-03-01

    The effect on plant growth of pre-inoculation of Pinus sylvestris with the ectomycorrhizal (ECM) edible basidiomycete Lactarius deliciosus (isolate D45) under controlled conditions, and the development on roots of this basidiomycete, were investigated in gamma-irradiated and unsterilized containers containing different forest soil cores or a perlite-vermiculite mixture. Five months after planting, L. deliciosus mycorrhizal plants exhibited greater growth than the non-mycorrhizal ones in all soil types, i.e. up to a 325% increase in shoot height in the sterilized soils. The experiment demonstrated the dependency of P. sylvestris seedlings upon ECM symbiosis for their survival in gamma-irradiated, microbiologically disturbed soil samples. Furthermore, in two soils, the growth of L. deliciosus-inoculated seedlings was greater in the sterilized soil samples than in the non-sterilized ones, i.e. 46% and 132% increase in shoot height under sterilized soil conditions. In containers randomly sampled from each soil type, the degree of root colonization by the inoculated isolate, calculated as the number of mycorrhizal root tips divided by the total number of root tips x100, ranged from 80% to 35%. Within the short term, the inoculated isolate developed rapidly on roots, dominated, and hampered ectomycorrhiza formation by various unidentified (but not Lactarius) resident ECM fungi in unsterilized soil types. Results indicate that the ECM species L. deliciosus is worth investigating to ascertain if other isolates benefit pine growth like the isolate D45, and are therefore also attractive candidates for forestry applications in the Mediterranean area.

  5. Molecular investigations of cytochrome c oxidase subunit I (COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae, in northern Europe and implications for its transmission between laying poultry farms.

    PubMed

    Oines, O; Brännström, S

    2011-12-01

    Samples of Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) from more than 49 Norwegian and Swedish laying poultry farms, and additional samples collected from Scottish, Finnish, Danish and Dutch layer farms, were compared genetically. Analysis of partial mitochondrial gene cytochrome c oxidase subunit I (COI) sequences of mites from Norway and Sweden revealed 32 haplotypes. Only single haplotypes were found on most farms, which suggests that infections are recycled within farms and that transmission routes are few. Both Norwegian and Swedish isolates were found in the two major haplogroups, but no haplotypes were shared between Norway and Sweden, indicating little or no recent exchange of mites between these countries. There appears to be no link between haplotypes and geographical location as identical haplotypes were found in both the northern and southern Swedish locations, and haplotypes were scattered in locations between these extremes. The current data suggest that wild birds in Sweden are not a reservoir for D. gallinae infection of layer farms as their mites were genetically distinct from D. gallinae of farm layer birds. Transmission of the poultry red mite in Scandinavia is thus likely to depend on synantropic factors such as the exchange of contaminated material or infested birds between farms or facilities.

  6. Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry.

    PubMed

    Trček, Janja; Barja, François

    2015-03-02

    Acetic acid bacteria have attracted much attention over the past few years, due mainly to their metabolic traits that are of interest to the biotechnology industry. In addition, it turns out that their ecological habitats are almost unlimited since they have been found as symbionts in different insects and also as emerging opportunistic human pathogens. Very surprising is the finding that they colonize niches considered anaerobic, disproving the generalized statement that they are strict aerobes. Since they have taken on different biological roles in our environment, more and more people are charged with the task of identifying them. However, this turns out to be not always easy, especially if we are using phenotypic approaches for identification. A substantial step forward in making the identification of acetic acid bacteria easier was made possible using molecular biological methods, which have been extensively tested since 2000. However, some molecular methods require expensive machines and experienced staff, and moreover the level of their discrimination varies. All these factors must be considered when selecting the most appropriate approach for identifying acetic acid bacteria. With this objective in mind, this review article discusses the benefits and drawbacks of molecular biological methods for identification of acetic acid bacteria, with a focus on the 16S-23S rRNA gene ITS regions and the recently described alternative method for identification of acetic acid bacteria, MALDI-TOF MS.

  7. International Education for Wisconsin.

    ERIC Educational Resources Information Center

    Moebius, Barbara

    1990-01-01

    Describes an international trade education program offered by Waukesha (WI) County Technical College. The program includes international business principles, international marketing, cultural awareness, business Spanish, international documentation, transportation, and finance. (JOW)

  8. Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

    PubMed Central

    McGregor, W G; Chen, R H; Lukash, L; Maher, V M; McCormick, J J

    1991-01-01

    To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene. PMID:2005888

  9. Community College Internal Auditors: Internal Audit Guidebook.

    ERIC Educational Resources Information Center

    Jones, Ronna; And Others

    This guidebook includes information compiled by the "Audit Manual" committee of Community College Internal Auditors (CCIA) from several California community college districts regarding their internal auditing practices. The first section of the guidebook discusses the purpose of internal audits, indicating that audits assist members of…

  10. Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells

    SciTech Connect

    McGregor, W.G.; Chen, Ruey-Hwa; Lukash, L.; Maher, V.M.; McCormick, J.J. )

    1991-04-01

    To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), the authors synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G, phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, they also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G[sub 1] and the repair-proficient mutants from S, [approximately]62% were G [center dot] C[r arrow]A [center dot] T. In the repair-proficient mutants from G[sub 1], 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G[sub 1], only 20% (3 of 15) were. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.

  11. Evaluation of a microfluidics-based platform and slab electrophoresis for determination of size, integrity and quantification of in vitro transcribed RNA used as a component in therapeutic drug manufacturing.

    PubMed

    Slagter-Jäger, Jacoba Grietje; Nicolette, Charles A; Tcherepanova, Irina Y

    2012-11-01

    Ribonucleic acid (RNA) is gaining utility as a key component of immunotherapeutics to transiently express antigens or to modulate endogenous gene expression for clinical applications. As a key ancillary component of clinical grade products, RNA requires a robust method for quality control. Here we evaluated the microfluidics based platform and slab electrophoresis for determination of integrity, concentration and size of four in vitro-transcribed RNA products with sizes of 1611, 808, 475 and 290 nucleotides (nts). Our data demonstrate that the Bioanalyzer can determine both size and integrity of the RNA, but the analysis suffers from a strong well position effect. For the RNAs tested, the integrity values obtained by the Bioanalyzer demonstrate a reverse correlation with the size of the molecule and are lower than those obtained using slab electrophoresis. Agarose gel electrophoresis produced the information on size of the RNA molecule with good precision, accuracy and reproducibility. We highlight observations which need to be taken into account when developing and qualifying a method of choice for assessment of in vitro-transcribed RNA using either approach.

  12. Surveying your internal customers.

    PubMed

    Weir, V L

    1998-06-01

    Internal customers often are overlooked when business techniques are applied. By applying common external customer satisfaction survey techniques to internal business functions, one hospital identified areas for improvement.

  13. Travelers' Health: International Adoption

    MedlinePlus

    ... a malaria-endemic area (See Chapter 3, Malaria ). Tuberculosis All internationally adopted children should be screened for tuberculosis (TB) after arriving in the United States. Internationally ...

  14. Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

    NASA Astrophysics Data System (ADS)

    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.

  15. BPPD Internal Application Checklists

    EPA Pesticide Factsheets

    EPA, Office of Pesticide Programs, BPPD internal application checklists for internal guidance to assist BPPD employees in their evaluation of applications submitted to BPPD by applicants and/or registrants.

  16. International Space Station Overview

    NASA Technical Reports Server (NTRS)

    Bates, William V., Jr.

    1999-01-01

    The overview of the International Space Station (ISS) is comprised of the program vision and mission; Space Station uses; definition of program phases; as well as descriptions and status of several scheduled International Space Station Overview assembly flights.

  17. Fishbone and internal kinks

    SciTech Connect

    Strauss, H.; Park, W.; Monticello, D.; Izzo, R.; White, R.; McGuire, K.; Manickam, J.; Goldston, R.

    1983-07-01

    The internal-kink mode, combined with neutral-beam heating and beam losses, appears to be responsible for the fishbone soft x-ray oscillations in PDX. Nonlinear simulations of both ideal and resistive kinks are presented and shown to be consistent with experimental observations. The internal kink may also be important in low-beta internal disruptions.

  18. Improving Internal Communication.

    ERIC Educational Resources Information Center

    Bonus, Thaddeus, Ed.

    Guidelines for developing the internal communications of colleges and universities, researching internal communication needs, and increasing information flow through traditional and nontraditional media are provided in 11 articles. Titles and authors include the following: "Work for an Open Internal Communication Policy" (Thaddeus Bonus); "Five…

  19. International utilization and operations

    NASA Technical Reports Server (NTRS)

    Goldberg, Stanley R.

    1989-01-01

    The international framework of the Space Station Freedom Program is described. The discussion covers the U.S. space policy, international agreements, international Station elements, overall program management structure, and utilization and operations management. Consideration is also given to Freedom's user community, Freedom's crew, pressurized payload and attached payload accommodations, utilization and operations planning, user integration, and user operations.

  20. National and International Views.

    ERIC Educational Resources Information Center

    Kilgour, David

    1992-01-01

    Discusses Canadian government policy dealing with internal matters of party politics and international questions of human rights. Describes the political situation in Burma as an oppressive military regime. Urges the Canadian government to take a firmer stand to influence the international community against tolerating regimes that are guilty of…

  1. The effects of sulfur intercalation on the optical properties of artificial `hackmanite', Na8[Al6Si6O24]Cl1.8S0.1; `sulfosodalite', Na8[Al6Si6O24]S; and natural tugtupite, Na8[Be2Al2Si8O24](Cl,S)2-δ

    NASA Astrophysics Data System (ADS)

    Warner, T. E.; Hutzen Andersen, J.

    2012-02-01

    The minerals `hackmanite' and tugtupite exhibit tenebrescence (reversible photochromism) and photoluminescence. These features are generally attributed to the presence of sulfide species within their structures. But how these optical properties might be affected by intercalating additional amounts of sulfur into their structures was until now unknown. Artificial `hackmanite', Na8[Al6Si6O24]Cl1.8S0.1, and `sulfosodalite', Na8[Al6Si6O24]S, were heated with sulfur in evacuated quartz-glass ampoules over the temperature range 450-1,050°C. This work has shown that sulfur intercalation into Na8[Al6Si6O24]Cl1.8S0.1 destroys the tenebrescence and induces a permanently pale blue and, at higher temperature, a pale green coloration. The effect on Na8[Al6Si6O24]S induced similar colorations but of a deeper hue. Annealing tugtupite, Na8[Be2Al2Si8O24](Cl,S)2-δ under a sulfur atmosphere over the range 600-700°C, destroyed the tenebrescence and resulted in a colorless tugtupite; but did not effect the photoluminescence. This suggests that the chemical species responsible for the tenebrescence in tugtupite is unlikely to be the same as that for the luminescence.

  2. Transcribing Speech: Practicalities, Philosophies and Prophesies

    ERIC Educational Resources Information Center

    Rahilly, Joan

    2011-01-01

    This article outlines the main practical and philosophical developments which have contributed to current approaches to phonetic transcription. Particular contributions from scholars in the field are highlighted as seminal in shaping transcription work. Consideration is also given to the ways in which insights from clinical transcription impact…

  3. Issues in Collecting and Transcribing Speech Samples.

    ERIC Educational Resources Information Center

    Louko, Linda J.; Edwards, Mary Louise

    2001-01-01

    This article identifies issues in phonetic transcription of speech samples for speech language pathologists. Important terms and concepts from articulatory/clinical phonetics are reviewed and guidelines are provided for streamlining the process of whole-word live transcriptions and for refining transcriptions by incorporating features from…

  4. The Ancestral Gene for Transcribed, Low-Copy Repeats in the Prader-Willi/Angleman Region Encodes a Large Protein Implicated in Protein Trafficking that is Deficient in Mice with Neuromuscular and

    SciTech Connect

    Ji, Y.

    1999-01-01

    Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

  5. Orientation-dependent influence of an intergenic enhancer on the promoter activity of the divergently transcribed mouse Shsp/alpha B-crystallin and Mkbp/HspB2 genes.

    PubMed

    Swamynathan, Shivalingappa K; Piatigorsky, Joram

    2002-12-20

    The mouse Shsp/alphaB-crystallin and Mkbp/HspB2 genes are closely linked and divergently transcribed. In this study, we have analyzed the contribution of the intergenic enhancer to Shsp/alphaB-crystallin and Mkbp/HspB2 promoter activity using dual-reporter vectors in transient transfection and transgenic mouse experiments. Deletion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 30- and 93-fold and Mkbp/HspB2 promoter activity by 6- and 10-fold in transiently transfected mouse lens alpha-TN4 and myoblast C2C12 cells, respectively. Surprisingly, inversion of the enhancer reduced Shsp/alphaB-crystallin promoter activity by 17-fold, but did not affect Mkbp/HspB2 promoter activity in the transfected cells. In contrast, enhancer activity was orientation-independent in combination with a heterologous promoter in transfected cells. Transgenic mouse experiments established the orientation dependence and Shsp/alphaB-crystallin promoter preference of the intergenic enhancer in its native context. The orientation dependence and preferential effect of the Shsp/alphaB-crystallin enhancer on the Shsp/alphaB-crystallin promoter provide an example of adaptive changes in gene regulation accompanying the functional diversification of duplicated genes during evolution.

  6. International safeguards data authentication

    SciTech Connect

    Melton, R.B.; Smith, C.E.; DeLand, S.M.; Manatt, D.R.

    1996-07-01

    The International Safeguards community is becoming increasingly reliant on information stored in electronic form. In international monitoring and related activities it must be possible to verify and maintain the integrity of this electronic information. This paper discusses the use of data authentication technology to assist in accomplishing this task. The paper provides background information, identifies the relevance to international safeguards, discusses issues related to export controls, algorithm patents, key management and the use of commercial vs. custom software.

  7. International Standard for Phenoxymethylpenicillin

    PubMed Central

    Humphrey, J. H.; Lightbown, J. W.; Mussett, Marjorie V.

    1959-01-01

    A batch of highly purified phenoxymethylpenicillin has been examined by eight laboratories in seven different countries, and has been assayed against the phenoxymethylpenicillin standard of the Food and Drug Administration of the US Department of Health, Education, and Welfare. The material examined has been established as the International Standard for Phenoxymethylpenicillin, and the International Unit of Phenoxymethylpenicillin is defined as the activity contained in 0.000590 mg of the International Standard. PMID:14405369

  8. International environmental policy

    SciTech Connect

    Caldwell, L.K.

    1990-01-01

    This report presents a survey of the global international movement for protection of the human environment. It describes the expanding dimensions of international environmental policy, clarifies that policy's present status, and provides a record of events of continuing historical significance. The author calls attention to the need for international agreements and proposals for such vital global environmental issues as climate change, disintegration of the stratospheric ozone layer, and long-range trans-boundary air pollution.

  9. EM International. Volume 1

    SciTech Connect

    Not Available

    1993-07-01

    It is the intent of EM International to describe the Office of Environmental Restoration and Waste Management`s (EM`s) various roles and responsibilities within the international community. Cooperative agreements and programs, descriptions of projects and technologies, and synopses of visits to international sites are all highlighted in this semiannual journal. Focus on EM programs in this issue is on international collaboration in vitrification projects. Technology highlights covers: in situ sealing for contaminated sites; and remote sensors for toxic pollutants. Section on profiles of countries includes: Arctic contamination by the former Soviet Union, and EM activities with Germany--cooperative arrangements.

  10. International migration, international relations and foreign policy.

    PubMed

    Mitchell, C

    1989-01-01

    Recent literature on migration, international relations, and foreign policy is reviewed in this article, stressing applications of global systems paradigms, studies of state entry and exit rules, and anatomies of domestic policy-setting processes on migration. After a concise assessment of the contemporary theory of global political economy, the paper argues for seeking mid-range generalizations on the international relations of migration. It also suggests that analysis begin with the policy-setting processes of the state. Especially through the use of comparative perspectives available from domestic policy making studies and from the field of international comparative public policy, this approach offers the opportunity to fix empirically the political roles of transnational social forces, which often present themselves as participants in domestic policy contests. Promising future directions in the study of state-to-state relations are also evaluated, with the anticipation that verifying regional or other intermediate patterns of world migration politics may contribute to more general theories of international political economy.

  11. International Cooperation at NASA

    NASA Astrophysics Data System (ADS)

    Tawney, Timothy; Feldstein, Karen

    International cooperation is a cornerstone principle of NASA’s activities, especially within the activities of the Science Mission Directorate. Nearly two thirds of the flight missions in which NASA leads or participates involve international cooperation. Numerous ground based activities also rely on international cooperation, whether because of unique expertise, unique geography, or the need for a global response. Going forward, in an era of tighter budgets and a more integrated global perspective, NASA and the rest of the space agencies around the world will be forced to work more closely together, in a broader array of activities than ever before, in order to be able to afford to push the boundaries of space exploration. The goal of this presentation is to provide an overview of NASA’s current international science cooperative activities. It will include a discussion of why NASA conducts international cooperation and look at the mechanisms through which international cooperation can occur at NASA, including peer-to-peer development of relationships. It will also discuss some of the limiting factors of international cooperation, such as export control, and ways in which to manage those constraints. Finally, the presentation would look at some of the present examples where NASA is working to increase international cooperation and improve coordination. Case studies will be used to demonstrate these mechanisms and concepts. For example, NASA continues to participate in international coordination groups such as the International Mars Exploration Working Group (IMEWG) and International Space Exploration Coordination Group (ISECG), but is expanding into new areas as well. NASA is one of the leaders in expanding and improving international coordination in the area of Near-Earth Object detection, characterization, and mitigation. Having participated in the first meetings of such groups as the International Asteroid Warning Network (IAWN) and Space Missions Planning

  12. International Education (Working Paper).

    ERIC Educational Resources Information Center

    Gruson, Edward S.

    The history, objectives, and funding patterns for international education are discussed. Attention is directed toward the language and area study centers of the U.S. Office of Education, undergraduate/graduate and scholarly exchange programs, and the support of advanced research in international studies. The main source of funds for language and…

  13. International Perspectives. Chapter 2.

    ERIC Educational Resources Information Center

    Acampora, Alfonso P., Ed.; Nebelkopf, Ethan, Ed.

    This document contains seven papers from the ninth World Conference of Therapeutic Communities that provide an international perspective on the therapeutic community (TC) movement as it is today. Papers include: (1) "What's Happening on an International Level" (William B. O'Brien); (2) "Therapeutic Communities of America"…

  14. International Resource Management.

    ERIC Educational Resources Information Center

    Schabel, H. G.

    The International Resource Management program enables undergraduate students of the University of Wisconsin, Stevens Point, College of Natural Resources to complete an academic minor in International Resource Management. The program attempts to alert students and faculty to global environmental issues and their interconnectedness with a variety of…

  15. International Student Success

    ERIC Educational Resources Information Center

    Smith, Clayton

    2016-01-01

    This article, with a focus on North American postsecondary education, identifies international students as a strategic enrollment management institutional priority; presents themes in the international student retention, satisfaction, and success research literature; and describes related best practices. It also presents the findings from an…

  16. Alberta's International Education Strategy.