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Sample records for 16s-23s rrna intergenic

  1. A report of cat scratch disease in Korea confirmed by PCR amplification of the 16S-23S rRNA intergenic region of Bartonella henselae.

    PubMed

    Suh, Borum; Chun, Jin-Kyoung; Yong, Dongeun; Lee, Yang Soon; Jeong, Seok Hoon; Yang, Woo Ick; Kim, Dong Soo

    2010-02-01

    We report a case of cat scratch disease in an 8-yr-old girl who presented with fever and enlargement of both axillary lymph nodes. Both aerobic and anaerobic cultures of the lymph node aspirate were negative for microbial growth. Gram staining and Warthin-Starry silver staining did not reveal any organism. Purified DNA from the PCR-amplicon of the 16S-23S rRNA intergenic region was sequenced and showed 99.7% identity with the corresponding sequence of Bartonella henselae strain Houston-1. Our findings suggest that the internal transcribed spacer is a reliable region for PCR identification of Bartonella species. In patients with lymphadenitis, a history of contact with cats or dogs necessitates the use of diagnostic approaches that employ not only the conventional staining and culture but also molecular methods to detect B. henselae. PMID:20197720

  2. The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies.

    PubMed

    Raviv, Ziv; Callison, S; Ferguson-Noel, N; Laibinis, V; Wooten, R; Kleven, S H

    2007-06-01

    Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies. PMID:17626483

  3. Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera.

    PubMed

    Daffonchio, Daniele; Cherif, Ameur; Brusetti, Lorenzo; Rizzi, Aurora; Mora, Diego; Boudabous, Abdellatif; Borin, Sara

    2003-09-01

    The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus: We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing. PMID:12957895

  4. Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

    PubMed

    Ghorashi, Seyed A; Bradbury, Janet M; Ferguson-Noel, Naola M; Noormohammadi, Amir H

    2013-12-27

    Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here. PMID:24238667

  5. 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. PMID:22510214

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

    PubMed Central

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

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  7. Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

    PubMed Central

    Kabadjova, Petia; Dousset, Xavier; Le Cam, Virginie; Prevost, Hervé

    2002-01-01

    A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763T and C. mobile DSM 4849T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766T and C. piscicola NCDO 2762T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNAIle and tRNAAla genes. The M-ISR included one tRNAAla gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing. PMID:12406725

  8. Specific Detection of Bradyrhizobium and Rhizobium Strains Colonizing Rice (Oryza sativa) Roots by 16S-23S Ribosomal DNA Intergenic Spacer-Targeted PCR

    PubMed Central

    Tan, Zhiyuan; Hurek, Thomas; Vinuesa, Pablo; Müller, Peter; Ladha, Jagdish K.; Reinhold-Hurek, Barbara

    2001-01-01

    In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial

  9. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

    PubMed Central

    Moreira, João Luiz S; Mota, Rodrigo M; Horta, Maria F; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

    2005-01-01

    Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus, Streptococcus, Weissella, Staphylococcus, and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. Conclusion Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies. PMID:15788104

  10. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

    PubMed Central

    Rijpens, N P; Jannes, G; Van Asbroeck, M; Rossau, R; Herman, L M

    1996-01-01

    The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods. PMID:8633866

  11. Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences

    PubMed Central

    Roth, Andreas; Fischer, Marga; Hamid, Mohamed E.; Michalke, Sabine; Ludwig, Wolfgang; Mauch, Harald

    1998-01-01

    Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification. PMID:9431937

  12. 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.

  13. 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

    2015-03-01

    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. PMID:25589227

  14. Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68°C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation†

    PubMed Central

    Ferris, Mike J.; Kühl, Michael; Wieland, Andrea; Ward, David M.

    2003-01-01

    We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68°C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O2 and oxygenic photosynthesis demonstrated the existence of physiologically distinct Synechococcus populations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths. PMID:12732563

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

    PubMed

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

    2001-02-01

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

  16. Molecular analysis of 16S-23S spacer regions of Acetobacter species.

    PubMed

    Kretová, M; Grones, J

    2005-01-01

    16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity. PMID:16408846

  17. 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus and A. xylinum, tRNA genes and antitermination sequences.

    PubMed

    Sievers, M; Alonso, L; Gianotti, S; Boesch, C; Teuber, M

    1996-08-15

    The 16S-23S ribosomal RNA spacer regions of Acetobacter europaeus DSM 6160, A. xylinum NCIB 11664 and A. xylinum CL27 were amplified by PCR. Specific PCR products were obtained from each strain and their nucleotide sequences determined. The spacer region of A. europaeus comprises 768 nucleotides (nt), that of A. xylinum 778 nt and that of A. xylinum CL27 759 nt. Genes encoding tRNAIle and tRNAAla were identified. Putative antitermination sequences were found between the tRNAAla sequence and the 5'-terminus of the 23S rRNA coding sequence. The boxA element has the nucleotide sequence TGCTCTTTGATA. Based on hybridization data of digested chromosomal DNA with spacer-specific probes, the copy number of the rrn operons on the chromosome of Acetobacter strains is estimated to be four. PMID:8759788

  18. Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

    PubMed Central

    Liguori, Andrew P.; Warrington, Stephanie D.; Ginther, Jennifer L.; Pearson, Talima; Bowers, Jolene; Glass, Mindy B.; Mayo, Mark; Wuthiekanun, Vanaporn; Engelthaler, David; Peacock, Sharon J.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Tuanyok, Apichai

    2011-01-01

    Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species. PMID:22195045

  19. Identification of Lactobacillus strains of goose origin using MALDI-TOF mass spectrometry and 16S-23S rDNA intergenic spacer PCR analysis.

    PubMed

    Dec, Marta; Urban-Chmiel, Renata; Gnat, Sebastian; Puchalski, Andrzej; Wernicki, Andrzej

    2014-04-01

    The objective of our study was to identify Lactobacillus sp. strains of goose origin using MALDI-TOF mass spectrometry, ITS-PCR and ITS-PCR/RFLP. All three techniques proved to be valuable tools for identification of avian lactobacilli and produced comparable classification results. Lactobacillus strains were isolated from 100% of geese aged 3 weeks to 4 years, but from only 25% of chicks aged 1-10 days. Among the 104 strains isolated, we distinguished 14 Lactobacillus species. The dominant species was Lactobacillus salivarius (35.6%), followed by Lactobacillus johnsonii (18.3%), Lactobacillus ingluviei (11.5%) and Lactobacillus agilis (7.7%). The intact-cell MALDI-TOF mass spectrometry enabled rapid species identification of the lactobacilli with minimal pretreatment. However, it produced more than one identification result for 11.5% examined strains (mainly of the species L. johnsonii). ITS-PCR distinguished 12 genotypes among the isolates, but was not able to differentiate closely related strains, i.e. between Lactobacillus amylovorus and Lactobacillus kitasatonis and between Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus zeae. These species were differentiated by ITS-PCR/RFLP using the restriction enzymes TaqI and MseI. The results obtained indicate that ITS-PCR and ITS-PCR/RFLP assays could be used not only for interspecific, but also for intraspecific, typing. PMID:24607713

  20. 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. PMID:25725268

  1. Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages.

    PubMed

    Blaiotta, Giuseppe; Pennacchia, Carmelina; Ercolini, Danilo; Moschetti, Giancarlo; Villani, Francesco

    2003-09-01

    Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains. PMID:14529185

  2. Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

    PubMed

    Trcek, Janja

    2005-10-01

    Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for

  3. PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile.

    PubMed Central

    Cartwright, C P; Stock, F; Beekmann, S E; Williams, E C; Gill, V J

    1995-01-01

    From January to March 1993, a suspected outbreak of antibiotic-associated diarrhea occurred on a pediatric oncology ward of the Clinical Center Hospital at the National Institutes of Health. Isolates of Clostridium difficile obtained from six patients implicated in this outbreak were typed by both PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) and restriction endonuclease analysis of genomic DNA. Comparable results were obtained with both methods; five of the six patients were infected with the same strain of C. difficile. Subsequent analysis of 102 C. difficile isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes. These data suggest that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C. difficile. PMID:7699038

  4. 16S–23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads

    PubMed Central

    Tokajian, Sima; Issa, Nahla; Salloum, Tamara; Ibrahim, Joe; Farah, Maya

    2016-01-01

    Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S–23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S–23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2–57.9 mol%. Five distinct ITS types were identified: ITSnone (without tRNA genes), ITSAla(TGC), ITSAla(TGC)+Ile(GAT), ITSIle(GAT)+Ala(TGC), and ITS Ile(GAT)+Pseudo. All of the identified tRNAAla(TGC) molecules consisted of 73 bases, and all of the tRNAIle(GAT) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S–23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence. PMID:26904019

  5. Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

    PubMed

    Grayson, T H; Alexander, S M; Cooper, L F; Gilpin, M L

    2000-07-01

    The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries. PMID:11016696

  6. Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage.

    PubMed Central

    Brow, M A; Oldenburg, M C; Lyamichev, V; Heisler, L M; Lyamicheva, N; Hall, J G; Eagan, N J; Olive, D M; Smith, L M; Fors, L; Dahlberg, J E

    1996-01-01

    We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive

  7. 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

  8. Comparative analysis of the genes encoding 23S-5S rRNA intergenic spacer regions of Lactobacillus casei-related strains.

    PubMed

    Chen, H; Lim, C K; Lee, Y K; Chan, Y N

    2000-03-01

    In this study, investigations into the 23S-5S rRNA intergenic spacer regions (ISRs) of the Lactobacillus casei group were performed. A 1.6 kb fragment, from Lactobacillus paracasei strain ATCC 27092, containing part of the 5S rRNA gene (60 bp), the 5S-23S spacer region (198 bp) and part of the 23S rRNA gene (1295 bp) was cloned and sequenced (GenBank no. AF098107). This fragment was used as a probe to determine the rRNA restriction fragment length polymorphism (RFLP) patterns of nine strains belonging to the Lactobacillus casei group, along with four other non-Lactobacillus casei lactobacilli species. A pair of PCR primers, 23-Fl and 5-Ru, was designed and used for PCR amplification of the 23S-5S rRNA ISRs of these strains. The ISR length and sequence polymorphisms provided additional information for the taxonomic study of the Lactobacillus casei group. The spacer-length polymorphism of Lactobacillus rhamnosus was distinct from those of the other strains and this observation is consistent with the classification of Lactobacillus rhamnosus proposed by Mori et al. For all Lactobacillus casei and Lactobacillus paracasei strains, two major bands (approx. 250 and 170 bp in size) were obtained except in the case of Lactobacillus paracasei subsp. tolerans strain NCIMB 9709T, which yielded only one amplified product (250 bp). The sequencing data of the PCR products of seven well-characterized Lactobacillus casei and Lactobacillus paracasei strains revealed the presence of a 76/80 bp insertion/deletion with some random, single-base substitutions between the longer and shorter spacers for each respective strain. A few base variations were also detected within different strains in this group although the overall sequence similarity was very high (95.9-99.5%). The rRNA RFLP and the spacer sequence of Lactobacillus casei type strain ATCC 393T exhibited unique identities in this cluster. On the other hand, Lactobacillus casei strain ATCC 334 showed a high level of similarity

  9. Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

    PubMed

    Sugawara, Ryota; Yamada, Sayumi; Tu, Zhihao; Sugawara, Akiko; Hoshiba, Toshihiro; Eisaka, Sadao; Yamaguchi, Akihiro

    2016-01-01

    Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions. PMID:27211917

  10. Differentiation of Debaryomyces hansenii and Candida famata by rRNA gene intergenic spacer fingerprinting and reassessment of phylogenetic relationships among D. hansenii, C. famata, D. fabryi, C. flareri (=D. subglobosus) and D. prosopidis: description of D. vietnamensis sp. nov. closely related to D. nepalensis.

    PubMed

    Nguyen, Huu-Vang; Gaillardin, Claude; Neuvéglise, Cécile

    2009-06-01

    The intergenic spacer rDNA amplification and AluI fingerprinting (IGSAF) method detected four distinct groups among 170 Debaryomyces hansenii strains: D. hansenii var. hansenii; Candida famata var. famata; D. hansenii var. fabryi and C. famata var. flareri. IGS sequence comparison of representative strains showed that D. hansenii var. hansenii and C. famata var. famata belonged to one species, whereas D. hansenii var. fabryi and C. famata var. flareri belonged to two different ones. This confirmed the following three species recently reinstated: D. hansenii (=C. famata), Debaryomyces fabryi and Debaryomyces subglobosus (=Candida flareri). Accordingly, growth at 37 degrees C may no longer be used to differentiate D. hansenii from D. fabryi. Riboflavin production is more specific for D. fabryi and D. subglobosus strains. IGSAF identified all the other 17 species of the genus Debaryomyces, six of them sharing with D. hansenii an rRNA gene unit harbouring two 5S rRNA genes. The phylogenetic tree established with IGS sequences was congruent with the one based on ACT1, GPD1 and COX2 sequences depicting a distinct D. hansenii clade close to the D. subglobosus, Debaryomyces prosopidis and D. fabryi clade. Description of Debaryomyces vietnamensis sp. nov. (type strain CBS 10535(T), MUCL 51648(T)), closely related to Debaryomyces nepalensis is given. PMID:19385997

  11. IntergenicDB: a database for intergenic sequences

    PubMed Central

    Notari, Daniel Luis; Molin, Aurione; Davanzo, Vanessa; Picolotto, Douglas; Ribeiro, Helena Graziottin; Silva, Scheila de Avila e

    2014-01-01

    A whole genome contains not only coding regions, but also non-coding regions. These are located between the end of a given coding region and the beginning of the following coding region. For this reason, the information about gene regulation process underlies in intergenic regions. There is no easy way to obtain intergenic regions from current available databases. IntergenicDB was developed to integrate data of intergenic regions and their gene related information from NCBI databases. The main goal of INTERGENICDB is to offer friendly database for intergenic sequences of bacterial genomes. Availability http://intergenicdb.bioinfoucs.com/ PMID:25097383

  12. Molecular Method for Bartonella Species Identification in Clinical and Environmental Samples▿

    PubMed Central

    García-Esteban, Coral; Gil, Horacio; Rodríguez-Vargas, Manuela; Gerrikagoitia, Xeider; Barandika, Jesse; Escudero, Raquel; Jado, Isabel; García-Amil, Cristina; Barral, Marta; García-Pérez, Ana L.; Bhide, Mangesh; Anda, Pedro

    2008-01-01

    A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously. PMID:18094134

  13. Genetic Diversity and Phylogeny of Rhizobia That Nodulate Acacia spp. in Morocco Assessed by Analysis of rRNA Genes

    PubMed Central

    Khbaya, Bouchaib; Neyra, Marc; Normand, Philippe; Zerhari, Karim; Filali-Maltouf, Abdelkarim

    1998-01-01

    Forty rhizobia nodulating four Acacia species (A. gummifera, A. raddiana, A. cyanophylla, and A. horrida) were isolated from different sites in Morocco. These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis. Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity. Three clusters were identified when 16S rDNA analysis was carried out. Two of these clusters include some isolates which nodulate, nonspecifically, the four Acacia species. These clusters, A and B, fit within the Sinorhizobium lineage and are closely related to S. meliloti and S. fredii, respectively. The third cluster appeared to belong to the Agrobacterium-Rhizobium galegae phylum and is more closely related to the Agrobacterium tumefaciens species. These relations were confirmed by sequencing a representative strain from each cluster. PMID:9835582

  14. Ribosomal operon intergenic sequence (IGS) heterogeneity in Campylobacter coli and Campylobacter jejuni

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter jejuni and Campylobacter coli are closely related species that can not be distinguished by their 16S or 23S rRNA genes. However, the intergenic sequence (IGS) fragment that is between the 16S and 23S genes is markedly different and characteristic for each species. A peculiarity of th...

  15. Cladistic biogeography of Juglans (Juglandaceae) based on chloroplast DNA intergenic spacer sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylogenetic utility of sequence variation from five chloroplast DNA intergenic spacer (IGS) regions: trnT-trnF, psbA-trnH, atpB-rbcL, trnV-16S rRNA, and trnS-trnfM was examined in the genus Juglans. A total of seventeen taxa representing the four sections within Juglans and an outgroup taxon, ...

  16. Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

    PubMed Central

    Liefting, L W; Andersen, M T; Beever, R E; Gardner, R C; Forster, R L

    1996-01-01

    Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data. PMID:8795200

  17. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

    PubMed Central

    Wang, Deguo; Liu, Yanhong

    2015-01-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  18. Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae.

    PubMed

    Wang, Deguo; Liu, Yanhong

    2015-06-01

    Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies. PMID:26016433

  19. Complete sequence and gene organization of the Nosema spodopterae rRNA gene.

    PubMed

    Tsai, Shu-Jen; Huang, Wei-Fone; Wang, Chung-Hsiung

    2005-01-01

    By sequencing the entire ribosomal RNA (rRNA) gene of Nosema spodopterae, we show here that its gene organization follows a pattern similar to the Nosema type species, Nosema bombycis, i.e. 5'-large subunit rRNA (2,497 bp)-internal transcribed spacer (185 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (277 bp)-5S rRNA (114 bp)-3'. Gene sequences and the secondary structures of large subunit rRNA, small subunit rRNA, and 5S rRNA are compared with the known corresponding sequences and structures of closely related microsporidia. The results suggest that the Nosema genus may be heterogeneous and that the rRNA gene organization may be a useful characteristic for determining which species are closely related to the type species. PMID:15702980

  20. Mycoplasmas hyorhinis in different regions of cuba. diagnosis

    PubMed Central

    Lobo, Evelyn; Poveda, Carlos; Gupta, Rakesh; Suarez, Alejandro; Hernández, Yenney; Ramírez, Ana; Poveda, José B.

    2011-01-01

    M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66% of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba. PMID:24031686

  1. Molecular analysis of a NOR site polymorphism in brown trout (Salmo trutta): organization of rDNA intergenic spacers.

    PubMed

    Castro, J; Sánchez, L; Martínez, P; Lucchini, S D; Nardi, I

    1997-12-01

    Using restriction endonuclease mapping, we have analyzed the organization of rDNA (DNA coding for ribosomal RNA (rRNA)) units in the salmonid fish Salmo trutta, as an initial step toward understand the molecular basis of a nucleolar organizer region (NOR) site polymorphism detected in this species. The size of the rDNA units ranged between 15 and 23 kb, with remarkable variation both within individuals and between populations. Three regions of internal tandem repetitiveness responsible for this length polymorphism were located to the intergenic spacers. NOR site polymorphic individuals showed a higher number of length classes, in some cases forming a complete 1 kb fragment ladder. The amount of rRNA genes was as much as 8-fold higher in polymorphic individuals compared with standard individuals. All individuals from the most polymorphic population showed a 14-kb insertion of unknown nature in a small proportion (below 25%) of the 28S rRNA genes. PMID:18464877

  2. Intergenic Locations of Rice Centromeric Chromatin

    PubMed Central

    Yan, Huihuang; Talbert, Paul B; Lee, Hye-Ran; Jett, Jamie; Henikoff, Steven; Chen, Feng; Jiang, Jiming

    2008-01-01

    Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions. PMID:19067486

  3. Sequencing of the Ribosomal Intergenic Spacer Region for Strain Identification of Porphyromonas gingivalis

    PubMed Central

    Rumpf, Robert W.; Griffen, Ann L.; Wen, Bo-Gui; Leys, Eugene J.

    1999-01-01

    The ribosomal intergenic spacer regions (ISRs) of 19 laboratory strains and 30 clinical samples of Porphyromonas gingivalis were amplified by PCR and sequenced to provide a strain identifier. The ISR is a variable region of DNA located between the conserved 16S and 23S rRNA genes. This makes it an ideal locus for differentiation of strains within a species: primers specific for the conserved flanking genes were used to amplify the ISR, which was then sequenced to identify the strain. We have constructed a P. gingivalis ISR sequence database to facilitate strain identification. ISR sequence analysis provides a strain identifier that can be easily reproduced among laboratories and catalogued for unambiguous comparison. PMID:10405432

  4. Extensive length variation in the ribosomal DNA intergenic spacer of yellow perch (Perca flavescens).

    PubMed

    Kakou, Bidénam; Angers, Bernard; Glémet, Hélène

    2016-03-01

    The intergenic spacer (IGS) is located between ribosomal RNA (rRNA) gene copies. Within the IGS, regulatory elements for rRNA gene transcription are found, as well as a varying number of other repetitive elements that are at the root of IGS length heterogeneity. This heterogeneity has been shown to have a functional significance through its effect on growth rate. Here, we present the structural organization of yellow perch (Perca flavescens) IGS based on its entire sequence, as well as the IGS length variation within a natural population. Yellow perch IGS structure has four discrete regions containing tandem repeat elements. For three of these regions, no specific length class was detected as allele size was seemingly normally distributed. However, for one repeat region, PCR amplification uncovered the presence of two distinctive IGS variants representing a length difference of 1116 bp. This repeat region was also devoid of any CpG sites despite a high GC content. Balanced selection may be holding the alleles in the population and would account for the high diversity of length variants observed for adjacent regions. Our study is an important precursor for further work aiming to assess the role of IGS length variation in influencing growth rate in fish. PMID:26841134

  5. Characteristics and significance of intergenic polyadenylated RNA transcription in Arabidopsis.

    PubMed

    Moghe, Gaurav D; Lehti-Shiu, Melissa D; Seddon, Alex E; Yin, Shan; Chen, Yani; Juntawong, Piyada; Brandizzi, Federica; Bailey-Serres, Julia; Shiu, Shin-Han

    2013-01-01

    The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these "translated" ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from "junk" DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes. PMID:23132786

  6. Structure of Intergenic Spacer IGS1 of Ribosomal Operon from Schistidium Mosses.

    PubMed

    Milyutina, I A; Ignatova, E A; Ignatov, M S; Goryunov, D V; Troitsky, A V

    2015-11-01

    The structure of the intergenic spacer 1 (IGS1) of the ribosomal operon from 12 species of Schistidium mosses was studied. In the IGS1 sequences of these species, three conserved regions and two areas of GC- and A-enriched repeats were identified. All of the studied mosses have a conserved pyrimidine-enriched motif at the 5'-end of IGS1. Species-specific nucleotide substitutions and insertions were found in the conserved areas. The repeated units contain single nucleotide substitutions that make unique the majority of repeated units. The positions of such repeats in IGS1 are species-specific, but their number can vary within the species and among operons of the same specimen. The comparison of IGS1 sequences from the Schistidium species and from representatives of ten other moss genera revealed the presence of common conserved motifs with similar localization. Presumably, these motifs are elements of termination of the pre-rRNA transcription and processing of rRNA. PMID:26615440

  7. PCR amplification of the 3' external transcribed and intergenic spacers of the ribosomal DNA repeat unit in three species of Saccharomyces.

    PubMed

    Molina, F I; Jong, S C; Huffman, J L

    1993-04-15

    Two spacer regions outside the ribosomal DNA (rDNA) transcriptional unit in three species of Saccharomyces, S. cerevisiae, S. carlsbergensis and S. pastorianus, were amplified using the polymerase chain reaction. These regions were composed of the 3' external transcribed spacer (ETS) and the intergenic spacer (IGS). Primers were developed from sequence alignments and by taking the reverse complement of a previously described sequence. The region amplified spanned base position 3110 on the 26S rRNA to base position 27 on the 5S rRNA of S. cerevisiae. Nine of the twelve strains used in this study exhibited different restriction profiles, showing that the spacers are highly variable between species. The results suggest that PCR fingerprinting of the non-coding spacer regions can be used to distinguish between closely related Saccharomyces species and may have potential in DNA profiling of other yeasts. PMID:8099889

  8. Detection and Identification of Gastrointestinal Lactobacillus Species by Using Denaturing Gradient Gel Electrophoresis and Species-Specific PCR Primers

    PubMed Central

    Walter, J.; Tannock, G. W.; Tilsala-Timisjarvi, A.; Rodtong, S.; Loach, D. M.; Munro, K.; Alatossava, T.

    2000-01-01

    Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. PMID:10618239

  9. Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

    PubMed Central

    Hotto, Amber M.; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B.

    2015-01-01

    RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3′ end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3′ extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation. PMID:25724636

  10. Pervasive Transcription of the Human Genome Produces Thousands of Previously Unidentified Long Intergenic Noncoding RNAs

    PubMed Central

    McManus, Michael T.

    2013-01-01

    Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements. PMID:23818866

  11. Diversity and Inheritance of Intergenic Spacer Sequences of 45S Ribosomal DNA among Accessions of Brassica oleracea L. var. capitata

    PubMed Central

    Yang, Kiwoung; Robin, Arif Hasan Khan; Yi, Go-Eun; Lee, Jonghoon; Chung, Mi-Young; Yang, Tae-Jin; Nou, Ill-Sup

    2015-01-01

    Ribosomal DNA (rDNA) of plants is present in high copy number and shows variation between and within species in the length of the intergenic spacer (IGS). The 45S rDNA of flowering plants includes the 5.8S, 18S and 25S rDNA genes, the internal transcribed spacer (ITS1 and ITS2), and the intergenic spacer 45S-IGS (25S-18S). This study identified six different types of 45S-IGS, A to F, which at 363 bp, 1121 bp, 1717 bp, 1969 bp, 2036 bp and 2111 bp in length, respectively, were much shorter than the reported reference IGS sequences in B. oleracea var. alboglabra. The shortest two IGS types, A and B, lacked the transcription initiation site, non-transcribed spacer, and external transcribed spacer. Functional behavior of those two IGS types in relation to rRNA synthesis is a subject of further investigation. The other four IGSs had subtle variations in the transcription termination site, guanine-cytosine (GC) content, and number of tandem repeats, but the external transcribed spacers of these four IGSs were quite similar in length. The 45S IGSs were found to follow Mendelian inheritance in a population of 15 F1s and their 30 inbred parental lines, which suggests that these sequences could be useful for development of new breeding tools. In addition, this study represents the first report of intra-specific (within subspecies) variation of the 45S IGS in B. oleracea. PMID:26633391

  12. Modular domains of the Dicistroviridae intergenic internal ribosome entry site

    PubMed Central

    Jang, Christopher J.; Jan, Eric

    2010-01-01

    The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae viral family can directly assemble 80S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. These functions are directed by two independently folded domains of the IGR IRES. One domain, composed of overlapping pseudoknots II and III (PKII/III), mediates ribosome recruitment. The second domain, composed of PKI, mimics a tRNA anticodon–codon interaction to position the ribosome at the ribosomal A-site. Although adopting a common secondary structure, the dicistrovirus IGR IRESs can be grouped into two classes based on distinct features within each domain. In this study, we report on the modularity of the IGR IRESs and show that the ribosome-binding domain and the tRNA anticodon mimicry domain are functionally interchangeable between the Type I and the Type II IGR IRESs. Using structural probing, ribosome-binding assays, and ribosome positioning analysis by toeprinting assays, we show that the chimeric IRESs fold properly, assemble 80S ribosomes, and can mediate IRES translation in rabbit reticulocyte lysates. We also demonstrate that the chimeric IRESs can stimulate the ribosome-dependent GTPase activity of eEF2, which suggests that the ribosome is primed for a step downstream from IRES binding. Overall, the results demonstrate that the dicistrovirus IGR IRESs are composed of two modular domains that work in concert to manipulate the ribosome and direct translation initiation. PMID:20423979

  13. Phylogeny of Porphyromonas gingivalis by Ribosomal Intergenic Spacer Region Analysis

    PubMed Central

    Rumpf, Robert W.; Griffen, Ann L.; Leys, Eugene J.

    2000-01-01

    Periodontitis has been associated with the presence of Porphyromonas gingivalis, and previous studies have shown phenotypic differences in the pathogenicities of strains of P. gingivalis. An accurate and comprehensive phylogeny of strains of P. gingivalis would be useful in determining if there is an evolutionary basis to pathogenicity in this species. Previous phylogenies of P. gingivalis strains based on random amplified polymorphic DNA (RAPD) analysis and multilocus enzyme electrophoresis (MLEE) show little agreement. While the 16S ribosomal gene is the standard for phylogenetic reconstruction among bacterial species, it is insufficiently variable for this purpose. In the present study, the phylogeny of P. gingivalis was constructed on the basis of the sequence of the most variable region of the ribosomal operon, the intergenic spacer region (ISR). Heteroduplex analysis of the ISR has been used to study the variability of P. gingivalis strains in periodontitis. In the present study, typing by heteroduplex analysis was compared to ISR sequence-based phylogeny and close agreement was observed. The two strains of P. gingivalis whose heteroduplex types are strongly associated with periodontitis were found to be closely related and were well separated from strains whose heteroduplex types are less strongly associated with disease, suggesting a relationship between pathogenicity and phylogeny. PMID:10790104

  14. Intergenic Alu exonisation facilitates the evolution of tissue-specific transcript ends.

    PubMed

    Tajnik, Mojca; Vigilante, Alessandra; Braun, Simon; Hänel, Heike; Luscombe, Nicholas M; Ule, Jernej; Zarnack, Kathi; König, Julian

    2015-12-01

    The 3' untranslated regions (3' UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3' UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3' UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3' UTRs. PMID:26400176

  15. Structure of rrn operons in pathogenic non-cultivable treponemes: sequence but not genomic position of intergenic spacers correlates with classification of Treponema pallidum and Treponema paraluiscuniculi strains

    PubMed Central

    Čejková, Darina; Zobaníková, Marie; Pospíšilová, Petra; Strouhal, Michal; Mikalová, Lenka; Weinstock, George M.

    2013-01-01

    This study examined the sequences of the two rRNA (rrn) operons of pathogenic non-cultivable treponemes, comprising 11 strains of T. pallidum ssp. pallidum (TPA), five strains of T. pallidum ssp. pertenue (TPE), two strains of T. pallidum ssp. endemicum (TEN), a simian Fribourg-Blanc strain and a rabbit T. paraluiscuniculi (TPc) strain. PCR was used to determine the type of 16S–23S ribosomal intergenic spacers in the rrn operons from 30 clinical samples belonging to five different genotypes. When compared with the TPA strains, TPc Cuniculi A strain had a 17 bp deletion, and the TPE, TEN and Fribourg-Blanc isolates had a deletion of 33 bp. Other than these deletions, only 17 heterogeneous sites were found within the entire region (excluding the 16S–23S intergenic spacer region encoding tRNA-Ile or tRNA-Ala). The pattern of nucleotide changes in the rrn operons corresponded to the classification of treponemal strains, whilst two different rrn spacer patterns (Ile/Ala and Ala/Ile) appeared to be distributed randomly across species/subspecies classification, time and geographical source of the treponemal strains. It is suggested that the random distribution of tRNA genes is caused by reciprocal translocation between repetitive sequences mediated by a recBCD-like system. PMID:23082031

  16. Species-specific repeat units in the intergenic spacer of the ribosomal RNA cistron of Anopheles aquasalis Curry.

    PubMed

    Perera, O P; Cockburn, A F; Mitchell, S E; Conn, J; Seawright, J A

    1998-11-01

    A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis. PMID:9840580

  17. The Mitochondrial Genome of Conus textile, coxI-coxII Intergenic Sequences and Conoidean Evolution

    PubMed Central

    Bandyopadhyay, Pradip K; Stevenson, Bradford J.; Ownby, John-Paul; Cady, Matthew T.; Watkins, Maren; Olivera, Baldomero M.

    2009-01-01

    The cone snails belong to the superfamily Conoidea, comprising ∼10,000 venomous marine gastropods. We determined the complete mitochondrial DNA sequence of Conus textile. The gene order is identical in Conus textile, Lophiotoma cerithiformis (another Conoidean gastropod), and the neogastropod Ilyanassa obsoleta, (not in the superfamily Conoidea). However, the intergenic interval between the coxI/coxII genes, was much longer in C. textile (165 bp) than in any other previously analyzed gastropod. We used the intergenic region to evaluate evolutionary patterns. In most neogastropods and three conidean families the intergenic interval is small (<30 nucleotides). Within Conus, the variation is from 130-170 bp, and each different clade within Conus has a narrower size distribution. In Conasprella, a subgenus traditionally assigned to Conus, the intergenic regions vary between 200-500 bp, suggesting that the species in Conasprella are not congeneric with Conus. The intergenic region was used for phylogenetic analysis of a group of fish-hunting Conus, despite the short length resolution was better than using standard markers. Thus, the coxI/coxII intergenic region can be used both to define evolutionary relationships between species in a clade, and to understand broad evolutionary patterns across the large superfamily Conoidea. PMID:17936021

  18. Chicken rRNA Gene Cluster Structure

    PubMed Central

    Dyomin, Alexander G.; Koshel, Elena I.; Kiselev, Artem M.; Saifitdinova, Alsu F.; Galkina, Svetlana A.; Fukagawa, Tatsuo; Kostareva, Anna A.

    2016-01-01

    Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5’ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3’ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity. PMID:27299357

  19. Role of the intergenic dinucleotide in vesicular stomatitis virus RNA transcription.

    PubMed Central

    Barr, J N; Whelan, S P; Wertz, G W

    1997-01-01

    To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in modulation of polymerase activity, we analyzed the RNA synthesis activities of bicistrionic genomic analogs that contained either the authentic N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide intergenic sequences, or no intergenic sequence at all. Quantitative measurements of the amounts of upstream, downstream, and readthrough mRNAs that were transcribed by these mutant templates showed that the behavior of the viral polymerase was profoundly affected by the nucleotide sequence that it encountered as it traversed the gene junction, although the polymerase was able to accommodate a remarkable degree of sequence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic sequence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation of downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analogs with altered intergenic sequences directed abundant synthesis of a readthrough transcript without correspondingly high levels of downstream mRNA, an observation inconsistent with the shunting model of VSV transcription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findings of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript. PMID:9032308

  20. Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.

    PubMed

    Purahong, Witoon; Stempfhuber, Barbara; Lentendu, Guillaume; Francioli, Davide; Reitz, Thomas; Buscot, François; Schloter, Michael; Krüger, Dirk

    2015-01-01

    Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

  1. Use of the CP and CPm Intergene Sequences to Discriminate CTV Strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop a rapid assay to distinguish potentially mild vs severe strains of Citrus tristeza virus. Multiple alignment performed on the coat protein (CP) and the minor coat protein (CPm) intergene sequences (~80-100 bp) from different CTV isolates revealed that severe strains (VT, ...

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

    PubMed Central

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

    1990-01-01

    Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence differences were observed between two H. momus populations. Comparison of rDNA primary sequence and rRNA secondary structures from H. momus with those from other well characterized organisms, demonstrated that the ascidians are more closely related to other chordates than invertebrates. The rDNA tandem repeat makes up a larger percentage (7%) of the genome of this animal than in other higher eukaryotes. The total length of the spacer and transcribed region in H. momus rDNA is small compared to most higher eukaryotes, being less than 8 kb, and the intergenic spacer region consists of smaller internal repeats. Comparative analysis of rDNA sequences has allowed the construction of secondary structures for the 18S, 5.8S and 26S rRNAs. Images PMID:2263465

  3. Molecular diversity of Renibacterium salmoninarum isolates determined by randomly amplified polymorphic DNA analysis.

    PubMed

    Grayson, T H; Atienzar, F A; Alexander, S M; Cooper, L F; Gilpin, M L

    2000-01-01

    The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture. PMID:10618262

  4. Molecular Diversity of Renibacterium salmoninarum Isolates Determined by Randomly Amplified Polymorphic DNA Analysis

    PubMed Central

    Grayson, T. Hilton; Atienzar, Franck A.; Alexander, Sarah M.; Cooper, Lynne F.; Gilpin, Martyn L.

    2000-01-01

    The molecular diversity among 60 isolates of Renibacterium salmoninarum which differ in place and date of isolation was investigated by using randomly amplified polymorphic DNA (RAPD) analysis. Isolates were grouped into 21 banding patterns which did not reflect the biological source. Four 16S-23S rRNA intergenic spacer (ITS1) sequence variations and two alleles of an exact tandem repeat locus, ETR-A, were the bases for formation of distinct groups within the RAPD clusters. This study provides evidence that the most common ITS1 sequence variant, SV1, possesses two copies of a 51-bp repeat unit at ETR-A and has been widely dispersed among countries which are associated with mainstream intensive salmonid culture. PMID:10618262

  5. Development of a loop-mediated isothermal amplification assay for rapid detection of Nocardia salmonicida, the causative agent of nocardiosis in fish.

    PubMed

    Xia, Liqun; Zhang, Honglian; Lu, Yishan; Cai, Jia; Wang, Bei; Jian, Jichang

    2015-03-01

    Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64°C. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10(3) CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis. PMID:25262681

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

    PubMed

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

    2006-05-01

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

  7. Associating disease-related genetic variants in intergenic regions to the genes they impact

    PubMed Central

    Ong, Cheng Soon

    2014-01-01

    We present a method to assist in interpretation of the functional impact of intergenic disease-associated SNPs that is not limited to search strategies proximal to the SNP. The method builds on two sources of external knowledge: the growing understanding of three-dimensional spatial relationships in the genome, and the substantial repository of information about relationships among genetic variants, genes, and diseases captured in the published biomedical literature. We integrate chromatin conformation capture data (HiC) with literature support to rank putative target genes of intergenic disease-associated SNPs. We demonstrate that this hybrid method outperforms a genomic distance baseline on a small test set of expression quantitative trait loci, as well as either method individually. In addition, we show the potential for this method to uncover relationships between intergenic SNPs and target genes across chromosomes. With more extensive chromatin conformation capture data becoming readily available, this method provides a way forward towards functional interpretation of SNPs in the context of the three dimensional structure of the genome in the nucleus. PMID:25374782

  8. Bacterial Diversity and Community Structure in an Aerated Lagoon Revealed by Ribosomal Intergenic Spacer Analyses and 16S Ribosomal DNA Sequencing

    PubMed Central

    Yu, Zhongtang; Mohn, William W.

    2001-01-01

    We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35°C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus Methylobacillus, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus Azospirillum. PMID:11282606

  9. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

    USGS Publications Warehouse

    Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  10. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans.

    PubMed

    Muller, Laura K; Lorch, Jeffrey M; Lindner, Daniel L; O'Connor, Michael; Gargas, Andrea; Blehert, David S

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was qualified further by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research. PMID:22962349

  11. Variable rRNA gene copies in extreme halobacteria

    SciTech Connect

    Sanz, J.L.; Marin, I.; Ramirez, L.; Amils, R. ); Abad, J.P.; Smith, C.L. )

    1988-08-25

    Using PFG electrophoresis techniques, the authors have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence. The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.

  12. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements.

    PubMed

    Schlesinger, Felix; Smith, Andrew D; Gingeras, Thomas R; Hannon, Gregory J; Hodges, Emily

    2013-10-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  13. Molecular Identification of Closely Related Candida Species Using Two Ribosomal Intergenic Spacer Fingerprinting Methods

    PubMed Central

    Cornet, Muriel; Sendid, Boualem; Fradin, Chantal; Gaillardin, Claude; Poulain, Daniel; Nguyen, Huu-Vang

    2011-01-01

    Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic. PMID:21227390

  14. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

    PubMed Central

    Schlesinger, Felix; Smith, Andrew D.; Gingeras, Thomas R.; Hannon, Gregory J.; Hodges, Emily

    2013-01-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  15. Mitochondrial intergenic COII/tRNA(Lys) 9-bp deletion, a biomarker for hepatocellular carcinoma?

    PubMed

    Ren, Weihua; Li, Yawei; Li, Rui; Feng, Hongbo; Wu, Shuangting; Mao, Yuhui; Huang, Lei

    2016-07-01

    The COII/tRNA(Lys) intergenic 9-bp deletion is one of the most commonly studied human mitochondrial DNA (mtDNA) polymorphisms. It consists of the loss of one of two tandemly repeated copies of the sequence CCCCCTCTA from a non-coding region located between cytochrome oxidase II (COII) and tRNA(Lys) gene. Most recently, case-control studies have shown a positive association between this deletion with hepatocellular cancer. In this study, we first performed a detailed analysis between this deletion and clinical diseases; moreover, we took the phylogenetic approach to examine the pathogenicity status of 9-bp deletion. PMID:26017042

  16. The CASC15 Long Intergenic Noncoding RNA Locus Is Involved in Melanoma Progression and Phenotype Switching.

    PubMed

    Lessard, Laurent; Liu, Michelle; Marzese, Diego M; Wang, Hongwei; Chong, Kelly; Kawas, Neal; Donovan, Nicholas C; Kiyohara, Eiji; Hsu, Sandy; Nelson, Nellie; Izraely, Sivan; Sagi-Assif, Orit; Witz, Isaac P; Ma, Xiao-Jun; Luo, Yuling; Hoon, Dave S B

    2015-10-01

    In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long noncoding RNAs in melanoma progression. We hypothesized that copy number alterations (CNAs) of intergenic nonprotein-coding domains could help identify long intergenic noncoding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 (cancer susceptibility candidate 15) lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and upregulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC (American Joint Committee on Cancer) stage III lymph node metastasis. Moreover, small interfering RNA (siRNA) knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma. PMID:26016895

  17. De Novo Identification of Regulatory Regions in Intergenic Spaces of Prokaryotic Genomes

    SciTech Connect

    Chain, P; Garcia, E; Mcloughlin, K; Ovcharenko, I

    2007-02-20

    This project was begun to implement, test, and experimentally validate the results of a novel algorithm for genome-wide identification of candidate transcription-factor binding sites in prokaryotes. Most techniques used to identify regulatory regions rely on conservation between different genomes or have a predetermined sequence motif(s) to perform a genome-wide search. Therefore, such techniques cannot be used with new genome sequences, where information regarding such motifs has not yet been discovered. This project aimed to apply a de novo search algorithm to identify candidate binding-site motifs in intergenic regions of prokaryotic organisms, initially testing the available genomes of the Yersinia genus. We retrofitted existing nucleotide pattern-matching algorithms, analyzed the candidate sites identified by these algorithms as well as their target genes to screen for meaningful patterns. Using properly annotated prokaryotic genomes, this project aimed to develop a set of procedures to identify candidate intergenic sites important for gene regulation. We planned to demonstrate this in Yersinia pestis, a model biodefense, Category A Select Agent pathogen, and then follow up with experimental evidence that these regions are indeed involved in regulation. The ability to quickly characterize transcription-factor binding sites will help lead to a better understanding of how known virulence pathways are modulated in biodefense-related organisms, and will help our understanding and exploration of regulons--gene regulatory networks--and novel pathways for metabolic processes in environmental microbes.

  18. Genome-Wide Analyses in Bacteria Show Small-RNA Enrichment for Long and Conserved Intergenic Regions

    PubMed Central

    Tsai, Chen-Hsun; Liao, Rick; Chou, Brendan; Palumbo, Michael

    2014-01-01

    Interest in finding small RNAs (sRNAs) in bacteria has significantly increased in recent years due to their regulatory functions. Development of high-throughput methods and more sophisticated computational algorithms has allowed rapid identification of sRNA candidates in different species. However, given their various sizes (50 to 500 nucleotides [nt]) and their potential genomic locations in the 5′ and 3′ untranslated regions as well as in intergenic regions, identification and validation of true sRNAs have been challenging. In addition, the evolution of bacterial sRNAs across different species continues to be puzzling, given that they can exert similar functions with various sequences and structures. In this study, we analyzed the enrichment patterns of sRNAs in 13 well-annotated bacterial species using existing transcriptome and experimental data. All intergenic regions were analyzed by WU-BLAST to examine conservation levels relative to species within or outside their genus. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicate that sRNAs are enriched in intergenic regions, which are longer and more conserved than the average intergenic regions in the corresponding bacterial genome. We also found that sRNA-coding regions have different conservation levels relative to their flanking regions. This work provides a way to analyze how noncoding RNAs are distributed in bacterial genomes and also shows conserved features of intergenic regions that encode sRNAs. These results also provide insight into the functions of regions surrounding sRNAs and into optimization of RNA search algorithms. PMID:25313390

  19. Distribution of 16S rRNA methylases among different species of Gram-negative bacilli with high-level resistance to aminoglycosides.

    PubMed

    Zhou, Y; Yu, H; Guo, Q; Xu, X; Ye, X; Wu, S; Guo, Y; Wang, M

    2010-11-01

    16S rRNA methylases confer high-level resistance to most aminoglycosides in Gram-negative bacteria. Seven 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, have been identified since 2003. We studied the distribution of methylase genes in more than 200 aminoglycoside-resistant Gram-negative clinical isolates collected in 2007 at our hospital in Shanghai, China. 16S rRNA methylase genes were amplified by polymerase chain reaction (PCR) among 217 consecutive clinical isolates of Gram-negative bacilli resistant to gentamicin and amikacin by a disk diffusion method. 16S rRNA methylase genes were present in 97.5% (193/198) of clinical isolates highly resistant to amikacin (≥512 μg/ml), with armA and rmtB detected in 67.2 and 30.3% of strains, respectively, while no 16S rRNA methylase genes were detected in 19 strains with amikacin minimum inhibitory concentration (MIC) ≤256 μg/ml. armA or rmtB genes were detected in 100% of 104 strains of Enterobacteriaceae, and these two genes were equally represented (49 vs. 55 strains). Genes for armA or rmtB were detected in 94.7% (89/94) of Acinetobacter baumannii and Pseudomonas aeruginosa strains, and armA was predominant (84 vs. 5 strains with rmtB). No rmtA, rmtC, rmtD or npmA genes were found. Enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) indicated that armA and rmtB genes were spread by both horizontal transfer and clonal dissemination. PMID:20614151

  20. Limitations and benefits of ARISA intra-genomic diversity fingerprinting.

    PubMed

    Popa, Radu; Popa, Rodica; Mashall, Matthew J; Nguyen, Hien; Tebo, Bradley M; Brauer, Suzanna

    2009-08-01

    Monitoring diversity changes and contamination in mixed cultures and simple microcosms is challenged by fast community structure dynamics, and the need for means allowing fast, cost-efficient and accurate identification of microorganisms at high phylogenetic resolution. The method we explored is a variant of Automated rRNA Intergenic Spacer Analysis based on Intra-Genomic Diversity Fingerprinting (ARISA-IGDF), and identifies phylotypes with multiple 16S-23S rRNA gene Intergenic Transcribed Spacers. We verified the effect of PCR conditions (annealing temperature, duration of final extension, number of cycles, group-specific primers and formamide) on ARISA-IGD fingerprints of 44 strains of Shewanella. We present a digitization algorithm and data analysis procedures needed to determine confidence in strain identification. Though using stringent PCR conditions and group-specific primers allow reasonably accurate identification of strains with three ARISA-IGD amplicons within the 82-1000 bp size range, ARISA-IGDF is best for phylotypes with >or=4 unambiguously different amplicons. This method allows monitoring the occurrence of culturable microbes and can be implemented in applications requiring high phylogenetic resolution, reproducibility, low cost and high throughput such as identifying contamination and monitoring the evolution of diversity in mixed cultures and low diversity microcosms and periodic screening of small microbial culture libraries. PMID:19538993

  1. Sequence conservation and functional constraint on intergenic spacers in reduced genomes of the obligate symbiont Buchnera.

    PubMed

    Degnan, Patrick H; Ochman, Howard; Moran, Nancy A

    2011-09-01

    Analyses of genome reduction in obligate bacterial symbionts typically focus on the removal and retention of protein-coding regions, which are subject to ongoing inactivation and deletion. However, these same forces operate on intergenic spacers (IGSs) and affect their contents, maintenance, and rates of evolution. IGSs comprise both non-coding, non-functional regions, including decaying pseudogenes at varying stages of recognizability, as well as functional elements, such as genes for sRNAs and regulatory control elements. The genomes of Buchnera and other small genome symbionts display biased nucleotide compositions and high rates of sequence evolution and contain few recognizable regulatory elements. However, IGS lengths are highly correlated across divergent Buchnera genomes, suggesting the presence of functional elements. To identify functional regions within the IGSs, we sequenced two Buchnera genomes (from aphid species Uroleucon ambrosiae and Acyrthosiphon kondoi) and applied a phylogenetic footprinting approach to alignments of orthologous IGSs from a total of eight Buchnera genomes corresponding to six aphid species. Inclusion of these new genomes allowed comparative analyses at intermediate levels of divergence, enabling the detection of both conserved elements and previously unrecognized pseudogenes. Analyses of these genomes revealed that 232 of 336 IGS alignments over 50 nucleotides in length displayed substantial sequence conservation. Conserved alignment blocks within these IGSs encompassed 88 Shine-Dalgarno sequences, 55 transcriptional terminators, 5 Sigma-32 binding sites, and 12 novel small RNAs. Although pseudogene formation, and thus IGS formation, are ongoing processes in these genomes, a large proportion of intergenic spacers contain functional sequences. PMID:21912528

  2. Use of the Coat Protein (CP) and minor CP Intergene Sequence to Discriminate Severe Strains of Citrus tristeza virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A rapid assay is a needed to differentiate mild vs severe strains of Citrus tristeza virus (CTV). Multiple alignment performed on the coat protein (CP) and the minor coat protein (CPm) intergene sequences (~80-100 bp) from different CTV isolates revealed that severe strains generally associated wit...

  3. Intergenic Sequence Ribotyping using a region neighboring dkgB links genovar to Kauffman-White serotype of Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Intergenic Sequence Ribotyping using a region neighboring dkgB links genovar to Kauffman-White serotype of Salmonella enterica Previous research identified that the 5S ribosomal (rrn) gene and associated flanking sequences that are closely linked to the dkgB gene of Salmonella enterica were highly ...

  4. Intergenic Sequence Ribotyping using a region neighboring dkgB links genovar to Kauffman-White serotype of Salmonella enterica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty six (36) unique sequences which varied in length from 258bp to 530bp were found for Salmonella enterica strains and isolates that are not present in public databases following BLAST analysis searches for similarity. The sequences were found by application of Intergenic Sequence Ribotyping (IS...

  5. Molecular identfication and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in West Alabama (USA) in 2009

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish during a disease outbreak in West Alabama in August 2009. Sequence analysis of 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the three strains revealed tha...

  6. Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in West Alabama in 2009

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three isolates (AL09-71, AL09-72, and AL09-73) of Aeromonas hydrophila were cultured from infected channel catfish during a disease outbreak in West Alabama in August 2009. Sequence analysis of 16S-23S rDNA intergenic spacer region (ISR), cpn60, gyrB, and rpoD genes of the three strains revealed tha...

  7. Molecular Analysis of Promoter and Intergenic Region Attenuator of the Vibrio vulnificus prx1ahpF Operon.

    PubMed

    Lee, Hyun Sung; Lim, Jong Gyu; Han, Kook; Lee, Younghoon; Choi, Sang Ho

    2015-08-01

    Prx1, an AhpF-dependent 2-Cys peroxiredoxin (Prx), was previously identified in Vibrio vulnificus, a facultative aerobic pathogen. In the present study, transcription of the V. vulnificus prx1ahpF genes, which are adjacently located on the chromosome, was evaluated by analyzing the promoter and intergenic region of the two genes. Northern blot analyses revealed that transcription of prx1ahpF results in two transcripts, the prx1 and prx1ahpF transcripts. Primer extension analysis and a point mutational analysis of the promoter region showed that the two transcripts are generated from a single promoter. In addition, the 3' end of the prx1 transcript at the prx1ahpF intergenic region was determined by a 3'RACE assay. These results suggested that the prx1ahpF genes are transcribed as an operon, and the prx1 transcript was produced by transcriptional termination in the intergenic region. RNA secondary structure prediction of the prx1ahpF intergenic region singled out a stem-loop structure without poly(U) tract, and a deletion analysis of the intergenic region showed that the atypical stem-loop structure acts as the transcriptional attenuator to result in the prx1 and prx1ahpF transcripts. The combined results demonstrate that the differential expression of prx1 and ahpF is accomplished by the cis-acting transcriptional attenuator located between the two genes and thereby leads to the production of a high level of Prx1 and a low level of AhpF. PMID:25824432

  8. Rapid Acquisition of Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus: Role of Hypermutation and Homologous Recombination

    PubMed Central

    Iguchi, Shigekazu; Mizutani, Tomonori; Hiramatsu, Keiichi; Kikuchi, Ken

    2016-01-01

    Background We previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance. Methods Using clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome. Results Strains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance. Conclusions Hypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case

  9. PCR amplification and characterization of the intergenic spacer region of the ribosomal DNA in Pyrenophora graminea.

    PubMed

    Pecchia, S; Mercatelli, E; Vannacci, G

    1998-09-01

    Successful amplification of the whole intergenic spacer region of the nuclear ribosomal repeat (IGS) in Pyrenophora graminea was obtained with a PCR-based assay. Single amplification products showed length differences. Depending on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates could be identified. The RFLP patterns of isolates obtained with the 6-base cutting enzymes Apal, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups. Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length. In order to characterize the long and short IGS-PCR products the restriction map is shown. The long product shows an additional HindIII site and a BglII site that is lacking in the short product. However, the latter shows a SacI site that is not present in the long IGS-PCR product. Therefore, the described PCR-RFLP analysis of the IGS appears to be a useful tool to resolve genetic variation between P. graminea isolates. PMID:9741081

  10. Transposable Element Insertions in Long Intergenic Non-Coding RNA Genes

    PubMed Central

    Kannan, Sivakumar; Chernikova, Diana; Rogozin, Igor B.; Poliakov, Eugenia; Managadze, David; Koonin, Eugene V.; Milanesi, Luciano

    2015-01-01

    Transposable elements (TEs) are abundant in mammalian genomes and appear to have contributed to the evolution of their hosts by providing novel regulatory or coding sequences. We analyzed different regions of long intergenic non-coding RNA (lincRNA) genes in human and mouse genomes to systematically assess the potential contribution of TEs to the evolution of the structure and regulation of expression of lincRNA genes. Introns of lincRNA genes contain the highest percentage of TE-derived sequences (TES), followed by exons and then promoter regions although the density of TEs is not significantly different between exons and promoters. Higher frequencies of ancient TEs in promoters and exons compared to introns implies that many lincRNA genes emerged before the split of primates and rodents. The content of TES in lincRNA genes is substantially higher than that in protein-coding genes, especially in exons and promoter regions. A significant positive correlation was detected between the content of TEs and evolutionary rate of lincRNAs indicating that inserted TEs are preferentially fixed in fast-evolving lincRNA genes. These results are consistent with the repeat insertion domains of LncRNAs hypothesis under which TEs have substantially contributed to the origin, evolution, and, in particular, fast functional diversification, of lincRNA genes. PMID:26106594

  11. A Novel Intergenic ETnII-β Insertion Mutation Causes Multiple Malformations in Polypodia Mice

    PubMed Central

    Lehoczky, Jessica A.; Thomas, Peedikayil E.; Patrie, Kevin M.; Owens, Kailey M.; Villarreal, Lisa M.; Galbraith, Kenneth; Washburn, Joe; Johnson, Craig N.; Gavino, Bryant; Borowsky, Alexander D.; Millen, Kathleen J.; Wakenight, Paul; Law, William; Van Keuren, Margaret L.; Gavrilina, Galina; Hughes, Elizabeth D.; Saunders, Thomas L.; Brihn, Lesil; Nadeau, Joseph H.; Innis, Jeffrey W.

    2013-01-01

    Mouse early transposon insertions are responsible for ∼10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5′ LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development. PMID:24339789

  12. Epistasis in intra- and inter-gene pool crosses of the common bean.

    PubMed

    Borel, J C; Ramalho, M A P; Abreu, A F B

    2016-01-01

    Epistasis has been shown to have an important role in the genetic control of several quantitative traits in the common bean. This study aimed to investigate the occurrence of epistasis in intra- and inter-pool gene crosses of the common bean. Four elite lines adapted to Brazilian conditions were used as parents, two from the Andean gene pool (ESAL 686; BRS Radiante) and two from the Mesoamerican gene pool (BRSMG Majestoso; BRS Valente). Four F2 populations were obtained: "A" (ESAL 686 x BRS Radiante), "B" (BRSMG Majestoso x BRS Valente), "C" (BRS Radiante x BRSMG Majestoso), and "D" (BRS Valente x ESAL 686). A random sample of F2 plants from each population was backcrossed to parents and F1 individuals, according to the triple test cross. Three types of progenies from each population were evaluated in contiguous trials. Seed yield and 100-seed weight were evaluated. Dominance genetic variance was predominant in most cases. However, the estimates of genetic variance may be biased by the occurrence of linkage disequilibrium and epistasis. Epistasis was detected for both traits; however, the occurrence differed among the populations and between the two traits. The results of this study reinforce the hypothesis that epistasis is present in the genetic control of traits in the common bean and suggest that the phenomenon is more frequent in inter-gene pool crosses than in intra-gene pool crosses. PMID:26985920

  13. Disruption of a Large Intergenic Noncoding RNA in Subjects with Neurodevelopmental Disabilities

    PubMed Central

    Talkowski, Michael E.; Maussion, Gilles; Crapper, Liam; Rosenfeld, Jill A.; Blumenthal, Ian; Hanscom, Carrie; Chiang, Colby; Lindgren, Amelia; Pereira, Shahrin; Ruderfer, Douglas; Diallo, Alpha B.; Lopez, Juan Pablo; Turecki, Gustavo; Chen, Elizabeth S.; Gigek, Carolina; Harris, David J.; Lip, Va; An, Yu; Biagioli, Marta; MacDonald, Marcy E.; Lin, Michael; Haggarty, Stephen J.; Sklar, Pamela; Purcell, Shaun; Kellis, Manolis; Schwartz, Stuart; Shaffer, Lisa G.; Natowicz, Marvin R.; Shen, Yiping; Morton, Cynthia C.; Gusella, James F.; Ernst, Carl

    2012-01-01

    Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders. PMID:23217328

  14. In silico prediction of long intergenic non-coding RNAs in sheep.

    PubMed

    Bakhtiarizadeh, Mohammad Reza; Hosseinpour, Batool; Arefnezhad, Babak; Shamabadi, Narges; Salami, Seyed Alireza

    2016-04-01

    Long non-coding RNAs (lncRNAs) are transcribed RNA molecules >200 nucleotides in length that do not encode proteins and serve as key regulators of diverse biological processes. Recently, thousands of long intergenic non-coding RNAs (lincRNAs), a type of lncRNAs, have been identified in mammalians using massive parallel large sequencing technologies. The availability of the genome sequence of sheep (Ovis aries) has allowed us genomic prediction of non-coding RNAs. This is the first study to identify lincRNAs using RNA-seq data of eight different tissues of sheep, including brain, heart, kidney, liver, lung, ovary, skin, and white adipose. A computational pipeline was employed to characterize 325 putative lincRNAs with high confidence from eight important tissues of sheep using different criteria such as GC content, exon number, gene length, co-expression analysis, stability, and tissue-specific scores. Sixty-four putative lincRNAs displayed tissues-specific expression. The highest number of tissues-specific lincRNAs was found in skin and brain. All novel lincRNAs that aligned to the human and mouse lincRNAs had conserved synteny. These closest protein-coding genes were enriched in 11 significant GO terms such as limb development, appendage development, striated muscle tissue development, and multicellular organismal development. The findings reported here have important implications for the study of sheep genome. PMID:27002388

  15. Conservation and diversity among the three-dimensional folds of the Dicistroviridae intergenic region IRESes.

    PubMed

    Pfingsten, Jennifer S; Costantino, David A; Kieft, Jeffrey S

    2007-07-27

    Internal ribosome entry site (IRES) RNAs are necessary for successful infection of many pathogenic viruses, but the details of the RNA structure-based mechanism used to bind and manipulate the ribosome remain poorly understood. The IRES RNAs from the Dicistroviridae intergenic region (IGR) are an excellent model system to understand the fundamental tenets of IRES function, requiring no protein factors to manipulate the ribosome and initiate translation. Here, we explore the architecture of four members of the IGR IRESes, representative of the two divergent classes of these IRES RNAs. Using biochemical and structural probing methods, we show that despite sequence variability they contain a common three-dimensional fold. The three-dimensional architecture of the ribosome binding domain from these IRESes is organized around a core helical scaffold, around which the rest of the RNA molecule folds. However, subtle variation in the folds of these IRESes and the presence of an additional secondary structure element suggest differences in the details of their manipulation of the large ribosomal subunit. Overall, the results demonstrate how a conserved three-dimensional RNA fold governs ribosome binding and manipulation. PMID:17544444

  16. RNA structure-based ribosome recruitment: lessons from the Dicistroviridae intergenic region IRESes.

    PubMed

    Pfingsten, Jennifer S; Kieft, Jeffrey S

    2008-07-01

    In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5' end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by many viruses of medical and economic importance. In this review, we briefly describe and compare mechanistic and structural groups of viral IRES RNAs, focusing on those IRESes that are capable of direct ribosome recruitment using specific RNA structures. We then discuss in greater detail some recent advances in our understanding of the intergenic region IRESes of the Dicistroviridae, which use the most streamlined ribosome-recruitment mechanism yet discovered. By combining these findings with knowledge of canonical translation and the behavior of other IRESes, mechanistic models of this RNA structure-based process are emerging. PMID:18515544

  17. The evolutionary landscape of intergenic trans-splicing events in insects

    PubMed Central

    Kong, Yimeng; Zhou, Hongxia; Yu, Yao; Chen, Longxian; Hao, Pei; Li, Xuan

    2015-01-01

    To explore the landscape of intergenic trans-splicing events and characterize their functions and evolutionary dynamics, we conduct a mega-data study of a phylogeny containing eight species across five orders of class Insecta, a model system spanning 400 million years of evolution. A total of 1,627 trans-splicing events involving 2,199 genes are identified, accounting for 1.58% of the total genes. Homology analysis reveals that mod(mdg4)-like trans-splicing is the only conserved event that is consistently observed in multiple species across two orders, which represents a unique case of functional diversification involving trans-splicing. Thus, evolutionarily its potential for generating proteins with novel function is not broadly utilized by insects. Furthermore, 146 non-mod trans-spliced transcripts are found to resemble canonical genes from different species. Trans-splicing preserving the function of ‘breakup' genes may serve as a general mechanism for relaxing the constraints on gene structure, with profound implications for the evolution of genes and genomes. PMID:26521696

  18. DNA methylation signatures of long intergenic noncoding RNAs in porcine adipose and muscle tissues

    PubMed Central

    Zhou, Zhong-Yin; Li, Aimin; Wang, Li-Gang; Irwin, David M; Liu, Yan-Hu; Xu, Dan; Han, Xu-Man; Wang, Lu; Wu, Shi-Fang; Wang, Li-Xian; Xie, Hai-Bing; Zhang, Ya-Ping

    2015-01-01

    Long intergenic noncoding RNAs (lincRNAs) are one of the major unexplored components of genomes. Here we re-analyzed a published methylated DNA immunoprecipitation sequencing (MeDIP-seq) dataset to characterize the DNA methylation pattern of pig lincRNA genes in adipose and muscle tissues. Our study showed that the methylation level of lincRNA genes was higher than that of mRNA genes, with similar trends observed in comparisons of the promoter, exon or intron regions. Different methylation pattern were observed across the transcription start sites (TSS) of lincRNA and protein-coding genes. Furthermore, an overlap was observed between many lincRNA genes and differentially methylated regions (DMRs) identified among different breeds of pigs, which show different fat contents, sexes and anatomic locations of tissues. We identify a lincRNA gene, linc-sscg3623, that displayed differential methylation levels in backfat between Min and Large White pigs at 60 and 120 days of age. We found that a demethylation process occurred between days 150 and 180 in the Min and Large White pigs, which was followed by remethylation between days 180 and 210. These results contribute to our understanding of the domestication of domestic animals and identify lincRNA genes involved in adipogenesis and muscle development. PMID:26493951

  19. DNA methylation signatures of long intergenic noncoding RNAs in porcine adipose and muscle tissues.

    PubMed

    Zhou, Zhong-Yin; Li, Aimin; Wang, Li-Gang; Irwin, David M; Liu, Yan-Hu; Xu, Dan; Han, Xu-Man; Wang, Lu; Wu, Shi-Fang; Wang, Li-Xian; Xie, Hai-Bing; Zhang, Ya-Ping

    2015-01-01

    Long intergenic noncoding RNAs (lincRNAs) are one of the major unexplored components of genomes. Here we re-analyzed a published methylated DNA immunoprecipitation sequencing (MeDIP-seq) dataset to characterize the DNA methylation pattern of pig lincRNA genes in adipose and muscle tissues. Our study showed that the methylation level of lincRNA genes was higher than that of mRNA genes, with similar trends observed in comparisons of the promoter, exon or intron regions. Different methylation pattern were observed across the transcription start sites (TSS) of lincRNA and protein-coding genes. Furthermore, an overlap was observed between many lincRNA genes and differentially methylated regions (DMRs) identified among different breeds of pigs, which show different fat contents, sexes and anatomic locations of tissues. We identify a lincRNA gene, linc-sscg3623, that displayed differential methylation levels in backfat between Min and Large White pigs at 60 and 120 days of age. We found that a demethylation process occurred between days 150 and 180 in the Min and Large White pigs, which was followed by remethylation between days 180 and 210. These results contribute to our understanding of the domestication of domestic animals and identify lincRNA genes involved in adipogenesis and muscle development. PMID:26493951

  20. Comparative Expression Dynamics of Intergenic Long Noncoding RNAs in the Genus Drosophila

    PubMed Central

    Nyberg, Kevin G.; Machado, Carlos A.

    2016-01-01

    Thousands of long noncoding RNAs (lncRNAs) have been annotated in eukaryotic genomes, but comparative transcriptomic approaches are necessary to understand their biological impact and evolution. To facilitate such comparative studies in Drosophila, we identified and characterized lncRNAs in a second Drosophilid—the evolutionary model Drosophila pseudoobscura. Using RNA-Seq and computational filtering of protein-coding potential, we identified 1,589 intergenic lncRNA loci in D. pseudoobscura. We surveyed multiple sex-specific developmental stages and found, like in Drosophila melanogaster, increasingly prolific lncRNA expression through male development and an overrepresentation of lncRNAs in the testes. Other trends seen in D. melanogaster, like reduced pupal expression, were not observed. Nonrandom distributions of female-biased and non-testis-specific male-biased lncRNAs between the X chromosome and autosomes are consistent with selection-based models of gene trafficking to optimize genomic location of sex-biased genes. The numerous testis-specific lncRNAs, however, are randomly distributed between the X and autosomes, and we cannot reject the hypothesis that many of these are likely to be spurious transcripts. Finally, using annotated lncRNAs in both species, we identified 134 putative lncRNA homologs between D. pseudoobscura and D. melanogaster and find that many have conserved developmental expression dynamics, making them ideal candidates for future functional analyses. PMID:27189981

  1. Comparative Expression Dynamics of Intergenic Long Noncoding RNAs in the Genus Drosophila.

    PubMed

    Nyberg, Kevin G; Machado, Carlos A

    2016-01-01

    Thousands of long noncoding RNAs (lncRNAs) have been annotated in eukaryotic genomes, but comparative transcriptomic approaches are necessary to understand their biological impact and evolution. To facilitate such comparative studies in Drosophila, we identified and characterized lncRNAs in a second Drosophilid-the evolutionary model Drosophila pseudoobscura Using RNA-Seq and computational filtering of protein-coding potential, we identified 1,589 intergenic lncRNA loci in D. pseudoobscura We surveyed multiple sex-specific developmental stages and found, like in Drosophila melanogaster, increasingly prolific lncRNA expression through male development and an overrepresentation of lncRNAs in the testes. Other trends seen in D. melanogaster, like reduced pupal expression, were not observed. Nonrandom distributions of female-biased and non-testis-specific male-biased lncRNAs between the X chromosome and autosomes are consistent with selection-based models of gene trafficking to optimize genomic location of sex-biased genes. The numerous testis-specific lncRNAs, however, are randomly distributed between the X and autosomes, and we cannot reject the hypothesis that many of these are likely to be spurious transcripts. Finally, using annotated lncRNAs in both species, we identified 134 putative lncRNA homologs between D. pseudoobscura and D. melanogaster and find that many have conserved developmental expression dynamics, making them ideal candidates for future functional analyses. PMID:27189981

  2. Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures[OPEN

    PubMed Central

    Zhu, Bo; Zhang, Wenli; Jiang, Jiming

    2015-01-01

    Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants. PMID:26373455

  3. Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary.

    PubMed

    Farkas, Szilvia L; Gál, János

    2009-07-01

    A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana- and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific Mycoplasma agassizii PCR targeting the 16S rRNA gene. Based on the analyses of partial sequences of the 16S rRNA gene, the Mycoplasma sp. of the ornate box turtle seemed to be identical with the recently described eastern box turtle (Terrapene carolina carolina) Mycoplasma sp. This is the first report of a novel chelonian adenovirus and a mycoplasma infection in an ornate box turtle (T. ornata ornata) in Europe. PMID:19375875

  4. Promoter of the Mycoplasma pneumoniae rRNA operon.

    PubMed Central

    Hyman, H C; Gafny, R; Glaser, G; Razin, S

    1988-01-01

    RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene. The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132. It was concluded that transcription of the rRNA operon of M. pneumoniae is initiated by a single promoter. The nucleotide sequence of the region is presented. Images PMID:2838465

  5. Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai)

    PubMed

    Kim, W S; Gardan, L; Rhim, S L; Geider, K

    1999-04-01

    A novel pathogen from Asian pears (Pyrus pyrifolia Nakai) was analysed by sequencing the 16S rDNA and the adjacent intergenic region, and the data were compared to related Enterobacteriaceae. The 16S rDNA of the Asian pear pathogen was almost identical with the sequence of Erwinia amylovora, in contrast to the 16S-23S rRNA intergenic transcribed spacer region of both species. A dendrogram was deduced from determined sequences of the spacer regions including those of several related species such as Erwinia amylovora, Enterobacter pyrinus, Pantoea stewartii subsp. stewartii and Escherichia coli. Dendrograms derived from 121 biochemical characteristics including Biotype 100 data placed the Asian pear pathogen close to Erwinia amylovora and more distantly to other members of the species Erwinia and to the species Pantoea and Enterobacter. Another DNA relatedness study was performed by DNA hybridizations and estimation of delta Tm values. The Asian pear strains constituted a tight DNA hybridization group (89-100%) and were barely related to strains of Erwinia amylovora (40-50%) with a delta Tm in the range of 5.2-6.8. The G + C content of DNA from the novel pathogen is 52 mol%. Therefore, it is proposed that strains isolated from Asian pears constitute a new species and the name Erwinia pyrifoliae is suggested; the type strain is strain Ep 16/96T (= CFBP 4172T = DSM 12163T). PMID:10319516

  6. Functional Implications of RNA Splicing for Human Long Intergenic Noncoding RNAs

    PubMed Central

    Chen, Feng-Chi; Pan, Chia-Lin; Lin, Hsuan-Yu

    2014-01-01

    Long intergenic noncoding RNAs (lincRNAs) have been suggested as playing important roles in human gene regulation. The majority of annotated human lincRNAs include multiple exons and are alternatively spliced. However, the connections between alternative RNA splicing (AS) and the functions/regulations of lincRNAs have remained elusive. In this study, we compared the sequence evolution and biological features between single-exonic lincRNAs and multi-exonic lincRNAs (SELs and MELs, respectively) that were present only in the hominoids (hominoid-specific) or conserved in primates (primate-conserved). The MEL exons were further classified into alternatively spliced exons (ASEs) and constitutively spliced exons (CSEs) for evolutionary analyses. Our results indicate that SELs and MELs differed significantly from each other. Firstly, in hominoid-specific lincRNAs, MELs (both CSEs and ASEs) evolved slightly more rapidly than SELs, which evolved approximately at the neutral rate. In primate-conserved lincRNAs, SELs and ASEs evolved slightly more slowly than CSEs and neutral sequences. The evolutionary path of hominid-specific lincRNAs thus seemed to have diverged from that of their more ancestral counterparts. Secondly, both of the exons and transcripts of SELs were significantly longer than those of MELs, and this was probably because SEL transcripts were more resistant to RNA splicing than MELs. Thirdly, SELs were physically closer to coding genes than MELs. Fourthly, SELs were more widely expressed in human tissues than MELs. These results suggested that SELs and MELs represented two biologically distinct groups of genes. In addition, the SEL–MEL and ASE–CSE differences implied that splicing might be important for the functionality or regulations of lincRNAs in primates. PMID:25574121

  7. Intergenic Sequence Comparison of Escherichia coli Isolates Reveals Lifestyle Adaptations but Not Host Specificity▿

    PubMed Central

    White, A. P.; Sibley, K. A.; Sibley, C. D.; Wasmuth, J. D.; Schaefer, R.; Surette, M. G.; Edge, T. A.; Neumann, N. F.

    2011-01-01

    Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyzed three promoter-containing intergenic regions from 284 diverse E. coli isolates in an attempt to identify molecular signatures associated with specific host types. Promoter sequences controlling production of curli fimbriae, flagella, and nutrient import yielded a phylogenetic tree with isolates clustered by established phylogenetic grouping (A, B1, B2, and D) but not by host source. Virulence genes were more prevalent in groups B2 and D isolates and in human isolates. Group B1 isolates, primarily from nonhuman sources, were the most genetically similar, indicating that they lacked molecular adaptations to specific host environments and were likely host generalists. Conversely, B2 isolates, primarily from human sources, displayed greater genetic distances and were more likely to be host adapted. In agreement with these hypotheses, prevalence of σS activity and the rdar morphotype, phenotypes associated with environmental survival, were significantly higher in B1 isolates than in B2 isolates. Based on our findings, we speculate that E. coli host specificity is not defined by genome-wide sequence changes but, rather, by the presence or absence of specific genes and associated promoter elements. Furthermore, the requirements for colonization of the human gastrointestinal tract may lead to E. coli lifestyle changes along with selection for increased virulence. PMID:21908635

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

    SciTech Connect

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

    2010-05-07

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

  9. Integrative Analysis of Normal Long Intergenic Non-Coding RNAs in Prostate Cancer.

    PubMed

    Bawa, Pushpinder; Zackaria, Sajna; Verma, Mohit; Gupta, Saurabh; Srivatsan, R; Chaudhary, Bibha; Srinivasan, Subhashini

    2015-01-01

    Recently, large numbers of normal human tissues have been profiled for non-coding RNAs and more than fourteen thousand long intergenic non-coding RNAs (lincRNAs) are found expressed in normal human tissues. The functional roles of these normal lincRNAs (nlincRNAs) in the regulation of protein coding genes in normal and disease biology are yet to be established. Here, we have profiled two RNA-seq datasets including cancer and matched non-neoplastic tissues from 12 individuals from diverse demography for both coding genes and nlincRNAs. We find 130 nlincRNAs significantly regulated in cancer, with 127 regulated in the same direction in the two datasets. Interestingly, according to Illumina Body Map, significant numbers of these nlincRNAs display baseline null expression in normal prostate tissues but are specific to other tissues such as thyroid, kidney, liver and testis. A number of the regulated nlincRNAs share loci with coding genes, which are either co-regulated or oppositely regulated in all cancer samples studied here. For example, in all cancer samples i) the nlincRNA, TCONS_00029157, and a neighboring tumor suppressor factor, SIK1, are both down regulated; ii) several thyroid-specific nlincRNAs in the neighborhood of the thyroid-specific gene TPO, are both up-regulated; and iii) the TCONS_00010581, an isoform of HEIH, is down-regulated while the neighboring EZH2 gene is up-regulated in cancer. Several nlincRNAs from a prostate cancer associated chromosomal locus, 8q24, are up-regulated in cancer along with other known prostate cancer associated genes including PCAT-1, PVT1, and PCAT-92. We observe that there is significant bias towards up-regulation of nlincRNAs with as high as 118 out of 127 up-regulated in cancer, even though regulation of coding genes is skewed towards down-regulation. Considering that all reported cancer associated lincRNAs (clincRNAs) are biased towards up-regulation, we conclude that this bias may be functionally relevant. PMID:25933431

  10. Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation

    PubMed Central

    Garcia, S; Kovařík, A

    2013-01-01

    In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S–5.8S–26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S–18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S–5.8S–26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants. PMID:23512008

  11. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor

    PubMed Central

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters. PMID:26657157

  12. Characterizations of the human parainfluenza type 2 virus gene encoding the L protein and the intergenic sequences.

    PubMed Central

    Kawano, M; Okamoto, K; Bando, H; Kondo, K; Tsurudome, M; Komada, H; Nishio, M; Ito, Y

    1991-01-01

    We cloned and determined the nucleotide sequences of cDNAs against genomic RNA encoding the L protein of human parainfluenza type 2 virus (PIV-2). The L gene is 6904 nucleotides long including the intergenic region at the HN-L junction and putative negative strand leader RNA, almost all of which is complementary to the positive strand leader RNA of PIV-2. The deduced L protein contains 2262 amino acids with a calculated molecular weight of 256,366. The L protein of PIV-2 shows 39.9, 28.9, 27.8 and 28.3% homologies with Newcastle disease virus (NDV), Sendai virus (SV), parainfluenza type 3 virus (PIV-3) and measles virus (MV), respectively. Although sequence data on other components of transcriptive complex, NP and P, suggested a closer relationship between PIV-2 and MV, as concerns the L protein, MV is closely related to another group as SV and PIV-3. From analysis of the alignment of the five l proteins, six blocks composed of conserved amino acids were found in the L proteins. The L protein of PIV-2 was detected in purified virions and virus-infected cells using antiserum directed against an oligopeptide corresponding to the amino terminal region. Primer extension analyses showed that the intergenic regions at the NP-P, P-M, M-F, F-HN and HN-L junctions are 4, 45, 28, 8 and 42 nucleotides long, respectively, indicating that the intergenic regions exhibit no conservation of length and sequence. Furthermore, the starting and ending sequences of paramyxoviruses were summarized. Images PMID:1645865

  13. Quantitative Northern Blot Analysis of Mammalian rRNA Processing.

    PubMed

    Wang, Minshi; Pestov, Dimitri G

    2016-01-01

    Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs. We also describe the ratio analysis of multiple precursors, a technique that facilitates the accurate assessment of changes in the efficiency of individual pre-rRNA processing steps. PMID:27576717

  14. Imprint of Ancient Evolution on rRNA Folding.

    PubMed

    Lanier, Kathryn A; Athavale, Shreyas S; Petrov, Anton S; Wartell, Roger; Williams, Loren Dean

    2016-08-23

    In a model describing the origin and evolution of the translation system, ribosomal RNA (rRNA) grew in size by accretion [Petrov, A. S., et al. (2015) History of the Ribosome and the Origin of Translation. Proc. Natl. Acad. Sci. U.S.A. 112, 15396-15401]. Large rRNAs were built up by iterative incorporation and encasement of small folded RNAs, in analogy with addition of new LEGOs onto the surface of a preexisting LEGO assembly. In this model, rRNA robustness in folding arises from inherited autonomy of local folding. We propose that rRNAs can be decomposed at various granularities, retaining folding mechanism and folding competence. To test these predictions, we disassembled Domain III of the large ribosomal subunit (LSU). We determined whether local rRNA structure, stability, and folding pathways are autonomous. Thermal melting, chemical footprinting, and circular dichroism were used to infer rules that govern folding of rRNA. We deconstructed Domain III of the LSU rRNA by mapping out its complex multistep melting pathway. We studied Domain III and two equal-size "sub-Domains" of Domain III. The combined results are consistent with a model in which melting transitions of Domain III are conserved upon cleavage into sub-Domains. Each of the eight melting transitions of Domain III corresponds in Tm and ΔH with a transition observed in one of the two isolated sub-Domains. The results support a model in which structure, stability, and folding mechanisms are dominated by local interactions and are unaffected by separation of the sub-Domains. Domain III rRNA is distinct from RNAs that form long-range cooperative interaction networks at early stages of folding or that do not fold reversibly. PMID:27428664

  15. Diversity of acetic acid bacteria present in healthy grapes from the Canary Islands.

    PubMed

    Valera, Maria José; Laich, Federico; González, Sara S; Torija, Maria Jesús; Mateo, Estibaliz; Mas, Albert

    2011-11-15

    The identification of acetic acid bacteria (AAB) from sound grapes from the Canary Islands is reported in the present study. No direct recovery of bacteria was possible in the most commonly used medium, so microvinifications were performed on grapes from Tenerife, La Palma and Lanzarote islands. Up to 396 AAB were isolated from those microvinifications and identified by 16S rRNA gene sequencing and phylogenetic analysis. With this method, Acetobacter pasteurianus, Acetobacter tropicalis, Gluconobacter japonicus and Gluconacetobacter saccharivorans were identified. However, no discrimination between the closely related species Acetobacter malorum and Acetobacter cerevisiae was possible. As previously described, 16S-23S rRNA gene internal transcribed spacer (ITS) region phylogenetic analysis was required to classify isolates as one of those species. These two species were the most frequently occurring, accounting for more than 60% of the isolates. For typing the AAB isolates, both the Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and (GTG)5-PCR techniques gave similar resolution. A total of 60 profiles were identified. Thirteen of these profiles were found in more than one vineyard, and only one profile was found on two different islands (Tenerife and La Palma). PMID:21903289

  16. Microcosm enrichment of biphenyl-degrading microbial communities from soils and sediments

    SciTech Connect

    Wagner-Doebler, I.; Bennasar, A.; Stroempl, C.; Bruemmer, I.; Eichner, C.; Grammel, I.; Moore, E.R.B.; Vancanneyt, M.

    1998-08-01

    A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 {times} 10{sup 9} and 2 {times} 10{sup 11} CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxy-biphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences.

  17. Aliterella atlantica gen. nov., sp. nov., and Aliterella antarctica sp. nov., novel members of coccoid Cyanobacteria.

    PubMed

    Rigonato, Janaina; Gama, Watson Arantes; Alvarenga, Danillo Oliveira; Branco, Luis Henrique Zanini; Brandini, Frederico Pereira; Genuário, Diego Bonaldo; Fiore, Marli Fatima

    2016-09-01

    Two Cyanobacteria isolated from South Atlantic Ocean continental shelf deep water and from a marine green algae inhabiting the Admiralty Bay, King George Island, Antarctica were investigated based on morphological and ultrastructural traits, phylogeny of 16S rRNA gene sequences, secondary structure of the 16S-23S internal transcribed spacer regions and phylogenomic analyses. The majority of these evaluations demonstrated that both strains differ from the genera of cyanobacteria with validly published names and, therefore, supported the description of the novel genus as Aliterella gen. nov. The identity and phylogeny of 16S rRNA gene sequences, together with the secondary structure of D1D1' and BoxB intergenic regions, further supported the two strains representing distinct species: Aliterella atlantica gen. nov., sp. nov. (type SP469036, strain CENA595T) and Aliterella antarctica sp. nov. (type SP469035, strain CENA408T). The phylogenomic analysis of A. atlantica sp. nov. CENA595T, based on 21 protein sequences, revealed that this genus belongs to the cyanobacterial order Chroococcidiopsidales. The isolation and cultivation of two geographically distant unicellular members of a novel cyanobacterial genus and the sequenced genome of the type strain bring new insights into the current classification of the coccoid group, and into the reconstruction of their evolutionary history. PMID:27054834

  18. Prevalence of Bartonella species in domestic cats in The Netherlands.

    PubMed Central

    Bergmans, A M; de Jong, C M; van Amerongen, G; Schot, C S; Schouls, L M

    1997-01-01

    Cats have been shown to provide the only known reservoir of Bartonella henselae, the causative agent of cat scratch disease. To determine the prevalence of Bartonella bacteremia and antibodies in Dutch cats, blood samples from 113 cats from shelters (sheltered cats), 50 pet cats, and 25 specific-pathogen-free (SPF) cats were analyzed. Culture and subsequent PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA intergenic region and 16S rRNA gene PCR-hybridization assays revealed a prevalence of Bartonella bacteremia in 22% of the sheltered cats and showed no bacteremia in the SPF cats. Three spacer RFLP types were found: types A, B, and G, with type B being predominant over types A and G. An important finding was the existence of mixtures of different Bartonella species. Bartonella DNA was detected in 7 of 27 DNA extracts from fleas combed from the sheltered cats (26%). Seropositivity was 50% for sheltered cats and 56% for pet cats, as determined by a B. henselae enzyme-linked immunoassay. PMID:9276397

  19. An extremophile Microbacterium strain and its protease production under alkaline conditions.

    PubMed

    Lü, Jin; Wu, Xiaodan; Jiang, Yali; Cai, Xiaofeng; Huang, Luyao; Yang, Yongbo; Wang, Huili; Zeng, Aibing; Li, Aiying

    2014-05-01

    Extremophiles are potential resources for alkaline protease production. In order to search for alkaline protease producers, we isolated and screened alkaliphilic microorganisms from alkaline saline environments. The microorganism HSL10 was identified as a member of the genus Microbacterium by morphological observation, Gram staining and sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region. By colony-forming unit counting under alkali or salt stress, it was further identified as an alkaliphilic microbe with mild halotolerance. In addition, it was capable of secreting alkaline proteases, evidenced by larger hydrolyzation zones in the skim milk-containing medium at pH 9.0 than at pH 7.0. Subsequently, we demonstrated that both NaCl and yeast extract significantly promoted protease production by HSL10. Finally, we established a sensitive colorimetric method for the detection of protease production by HSL10 under neutral and alkaline conditions, by using the Bradford reagent for substrate staining to improve the contrast between the hydrolyzation zone and the substrate background on agar plates. HSL10 was the first example of an alkaliphilic protease-producing member in Microbacterium, and its isolation and characterization have both academic and commercial importance. PMID:23686381

  20. Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.

    PubMed

    Grayson, T H; Cooper, L F; Atienzar, F A; Knowles, M R; Gilpin, M L

    1999-03-01

    Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences. PMID:10049848

  1. Molecular Differentiation of Renibacterium salmoninarum Isolates from Worldwide Locations

    PubMed Central

    Grayson, Thomas H.; Cooper, Lynne F.; Atienzar, Franck A.; Knowles, Mark R.; Gilpin, Martyn L.

    1999-01-01

    Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences. PMID:10049848

  2. Identification and Functional Prediction of Large Intergenic Noncoding RNAs (lincRNAs) in Rainbow Trout (Oncorhynchus mykiss).

    PubMed

    Wang, Jian; Fu, Liyuan; Koganti, Prasanthi P; Wang, Lei; Hand, Jacqelyn M; Ma, Hao; Yao, Jianbo

    2016-04-01

    Long noncoding RNAs (lncRNAs) have been recognized in recent years as key regulators of diverse cellular processes. Genome-wide large-scale projects have uncovered thousands of lncRNAs in many model organisms. Large intergenic noncoding RNAs (lincRNAs) are lncRNAs that are transcribed from intergenic regions of genomes. To date, no lincRNAs in non-model teleost fish have been reported. In this report, we present the first reference catalog of 9674 rainbow trout lincRNAs based on analysis of RNA-Seq data from 15 tissues. Systematic analysis revealed that lincRNAs in rainbow trout share many characteristics with those in other mammalian species. They are shorter and lower in exon number and expression level compared with protein-coding genes. They show tissue-specific expression pattern and are typically co-expressed with their neighboring genes. Co-expression network analysis suggested that many lincRNAs are associated with immune response, muscle differentiation, and neural development. The study provides an opportunity for future experimental and computational studies to uncover the functions of lincRNAs in rainbow trout. PMID:26864089

  3. All-Trans Retinoic Acid Induces Expression of a Novel Intergenic Long Noncoding RNA in Adult rat Primary Hippocampal Neurons.

    PubMed

    Kour, Sukhleen; Rath, Pramod C

    2016-02-01

    Around 90% of the mammalian genome undergoes pervasive transcription into various types of small and long regulatory noncoding RNAs, whereas only ∼ 1.5% codes for proteins. Long noncoding RNAs (lncRNAs) constitute diverse classes of sense- and antisense transcripts that are abundantly expressed in the mammalian central nervous system (CNS) in cell type- and developmental stage-specific manners. They are implicated in brain development, differentiation, neuronal plasticity, and other cognitive functions. Mammalian brain requires the vitamin A metabolite all-trans retinoic acid (atRA) for its normal development, differentiation, and cell-fate determination. However, its role in adult brain function is less understood. Here, we report atRA-mediated transcriptional upregulation of endogenous expression of a novel long intergenic noncoding RNA-rat brain expressed (LINC-RBE) in cultured primary hippocampal neurons from adult rat. We have previously reported LINC-RBE as an intergenic, simple repeat sequence containing lncRNA highly expressed in the rat brain. This is a first-time report of involvement of atRA in transcriptional upregulation of lncRNA expression in rat hippocampal neurons. Therefore, it may be involved in regulation of brain function and disease. PMID:26572536

  4. The CASC15 long intergenic non-coding RNA locus is involved in melanoma progression and phenotype-switching

    PubMed Central

    Lessard, Laurent; Liu, Michelle; Marzese, Diego M.; Wang, Hongwei; Chong, Kelly; Kawas, Neal; Donovan, Nicholas C; Kiyohara, Eiji; Hsu, Sandy; Nelson, Nellie; Izraely, Sivan; Sagi-Assif, Orit; Witz, Isaac P; Ma, Xiao-Jun; Luo, Yuling; Hoon, Dave SB

    2015-01-01

    In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long non-coding RNAs in melanoma progression. We hypothesized that copy number alterations of intergenic non-protein coding domains could help identify long intergenic non-coding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and up-regulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC stage III lymph node metastasis. Moreover, siRNA knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma. PMID:26016895

  5. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical

  6. rRNA fragmentation induced by a yeast killer toxin.

    PubMed

    Kast, Alene; Klassen, Roland; Meinhardt, Friedhelm

    2014-02-01

    Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins. PMID:24308908

  7. Regulation of Arabidopsis thaliana 5S rRNA Genes.

    PubMed

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

    2007-05-01

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

  8. Capturing intergenerativity: the use of student reflective journals to identify learning within an undergraduate course in gerontological nursing.

    PubMed

    Davies, Susan M; Reitmaier, Amy B; Smith, Linda Reveling; Mangan-Danckwart, Deborah

    2013-03-01

    The benefits of intergenerational contact between older and young adults have been demonstrated; yet, nursing programs have underexplored the potential of such relationships for enhancing student learning. This article presents an analysis of student reflective journals as part of an evaluation of an undergraduate gerontological nursing course. The course aims to create positive learning experiences by involving older adults as partners in student learning. Older adults are recruited to receive visits from a designated student to share aspects of their life and experiences. Students write reflective journals based on these visits as a method of evaluating their learning. A framework analysis of 80 journals completed by 59 students identified four major themes representing the impact of these visits on student learning: becoming aware, making connections, seeing the unique person, and valuing intergenerational relationships. The analysis suggests the relevance of the concept of intergenerativity in illuminating shared benefits of the practicum experience. PMID:23402281

  9. Genome-wide CpG island methylation and intergenic demethylation propensities vary among different tumor sites.

    PubMed

    Lee, Seung-Tae; Wiemels, Joseph L

    2016-02-18

    The epigenetic landscape of cancer includes both focal hypermethylation and broader hypomethylation in a genome-wide manner. By means of a comprehensive genomic analysis on 6637 tissues of 21 tumor types, we here show that the degrees of overall methylation in CpG island (CGI) and demethylation in intergenic regions, defined as 'backbone', largely vary among different tumors. Depending on tumor type, both CGI methylation and backbone demethylation are often associated with clinical, epidemiological and biological features such as age, sex, smoking history, anatomic location, histological type and grade, stage, molecular subtype and biological pathways. We found connections between CGI methylation and hypermutability, microsatellite instability, IDH1 mutation, 19p gain and polycomb features, and backbone demethylation with chromosomal instability, NSD1 and TP53 mutations, 5q and 19p loss and long repressive domains. These broad epigenetic patterns add a new dimension to our understanding of tumor biology and its clinical implications. PMID:26464434

  10. H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA

    PubMed Central

    Larochelle, Marc; Gaudreau, Luc

    2003-01-01

    H2A.Z has been shown to regulate transcription in yeast, and that function resides in its C-terminal region as the reciprocal portion of H2A cannot substitute for the latter. We show that fusion of a transcriptional activating region to the C-terminal region of H2A, which is substituted for that of H2A.Z, can allow the chimera to fulfil the special role of H2A.Z in positive gene regulation, as well as complement growth deficiencies of htz1Δ cells. We further show that the ‘transcription’ function of H2A.Z is linked to its ability to preferentially localize to certain intergenic DNA regions. Our results suggest that H2A.Z modulates functional interactions with transcription regulatory components, and thus increases its localization to promoters where it helps poise chromatin for gene activation. PMID:12941702

  11. Genome-wide CpG island methylation and intergenic demethylation propensities vary among different tumor sites

    PubMed Central

    Lee, Seung-Tae; Wiemels, Joseph L.

    2016-01-01

    The epigenetic landscape of cancer includes both focal hypermethylation and broader hypomethylation in a genome-wide manner. By means of a comprehensive genomic analysis on 6637 tissues of 21 tumor types, we here show that the degrees of overall methylation in CpG island (CGI) and demethylation in intergenic regions, defined as ‘backbone’, largely vary among different tumors. Depending on tumor type, both CGI methylation and backbone demethylation are often associated with clinical, epidemiological and biological features such as age, sex, smoking history, anatomic location, histological type and grade, stage, molecular subtype and biological pathways. We found connections between CGI methylation and hypermutability, microsatellite instability, IDH1 mutation, 19p gain and polycomb features, and backbone demethylation with chromosomal instability, NSD1 and TP53 mutations, 5q and 19p loss and long repressive domains. These broad epigenetic patterns add a new dimension to our understanding of tumor biology and its clinical implications. PMID:26464434

  12. Conservation of the Exon-Intron Structure of Long Intergenic Non-Coding RNA Genes in Eutherian Mammals.

    PubMed

    Chernikova, Diana; Managadze, David; Glazko, Galina V; Makalowski, Wojciech; Rogozin, Igor B

    2016-01-01

    The abundance of mammalian long intergenic non-coding RNA (lincRNA) genes is high, yet their functions remain largely unknown. One possible way to study this important question is to use large-scale comparisons of various characteristics of lincRNA with those of protein-coding genes for which a large body of functional information is available. A prominent feature of mammalian protein-coding genes is the high evolutionary conservation of the exon-intron structure. Comparative analysis of putative intron positions in lincRNA genes from various mammalian genomes suggests that some lincRNA introns have been conserved for over 100 million years, thus the primary and/or secondary structure of these molecules is likely to be functionally important. PMID:27429005

  13. Alternate rRNA secondary structures as regulators of translation.

    PubMed

    Feng, Shu; Li, Heng; Zhao, Jing; Pervushin, Konstantin; Lowenhaupt, Ky; Schwartz, Thomas U; Dröge, Peter

    2011-02-01

    Structural dynamics of large molecular assemblies are intricately linked to function. For ribosomes, macromolecular changes occur especially during mRNA translation and involve participation of ribosomal RNA. Without suitable probes specific to RNA secondary structure, however, elucidation of more subtle dynamic ribosome structure-function relationships, especially in vivo, remains challenging. Here we report that the Z-DNA- and Z-RNA-binding domain Zα, derived from the human RNA editing enzyme ADAR1-L, binds with high stability to specific rRNA segments of Escherichia coli and human ribosomes. Zα impaired in Z-RNA recognition does not associate with ribosomes. Notably, Zα(ADAR1)-ribosome interaction blocks translation in vitro and in vivo, with substantial physiological consequences. Our study shows that ribosomes can be targeted by a protein that specifically recognizes an alternate rRNA secondary structure, and suggests a new mechanism of translational regulation on the ribosome. PMID:21217697

  14. Evolutionary history of the COII/tRNALys intergenic 9 base pair deletion in human mitochondrial DNAs from the Pacific.

    PubMed

    Redd, A J; Takezaki, N; Sherry, S T; McGarvey, S T; Sofro, A S; Stoneking, M

    1995-07-01

    Length changes in human mitochondrial DNA (mtDNA) are potentially useful markers for inferring the evolutionary history of populations. One such length change is a nine base pair (9-bp) deletion that is located in the intergenic region between the COII gene and the Lysine tRNA gene (COII/tRNALys intergenic region). This deletion has been used as a genetic marker to trace descent from peoples of East Asian origin. A geographic cline of the deletion frequency across modern Pacific Islander populations suggests that the deletion may be useful for tracing prehistoric Polynesian origins and affinities. Mitochondrial DNA sequence variation within two variable segments of the control region (CR) permits a number of inferences regarding the evolutionary history of the 9-bp deletion that cannot be determined from frequency data alone. We obtained CR sequences from 74 mtDNAs with the 9-bp deletion from Indonesia, coastal Papua New Guinea (PNG), and American Samoa. Phylogenetic and pairwise distribution analysis of these CR sequences pooled with previously published CR sequences reveals that the deletion arose independently in Africa and Asia and suggests possible multiple origins of the deletion in Asia. A clinal increase of the frequency of the 9-bp deletion across the three Pacific populations is associated with a decrease in CR sequence diversity, consistent with founder events. Furthermore, analysis of pairwise difference distributions indicates an expansion time of proto-Polynesians that began 5,500 yr ago from Southeast Asia. These results are consistent with the express train model of Polynesian origins. PMID:7659016

  15. Genic and Intergenic SSR Database Generation, SNPs Determination and Pathway Annotations, in Date Palm (Phoenix dactylifera L.)

    PubMed Central

    2016-01-01

    The present investigation was carried out aiming to use the bioinformatics tools in order to identify and characterize, simple sequence repeats within the third Version of the date palm genome and develop a new SSR primers database. In addition single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions were recognized. Moreover, the pathways for the sequences assigned by SSR primers, the biological functions and gene interaction were determined. A total of 172,075 SSR motifs was identified on date palm genome sequence with a frequency of 450.97 SSRs per Mb. Out of these, 130,014 SSRs (75.6%) were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, only 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. A total of 111,403 of SSR primer pairs were designed, that represents 291.9 SSR primers per Mb. Out of the 111,403, only 31,380 SSR primers were in the genic regions, while 80,023 primers were in the intergenic regions. A number of 250,507 SNPs were recognized in 84,172 SSR flanking regions, which represents 75.55% of the total SSR flanking regions. Out of 12,274 genes only 463 genes comprising 896 SSR primers were mapped onto 111 pathways using KEGG data base. The most abundant enzymes were identified in the pathway related to the biosynthesis of antibiotics. We tested 1031 SSR primers using both publicly available date palm genome sequences as templates in the in silico PCR reactions. Concerning in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars. PMID:27434138

  16. Diversification of the light-harvesting complex gene family via intra- and intergenic duplications in the coral symbiotic alga Symbiodinium.

    PubMed

    Maruyama, Shinichiro; Shoguchi, Eiichi; Satoh, Nori; Minagawa, Jun

    2015-01-01

    The light-harvesting complex (LHC) is an essential component in light energy capture and transduction to facilitate downstream photosynthetic reactions in plant and algal chloroplasts. The unicellular dinoflagellate alga Symbiodinium is an endosymbiont of cnidarian animals, including corals and sea anemones, and provides carbohydrates generated through photosynthesis to host animals. Although Symbiodinium possesses a unique LHC gene family, called chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC), its genome-level diversity and evolutionary trajectories have not been investigated. Here, we describe a phylogenetic analysis revealing that many of the LHCs are encoded by highly duplicated genes with multi-subunit polyprotein structures in the nuclear genome of Symbiodinium minutum. This analysis provides an extended list of the LHC gene family in a single organism, including 80 loci encoding polyproteins composed of 145 LHC subunits recovered in the phylogenetic tree. In S. minutum, 5 phylogenetic groups of the Lhcf-type gene family, which is exclusively conserved in algae harboring secondary plastids of red algal origin, were identified. Moreover, 5 groups of the Lhcr-type gene family, of which members are known to be associated with PSI in red algal plastids and secondary plastids of red algal origin, were identified. Notably, members classified within a phylogenetic group of the Lhcf-type (group F1) are highly duplicated, which may explain the presence of an unusually large number of LHC genes in this species. Some gene units were homologous to other units within single loci of the polyprotein genes, whereas intergenic homologies between separate loci were conspicuous in other cases, implying that gene unit 'shuffling' by gene conversion and/or genome rearrangement might have been a driving force for diversification. These results suggest that vigorous intra- and intergenic gene duplication events have resulted in the genomic framework of

  17. Diversification of the Light-Harvesting Complex Gene Family via Intra- and Intergenic Duplications in the Coral Symbiotic Alga Symbiodinium

    PubMed Central

    Maruyama, Shinichiro; Shoguchi, Eiichi; Satoh, Nori; Minagawa, Jun

    2015-01-01

    The light-harvesting complex (LHC) is an essential component in light energy capture and transduction to facilitate downstream photosynthetic reactions in plant and algal chloroplasts. The unicellular dinoflagellate alga Symbiodinium is an endosymbiont of cnidarian animals, including corals and sea anemones, and provides carbohydrates generated through photosynthesis to host animals. Although Symbiodinium possesses a unique LHC gene family, called chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC), its genome-level diversity and evolutionary trajectories have not been investigated. Here, we describe a phylogenetic analysis revealing that many of the LHCs are encoded by highly duplicated genes with multi-subunit polyprotein structures in the nuclear genome of Symbiodinium minutum. This analysis provides an extended list of the LHC gene family in a single organism, including 80 loci encoding polyproteins composed of 145 LHC subunits recovered in the phylogenetic tree. In S. minutum, 5 phylogenetic groups of the Lhcf-type gene family, which is exclusively conserved in algae harboring secondary plastids of red algal origin, were identified. Moreover, 5 groups of the Lhcr-type gene family, of which members are known to be associated with PSI in red algal plastids and secondary plastids of red algal origin, were identified. Notably, members classified within a phylogenetic group of the Lhcf-type (group F1) are highly duplicated, which may explain the presence of an unusually large number of LHC genes in this species. Some gene units were homologous to other units within single loci of the polyprotein genes, whereas intergenic homologies between separate loci were conspicuous in other cases, implying that gene unit ‘shuffling’ by gene conversion and/or genome rearrangement might have been a driving force for diversification. These results suggest that vigorous intra- and intergenic gene duplication events have resulted in the genomic framework of

  18. Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

    PubMed Central

    Osterås, M; Stanley, J; Finan, T M

    1995-01-01

    Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum. PMID:7559334

  19. Genic and Intergenic SSR Database Generation, SNPs Determination and Pathway Annotations, in Date Palm (Phoenix dactylifera L.).

    PubMed

    Mokhtar, Morad M; Adawy, Sami S; El-Assal, Salah El-Din S; Hussein, Ebtissam H A

    2016-01-01

    The present investigation was carried out aiming to use the bioinformatics tools in order to identify and characterize, simple sequence repeats within the third Version of the date palm genome and develop a new SSR primers database. In addition single nucleotide polymorphisms (SNPs) that are located within the SSR flanking regions were recognized. Moreover, the pathways for the sequences assigned by SSR primers, the biological functions and gene interaction were determined. A total of 172,075 SSR motifs was identified on date palm genome sequence with a frequency of 450.97 SSRs per Mb. Out of these, 130,014 SSRs (75.6%) were located within the intergenic regions with a frequency of 499 SSRs per Mb. While, only 42,061 SSRs (24.4%) were located within the genic regions with a frequency of 347.5 SSRs per Mb. A total of 111,403 of SSR primer pairs were designed, that represents 291.9 SSR primers per Mb. Out of the 111,403, only 31,380 SSR primers were in the genic regions, while 80,023 primers were in the intergenic regions. A number of 250,507 SNPs were recognized in 84,172 SSR flanking regions, which represents 75.55% of the total SSR flanking regions. Out of 12,274 genes only 463 genes comprising 896 SSR primers were mapped onto 111 pathways using KEGG data base. The most abundant enzymes were identified in the pathway related to the biosynthesis of antibiotics. We tested 1031 SSR primers using both publicly available date palm genome sequences as templates in the in silico PCR reactions. Concerning in vitro validation, 31 SSR primers among those used in the in silico PCR were synthesized and tested for their ability to detect polymorphism among six Egyptian date palm cultivars. All tested primers have successfully amplified products, but only 18 primers detected polymorphic amplicons among the studied date palm cultivars. PMID:27434138

  20. Analysis of the CYP21A2 gene with intergenic recombination and multiple gene deletions in the RCCX module.

    PubMed

    Chang, Shwu-Fen; Lee, Hsien-Hsiung

    2011-01-01

    The most frequent bimodular RCCX module of the RP1-C4A-CYP21A1P-TNXA-RP2-C4B-CYP21A2-TNXB gene sequence is located on chromosome 6p21.3. To determine RCCX alterations, we used the polymerase chain reaction (PCR) product containing the tenascin B (TNXB) and CYP21A2 genes with TaqI digestion and Southern blot analysis with AseI and NdeI endonuclease digestion of genomic DNA from congenital adrenal hyperplasia patients with common mutations resulting from an intergenic conversion of CYP21A1P, such as an I2 splice, I172N, V281L, F306-L307insT, Q318X, and R356W, and dual mutations of I236N/V237E in the CYP21A2 gene. The results showed that a 3.7-kb fragment of the CYP21A2 gene was detected in each case, and 21.6- and 11.3-kb DNA fragments were found in the RCCX region by a Southern blot analysis with these corresponding mutations. However, the IVS2-12A/C- > G (I2 splice) haplotype in combination with the 707-714delGAGACTAC (without the P30L mutation) mutation produced a 3.2-kb TaqI fragment in the PCR product analysis and a specific 9.3-kb fragment by the Southern blot method. Therefore, we concluded that the rearrangement in the RCCX region resulting from processing of either an intergenic recombination or multiple gene deletions can be identified by the PCR analysis and Southern blot method based on a fragment-distinguishing configuration without a family study. PMID:21117955

  1. Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single strand conformation polymorphism (SSCP). Diversity indexes sh...

  2. Robust Computational Analysis of rRNA Hypervariable Tag Datasets

    PubMed Central

    Sipos, Maksim; Jeraldo, Patricio; Chia, Nicholas; Qu, Ani; Dhillon, A. Singh; Konkel, Michael E.; Nelson, Karen E.; White, Bryan A.; Goldenfeld, Nigel

    2010-01-01

    Next-generation DNA sequencing is increasingly being utilized to probe microbial communities, such as gastrointestinal microbiomes, where it is important to be able to quantify measures of abundance and diversity. The fragmented nature of the 16S rRNA datasets obtained, coupled with their unprecedented size, has led to the recognition that the results of such analyses are potentially contaminated by a variety of artifacts, both experimental and computational. Here we quantify how multiple alignment and clustering errors contribute to overestimates of abundance and diversity, reflected by incorrect OTU assignment, corrupted phylogenies, inaccurate species diversity estimators, and rank abundance distribution functions. We show that straightforward procedural optimizations, combining preexisting tools, are effective in handling large () 16S rRNA datasets, and we describe metrics to measure the effectiveness and quality of the estimators obtained. We introduce two metrics to ascertain the quality of clustering of pyrosequenced rRNA data, and show that complete linkage clustering greatly outperforms other widely used methods. PMID:21217830

  3. A cis-regulatory sequence from a short intergenic region gives rise to a strong microbe-associated molecular pattern-responsive synthetic promoter.

    PubMed

    Lehmeyer, Mona; Hanko, Erik K R; Roling, Lena; Gonzalez, Lilian; Wehrs, Maren; Hehl, Reinhard

    2016-06-01

    The high gene density in Arabidopsis thaliana leaves only relatively short intergenic regions for potential cis-regulatory sequences. To learn more about the regulation of genes harbouring only very short upstream intergenic regions, this study investigates a recently identified novel microbe-associated molecular pattern (MAMP)-responsive cis-sequence located within the 101 bp long intergenic region upstream of the At1g13990 gene. It is shown that the cis-regulatory sequence is sufficient for MAMP-responsive reporter gene activity in the context of its native promoter. The 3' UTR of the upstream gene has a quantitative effect on gene expression. In context of a synthetic promoter, the cis-sequence is shown to achieve a strong increase in reporter gene activity as a monomer, dimer and tetramer. Mutation analysis of the cis-sequence determined the specific nucleotides required for gene expression activation. In transgenic A. thaliana the synthetic promoter harbouring a tetramer of the cis-sequence not only drives strong pathogen-responsive reporter gene expression but also shows a high background activity. The results of this study contribute to our understanding how genes with very short upstream intergenic regions are regulated and how these regions can serve as a source for MAMP-responsive cis-sequences for synthetic promoter design. PMID:26833485

  4. A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting f...

  5. Evaluation of a dkgB linked intergenic sequence ribotyping (ISR) method for assigning serotype to Salmonella enterica isolated from poultry environmental samples.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Kauffman White (KW) serotyping method requires more than 250 antisera to characterize more than 2,500 Salmonella serovars. The complexity of serotyping could be overcome using molecular methods. In this study, a dkgB-linked intergenic sequence ribotyping (ISR) method that generates sequence occu...

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

    PubMed

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

    2005-09-01

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

  7. Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting

    PubMed Central

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  8. Microdiversity of deep-sea Bacillales isolated from Tyrrhenian sea sediments as revealed by ARISA, 16S rRNA gene sequencing and BOX-PCR fingerprinting.

    PubMed

    Ettoumi, Besma; Guesmi, Amel; Brusetti, Lorenzo; Borin, Sara; Najjari, Afef; Boudabous, Abdellatif; Cherif, Ameur

    2013-01-01

    With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments. PMID:24005887

  9. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  10. Phylogenetic analysis of oryx species using partial sequences of mitochondrial rRNA genes.

    PubMed

    Khan, H A; Arif, I A; Al Farhan, A H; Al Homaidan, A A

    2008-01-01

    We conducted a comparative evaluation of 12S rRNA and 16S rRNA genes of the mitochondrial genome for molecular differentiation among three oryx species (Oryx leucoryx, Oryx dammah and Oryx gazella) with respect to two closely related outgroups, addax and roan. Our findings showed the failure of 12S rRNA gene to differentiate between the genus Oryx and addax, whereas a 342-bp partial sequence of 16S rRNA accurately grouped all five taxa studied, suggesting the utility of 16S rRNA segment for molecular phylogeny of oryx at the genus and possibly species levels. PMID:19048493

  11. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  12. Interactions of aminoglycoside antibiotics with rRNA.

    PubMed

    Trylska, Joanna; Kulik, Marta

    2016-08-15

    Aminoglycoside antibiotics are protein synthesis inhibitors applied to treat infections caused mainly by aerobic Gram-negative bacteria. Due to their adverse side effects they are last resort antibiotics typically used to combat pathogens resistant to other drugs. Aminoglycosides target ribosomes. We describe the interactions of aminoglycoside antibiotics containing a 2-deoxystreptamine (2-DOS) ring with 16S rRNA. We review the computational studies, with a focus on molecular dynamics (MD) simulations performed on RNA models mimicking the 2-DOS aminoglycoside binding site in the small ribosomal subunit. We also briefly discuss thermodynamics of interactions of these aminoglycosides with their 16S RNA target. PMID:27528743

  13. Growth rate regulation of rRNA content of a marine Synechococcus (cyanobacterium) strain

    SciTech Connect

    Binder, B.J.; Liu, Y.C.

    1998-09-01

    The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined. A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates. The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells. The relationship between growth rate and cellular rRNA content comprised three phases: (1) at low growth rates, rRNA cell{sup {minus}1} remained approximately constant; (2) at intermediate rates, rRNA cell{sup {minus}1} increased proportionally with growth rate; and (3) at the highest, light-saturated rates, rRNA cell{sup {minus}1} dropped abruptly. Total cellular RNA was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate. Mean cell volume and rRNA concentration were related to growth rate in a manner similar to rRNA cell{sup {minus}1}, although the overall magnitude linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates. Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.

  14. 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

  15. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules.

    PubMed

    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

  16. Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

    PubMed

    Su, Y-L; Feng, J; Li, Y-W; Bai, J-S; Li, A-X

    2016-02-01

    Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish. PMID:25858765

  17. Detection of Bartonella henselae and Bartonella clarridgeiae DNA in hepatic specimens from two dogs with hepatic disease.

    PubMed

    Gillespie, Tracey N; Washabau, Robert J; Goldschmidt, Michael H; Cullen, John M; Rogala, Allison R; Breitschwerdt, Edward B

    2003-01-01

    A 4-year-old Basset Hound and a 6-year-old Doberman Pinscher were referred for diagnostic evaluation following documentation of persistently increased hepatic enzyme activities and hepatic dysfunction. Histologic evaluation of hepatic biopsy specimens from the 2 dogs revealed granulomatous hepatitis in the Basset Hound and lymphocytic hepatitis with fibrosis and copper accumulation in the Doberman Pinscher. No etiologic agents were identified histologically. Bartonella henselae DNA was subsequently amplified from hepatic tissue from the Basset Hound and Bartonella clarridgeiae was amplified from hepatic tissue from the Doberman Pinscher. Amplification was performed with a polymerase chain reaction assay incorporating primers that target a portion of the 16S-23S rRNA intergenic spacer region. Both dogs were treated with azithromycin, in combination with a variety of other medications and herbal treatments, and improved clinically. Identification of Bartonella DNA in these dogs indicates the need for future prospective studies to determine the clinical relevance of Bartonella spp infection in dogs with hepatic disease. PMID:12523479

  18. Diversity and antimicrobial properties of lactic acid bacteria isolated from rhizosphere of olive trees and desert truffles of Tunisia.

    PubMed

    Fhoula, Imene; Najjari, Afef; Turki, Yousra; Jaballah, Sana; Boudabous, Abdelatif; Ouzari, Hadda

    2013-01-01

    A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota. PMID:24151598

  19. Bacterial Functional Redundancy along a Soil Reclamation Gradient

    PubMed Central

    Yin, Bei; Crowley, David; Sparovek, Gerd; De Melo, Wanderley Jose; Borneman, James

    2000-01-01

    A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU). BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site. The number and diversity of bacterial groups responding to four carbon substrates (l-serine, l-threonine, sodium citrate, and α-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning. PMID:11010883

  20. Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.

    PubMed

    Ding, Z F; Xia, S Y; Xue, H; Tang, J Q; Ren, Q; Gu, W; Meng, Q G; Wang, W

    2015-09-01

    Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen. PMID:25167936

  1. Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease).

    PubMed Central

    Kordick, D L; Hilyard, E J; Hadfield, T L; Wilson, K H; Steigerwalt, A G; Brenner, D J; Breitschwerdt, E B

    1997-01-01

    Shortly after adopting a 6-week-old cat, a veterinarian was bitten on the left index finger. Within 3 weeks, he developed headache, fever, and left axillary lymphadenopathy. Initial blood cultures from the cat and veterinarian were sterile. Repeat cultures from the cat grew Bartonella-like organisms with lophotrichous flagella. Sera from the veterinarian were not reactive against Bartonella henselae, B. quintana, or B. elizabethae antigens but were seroreactive (reciprocal titer, 1,024) against the feline isolate. Sequential serum samples from the cat were reactive against antigens of B. henselae (titer, 1,024), B. quintana (titer, 128), and the feline isolate (titer, 2,048). Phenotypic and genotypic characterization of this and six additional feline isolates, including microscopic evaluation, biochemical analysis, 16S rRNA gene sequencing, DNA-DNA hybridization, and PCR-restriction fragment length polymorphism of the 16S gene, 16S-23S intergenic spacer region, and citrate synthase gene identified the isolates as B. clarridgeiae. This is the first report of cat scratch disease associated with B. clarridgeiae. PMID:9196200

  2. Diversity and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Rhizosphere of Olive Trees and Desert Truffles of Tunisia

    PubMed Central

    Najjari, Afef; Turki, Yousra; Jaballah, Sana; Boudabous, Abdelatif; Ouzari, Hadda

    2013-01-01

    A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota. PMID:24151598

  3. Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains▿

    PubMed Central

    Kim, Hye-Sook; Ma, Bing; Perna, Nicole T.; Charkowski, Amy O.

    2009-01-01

    Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers. PMID:19411432

  4. Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

    PubMed Central

    Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

    2006-01-01

    A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

  5. The rRNA evolution and procaryotic phylogeny

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1986-01-01

    Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

  6. Higher-order structure of rRNA

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Woese, C. R.

    1986-01-01

    A comparative search for phylogenetically covarying basepair replacements within potential helices has been the only reliable method to determine the correct secondary structure of the 3 rRNAs, 5S, 16S, and 23S. The analysis of 16S from a wide phylogenetic spectrum, that includes various branches of the eubacteria, archaebacteria, eucaryotes, in addition to the mitochondria and chloroplast, is beginning to reveal the constraints on the secondary structures of these rRNAs. Based on the success of this analysis, and the assumption that higher order structure will also be phylogenetically conserved, a comparative search was initiated for positions that show co-variation not involved in secondary structure helices. From a list of potential higher order interactions within 16S rRNA, two higher-order interactions are presented. The first of these interactions involves positions 570 and 866. Based on the extent of phylogenetic covariation between these positions while maintaining Watson-Crick pairing, this higher-order interaction is considered proven. The other interaction involves a minimum of six positions between the 1400 and 1500 regions of the 16S rRNA. Although these patterns of covariation are not as striking as the 570/866 interaction, the fact that they all exist in an anti-parallel fashion and that experimental methods previously implicated these two regions of the molecule in tRNA function suggests that these interactions be given serious consideration.

  7. Chromosomal Organization of Rrna Operons in Bacillus Subtilis

    PubMed Central

    Jarvis, E. D.; Widom, R. L.; LaFauci, G.; Setoguchi, Y.; Richter, I. R.; Rudner, R.

    1988-01-01

    Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70° on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent ``hot spots'' of plasmid insertion. PMID:2465199

  8. Functional analysis of long intergenic non-coding RNAs in phosphate-starved rice using competing endogenous RNA network

    PubMed Central

    Xu, Xi-Wen; Zhou, Xiong-Hui; Wang, Rui-Ru; Peng, Wen-Lei; An, Yue; Chen, Ling-Ling

    2016-01-01

    Long intergenic non-coding RNAs (lincRNAs) may play widespread roles in gene regulation and other biological processes, however, a systematic examination of the functions of lincRNAs in the biological responses of rice to phosphate (Pi) starvation has not been performed. Here, we used a computational method to predict the functions of lincRNAs in Pi-starved rice. Overall, 3,170 lincRNA loci were identified using RNA sequencing data from the roots and shoots of control and Pi-starved rice. A competing endogenous RNA (ceRNA) network was constructed for each tissue by considering the competing relationships between lincRNAs and genes, and the correlations between the expression levels of RNAs in ceRNA pairs. Enrichment analyses showed that most of the communities in the networks were related to the biological processes of Pi starvation. The lincRNAs in the two tissues were individually functionally annotated based on the ceRNA networks, and the differentially expressed lincRNAs were biologically meaningful. For example, XLOC_026030 was upregulated from 3 days after Pi starvation, and its functional annotation was ‘cellular response to Pi starvation’. In conclusion, we systematically annotated lincRNAs in rice and identified those involved in the biological response to Pi starvation. PMID:26860696

  9. The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons.

    PubMed

    Hopp, Crystal M; Gardner, Jeffrey F; Salyers, Abigail A

    2015-09-01

    CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes. PMID:26212728

  10. Detection of spatial and temporal influences on bacterial communities in an urban stream by automated ribosomal intergenic ribosomal spacer analysis.

    PubMed

    Or, Amitai; Gophna, Uri

    2011-01-01

    The Yarqon is the largest urban river in Israel, and is a slow-flowing stream whose water originates mostly from wastewater treatment plants. Thus, its microbial community is expected to be heavily impacted both by anthropogenic factors and by seasonal temporal variation. In order to identify the main factors that influence the bacterial community, and their spatial-temporal variation, 50 samples were collected representing five different time points and eleven locations. Samples were analyzed for biotic and a-biotic parameters and the bacterial populations were analyzed by Automated Ribosomal Intergenic Spacer Analysis (ARISA). Bacterial richness and diversity were calculated and compared across samples. Canonical Correspondence Analysis (CCA) showed that ARISA clustered the samples according to temporal variation. Molecular fingerprinting analysis provided a snapshot of the microbial community and showed good correlation with geochemical parameters, despite the rapid changes of the Mediterranean environment and the anthropogenic impact. Molecular fingerprinting methods based on natural fragment length polymorphisms may therefore represent a supplementary approach for stream monitoring, alongside physico-chemical measurements. PMID:21869567

  11. Evaluation of automated ribosomal intergenic spacer analysis for bacterial fingerprinting of rumen microbiome compared to pyrosequencing technology.

    PubMed

    Jami, Elie; Shterzer, Naama; Mizrahi, Itzhak

    2014-01-01

    The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA), an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data. PMID:25437610

  12. Evaluation of Automated Ribosomal Intergenic Spacer Analysis for Bacterial Fingerprinting of Rumen Microbiome Compared to Pyrosequencing Technology

    PubMed Central

    Jami, Elie; Shterzer, Naama; Mizrahi, Itzhak

    2014-01-01

    The mammalian gut houses a complex microbial community which is believed to play a significant role in host physiology. In recent years, several microbial community analysis methods have been implemented to study the whole gut microbial environment, in contrast to classical microbiological methods focusing on bacteria which can be cultivated. One of these is automated ribosomal intergenic spacer analysis (ARISA), an inexpensive and popular way of analyzing bacterial diversity and community fingerprinting in ecological samples. ARISA uses the natural variability in length of the DNA fragment found between the 16S and 23S genes in different bacterial lineages to infer diversity. This method is now being supplanted by affordable next-generation sequencing technologies that can also simultaneously annotate operational taxonomic units for taxonomic identification. We compared ARISA and pyrosequencing of samples from the rumen microbiome of cows, previously sampled at different stages of development and varying in microbial complexity using several ecological parameters. We revealed close agreement between ARISA and pyrosequencing outputs, especially in their ability to discriminate samples from different ecological niches. In contrast, the ARISA method seemed to underestimate sample richness. The good performance of the relatively inexpensive ARISA makes it relevant for straightforward use in bacterial fingerprinting analysis as well as for quick cross-validation of pyrosequencing data. PMID:25437610

  13. Identification of chimeric TSNAX-DISC1 resulting from intergenic splicing in endometrial carcinoma through high-throughput RNA sequencing.

    PubMed

    Li, Na; Zheng, Jian; Li, Hua; Deng, Jieqiong; Hu, Min; Wu, Hongchun; Li, Wei; Li, Fang; Lan, Xun; Lu, Jiachun; Zhou, Yifeng

    2014-12-01

    Gene fusion is among the primary processes that generate new genes and has been well characterized as potent pathway of oncogenesis. Here, by high-throughput RNA sequencing in nine paired human endometrial carcinoma (EC) and matched non-cancerous tissues, we obtained that chimeric translin-associated factor X-disrupted-in-schizophrenia 1 (TSNAX-DISC1) occurred significantly upregulated in multiple EC samples. Experimental investigation showed that TSNAX-DISC1 appears to be formed by splicing without chromosomal rearrangement. The chimera expression inversely correlated with the binding of CCCTC-binding factor (CTCF) to the insulators. Subsequent investigations indicate that long intergenic non-coding RNA lincRNA-NR_034037, separating TSNAX from DISC1, regulates TSNAX -DISC1 production and TSNAX/DISC1 expression levels by extricating CTCF from insulators. Dysregulation of TSNAX influences steroidogenic factor-1-stimulated transcription on the StAR promoter, altering progesterone actions, implying the association with cancer. Together, these results advance our understanding of the mechanism in which lincRNA-NR_034037 regulates TSNAX-DISC1 formation programs that tightly regulate EC development. PMID:25239642

  14. Identification of Cistanche species (Orobanchaceae) based on sequences of the plastid psbA-trnH intergenic region.

    PubMed

    Han, Jian-Ping; Song, Jing-Yuan; Liu, Chang; Chen, Jun; Qian, Jun; Zhu, Ying-Jie; Shi, Lin-Chun; Yao, Hui; Chen, Shi-Lin

    2010-01-01

    The dried succulent stems of Cistanche (Cistanche deserticola Y. C. Ma and Cistanche tubulosa Wight.) are one of the most widely used components of traditional Chinese medicines. However, it is often confused and substituted with the roots of Orobanche pycnostachya, Boschniakia rossica (Cham. & Schltdl.) Standl., Cistanche sinensis Beck, and Cistanche salsa (C. A. Mey.) Beck. In this study, we identified psbA-trnH regions from species and tested their suitable for the identification of the above mentioned taxa. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Cistanche species. Additionally, the average genetic distance of psbA-trnH ranging from 0.077% to 0.743%. In contrast, the intra-specific variation among Cistanche species was found to be significantly different from those of other species, with percentages of variation studied ranged from 0% to 0.007%. The sequence difference between the psbA-trnH sequences of Cistanche species and Orobanche pycnostachya ranged from 0.979% to 1.149%. The distance between the Cistanche species and Boschniakia rossica ranged from 1.066% to 1.224%. Our results suggest that the psbA-trnH intergenic spacer region represent a barcode that can be used to identify Cistanche species and other morphologically undistinguishable species. PMID:21348418

  15. Regulation of miR-200c/141 expression by intergenic DNA-looping and transcriptional read-through.

    PubMed

    Batista, Luciana; Bourachot, Brigitte; Mateescu, Bogdan; Reyal, Fabien; Mechta-Grigoriou, Fatima

    2016-01-01

    The miR-200 family members have been implicated in stress responses and ovarian tumorigenesis. Here, we find that miR-200c/141 transcription is intimately linked to the transcription of the proximal upstream gene PTPN6 (SHP1) in all physiological conditions tested. PTPN6 and miR-200c/141 are transcriptionally co-regulated by two complementary mechanisms. First, a bypass of the regular PTPN6 polyadenylation signal allows the transcription of the downstream miR-200c/141. Second, the promoters of the PTPN6 and miR-200c/141 transcription units physically interact through a 3-dimensional DNA loop and exhibit similar epigenetic regulation. Our findings highlight that transcription of intergenic miRNAs is a novel outcome of transcriptional read-through and reveal a yet unexplored type of DNA loop associating two closely located promoters. These mechanisms have significant relevance in ovarian cancers and stress response, pathophysiological conditions in which miR-200c/141 exert key functions. PMID:26725650

  16. An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Dong, Dong; Wang, Guangce; Zhang, Baoyu; Peng, Guang; Xu, Pu; Tang, Xiaorong

    2009-09-01

    An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

  17. Correlation of maple sap composition with bacterial and fungal communities determined by multiplex automated ribosomal intergenic spacer analysis (MARISA).

    PubMed

    Filteau, Marie; Lagacé, Luc; LaPointe, Gisèle; Roy, Denis

    2011-08-01

    During collection, maple sap is contaminated by bacteria and fungi that subsequently colonize the tubing system. The bacterial microbiota has been more characterized than the fungal microbiota, but the impact of both components on maple sap quality remains unclear. This study focused on identifying bacterial and fungal members of maple sap and correlating microbiota composition with maple sap properties. A multiplex automated ribosomal intergenic spacer analysis (MARISA) method was developed to presumptively identify bacterial and fungal members of maple sap samples collected from 19 production sites during the tapping period. Results indicate that the fungal community of maple sap is mainly composed of yeast related to Mrakia sp., Mrakiella sp., Guehomyces pullulans, Cryptococcus victoriae and Williopsis saturnus. Mrakia, Mrakiella and Guehomyces peaks were identified in samples of all production sites and can be considered dominant and stable members of the fungal microbiota of maple sap. A multivariate analysis based on MARISA profiles and maple sap chemical composition data showed correlations between Candida sake, Janthinobacterium lividum, Williopsis sp., Leuconostoc mesenteroides, Mrakia sp., Rhodococcus sp., Pseudomonas tolaasii, G. pullulans and maple sap composition at different flow periods. This study provides new insights on the relationship between microbial community and maple sap quality. PMID:21569942

  18. Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes

    PubMed Central

    Blok, V. C.; Phillips, M. S.; Fargette, M.

    1997-01-01

    The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications. PMID:19274129

  19. Comparison of the bacterial community structure within the equine hindgut and faeces using Automated Ribosomal Intergenic Spacer Analysis (ARISA).

    PubMed

    Sadet-Bourgeteau, S; Philippeau, C; Dequiedt, S; Julliand, V

    2014-12-01

    The horse's hindgut bacterial ecosystem has often been studied using faecal samples. However few studies compared both bacterial ecosystems and the validity of using faecal samples may be questionable. Hence, the present study aimed to compare the structure of the equine bacterial community in the hindgut (caecum, right ventral colon) and faeces using a fingerprint technique known as Automated Ribosomal Intergenic Spacer Analysis (ARISA). Two DNA extraction methods were also assessed. Intestinal contents and faeces were sampled 3 h after the morning meal on four adult fistulated horses fed meadow hay and pelleted concentrate. Irrespective of the intestinal segment, Principal Component Analysis of ARISA profiles showed a strong individual effect (P<0.0001). However, across the study, faecal bacterial community structure significantly (P<0.001) differed from those of the caecum and colon, while there was no difference between the two hindgut communities. The use of a QIAamp(®) DNA Stool Mini kit increased the quality of DNA extracted irrespective of sample type. The differences observed between faecal and hindgut bacterial communities challenge the use of faeces as a representative for hindgut activity. Further investigations are necessary to compare bacterial activity between the hindgut and faeces in order to understand the validity of using faecal samples. PMID:25075719

  20. Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation.

    PubMed

    Finger, S Alison; Velapatiño, Billie; Kosek, Margaret; Santivañez, Livia; Dailidiene, Daiva; Quino, Willi; Balqui, Jacqueline; Herrera, Phabiola; Berg, Douglas E; Gilman, Robert H

    2006-07-01

    We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation. PMID:16820463

  1. A p53-bound enhancer region controls a long intergenic noncoding RNA required for p53 stress response.

    PubMed

    Melo, C A; Léveillé, N; Rooijers, K; Wijchers, P J; Geeven, G; Tal, A; Melo, S A; de Laat, W; Agami, R

    2016-08-18

    Genome-wide chromatin studies identified the tumor suppressor p53 as both a promoter and an enhancer-binding transcription factor. As an enhancer factor, p53 can induce local production of enhancer RNAs, as well as transcriptional activation of distal neighboring genes. Beyond the regulation of protein-coding genes, p53 has the capacity to regulate long intergenic noncoding RNA molecules (lincRNAs); however, their importance to the p53 tumor suppressive function remains poorly characterized. Here, we identified and characterized a novel p53-bound intronic enhancer that controls the expression of its host, the lincRNA00475 (linc-475). We demonstrate the requirement of linc-475 for the proper induction of a p53-dependent cell cycle inhibitory response. We further confirm the functional importance of linc-475 in the maintenance of CDKN1A/p21 levels, a cell cycle inhibitor and a major p53 target gene, following p53 activation. Interestingly, loss of linc-475 reduced the binding of both p53 and RNA polymerase II (RNAPII) to the promoter of p21, attenuating its transcription rate following p53 activation. Altogether, our data suggest a direct role of p53-bound enhancer domains in the activation of lincRNAs required for an efficient p53 transcriptional response. PMID:26776159

  2. The Mitochondrial Genome of the Leaf-Cutter Ant Atta laevigata: A Mitogenome with a Large Number of Intergenic Spacers

    PubMed Central

    Rodovalho, Cynara de Melo; Lyra, Mariana Lúcio; Ferro, Milene; Bacci, Maurício

    2014-01-01

    In this paper we describe the nearly complete mitochondrial genome of the leaf-cutter ant Atta laevigata, assembled using transcriptomic libraries from Sanger and Illumina next generation sequencing (NGS), and PCR products. This mitogenome was found to be very large (18,729 bp), given the presence of 30 non-coding intergenic spacers (IGS) spanning 3,808 bp. A portion of the putative control region remained unsequenced. The gene content and organization correspond to that inferred for the ancestral pancrustacea, except for two tRNA gene rearrangements that have been described previously in other ants. The IGS were highly variable in length and dispersed through the mitogenome. This pattern was also found for the other hymenopterans in particular for the monophyletic Apocrita. These spacers with unknown function may be valuable for characterizing genome evolution and distinguishing closely related species and individuals. NGS provided better coverage than Sanger sequencing, especially for tRNA and ribosomal subunit genes, thus facilitating efforts to fill in sequence gaps. The results obtained showed that data from transcriptomic libraries contain valuable information for assembling mitogenomes. The present data also provide a source of molecular markers that will be very important for improving our understanding of genomic evolutionary processes and phylogenetic relationships among hymenopterans. PMID:24828084

  3. A New Intergenic α-Globin Deletion (α-α(Δ125)) Found in a Kabyle Population.

    PubMed

    Rabbind Singh, Amrathlal; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-03-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact. PMID:26911300

  4. Molecular typing among beef isolates of Escherichia coli using consensus repetitive intergenic enterobacteria-polymerase chain reaction (ERIC-PCR)

    NASA Astrophysics Data System (ADS)

    Zoolkifli, Nurliyana Wan; Mutalib, Sahilah Abd

    2013-11-01

    Genomic DNA of Escherichia coli were characterized by enterobacterial repetitive intergenic consensus-Polymerase chain reaction (ERIC-PCR) and the presence of Shiga toxin gene-I (Stx1) and Shiga toxin gene-2 (Stx2). These isolates were originated from imported raw beef which are come from two countries namely Australia and India. The isolation of E. coli was conducted by using Eosin Methylene Blue Agar (EMBA). A total of 94 strains had been isolated from 30 samples of imported raw beefand 42 strains had been detected positively E. coli by doing biochemical tests. All strains had been tested and the results of biochemical tests showed that 3 strains were from Australia samples while the other 39 strains were from India samples. The biochemical tests used are Indole test, Methyl Red test, Voges-Proskauer test and Citrate test. All the 42 strains were examined for Shiga toxin (stx1 and stx2) gene detection by two pair primers which are stx2F (5'-TTCTTCGGTATCCTATTCCC-3'), stx2R (5'-ATGCATCTCTGGTCATTGTA-3'), stx1F (5'-CAGTTAATGTGGTGGCGAAG-3'), and stx1R (5'-CTGTCACAGTAACAACCGT-3'). The results showed that none of the strains are positive for Shiga toxin gene. Application of ERIC-PCR method towards E. coli had successfully shown the high diversity polymorphism in 21 different genome types of DNA with primers ERIC1R (5'- CACTTAGGGGTCCTCGAATGTA- 3') and ERIC2R (5'- AAGTAAGTGACTGGGGTGACGC- 3').

  5. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Liu, Zhong; Zhao, Rui; Giles, Keith E.

    2016-01-01

    It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells. PMID:27299313

  6. Length polymorphisms in tRNA intergenic spacers detected by using the polymerase chain reaction can distinguish streptococcal strains and species.

    PubMed Central

    McClelland, M; Petersen, C; Welsh, J

    1992-01-01

    Intergenic tRNA spacers from strains of streptococcal groups A, B, and G were amplified by using the polymerase chain reaction (PCR) at low stringency with consensus tRNA gene primers. Cloning and sequencing showed that many of the homologous intergenic spacers differed in length between species. The sequences of the tRNA genes that flank these polymorphic spacers were determined and used to synthesize fully complementary primers. With these primers at high stringency, PCR products which varied in lengths from 53 to 71 bp, depending on the species or strain, were obtained from streptococcal DNAs, even in the presence of a 1,000-fold mass excess of human DNA. PCR products, the lengths of which could also be used for classification, were obtained at high stringency from a few genera closely related to Streptococcus. No products were obtained from genomic DNAs from more distantly related genera. Production of species- or strain-specific tRNA intergenic length polymorphisms with primers that generate characteristic products from a variety of species within the same genus should be applicable to many organisms, including those that would otherwise be difficult to culture or identify. Images PMID:1378058

  7. Exchange of Spacer Regions between Rrna Operons in Escherichia Coli

    PubMed Central

    Harvey, S.; Hill, C. W.

    1990-01-01

    The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala 1 B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala 1 B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids. PMID:2168847

  8. LincSNP: a database of linking disease-associated SNPs to human large intergenic non-coding RNAs

    PubMed Central

    2014-01-01

    Background Genome-wide association studies (GWAS) have successfully identified a large number of single nucleotide polymorphisms (SNPs) that are associated with a wide range of human diseases. However, many of these disease-associated SNPs are located in non-coding regions and have remained largely unexplained. Recent findings indicate that disease-associated SNPs in human large intergenic non-coding RNA (lincRNA) may lead to susceptibility to diseases through their effects on lincRNA expression. There is, therefore, a need to specifically record these SNPs and annotate them as potential candidates for disease. Description We have built LincSNP, an integrated database, to identify and annotate disease-associated SNPs in human lincRNAs. The current release of LincSNP contains approximately 140,000 disease-associated SNPs (or linkage disequilibrium SNPs), which can be mapped to around 5,000 human lincRNAs, together with their comprehensive functional annotations. The database also contains annotated, experimentally supported SNP-lincRNA-disease associations and disease-associated lincRNAs. It provides flexible search options for data extraction and searches can be performed by disease/phenotype name, SNP ID, lincRNA name and chromosome region. In addition, we provide users with a link to download all the data from LincSNP and have developed a web interface for the submission of novel identified SNP-lincRNA-disease associations. Conclusions The LincSNP database aims to integrate disease-associated SNPs and human lincRNAs, which will be an important resource for the investigation of the functions and mechanisms of lincRNAs in human disease. The database is available at http://bioinfo.hrbmu.edu.cn/LincSNP. PMID:24885522

  9. Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage

    PubMed Central

    Pearson, Mark J.; Philp, Ashleigh M.; Heward, James A.; Roux, Benoit T.; Walsh, David A.; Davis, Edward T.; Lindsay, Mark A.

    2016-01-01

    Objective To identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA. Methods OA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex. Results RNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines. Conclusion The inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role

  10. Detection of Clostridium tyrobutyricum in milk to prevent late blowing in cheese by automated ribosomal intergenic spacer analysis.

    PubMed

    Panelli, Simona; Brambati, Eva; Bonacina, Cesare; Feligini, Maria

    2013-10-01

    Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 10(6) to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process. PMID:24106762

  11. Genome sequencing and annotation of Proteus sp. SAS71

    PubMed Central

    Selim, Samy; Hassan, Sherif; Hagagy, Nashwa

    2015-01-01

    We report draft genome sequence of Proteus sp. strain SAS71, isolated from water spring in Aljouf region, Saudi Arabia. The draft genome size is 3,037,704 bp with a G + C content of 39.3% and contains 6 rRNA sequence (single copies of 5S, 16S & 23S rRNA). The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LDIU00000000. PMID:26697338

  12. Insights into the phylogenetic positions of photosynthetic bacteria obtained from 5S rRNA and 16S rRNA sequence data

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1985-01-01

    Comparisons of complete 16S ribosomal ribonucleic acid (rRNA) sequences established that the secondary structure of these molecules is highly conserved. Earlier work with 5S rRNA secondary structure revealed that when structural conservation exists the alignment of sequences is straightforward. The constancy of structure implies minimal functional change. Under these conditions a uniform evolutionary rate can be expected so that conditions are favorable for phylogenetic tree construction.

  13. Characterization of rhizobia from legumes of agronomic interest grown in semi-arid areas of Central Spain relates genetic differences to soil properties.

    PubMed

    Ruiz-Díez, Beatriz; Fajardo, Susana; Felipe, María del Rosario de; Fernández-Pascual, Mercedes

    2012-02-01

    A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well-adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty-six isolates were obtained from root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty-six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty-nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment. PMID:21953333

  14. Secondary structure of mouse 28S rRNA and general model for the folding of the large rRNA in eukaryotes.

    PubMed Central

    Michot, B; Hassouna, N; Bachellerie, J P

    1984-01-01

    We present a secondary structure model for the entire sequence of mouse 28S rRNA (1) which is based on an extensive comparative analysis of the available eukaryotic sequences, i.e. yeast (2, 3), Physarum polycephalum (4), Xenopus laevis (5) and rat (6). It has been derived with close reference to the models previously proposed for yeast 26S rRNA (2) and for prokaryotic 23S rRNA (7-9). Examination of the recently published eukaryotic sequences confirms that all pro- and eukaryotic large rRNAs share a largely conserved secondary structure core, as already apparent from the previous analysis of yeast 26S rRNA (2). These new comparative data confirm most features of the yeast model (2). They also provide the basis for a few modifications and for new proposals which extend the boundaries of the common structural core (now representing about 85% of E. coli 23S rRNA length) and bring new insights for tracing the structural evolution, in higher eukaryotes, of the domains which have no prokaryotic equivalent and are inserted at specific locations within the common structural core of the large subunit rRNA. PMID:6374617

  15. Ribosome heterogeneity in tumorigenesis: the rRNA point of view

    PubMed Central

    Marcel, Virginie; Catez, Frédéric; Diaz, Jean-Jacques

    2015-01-01

    The "specialized ribosome" concept proposes that ribosome variants are produced and differentially regulate translation. Examples supporting this notion demonstrated heterogeneity of ribosomal protein composition. However, ribosome translational activity is carried out by rRNA. We, and others, recently showed that rRNA heterogeneity regulates translation to generate distinct translatomes promoting tumorigenesis. PMID:27305893

  16. Tetrathiobacter kashmirensis Strain CA-1 16S rRNA gene complete sequence.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used 1326 base pair 16S rRNA gene sequence methods to confirm the identification of a bacterium as Tetrathiobacter kashmirensis. Morphological, biochemical characteristics, and fatty acid profiles are consistent with the 16S rRNA gene sequence identification of the bacterium. The isolate...

  17. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  18. Strain identification and 5S rRNA gene characterization of the hyperthermophilic archaebacterium Sulfolobus acidocaldarius.

    PubMed Central

    Durovic, P; Kutay, U; Schleper, C; Dennis, P P

    1994-01-01

    A commonly used laboratory Sulfolobus strain has been unambiguously identified as Sulfolobus acidocaldarius DSM639. The 5S rRNA gene from this strain was cloned and sequenced. It differs at 17 of 124 positions from the identical 5S rRNA sequences from Sulfolobus solfataricus and a strain apparently misidentified as S. acidocaldarius. Analysis of the transcripts from the 5S rRNA gene failed to identify any precursor extending a significant distance beyond the 5' or 3' boundary of the 5S rRNA-coding sequence. This result suggests that the primary transcript of the 5S rRNA gene corresponds in length (within 1 or 2 nucleotides) to the mature 5S rRNA sequence found in 50S ribosomal subunits. Images PMID:8288546

  19. Evidence for the presence of 5S rRNA in mammalian mitochondria.

    PubMed

    Magalhães, P J; Andreu, A L; Schon, E A

    1998-09-01

    Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified. PMID:9725900

  20. Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

    PubMed Central

    Kiparisov, S.; Sergiev, P. V.; Dontsova, O. A.; Petrov, A.; Meskauskas, A.; Dinman, J. D.

    2005-01-01

    5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression. PMID:16047201

  1. Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

    PubMed Central

    Pontvianne, Frederic; Blevins, Todd; Chandrasekhara, Chinmayi; Mozgová, Iva; Hassel, Christiane; Pontes, Olga M.F.; Tucker, Sarah; Mokroš, Petr; Muchová, Veronika; Fajkus, Jiří; Pikaard, Craig S.

    2013-01-01

    Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic–nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state. PMID:23873938

  2. Genetic diversity, inter-gene pool introgression and nutritional quality of common beans (Phaseolus vulgaris L.) from Central Africa.

    PubMed

    Blair, Matthew W; González, Laura F; Kimani, Paul M; Butare, Louis

    2010-07-01

    The Great Lakes region of Central Africa is a major producer of common beans in Africa. The region is known for high population density and small average farm size. The common bean represents the most important legume crop of the region, grown on over a third of the cultivated land area, and the per capita consumption is among the highest in the world for the food crop. The objective of this study was to evaluate the genetic diversity in a collection of 365 genotypes from the Great Lakes region of Central Africa, including a large group of landraces from Rwanda as well as varieties from primary centers of diversity and from neighboring countries of Central Africa, such as the Democratic Republic of Congo and Uganda, using 30 fluorescently labeled microsatellite markers and automated allele detection. In addition, the landraces were evaluated for their seed iron and zinc concentration to determine if genetic diversity influenced nutritional quality. Principal coordinate and neighbor-joining analyses allowed the separation of the landraces into 132 Andean and 195 Mesoamerican (or Middle American) genotypes with 32 landraces and 6 varieties intermediate between the gene pools and representing inter-gene pool introgression in terms of seed characteristics and alleles. Genetic diversity and the number of alleles were high for the collection, reflecting the preference for a wide range of seed types in the region and no strong commercial class preference, although red, red mottled and brown seeded beans were common. Observed heterozygosity was also high and may be explained by the common practice of maintaining seed and plant mixtures, a coping strategy practiced by Central African farmers to reduce the effects of abiotic and biotic stresses. Finally, nutritional quality differed between the gene pools with respect to seed iron and zinc concentration, while genotypes from the intermediate group were notably high in both minerals. In conclusion, this study has shown that

  3. Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone.

    PubMed

    Melia, Tisha; Hao, Pengying; Yilmaz, Feyza; Waxman, David J

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the

  4. An evaluation of logic regression-based biomarker discovery across multiple intergenic regions for predicting host specificity in Escherichia coli.

    PubMed

    Zhi, Shuai; Li, Qiaozhi; Yasui, Yutaka; Banting, Graham; Edge, Thomas A; Topp, Edward; McAllister, Tim A; Neumann, Norman F

    2016-10-01

    Several studies have demonstrated that E. coli appears to display some level of host adaptation and specificity. Recent studies in our laboratory support these findings as determined by logic regression modeling of single nucleotide polymorphisms (SNP) in intergenic regions (ITGRs). We sought to determine the degree of host-specific information encoded in various ITGRs across a library of animal E. coli isolates using both whole genome analysis and a targeted ITGR sequencing approach. Our findings demonstrated that ITGRs across the genome encode various degrees of host-specific information. Incorporating multiple ITGRs (i.e., concatenation) into logic regression model building resulted in greater host-specificity and sensitivity outcomes in biomarkers, but the overall level of polymorphism in an ITGR did not correlate with the degree of host-specificity encoded in the ITGR. This suggests that distinct SNPs in ITGRs may be more important in defining host-specificity than overall sequence variation, explaining why traditional unsupervised learning phylogenetic approaches may be less informative in terms of revealing host-specific information encoded in DNA sequence. In silico analysis of 80 candidate ITGRs from publically available E. coli genomes was performed as a tool for discovering highly host-specific ITGRs. In one ITGR (ydeR-yedS) we identified a SNP biomarker that was 98% specific for cattle and for which 92% of all E. coli isolates originating from cattle carried this unique biomarker. In the case of humans, a host-specific biomarker (98% specificity) was identified in the concatenated ITGR sequences of rcsD-ompC, ydeR-yedS, and rclR-ykgE, and for which 78% of E. coli originating from humans carried this biomarker. Interestingly, human-specific biomarkers were dominant in ITGRs regulating antibiotic resistance, whereas in cattle host-specific biomarkers were found in ITGRs involved in stress regulation. These data suggest that evolution towards host

  5. Hepatic Long Intergenic Noncoding RNAs: High Promoter Conservation and Dynamic, Sex-Dependent Transcriptional Regulation by Growth Hormone

    PubMed Central

    Melia, Tisha; Hao, Pengying; Yilmaz, Feyza

    2015-01-01

    Long intergenic noncoding RNAs (lincRNAs) are increasingly recognized as key chromatin regulators, yet few studies have characterized lincRNAs in a single tissue under diverse conditions. Here, we analyzed 45 mouse liver RNA sequencing (RNA-Seq) data sets collected under diverse conditions to systematically characterize 4,961 liver lincRNAs, 59% of them novel, with regard to gene structures, species conservation, chromatin accessibility, transcription factor binding, and epigenetic states. To investigate the potential for functionality, we focused on the responses of the liver lincRNAs to growth hormone stimulation, which imparts clinically relevant sex differences to hepatic metabolism and liver disease susceptibility. Sex-biased expression characterized 247 liver lincRNAs, with many being nuclear RNA enriched and regulated by growth hormone. The sex-biased lincRNA genes are enriched for nearby and correspondingly sex-biased accessible chromatin regions, as well as sex-biased binding sites for growth hormone-regulated transcriptional activators (STAT5, hepatocyte nuclear factor 6 [HNF6], FOXA1, and FOXA2) and transcriptional repressors (CUX2 and BCL6). Repression of female-specific lincRNAs in male liver, but not that of male-specific lincRNAs in female liver, was associated with enrichment of H3K27me3-associated inactive states and poised (bivalent) enhancer states. Strikingly, we found that liver-specific lincRNA gene promoters are more highly species conserved and have a significantly higher frequency of proximal binding by liver transcription factors than liver-specific protein-coding gene promoters. Orthologs for many liver lincRNAs were identified in one or more supraprimates, including two rat lincRNAs showing the same growth hormone-regulated, sex-biased expression as their mouse counterparts. This integrative analysis of liver lincRNA chromatin states, transcription factor occupancy, and growth hormone regulation provides novel insights into the

  6. Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

    PubMed Central

    2011-01-01

    Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease. PMID:21669002

  7. Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants.

    PubMed

    Lee, Chunghee; Wen, Jun

    2004-06-01

    Sequences of the chloroplast trnC-trnD region and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were obtained for all species of Panax L. (the ginseng plant genus, Araliaceae) to reconstruct phylogenetic relationships. The trnC-trnD phylogeny is congruent with the ITS phylogeny for the diploid taxa of Panax. This study is the first use of the trnC-trnD sequence data for phylogenetic analysis at the interspecific level. We evaluated this DNA region for its phylogenetic utility at the lower taxonomic level for flowering plants. The trnC-trnD region includes the trnC-petN intergenic spacer, the petN gene, the petN-psbM intergenic spacer, the psbM gene, and the psbM-trnD intergenic spacer. The petN and psbM genes are small, 90 and 104-114 bp across angiosperms, respectively, and have conserved sequences. We have designed universal amplification and sequencing primers within these two genes. Using these primers, we have successfully amplified the entire trnC-trnD region for a diversity of flowering plant groups, including Aralia L. (Araliaceae), Calycanthus L. (Calycanthaceae), Corylus L. (Betulaceae), Hamamelis L. (Hamamelidaceae), Hydrocotyle L. (Apiaceae), Illigera Blume (Hernandiaceae), Nelumbo Adans. (Nelumbonaceae), Nolana L. ex L.f. (Solanaceae), Prunus L. (Rosaceae), and Staphylea L. (Staphyleaceae). In Panax, the trnC-trnD region provides a similar number of informative phylogenetic characters as the ITS regions and a slightly higher number of informative characters than the chloroplast ndhF gene. We thus demonstrate the utility of the trnC-trnD region for lower-level phylogenetic studies in flowering plants. PMID:15120387

  8. Assessing host-specificity of Escherichia coli using a supervised learning logic-regression-based analysis of single nucleotide polymorphisms in intergenic regions.

    PubMed

    Zhi, Shuai; Li, Qiaozhi; Yasui, Yutaka; Edge, Thomas; Topp, Edward; Neumann, Norman F

    2015-11-01

    Host specificity in E. coli is widely debated. Herein, we used supervised learning logic-regression-based analysis of intergenic DNA sequence variability in E. coli in an attempt to identify single nucleotide polymorphism (SNP) biomarkers of E. coli that are associated with natural selection and evolution toward host specificity. Seven-hundred and eighty strains of E. coli were isolated from 15 different animal hosts. We utilized logic regression for analyzing DNA sequence data of three intergenic regions (flanked by the genes uspC-flhDC, csgBAC-csgDEFG, and asnS-ompF) to identify genetic biomarkers that could potentially discriminate E. coli based on host sources. Across 15 different animal hosts, logic regression successfully discriminated E. coli based on animal host source with relatively high specificity (i.e., among the samples of the non-target animal host, the proportion that correctly did not have the host-specific marker pattern) and sensitivity (i.e., among the samples from a given animal host, the proportion that correctly had the host-specific marker pattern), even after fivefold cross validation. Permutation tests confirmed that for most animals, host specific intergenic biomarkers identified by logic regression in E. coli were significantly associated with animal host source. The highest level of biomarker sensitivity was observed in deer isolates, with 82% of all deer E. coli isolates displaying a unique SNP pattern that was 98% specific to deer. Fifty-three percent of human isolates displayed a unique biomarker pattern that was 98% specific to humans. Twenty-nine percent of cattle isolates displayed a unique biomarker that was 97% specific to cattle. Interestingly, even within a related host group (i.e., Family: Canidae [domestic dogs and coyotes]), highly specific SNP biomarkers (98% and 99% specificity for dog and coyotes, respectively) were observed, with 21% of dog E. coli isolates displaying a unique dog biomarker and 61% of coyote isolates

  9. 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

  10. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

    PubMed

    Small, J; Call, D R; Brockman, F J; Straub, T M; Chandler, D P

    2001-10-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  11. Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria

    PubMed Central

    Valach, Matus; Moreira, Sandrine; Kiethega, Georgette N.; Burger, Gertraud

    2014-01-01

    Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events. PMID:24259427

  12. Depletion of pre-16S rRNA in starved Escherichia coli cells.

    PubMed

    Cangelosi, G A; Brabant, W H

    1997-07-01

    Specific hybridization assays for intermediates in rRNA synthesis (pre-rRNA) may become useful for monitoring the growth activity of individual microbial species in complex natural systems. This possibility depends upon the assumption that rRNA processing in microbial cells continues after growth and pre-rRNA synthesis cease, resulting in drainage of the pre-rRNA pool. This is not the case in many eukaryotic cells, but less is known about the situation in bacteria. Therefore, we used DNA probes to measure steady-state cellular pre-16S rRNA pools during growth state transitions in Escherichia coli. Pre-16S rRNA became undetectable when cells entered the stationary phase on rich medium and was replenished upon restoration of favorable growth conditions. These fluctuations were of much greater magnitude than concurrent fluctuations in the mature 16S rRNA pool. The extent of pre-16S rRNA depletion depended upon the circumstances limiting growth. It was significantly more pronounced in carbon-energy-starved cells than in nitrogen-starved cells or in cells treated with energy uncouplers. In the presence of the transcriptional inhibitor rifampin, rates of pre-16S rRNA depletion in carbon-energy-starved cells and nitrogen-starved cells were similar, suggesting that the difference between these conditions resides primarily at the level of pre-rRNA synthesis. Chloramphenicol, which inhibits the final steps in rRNA maturation, halted pre-16S rRNA depletion under all conditions. The data show that E. coli cells continue to process pre-rRNA after growth and rrn operon transcription cease, leading to drainage of the pre-rRNA pool. This supports the feasibility of using pre-rRNA-targeted probes to monitor bacterial growth in natural systems, with the caveat that patterns of pre-rRNA depletion vary with the conditions limiting growth. PMID:9226253

  13. Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes.

    PubMed

    Zhu, Luchang; Olsen, Randall J; Horstmann, Nicola; Shelburne, Samuel A; Fan, Jia; Hu, Ye; Musser, James M

    2016-07-01

    Variable-number tandem-repeat (VNTR) polymorphisms are ubiquitous in bacteria. However, only a small fraction of them has been functionally studied. Here, we report an intergenic VNTR polymorphism that confers an altered level of toxin production and increased virulence in Streptococcus pyogenes The nature of the polymorphism is a one-unit deletion in a three-tandem-repeat locus upstream of the rocA gene encoding a sensor kinase. S. pyogenes strains with this type of polymorphism cause human infection and produce significantly larger amounts of the secreted cytotoxins S. pyogenes NADase (SPN) and streptolysin O (SLO). Using isogenic mutant strains, we demonstrate that deleting one or more units of the tandem repeats abolished RocA production, reduced CovR phosphorylation, derepressed multiple CovR-regulated virulence factors (such as SPN and SLO), and increased virulence in a mouse model of necrotizing fasciitis. The phenotypic effect of the VNTR polymorphism was nearly the same as that of inactivating the rocA gene. In summary, we identified and characterized an intergenic VNTR polymorphism in S. pyogenes that affects toxin production and virulence. These new findings enhance understanding of rocA biology and the function of VNTR polymorphisms in S. pyogenes. PMID:27141081

  14. Bacterial interspersed mosaic elements (BIMEs) are a major source of sequence polymorphism in Escherichia coli intergenic regions including specific associations with a new insertion sequence.

    PubMed

    Bachellier, S; Clément, J M; Hofnung, M; Gilson, E

    1997-03-01

    A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS 1397. Remarkably, 14 out of IS 1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS 1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed. PMID:9055066

  15. Replication origins and a sequence involved in coordinate induction of the immediate-early gene family are conserved in an intergenic region of herpes simplex virus.

    PubMed Central

    Whitton, J L; Clements, J B

    1984-01-01

    We have determined the structure of the 5' portion of herpes simplex virus type 2 (HSF-2) immediate-early (IE) mRNA-3 and have obtained the DNA sequence specifying the N terminus of its encoded polypeptide, Vmw182, its untranslated leader and the intergenic region between IEmRNAs-3 & 4/5. Comparison of the HSV-2 intergenic sequences with the HSV-1 equivalent region identifies several conserved regions: (1) an AT-rich element with core consensus TAATGARAT which is likely to be the 'activator' sequence through which coordinate induction of the IE gene family is mediated. (2) GC-rich and GA-rich tracts, found in a wide variety of eukaryotic promoters, which vary in position and orientation between HSV-2 and HSV-1 and which represent modulators of transcription. (3) TATA homologies present 15-25 base pairs (bp) upstream of mRNA 5' termini. (4) a 137bp direct repeat in HSV-2 which contains sequence almost identical to the HSV-1 replication origin. Images PMID:6322134

  16. Specific binding of Thiobacillus ferrooxidans RbcR to the intergenic sequence between the rbc operon and the rbcR gene.

    PubMed Central

    Kusano, T; Sugawara, K

    1993-01-01

    The presence of two sets (rbcL1-rbcS1 and rbcL2-rbcS2) of rbc operons has been demonstrated in Thiobacillus ferrooxidans Fe1 (T. Kusano, T. Takeshima, C. Inoue, and K. Sugawara, J. Bacteriol. 173:7313-7323, 1991). A possible regulatory gene, rbcR, 930 bp long and possibly translated into a 309-amino-acid protein, was found upstream from the rbcL1 gene as a single copy. The gene is located divergently to rbcL1 with a 144-bp intergenic sequence. As in the cases of the Chromatium vinosum RbcR and Alcaligenes eutrophus CfxR, T. ferrooxidans RbcR is thought to be a new member of the LysR family, and these proteins share 46.5 and 42.8% identity, respectively. Gel mobility shift assays showed that T. ferrooxidans RbcR, produced in Escherichia coli, binds specifically to the intergenic sequence between rbcL1 and rbcR. Footprinting and site-directed mutagenesis experiments further demonstrated that RbcR binds to overlapping promoter elements of the rbcR and rbcL1 genes. The above data strongly support the participation of RbcR in regulation of the rbcL1-rbcS1 operon and the rbcR gene in T. ferrooxidans. Images PMID:8432695

  17. Identification of SmtB/ArsR cis elements and proteins in archaea using the Prokaryotic InterGenic Exploration Database (PIGED).

    PubMed

    Bose, Michael; Slick, David; Sarto, Mickey J; Murphy, Patrick; Roberts, David; Roberts, Jacqueline; Barber, Robert D

    2006-08-01

    Microbial genome sequencing projects have revealed an apparently wide distribution of SmtB/ArsR metal-responsive transcriptional regulators among prokaryotes. Using a position-dependent weight matrix approach, prokaryotic genome sequences were screened for SmtB/ArsR DNA binding sites using data derived from intergenic sequences upstream of orthologous genes encoding these regulators. Sixty SmtB/ArsR operators linked to metal detoxification genes, including nine among various archaeal species, are predicted among 230 annotated and draft prokaryotic genome sequences. Independent multiple sequence alignments of putative operator sites and corresponding winged helix-turn-helix motifs define sequence signatures for the DNA binding activity of this SmtB/ArsR subfamily. Prediction of an archaeal SmtB/ArsR based upon these signature sequences is confirmed using purified Methanosarcina acetivorans C2A protein and electrophoretic mobility shift assays. Tools used in this study have been incorporated into a web application, the Prokaryotic InterGenic Exploration Database (PIGED; http://bioinformatics.uwp.edu/~PIGED/home.htm), facilitating comparable studies. Use of this tool and establishment of orthology based on DNA binding signatures holds promise for deciphering potential cellular roles of various archaeal winged helix-turn-helix transcriptional regulators. PMID:16877320

  18. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications.

    PubMed

    Herzog, M; Maroteaux, L

    1986-11-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  19. Dinoflagellate 17S rRNA sequence inferred from the gene sequence: Evolutionary implications

    PubMed Central

    Herzog, Michel; Maroteaux, Luc

    1986-01-01

    We present the complete sequence of the nuclear-encoded small-ribosomal-subunit RNA inferred from the cloned gene sequence of the dinoflagellate Prorocentrum micans. The dinoflagellate 17S rRNA sequence of 1798 nucleotides is contained in a family of 200 tandemly repeated genes per haploid genome. A tentative model of the secondary structure of P. micans 17S rRNA is presented. This sequence is compared with the small-ribosomal-subunit rRNA of Xenopus laevis (Animalia), Saccharomyces cerevisiae (Fungi), Zea mays (Planta), Dictyostelium discoideum (Protoctista), and Halobacterium volcanii (Monera). Although the secondary structure of the dinoflagellate 17S rRNA presents most of the eukaryotic characteristics, it contains sufficient archaeobacterial-like structural features to reinforce the view that dinoflagellates branch off very early from the eukaryotic lineage. PMID:16578795

  20. Diversity of 5S rRNA genes within individual prokaryotic genomes

    PubMed Central

    Pei, Anna; Li, Hongru; Oberdorf, William E; Alekseyenko, Alexander V.; Parsons, Tamasha; Yang, Liying; Gerz, Erika A.; Lee, Peng; Xiang, Charlie; Nossa, Carlos W.; Pei, Zhiheng

    2012-01-01

    We examined intragenomic variation of paralogous 5S rRNA genes to evaluate the concept of ribosomal constraints. In a dataset containing 1168 genomes from 779 unique species, 96 species exhibited >3% diversity. Twenty seven species with >10% diversity contained a total of 421 mismatches between all pairs of the most dissimilar copies of 5S rRNA genes. The large majority (401 of 421) the diversified positions were conserved at the secondary structure level. The high diversity was associated with partial rRNA operon, split operon, or spacer length-related divergence. In total, these findings indicated that there were tight ribosomal constraints on paralogous 5S rRNA genes in a genome despite of the high degree of diversity at the primary structure level. There is supplementary material. PMID:22765222

  1. An Archaea 5S rRNA analog is stably expressed in Escherichia coli

    NASA Technical Reports Server (NTRS)

    Yang, Y.; Fox, G. E.

    1996-01-01

    Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

  2. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit.

    PubMed Central

    Hartmann, R K; Erdmann, V A

    1989-01-01

    A cloned 16S rRNA gene from the extreme thermophilic eubacterium Thermus thermophilus HB8 was used to characterize the in vivo expression of the 16S rRNA genes in this organism by nuclease S1 mapping. The gene represents an isolated transcription unit encoding solely 16S rRNA. Under exponential growth conditions, transcription was initiated at a single promoter, which represents the structural equivalent of Escherichia coli rrn P2 promoters. The promoter-leader region was very similar to the E. coli rrn P2 promoter-leader segment that is responsible for antitermination. The T. thermophilus leader region was approximately 85 nucleotides shorter than its E. coli P2 counterpart. Potential processing intermediates were correlated with a proposed secondary structure of T. thermophilus pre-16S rRNA. Images PMID:2722737

  3. Inter- and intraspecies identification of Bartonella (Rochalimaea) species.

    PubMed

    Roux, V; Raoult, D

    1995-06-01

    Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp. Restriction fragment length polymorphism after PCR amplification of the 16S-23S rRNA gene ITS may be useful for rapid species identification, and PFGE could be an efficient method for isolate identification. PMID:7650189

  4. Pantanalinema gen. nov. and Alkalinema gen. nov.: novel pseudanabaenacean genera (Cyanobacteria) isolated from saline-alkaline lakes.

    PubMed

    Vieira Vaz, Marcelo Gomes Marçal; Genuário, Diego Bonaldo; Andreote, Ana Paula Dini; Malone, Camila Francieli Silva; Sant'Anna, Célia Leite; Barbiero, Laurent; Fiore, Marli Fátima

    2015-01-01

    The genus Leptolyngbya Anagnostidis & Komárek (1988) was described from a set of strains identified as 'LPP-group B'. The morphology within this group is not particularly informative and underestimates the group's genetic diversity. In the present study, two new pseudanabaenacean genera related to Leptolyngbya morphotypes, Pantanalinema gen. nov. and Alkalinema gen. nov., are described under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants, based on a polyphasic approach. Pantanalinema gen. nov. (type species Pantanalinema rosaneae sp. nov.) has sheaths and trichomes with slight gliding motility, which distinguish this genus from Alkalinema gen. nov. (type species Alkalinema pantanalense sp. nov.), which possesses trichomes arranged in an ornate (interwoven) pattern. 16S rRNA gene sequences of strains of Pantanalinema and Alkalinema exhibited low identity to each other (≤91.6 %) and to other sequences from known pseudanabaenacean genera (≤94.3 and 93.7 %, respectively). In a phylogenetic reconstruction, six sequences from strains of Pantanalinema and four from strains of Alkalinema formed two separate and robust clades (99 % bootstrap value), with the genera Oculatella and Phormidesmis, respectively, as the closest related groups. 16S-23S rRNA intergenic spacer sequences and secondary structures of strains of Pantanalinema and Alkalinema did not correspond to any previous descriptions. The strains of Pantanalinema and Alkalinema were able to survive and produce biomass at a range of pH (pH 4-11) and were also able to alter the culture medium to pH values ranging from pH 8.4 to 9.9. These data indicate that cyanobacterial communities in underexplored environments, such as the Pantanal wetlands, are promising sources of novel taxa. PMID:25351877

  5. Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus.

    PubMed

    Ramírez-Bahena, Martha Helena; Peix, Alvaro; Rivas, Raúl; Camacho, María; Rodríguez-Navarro, Dulce N; Mateos, Pedro F; Martínez-Molina, Eustoquio; Willems, Anne; Velázquez, Encarna

    2009-08-01

    Several strains isolated from the legume Pachyrhizus erosus were characterized on the basis of diverse genetic, phenotypic and symbiotic approaches. These novel strains formed two groups closely related to Bradyrhizobium elkanii according to their 16S rRNA gene sequences. Strains PAC48T and PAC68T, designated as the type strains of these two groups, presented 99.8 and 99.1% similarity, respectively, in their 16S rRNA gene sequences with respect to B. elkanii USDA 76T. In spite of these high similarity values, the analysis of additional phylogenetic markers such as atpD and glnII genes and the 16S-23S intergenic spacer (ITS) showed that strains PAC48T and PAC68T represented two separate novel species of the genus Bradyrhizobium with B. elkanii as their closest relative. Phenotypic differences among the novel strains isolated from Pachyrhizus and B. elkanii were found regarding the assimilation of carbon sources and antibiotic resistance. All these differences were congruent with DNA-DNA hybridization analysis which revealed 21% genetic relatedness between strains PAC48T and PAC68T and 46% and 25%, respectively, between these strains and B. elkanii LMG 6134T. The nodD and nifH genes of strains PAC48T and PAC68T were phylogenetically divergent from those of bradyrhizobia species that nodulate soybean. Soybean was not nodulated by the novel Pachyrhizus isolates. Based on the genotypic and phenotypic data obtained in this study, the new strains represent two novel species for which the names Bradyrhizobium pachyrhizi sp. nov. (type strain PAC48T=LMG 24246T=CECT 7396T) and Bradyrhizobium jicamae sp. nov. (type strain PAC68T=LMG 24556T=CECT 7395T) are proposed. PMID:19567584

  6. Lactobacillus brantae sp. nov., isolated from faeces of Canada geese (Branta canadensis).

    PubMed

    Volokhov, Dmitriy V; Amselle, Megan; Beck, Brian J; Popham, David L; Whittaker, Paul; Wang, Hua; Kerrigan, Elizabeth; Chizhikov, Vladimir E

    2012-09-01

    Three strains of lactic acid bacteria (LAB) were isolated from the faeces of apparently healthy wild Canada geese (Branta canadensis) in 2010 by cultivating faecal LAB on Rogosa SL agar under aerobic conditions. These three isolates were found to share 99.9 % gene sequence similarity of their 16S rRNA, their 16S-23S intergenic transcribed spacer region (ITS), partial 23S rRNA, rpoB, rpoC, rpoA and pheS gene sequences. However, the three strains exhibited lower levels of sequence similarity of these genetic targets to all known LAB, and the phylogenetically closest species to the geese strains were Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus saniviri. In comparison to L. casei ATCC 393(T), L. paracasei ATCC 25302(T), L. rhamnosus ATCC 7469(T) and L. saniviri DSM 24301(T), the novel isolates reacted uniquely in tests for cellobiose, galactose, mannitol, citric acid, aesculin and dextrin, and gave negative results in tests for l-proline arylamidase and l-pyrrolydonyl-arylamidase, and in the Voges-Proskauer test. Biochemical tests for cellobiose, aesculin, galactose, gentiobiose, mannitol, melezitose, ribose, salicin, sucrose, trehalose, raffinose, turanose, amygdalin and arbutin could be used for differentiation between L. saniviri and the novel strains. On the basis of phenotypic and genotypic characteristics, and phylogenetic data, the three isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus brantae sp. nov. is proposed. The type strain is SL1108(T) (= ATCC BAA-2142(T) = LMG 26001(T) = DSM 23927(T)) and two additional strains are SL1170 and SL60106. PMID:22021580

  7. Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis.

    PubMed Central

    Houge, G; Robaye, B; Eikhom, T S; Golstein, J; Mellgren, G; Gjertsen, B T; Lanotte, M; Døskeland, S O

    1995-01-01

    Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes. PMID:7891700

  8. Prevalence of Mitochondrial 12S rRNA Mutations Associated with Aminoglycoside Ototoxicity

    ERIC Educational Resources Information Center

    Guan, Min-Xin

    2005-01-01

    The mitochondrial DNA (mtDNA) 12S rRNA is a hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss. Of those, the homoplasmic A1555G and C1494T mutations at a highly conserved decoding region of the 12S rRNA have been associated with hearing loss. These two mutations account for a significant number of…

  9. Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses

    PubMed Central

    Stoner, D. L.; Browning, C. K.; Bulmer, D. K.; Ward, T. E.; MacDonell, M. T.

    1996-01-01

    This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the genera Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed. PMID:16535333

  10. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers

    PubMed Central

    Liu, Zongzhi; DeSantis, Todd Z.; Andersen, Gary L.; Knight, Rob

    2008-01-01

    The recent introduction of massively parallel pyrosequencers allows rapid, inexpensive analysis of microbial community composition using 16S ribosomal RNA (rRNA) sequences. However, a major challenge is to design a workflow so that taxonomic information can be accurately and rapidly assigned to each read, so that the composition of each community can be linked back to likely ecological roles played by members of each species, genus, family or phylum. Here, we use three large 16S rRNA datasets to test whether taxonomic information based on the full-length sequences can be recaptured by short reads that simulate the pyrosequencer outputs. We find that different taxonomic assignment methods vary radically in their ability to recapture the taxonomic information in full-length 16S rRNA sequences: most methods are sensitive to the region of the 16S rRNA gene that is targeted for sequencing, but many combinations of methods and rRNA regions produce consistent and accurate results. To process large datasets of partial 16S rRNA sequences obtained from surveys of various microbial communities, including those from human body habitats, we recommend the use of Greengenes or RDP classifier with fragments of at least 250 bases, starting from one of the primers R357, R534, R798, F343 or F517. PMID:18723574

  11. Eukaryote-specific rRNA expansion segments function in ribosome biogenesis.

    PubMed

    Ramesh, Madhumitha; Woolford, John L

    2016-08-01

    The secondary structure of ribosomal RNA (rRNA) is largely conserved across all kingdoms of life. However, eukaryotes have evolved extra blocks of rRNA sequences, relative to those of prokaryotes, called expansion segments (ES). A thorough characterization of the potential roles of ES remains to be done, possibly because of limitations in the availability of robust systems to study rRNA mutants. We sought to systematically investigate the potential functions, if any, of the ES in 25S rRNA of Saccharomyces cerevisiae by deletion mutagenesis. We deleted 14 of the 16 different eukaryote-specific ES in yeast 25S rRNA individually and assayed their phenotypes. Our results show that all but two of the ES tested are necessary for optimal growth and are required for production of 25S rRNA, suggesting that ES play roles in ribosome biogenesis. Further, we classified expansion segments into groups that participate in early nucleolar, middle, and late nucleoplasmic steps of ribosome biogenesis, by assaying their pre-rRNA processing phenotypes. This study is the first of its kind to systematically identify the functions of eukaryote-specific expansion segments by showing that they play roles in specific steps of ribosome biogenesis. The catalog of phenotypes we identified, combined with previous investigations of the roles ribosomal proteins in large subunit biogenesis, leads us to infer that assembling ribosomes are composed of distinct RNA and protein structural neighborhood clusters that participate in specific steps of ribosome biogenesis. PMID:27317789

  12. Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

    PubMed Central

    Suenaga, Hikaru; Liu, Rui; Shiramasa, Yuko; Kanagawa, Takahiro

    2005-01-01

    We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities. PMID:16085888

  13. Mutations of mitochondrial 12S rRNA in gastric carcinoma and their significance

    PubMed Central

    Han, Cheng-Bo; Ma, Jia-Ming; Xin, Yan; Mao, Xiao-Yun; Zhao, Yu-Jie; Wu, Dong-Ying; Zhang, Su-Min; Zhang, Yu-Kui

    2005-01-01

    AIM: To detect the variations of mitochondrial 12S rRNA in patients with gastric carcinoma, and to study their significance and the relationship between these variations and the genesis of gastric carcinoma. METHODS: PCR amplified mitochondrial 12S rRNA of 44 samples including 22 from gastric carcinoma tissues and 22 from adjacent normal tissues, was detected by direct DNA sequencing. Then laser capture microdissection technique (LCM) was used to separate the cancerous cells and dysplasia cells with specific mutations. Denaturing high performance liquid chromatography (DHPLC) plus allele-specific PCR (AS-PCR), nest-PCR and polyacrylamide gel electrophoresis (PAGE) were used to further evaluate this mutant property and quantitative difference of mutant type between cancerous and dysplasia cells. Finally, RNAdraw biosoft was used to analyze the RNA secondary structure of mutant-type 12S rRNA. RESULTS: Compared with Mitomap database, some new variations were found, among which np652 G insertion and np716 T-G transversion were found only in cancerous tissues. There was a statistic difference in the frequency of 12S rRNA variation between intestinal type (12/17, 70.59%) and diffusive type (5/17, 29.41%) of gastric carcinoma (P<0.05). DHPLC analysis showed that 12S rRNA np652 G insertion and np716 T-G transversion were heteroplasmic mutations. The frequency of 12S rRNA variation in cancerous cells was higher than that in dysplasia cells (P<0.01). 12S rRNA np652 G insertion showed obviously negative effects on the stability of 12S rRNA secondary structure, while others such as T-G transversion did not. CONCLUSION: The mutations of mitochondrial 12S rRNA may be associated with the occurrence of intestinal-type gastric carcinoma. Most variations exist both in gastric carcinomas and in normal tissues, and they might not be the characteristics of tumors. However, np652 G insertion and np716 T-G transversion may possess some molecular significance in gastric carcinogenesis

  14. Phylogenetic relationships of Carpha and its relatives (Schoeneae, Cyperaceae) inferred from chloroplast trnL intron and trnL-trnF intergenic spacer sequences.

    PubMed

    Zhang, Xiufu; Marchant, Adam; Wilson, Karen L; Bruhl, Jeremy J

    2004-05-01

    Within the tribe Schoeneae (Cyperaceae), the relationships between Carpha and its relatives have not been certain, and the limits and definition of Carpha have been controversial. Further, the relationships of species within Carpha have been unclear. In this study, cladistic analyses based on chloroplast trnL intron and trnL-trnF intergenic spacer sequence data were undertaken to estimate phylogenetic relationships in and around Carpha. This study found that Trianoptiles is sister to Carpha; Ptilothrix is sister to Cyathochaeta rather than to Carpha as suggested by some former authors; and Gymnoschoenus is distant from Carpha and its close relatives. The merging of Schoenoides back into Oreobolus is supported. The findings also revealed the non-monophyletic status of Costularia and of Schoenus, and indicated the phylogenetic relationships of species within Carpha. PMID:15062800

  15. Chloroplast Genome Differences between Asian and American Equisetum arvense (Equisetaceae) and the Origin of the Hypervariable trnY-trnE Intergenic Spacer

    PubMed Central

    Kim, Hyoung Tae; Kim, Ki-Joong

    2014-01-01

    Comparative analyses of complete chloroplast (cp) DNA sequences within a species may provide clues to understand the population dynamics and colonization histories of plant species. Equisetum arvense (Equisetaceae) is a widely distributed fern species in northeastern Asia, Europe, and North America. The complete cp DNA sequences from Asian and American E. arvense individuals were compared in this study. The Asian E. arvense cp genome was 583 bp shorter than that of the American E. arvense. In total, 159 indels were observed between two individuals, most of which were concentrated on the hypervariable trnY-trnE intergenic spacer (IGS) in the large single-copy (LSC) region of the cp genome. This IGS region held a series of 19 bp repeating units. The numbers of the 19 bp repeat unit were responsible for 78% of the total length difference between the two cp genomes. Furthermore, only other closely related species of Equisetum also show the hypervariable nature of the trnY-trnE IGS. By contrast, only a single indel was observed in the gene coding regions: the ycf1 gene showed 24 bp differences between the two continental individuals due to a single tandem-repeat indel. A total of 165 single-nucleotide polymorphisms (SNPs) were recorded between the two cp genomes. Of these, 52 SNPs (31.5%) were distributed in coding regions, 13 SNPs (7.9%) were in introns, and 100 SNPs (60.6%) were in intergenic spacers (IGS). The overall difference between the Asian and American E. arvense cp genomes was 0.12%. Despite the relatively high genetic diversity between Asian and American E. arvense, the two populations are recognized as a single species based on their high morphological similarity. This indicated that the two regional populations have been in morphological stasis. PMID:25157804

  16. Chloroplast genome differences between Asian and American Equisetum arvense (Equisetaceae) and the origin of the hypervariable trnY-trnE intergenic spacer.

    PubMed

    Kim, Hyoung Tae; Kim, Ki-Joong

    2014-01-01

    Comparative analyses of complete chloroplast (cp) DNA sequences within a species may provide clues to understand the population dynamics and colonization histories of plant species. Equisetum arvense (Equisetaceae) is a widely distributed fern species in northeastern Asia, Europe, and North America. The complete cp DNA sequences from Asian and American E. arvense individuals were compared in this study. The Asian E. arvense cp genome was 583 bp shorter than that of the American E. arvense. In total, 159 indels were observed between two individuals, most of which were concentrated on the hypervariable trnY-trnE intergenic spacer (IGS) in the large single-copy (LSC) region of the cp genome. This IGS region held a series of 19 bp repeating units. The numbers of the 19 bp repeat unit were responsible for 78% of the total length difference between the two cp genomes. Furthermore, only other closely related species of Equisetum also show the hypervariable nature of the trnY-trnE IGS. By contrast, only a single indel was observed in the gene coding regions: the ycf1 gene showed 24 bp differences between the two continental individuals due to a single tandem-repeat indel. A total of 165 single-nucleotide polymorphisms (SNPs) were recorded between the two cp genomes. Of these, 52 SNPs (31.5%) were distributed in coding regions, 13 SNPs (7.9%) were in introns, and 100 SNPs (60.6%) were in intergenic spacers (IGS). The overall difference between the Asian and American E. arvense cp genomes was 0.12%. Despite the relatively high genetic diversity between Asian and American E. arvense, the two populations are recognized as a single species based on their high morphological similarity. This indicated that the two regional populations have been in morphological stasis. PMID:25157804

  17. Evidence for Regulation of ECM3 Expression by Methylation of Histone H3 Lysine 4 and Intergenic Transcription in Saccharomyces cerevisiae

    PubMed Central

    Raupach, Elizabeth A.; Martens, Joseph A.; Arndt, Karen M.

    2016-01-01

    Transcription of nonprotein-coding DNA is widespread in eukaryotes and plays important regulatory roles for many genes, including genes that are misregulated in cancer cells. Its pervasiveness presents the potential for a wealth of diverse regulatory roles for noncoding transcription. We previously showed that the act of transcribing noncoding DNA (ncDNA) across the promoter of the protein-coding SER3 gene in Saccharomyces cerevisiae positions nucleosomes over the upstream activating sequences, leading to strong repression of SER3 transcription. To explore the possibility of other regulatory roles for ncDNA transcription, we selected six candidate S. cerevisiae genes that express ncRNAs over their promoters and analyzed the regulation of one of these genes, ECM3, in detail. Because noncoding transcription can lead to changes in the local chromatin landscape that impinge on the expression of nearby coding genes, we surveyed the effects of various chromatin regulators on the expression of ECM3. These analyses identified roles for the Paf1 complex in positively regulating ECM3 transcription through methylation of histone H3 at lysine 4 (K4) and for Paf1 in controlling the pattern of intergenic transcription at this locus. By deleting a putative promoter for the noncoding transcription unit that lies upstream of ECM3, we provide evidence for a positive correlation between intergenic transcription and ECM3 expression. Our results are consistent with a model in which cotranscriptional methylation of histone H3 K4, mediated by the Paf1 complex and noncoding transcription, leads to activation of ECM3 transcription. PMID:27449519

  18. beta-Cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenic consensus sequence.

    PubMed

    Abdou, S; Collomb, J; Sallas, F; Marsura, A; Finance, C

    1997-01-01

    The ability of cyclodextrins to enhance the antiviral activity of a phosphodiester oligodeoxynucleotide has been investigated. A 18-mer oligodeoxynucleotide complementary to the initiation region of the mRNA coding for the spike protein and containing the intergenic consensus sequence of an enteric coronavirus has been tested for antiviral action against virus growth in human adenocarcinoma cells. The phosphodiester oligodeoxynucleotide only showed a limited effect on virus growth rate (from 12 to 34% viral inhibition in cells treated with 7.5 to 25 microM oligodeoxynucleotide, respectively, at a multiplicity of infection of 0.1 infectious particle per cell). In the same conditions, the phosphorothioate analogue exhibited stronger antiviral activity, the inhibition increased from 56 to 90%. The inhibitory effect of this analogue was antisense and sequence-specific. Northern blot analysis showed that the sequence-dependent mechanism of action appears to be the inhibition of mRNA transcription. We conclude that the coronavirus intergenic consensus sequence is a good target for an antisense oligonucleotide antiviral action. The properties of the phosphodiester oligonucleotide was improved after its complexation with cyclodextrins. The most important increase of the antiviral activity (90% inhibition) was obtained with only 7.5 microM oligonucleotide complexed to a cyclodextrin derivative, 6-deoxy-6-S-beta-D-galactopyranosyl-6-thio-cyclomalto-heptaose+ ++ in a molar ratio of 1:100. These studies suggest that the use of cyclodextrin derivatives as carrier for phosphodiester oligonucleotides delivery may be an effective method for increasing the therapeutic potential of these compounds in viral infections. PMID:9672621

  19. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence.

    PubMed

    Hao, Huijing; Liang, Junrong; Duan, Ran; Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  20. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

    PubMed Central

    Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  1. 'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.

    PubMed

    Teixeira, Diva do Carmo; Saillard, Colette; Eveillard, Sandrine; Danet, Jean Luc; da Costa, Paulo Inácio; Ayres, Antonio Juliano; Bové, Joseph

    2005-09-01

    Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan

  2. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction

    PubMed Central

    Fritz, Brian R.; Jewett, Michael C.

    2014-01-01

    In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3′ modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

  3. Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

    PubMed Central

    Orsi, William; Biddle, Jennifer F.; Edgcomb, Virginia

    2013-01-01

    The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface. PMID:23418556

  4. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  5. Chromosomal localization and sequence variation of 5S rRNA gene in five Capsicum species.

    PubMed

    Park, Y K; Park, K C; Park, C H; Kim, N S

    2000-02-29

    Chromosomal localization and sequence analysis of the 5S rRNA gene were carried out in five Capsicum species. Fluorescence in situ hybridization revealed that chromosomal location of the 5S rRNA gene was conserved in a single locus at a chromosome which was assigned to chromosome 1 by the synteny relationship with tomato. In sequence analysis, the repeating units of the 5S rRNA genes in the Capsicum species were variable in size from 278 bp to 300 bp. In sequence comparison of our results to the results with other Solanaceae plants as published by others, the coding region was highly conserved, but the spacer regions varied in size and sequence. T stretch regions, just after the end of the coding sequences, were more prominant in the Capsicum species than in two other plants. High G x C rich regions, which might have similar functions as that of the GC islands in the genes transcribed by RNA PolII, were observed after the T stretch region. Although we could not observe the TATA like sequences, an AT rich segment at -27 to -18 was detected in the 5S rRNA genes of the Capsicum species. Species relationship among the Capsicum species was also studied by the sequence comparison of the 5S rRNA genes. While C. chinense, C. frutescens, and C. annuum formed one lineage, C. baccatum was revealed to be an intermediate species between the former three species and C. pubescens. PMID:10774742

  6. Decreases in average bacterial community rRNA operon copy number during succession.

    PubMed

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-05-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution. PMID:26565722

  7. Tax4Fun: predicting functional profiles from metagenomic 16S rRNA data

    PubMed Central

    Aßhauer, Kathrin P.; Wemheuer, Bernd; Daniel, Rolf; Meinicke, Peter

    2015-01-01

    Motivation: The characterization of phylogenetic and functional diversity is a key element in the analysis of microbial communities. Amplicon-based sequencing of marker genes, such as 16S rRNA, is a powerful tool for assessing and comparing the structure of microbial communities at a high phylogenetic resolution. Because 16S rRNA sequencing is more cost-effective than whole metagenome shotgun sequencing, marker gene analysis is frequently used for broad studies that involve a large number of different samples. However, in comparison to shotgun sequencing approaches, insights into the functional capabilities of the community get lost when restricting the analysis to taxonomic assignment of 16S rRNA data. Results: Tax4Fun is a software package that predicts the functional capabilities of microbial communities based on 16S rRNA datasets. We evaluated Tax4Fun on a range of paired metagenome/16S rRNA datasets to assess its performance. Our results indicate that Tax4Fun provides a good approximation to functional profiles obtained from metagenomic shotgun sequencing approaches. Availability and implementation: Tax4Fun is an open-source R package and applicable to output as obtained from the SILVAngs web server or the application of QIIME with a SILVA database extension. Tax4Fun is freely available for download at http://tax4fun.gobics.de/. Contact: kasshau@gwdg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25957349

  8. Interactions between 23S rRNA and tRNA in the ribosomal E site.

    PubMed Central

    Bocchetta, M; Xiong, L; Shah, S; Mankin, A S

    2001-01-01

    Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold. PMID:11214181

  9. Sequence and phylogenetic analysis of SSU rRNA gene of five microsporidia.

    PubMed

    Dong, ShiNan; Shen, ZhongYuan; Xu, Li; Zhu, Feng

    2010-01-01

    The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia. PMID:19768503

  10. Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

    PubMed

    Perrin-Pecontal, P; Gouy, M; Nigon, V M; Trabuchet, G

    1992-01-01

    A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human. PMID:1556740