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Sample records for ribosomal dna intergenic

  1. Unusual structure of ribosomal DNA in the copepod Tigriopus californicus: intergenic spacer sequences lack internal subrepeats.

    PubMed

    Burton, R S; Metz, E C; Flowers, J M; Willett, C S

    2005-01-03

    Eukaryotic nuclear ribosomal DNA (rDNA) is typically arranged as a series of tandem repeats coding for 18S, 5.8S, and 28S ribosomal RNAs. Transcription of rDNA repeats is initiated in the intergenic spacer (IGS) region upstream of the 18S gene. The IGS region itself typically consists of a set of subrepeats that function as transcriptional enhancers. Two important evolutionary forces have been proposed to act on the IGS region: first, selection may favor changes in the number of subrepeats that adaptively adjust rates of rDNA transcription, and second, coevolution of IGS sequence with RNA polymerase I transcription factors may lead to species specificity of the rDNA transcription machinery. To investigate the potential role of these forces on population differentiation and hybrid breakdown in the intertidal copepod Tigriopus californicus, we have characterized the rDNA of five T. californicus populations from the Pacific Coast of North America and one sample of T. brevicornicus from Scotland. Major findings are as follows: (1) the structural genes for 18S and 28S are highly conserved across T. californicus populations, in contrast to other nuclear and mitochondrial DNA (mtDNA) genes previously studied in these populations. (2) There is extensive differentiation among populations in the IGS region; in the extreme, no homology is observed across the IGS sequences (>2 kb) from the two Tigriopus species. (3) None of the Tigriopus IGS sequences have the subrepeat structure common to other eukaryotic IGS regions. (4) Segregation of rDNA in laboratory crosses indicates that rDNA is located on at least two separate chromosomes in T. californicus. These data suggest that although IGS length polymorphism does not appear to play the adaptive role hypothesized in some other eukaryotic systems, sequence divergence in the rDNA promoter region within the IGS could lead to population specificity of transcription in hybrids.

  2. Development of Specific Nested Oligonucleotide PCR Primers for the Streptococcus iniae 16S-23S Ribosomal DNA Intergenic Spacer

    PubMed Central

    Berridge, Brian R.; Fuller, Jeffrey D.; de Azavedo, Joyce; Low, Donald E.; Bercovier, Herve; Frelier, Paul F.

    1998-01-01

    Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer. PMID:9705438

  3. Identification and detection of Trypanosoma cruzi by using a DNA amplification fingerprint obtained from the ribosomal intergenic spacer.

    PubMed Central

    González, N; Galindo, I; Guevara, P; Novak, E; Scorza, J V; Añez, N; Da Silveira, J F; Ramírez, J L

    1994-01-01

    We designed a PCR assay targeted on repeated elements of the ribosomal intergenic spacer which produces highly polymorphic DNA band patterns for different strains of Trypanosoma cruzi. By labeling the PCR products with digoxigenin and by chemiluminescence detection, we improved the assay sensitivity by three orders of magnitude to get T. cruzi strain fingerprints in feces of the trypanosome-infected triatomine bug vector. We also developed a capture assay for the digoxigenin-labeled PCR products that allowed us to detect T. cruzi in triatomine bug vector feces and in human serum samples with a solid support. Images PMID:8126172

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

    PubMed

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

    2015-12-03

    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.

  5. 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

  6. Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals.

    PubMed

    Radeva, Galina; Selenska-Pobell, Sonja

    2005-11-01

    Bacterial diversity was assessed in water samples collected from several uranium mining wastes in Ger many and in the United States by using 16S rDNA and ribosomal intergenic spacer amplification retrievals. The results obtained using the 16S rDNA retrieval showed that the samples collected from the uranium mill tailings of Schlema/Alberoda, Germany, were predominated by Nitrospina-like bacteria, whereas those from the mill tailings of Shiprock, New Mexico, USA, were predominated by gamma-Pseudomonas and Frauteria spp. Additional smaller populations of the Cytophaga-Flavobacterium-Bacteroides group and alpha- and delta-Proteobacteria were identified in the Shiprock samples as well. Proteobacteria and Cytophaga-Flavobacterium-Bacteroides were also found in the third uranium mill tailings studied, Gittersee/Coschütz, Germany, but the groups of the predominant clones were rather small. Most of the clones of the Gittersee/Coschütz samples represented individual sequences, which indicates a high level of bacterial diversity. The samples from the fourth uranium waste studied, Steinsee Deponie B1, Germany, were predominantly occupied by Acinetobacter spp. The ribosomal intergenic spacer amplification retrieval provided results complementary to those obtained by the 16S rDNA analyses. For instance, in the Shiprock samples, an additional predominant bacterial group was identified and affiliated with Nitrosomonas sp., whereas in the Gittersee/Coschütz samples, anammox populations were identified that were not retrieved by the applied 16S rDNA approach.

  7. Genetic variability and geographical diversity of the main Chagas' disease vector Panstrongylus megistus (Hemiptera: Triatominae) in Brazil based on ribosomal DNA intergenic sequences.

    PubMed

    Cavassin, Francelisse Bridi; Kuehn, Christian Collins; Kopp, Rogério Luiz; Thomaz-Soccol, Vanette; Da Rosa, João Aristeu; Luz, Ennio; Mas-Coma, Santiago; Bargues, María Dolores

    2014-05-01

    Studies were made on the ribosomal DNA intergenic region, comprising complete internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 sequences, of populations of the triatomine Panstrongylus megistus, the most important vector of Chagas' disease in Brazil since Triatoma infestans eradication. Specimens were from 26 localities of Rio Grande do Sul, Santa Catarina, Paraná, São Paulo, Minas Gerais, Bahia, and Sergipe states. In total, 21 ITS-1 and 12 ITS-2 haplotypes were found. Nucleotide differences were higher in ITS-1 (3.00%) than in ITS-2 (1.33%). The intergenic region was 1,513-1,522-bp-long (mean 1,516.9 bp), providing 26 combined haplotypes. The combination of microsatellites found in both ITSs may be of applied usefulness, to assess interpopulation specimen exchange and potential recolonizations after vector elimination by control implementation. Network results suggest that São Paulo may be considered one of the spreading centers of this species. Molecular clock datation suggests that P. megistus populations are diversifying at least since 4.54 million years ago, with diversification still ongoing today by geographical isolation of populations. Evidence is provided about the relationship of genetic diversity with geographical spread that characterizes a major vector and explains its ability to colonize distant areas and different ecotopes, including human habitats, and consequently its importance in Chagas' disease epidemiology.

  8. Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes

    PubMed Central

    Mochizuki, Takashi; Ishizaki, Hiroshi; Barton, Richard C.; Moore, Mary K.; Jackson, Colin J.; Kelly, Steven L.; Evans, E. Glyn V.

    2003-01-01

    Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates. PMID:14532186

  9. 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

  10. Host selectivity and genetic variation of Discula umbrinella isolates from two oak species: analyses of intergenic spacer region sequences of ribosomal DNA.

    PubMed

    Cohen, Susan D

    2006-10-01

    Discula umbrinella, a fungal endophyte of oak species, colonizes and reproduces on leaves of Quercus alba and Q. rubra in forest ecosystems. Twenty-nine isolates collected from leaves of both oak species (16 from Q. alba and 13 from Q. rubra) were assayed for oak species preference and genetic variation based on primer-specific polymerase chain reactions for the intergenic spacer region (IGS) of ribosomal DNA. DNA sequencing of the polymerase chain reaction products revealed a 10-bp insertion (237-247 bp) at the 3' end of the IGS region present in nine isolates and absent in 20 of the isolates. Phylogenetic analysis of the IGS region using the neighbor-joining method identified IGS groups (groups I-V) based on single nucleotide sequence differences. Host selectivity and geographic origin of isolates were correlated in some instances with the IGS groups. Isolates within each IGS group were further analyzed for nucleotide polymorphisms to confirm genotype identity and genotype diversity. Ten different genotypes (Va-Vj) were identified among the isolates analyzed. Genotype diversity was greatest in IGS groups I, IV, and V. Seventy percent of the genotypes (Vc, Vd, Ve, Vf, Vg,Vi, and Vj) contained isolates with single tree species preferences.

  11. Cytomolecular Analysis of Ribosomal DNA Evolution in a Natural Allotetraploid Brachypodium hybridum and Its Putative Ancestors—Dissecting Complex Repetitive Structure of Intergenic Spacers

    PubMed Central

    Borowska-Zuchowska, Natalia; Kwasniewski, Miroslaw; Hasterok, Robert

    2016-01-01

    Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level. PMID:27790225

  12. Cytomolecular Analysis of Ribosomal DNA Evolution in a Natural Allotetraploid Brachypodium hybridum and Its Putative Ancestors-Dissecting Complex Repetitive Structure of Intergenic Spacers.

    PubMed

    Borowska-Zuchowska, Natalia; Kwasniewski, Miroslaw; Hasterok, Robert

    2016-01-01

    Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n = 30), which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n = 10) and B. stacei (2n = 20). Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs) from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level.

  13. The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

    PubMed

    Schnare, M N; Collings, J C; Spencer, D F; Gray, M W

    2000-09-15

    In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

  14. 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

  15. Potential application of ribosomal intergenic spacer analysis to the microbial community analysis of agronomic products.

    PubMed

    Ikeda, Seishi; Fujimura, Tatsuhito; Ytow, Nozomi

    2005-07-13

    Ribosomal intergenic spacer analysis (RISA) has been applied to the microbial community analysis of agronomic products in combination with a simple and rapid DNA extraction method, consisting of a one-step extraction and two-step purification, for a variety of agronomic products. RISA appears to be a useful tool for the study of the community structures of food-associated microbes and their use as a unique fingerprinting signature for each agronomic product. Sequencing analyses of amplicons generated from RISA suggest that this method can detect conventional microbes. In the case of RISA of wasabi paste DNA, the sequences of the amplicons showed high similarity to the plant pathogen Xanthomonas campestris and the soil bacterium Bacillus subtilis, whereas several food-associated bacteria (Lactococcus lactis, Lactococcus raffinolactis, and Lactococcus sakei) were detected using this technique in sausage DNA. Unexpectedly, the sequencing analyses also revealed the presence of several microbes that possessed high similarity to human bacterial pathogens such as Weissella confusa and Yersinia pestis. The results suggest that RISA will be a useful method for routine microbial community analysis in agronomic products.

  16. Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

    PubMed Central

    Ranjard, L.; Poly, F.; Lata, J.-C.; Mougel, C.; Thioulouse, J.; Nazaret, S.

    2001-01-01

    Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi. PMID:11571146

  17. Molecular characterization of ribosomal intergenic spacer in the tadpole shrimp Triops cancriformis (Crustacea, Branchiopoda, Notostraca).

    PubMed

    Luchetti, Andrea; Scanabissi, Franca; Mantovani, Barbara

    2006-08-01

    Nuclear ribosomal DNA constitutes a multigene family, with tandemly arranged units linked by an intergenic spacer (IGS), which contains initiation/termination transcription signals and usually tandemly arranged subrepeats. The structure and variability of the IGS region are analyzed here in hermaphroditic and parthenogenetic populations of the "living fossil" Triops cancriformis (Branchiopoda, Notostraca). The results indicate the presence of concerted evolution at the population level for this G+C-rich IGS region as a whole, with the major amount of genetic variability found outside the subrepeat region. The subrepeats region is composed of 3 complete repeats (a, c, d) intermingled with 3 repeat fragments (b, e, f) and unrelated sequences. The most striking datum is the absolute identity of subrepeats (except type d) occupying the same position in different individuals/populations. A putative promoter sequence is present upstream of the 18S rRNA gene, but not in subrepeats, which is at variance with other arthropod IGSs. The absence of a promoter sequence in the subrepeats and subrepeat sequence conservation suggests that this region acts as an enhancer simply by its repetitive nature, as observed in some vertebrates. The putative external transcribed spacer (840 bp) shows hairpin structures, as in yeasts, protozoans, Drosophila, and vertebrates.

  18. Characterization of North American Armillaria species: Genetic relationships determined by ribosomal DNA sequences and AFLP markers

    Treesearch

    M. -S. Kim; N. B. Klopfenstein; J. W. Hanna; G. I. McDonald

    2006-01-01

    Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS-1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data,...

  19. A comparison of fungal communities from four salt marsh plants using automated ribosomal intergenic spacer analysis (ARISA).

    PubMed

    Torzilli, Albert P; Sikaroodi, Masoumeh; Chalkley, David; Gillevet, Patrick M

    2006-01-01

    Fungal decomposers are important contributors to the detritus-based food webs of salt marsh ecosystems. Knowing the composition of salt marsh fungal communities is essential in understanding how detritus processing is affected by changes in community dynamics. Automated ribosomal intergenic spacer analysis (ARISA) was used to examine the composition of fungal communities associated with four temperate salt marsh plants, Spartina alterniflora (short and tall forms), Juncus roemerianus, Distichlis spicata and Sarcocornia perennis. Plant tissues were homogenized and subjected to a particle-filtration protocol that yielded 106 microm particulate fractions, which were used as a source of fungal isolates and fungal DNA. Genera identified from sporulating cultures demonstrated that the 106 microm particles from each host plant were reliable sources of fungal DNA for ARISA. Analysis of ARISA data by principal component analysis (PCA), principal coordinate analysis (PCO) and species diversity comparisons indicated that the fungal communities from the two grasses, S. alterniflora and D. spicata were more similar to each other than they were to the distinct communities associated with J. roemerianus and S. perennis. Principal component analysis also showed no consistent, seasonal pattern in the composition of these fungal communities. Comparisons of ARISA fingerprints from the different fungal communities and those from pure cultures of selected Spartina ascomycetes supported the host/substrate specificity observed for the fungal communities.

  20. Combined eukaryotic and bacterial community fingerprinting of natural freshwater biofilms using automated ribosomal intergenic spacer analysis.

    PubMed

    Fechner, Lise C; Vincent-Hubert, Françoise; Gaubert, Philippe; Bouchez, Théodore; Gourlay-Francé, Catherine; Tusseau-Vuillemin, Marie-Hélène

    2010-12-01

    Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested it on natural biofilms. Additionally, we tested universal primers, used previously to perform ARISA on fungal communities. Cloning and sequencing showed that the universal primer set amplified various eukaryotes, whereas the new set was diatom specific. The new set amplified a wider variety of diatoms. Therefore, the universal set is appropriate to study the general eukaryotic population shifts in biofilms, whereas the new set is more appropriate to study diatoms specifically. We used both primer sets, along with a bacterial set, to study the population shifts in natural river biofilms. Principal component analysis of the ARISA fingerprints revealed seasonal shifts that did not coincide for bacterial and eukaryotic communities. Therefore, the use of both eukaryotic and bacterial primers provides a useful insight to assess microbial succession in biofilms.

  1. 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.

  2. Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

    PubMed Central

    HATAKEYAMA, YOSHINORI; SHIBUYA, NORIHIRO; NISHIYAMA, TAKASHI; NAKASHIMA, NOBUHIKO

    2004-01-01

    The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses. PMID:15100433

  3. Comparison of Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Aquatic Bacterial Communities: an Ecological Perspective▿

    PubMed Central

    Jones, Stuart E.; Shade, Ashley L.; McMahon, Katherine D.; Kent, Angela D.

    2007-01-01

    Two primer sets for automated ribosomal intergenic spacer analysis (ARISA) were used to assess the bacterial community composition (BCC) in Lake Mendota, Wisconsin, over 3 years. Correspondence analysis revealed differences in community profiles generated by different primer sets, but overall ecological patterns were conserved in each case. ARISA is a powerful tool for evaluating BCC change through space and time, regardless of the specific primer set used. PMID:17122397

  4. Evolutionary Mobility of the Ribosomal DNA Array in Yeasts

    PubMed Central

    Proux-Wéra, Estelle; Byrne, Kevin P.; Wolfe, Kenneth H.

    2013-01-01

    The ribosomal DNA (rDNA) of eukaryotes is organized as large tandem arrays. Here, we compare the genomic locations of rDNA among yeast species and show that, despite its huge size (>1 Mb), the rDNA array has moved around the genome several times within the family Saccharomycetaceae. We identify an ancestral, nontelomeric, rDNA site that is conserved across many species including Saccharomyces cerevisiae. Within the genus Lachancea, however, the rDNA apparently transposed from the ancestral site to a new site internal to a different chromosome, becoming inserted into a short intergenic region beside a tRNA gene. In at least four other yeast lineages, the rDNA moved from the ancestral site to telomeric locations. Remarkably, both the ancestral rDNA site and the new site in Lachancea are adjacent to protein-coding genes whose products maintain the specialized chromatin structure of rDNA (HMO1 and CDC14, respectively). In almost every case where the rDNA was lost from the ancestral site, the entire array disappeared without any other rearrangements in the region, leaving just an intergenic spacer of less than 2 kb. The mechanism by which this large and complex locus moves around the genome is unknown, but we speculate that it may involve the formation of double-strand DNA breaks by Fob1 protein or the formation of extrachromosomal rDNA circles. PMID:23419706

  5. Ribosomal Intergenic Spacer 1 Based Characterization of Button Mushroom (Agaricus bisporus) Strains.

    PubMed

    Kwon, Hyuk Woo; Choi, Min Ah; Kim, Dae Wook; Oh, Youn-Lee; Hyun, Min Woo; Kong, Won-Sik; Kim, Seong Hwan

    2016-12-01

    Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom.

  6. Ribosomal Intergenic Spacer 1 Based Characterization of Button Mushroom (Agaricus bisporus) Strains

    PubMed Central

    Kwon, Hyuk Woo; Choi, Min Ah; Kim, Dae Wook; Oh, Youn-Lee; Hyun, Min Woo; Kong, Won-Sik

    2016-01-01

    Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom. PMID:28154490

  7. Ribosomal intergenic spacer analysis as a tool for monitoring methanogenic Archaea changes in an anaerobic digester.

    PubMed

    Ciesielski, Slawomir; Bułkowska, Katarzyna; Dabrowska, Dorota; Kaczmarczyk, Dariusz; Kowal, Przemyslaw; Możejko, Justyna

    2013-08-01

    The applicability of a newly-designed PCR primer pair in examination of methanogenic Archaea in a digester treating plant biomass was evaluated by Ribosmal Intergenic Spacer Analysis (RISA). To find a suitable approach, three variants of RISA were tested: (1) standard, polyacrylamide gel-based, (2) automated, utilized capillary electrophoresis (GA-ARISA), and (3) automated microfluidics-based (MF-ARISA). All three techniques yielded a consistent picture of archaeal community structure changes during anaerobic digestion monitored for more than 6 weeks. While automated variants were more practical for handling and rapid analysis of methanogenic Archaea, the gel-based technique was advantageous when micro-organism identification was required. A DNA-sequence analysis of dominant bands extracted from the gel revealed that the main role in methane synthesis was played by micro-organisms affiliated with Methanosarcina barkeri. The obtained results revealed that RISA is a robust method allowing for detailed analysis of archaeal community structure during organic biomass conversion into biogas. In addition, our results showed that GA-ARISA has a higher resolution and reproducibility than other variants of RISA and could be used as a technique for tracking changes in methanogenic Archaea in an anaerobic digester.

  8. Heterogeneity within the gram-positive anaerobic cocci demonstrated by analysis of 16S-23S intergenic ribosomal RNA polymorphisms.

    PubMed

    Hill, K E; Davies, C E; Wilson, M J; Stephens, P; Lewis, M A O; Hall, V; Brazier, J; Thomas, D W

    2002-11-01

    Peptostreptococci are gram-positive, strictly anaerobic bacteria which, although regarded as members of the commensal human microflora, are also frequently isolated from sites of clinical infection. The study of this diverse group of opportunist pathogens has been hindered by an inadequate taxonomy and the lack of a valid identification scheme. Recent re-classification of the Peptostreptococcus family into five distinct genus groups has helped to clarify the situation. However, this has been on the basis of 16S rRNA sequence determinations, which are both time-consuming and expensive. The aim of the present study was to evaluate the use of PCR-amplified ribosomal DNA spacer polymorphisms for the rapid differentiation of the currently recognised taxa within the group of anaerobic gram-positive cocci. A collection comprising 19 reference strains with representatives of each of the 15 species, two close relatives and two of the well-characterised groups, together with 38 test strains was studied. All strains were identified to species group level by phenotypic means. Amplification of the 16S-23S intergenic spacer region (ISR) with universal primers produced distinct banding patterns for all the 19 reference strains and the patterns could be differentiated easily visually. However, of the 38 test strains, less than half could be speciated from ISR analysis alone. Only five groups produced correlating banding patterns for all members tested (Peptoniphilus lacrimalis, P. ivorii, Anaerococcus octavius, Peptostreptococcus anaerobius and Micromonas micros). For other species, either the type strain differed significantly from other species members (e.g., A. hydrogenalis) or there appeared to be considerable intra-species variation (e.g., A. vaginalis). Partial 16S rRNA gene sequences for the 'trisimilis' and 'betaGAL' groups showed that both are most closely related to the Anaerococcus group. This work highlights the heterogeneous nature of a number of Peptostreptococcus

  9. Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni

    USDA-ARS?s Scientific Manuscript database

    Campylobacter jejuni and Campylobacter coli are closely related species that can not be distinguished by their 16S or 23S rRNA gene sequences. However, the intergenic sequence region (ISR) that is between the 16S and 23S genes is markedly different and characteristic for each species. A peculiarit...

  10. 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.

  11. [Comparative characteristics of intergenic spacers of ribosomal RNA gene cluster in mosquitoes of the genus Culex (Diptera: Culicidae)].

    PubMed

    Shaĭkevich, E V; Zagoskin, M V; Mukha, D V

    2013-01-01

    Nucleotide sequences of intergenic spacer of ribosomal RNA gene cluster (rIGS) were identified in mosquitoes Culex modestus, Culex torrentium and Culex pipiens pallens. The level of interpopulation variability of the rIGS in the subspecies C. pipiens pipiens (form pipiens--mosquitoes that inhabit the open waters, and form molestus--mosquitoes that inhabit basements) living in Russia was estimated. No extensive repetitive sequences characteristic of the rIGS of all previously described species of mosquitoes were found within the rIGS of Culex mosquitoes. At the same time, evolutionarily conserved motifs and relatively short degenerate sequences of different classes of transposable elements, as well as multiple blocks of variable microsatellite repeats were identified. Our data demonstrated that the rIGS of Culex mosquitoes can be considered as a promising molecular marker for the analysis of population and phylogenetic relationships within this group of insects.

  12. Cultivation-Independent Characterization of Methylobacterium Populations in the Plant Phyllosphere by Automated Ribosomal Intergenic Spacer Analysis▿ †

    PubMed Central

    Knief, Claudia; Frances, Lisa; Cantet, Franck; Vorholt, Julia A.

    2008-01-01

    Bacteria of the genus Methylobacterium are widespread in the environment, but their ecological role in ecosystems, such as the plant phyllosphere, is not very well understood. To gain better insight into the distribution of different Methylobacterium species in diverse ecosystems, a rapid and specific cultivation-independent method for detection of these organisms and analysis of their community structure is needed. Therefore, 16S rRNA gene-targeted primers specific for this genus were designed and evaluated. These primers were used in PCR in combination with a reverse primer that binds to the tRNAAla gene, which is located upstream of the 23S rRNA gene in the 16S-23S intergenic spacer (IGS). PCR products that were of different lengths were obtained due to the length heterogeneity of the IGS of different Methylobacterium species. This length variation allowed generation of fingerprints of Methylobacterium communities in environmental samples by automated ribosomal intergenic spacer analysis. The Methylobacterium communities on leaves of different plant species in a natural field were compared using this method. The new method allows rapid comparisons of Methylobacterium communities and is thus a useful tool to study Methylobacterium communities in different ecosystems. PMID:18263752

  13. 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. Copyright © 2012 Elsevier Inc

  14. Characterization of IS6110 insertions in the dnaA-dnaN intergenic region of Mycobacterium tuberculosis clinical isolates.

    PubMed

    Turcios, L; Casart, Y; Florez, I; de Waard, J; Salazar, L

    2009-02-01

    Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to be clonally related. The presence of IS6110 in the dnaA-dnaN intergenic region, one preferential locus for the integration of IS6110, was evaluated in 125 M. tuberculosis isolates. Five isolates had IS6110 inserted in this region, and two consisted of a mix of isogenic strains that putatively have evolved during a single infection. Strains from the same isolate had identical spoligo and mycobacterial interspersed repetitive unit-variable-number tandem repeat profiles, but had slight variations in IS6110 RFLP patterns, due to the presence of IS6110 in the dnaA-dnaN intergenic region. Duplication of the dnaA-dnaN intergenic region was found in one isogenic strain.

  15. 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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  17. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-15

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen the yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  18. The Epigenetic Pathways to Ribosomal DNA Silencing

    PubMed Central

    Srivastava, Rakesh; Srivastava, Rashmi

    2016-01-01

    SUMMARY Heterochromatin is the transcriptionally repressed portion of eukaryotic chromatin that maintains a condensed appearance throughout the cell cycle. At sites of ribosomal DNA (rDNA) heterochromatin, epigenetic states contribute to gene silencing and genome stability, which are required for proper chromosome segregation and a normal life span. Here, we focus on recent advances in the epigenetic regulation of rDNA silencing in Saccharomyces cerevisiae and in mammals, including regulation by several histone modifications and several protein components associated with the inner nuclear membrane within the nucleolus. Finally, we discuss the perturbations of rDNA epigenetic pathways in regulating cellular aging and in causing various types of diseases. PMID:27250769

  19. Ribosomal RNA gene silencing in interpopulation hybrids of Tigriopus californicus: nucleolar dominance in the absence of intergenic spacer subrepeats.

    PubMed

    Flowers, Jonathan M; Burton, Ronald S

    2006-07-01

    A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in F1 and F2 interpopulation hybrids of this species. In the F2 generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants.

  20. Ribosomal RNA Gene Silencing in Interpopulation Hybrids of Tigriopus californicus: Nucleolar Dominance in the Absence of Intergenic Spacer Subrepeats

    PubMed Central

    Flowers, Jonathan M.; Burton, Ronald S.

    2006-01-01

    A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in F1 and F2 interpopulation hybrids of this species. In the F2 generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants. PMID:16648582

  1. Comparison of soil fungal community structure in different peanut rotation sequences using ribosomal intergenic spacer analysis in relation to aflatoxin-producing fungi.

    PubMed

    Sudini, H; Arias, C R; Liles, M R; Bowen, K L; Huettel, R N

    2011-01-01

    The present study focuses on determining soil fungal community structure in different peanut-cropping sequences by using a high-resolution DNA fingerprinting technique: ribosomal intergenic spacer analysis (RISA). This study was initiated to determine fungal community profiles in four peanut-cropping sequences (continuous peanut, 4 years of continuous bahiagrass followed by peanut, peanut-corn-cotton, and peanut-cotton rotations), with a special focus to evaluate whether the profiles under investigation may have also indicated microbial differences that could affect Aspergillus flavus populations. Results indicated 75% similarities among fungal communities from the same cropping sequences as well as with similar times of sampling. Polymerase chain reaction (PCR)-based detection of A. flavus directly from these soils was carried out using A. flavus-specific primers (FLA1 and FLA2) and also through quantitative estimation on A. flavus and A. parasiticus agar medium. Population levels of A. flavus in soil samples ranged from zero to 1.2 × 10(3) CFU g(-1) of soil (based on culturable methods); however, the fungus was not detected with A. flavus-specific primers. The minimum threshold limit at which these aflatoxin-producing fungi could be detected from the total soil genomic DNA was determined through artificial inoculation of samples with 10-fold increases in concentrations. The results indicated that a minimum population density of 2.6 × 10(6) CFU g(-1) of soil is required for PCR detection in our conditions. These results are useful in further determining the relative population levels of these fungi in peanut soils with other soil fungi. This is a new approach to understanding soil fungal communities and how they might change over time and under different rotation systems.

  2. Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products.

    PubMed

    Martin, F; Vairelles, D; Henrion, B

    1993-10-01

    Capillary electrophoresis (CE) provides a rapid and automated technique for the analysis of subnanogram amounts of DNA fragments generated by the polymerase chain reaction (PCR). Here we describe the implementation of size-selective CE for DNA profiling and restriction fragment length polymorphism analysis of amplified polymorphic spacers of ribosomal DNA from fungi. Separations of unpurified and isopropanol-precipitated PCR-amplified DNA fragments in the size range of 20-1600 base pairs were achieved in less than 20 min with the use of hydroxypropyl methylcellulose as a sieving medium. The amplified internal transcribed spacer (ITS) and intergenic spacer (IGS) of RNA genes could be sized by coelectrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation. This, together with the strictly controlled conditions of CE, permit the discrimination of amplified rDNA fragments differing only a few base pairs in length. Inter- and intraspecific variation in the size and number of restriction sites of the amplified rDNA spacers from the ectomycorrhizal basidiomycetes Laccaria laccata and Laccaria bicolor was observed and most strains could thus be reliably genotyped by PCR-CE. Multiple amplified IGS fragments of heterogeneous size were detected in several strains. This polymorphism is due to the occurrence of 5S rDNA subrepeats (i.e., multiple annealing of primer) within IGS. With CE, in contrast to slab gel electrophoresis, run times are short, the capillary can be reused, and full automation is feasible. Data acquisition and analysis are computer-controlled, which facilitates the locus identification and reduces error especially when large numbers of PCR products must be analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates.

    PubMed

    Ryberg, Anna; Olsson, Crister; Ahrné, Siv; Monstein, Hans-Jürg

    2011-02-01

    Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)(5)- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)(5) and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)(5) and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)(5) and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. CtBP Levels Control Intergenic Transcripts, PHO/YY1 DNA Binding, and PcG Recruitment to DNA

    PubMed Central

    Basu, Arindam; Atchison, Michael L.

    2013-01-01

    Carboxy-terminal binding protein (CtBP) is a well-known corepressor of several DNA binding transcription factors in Drosophila as well as in mammals. CtBP is implicated in Polycomb Group (PcG) complex-mediated transcriptional repression because it can bind to some PcG proteins, and mutation of the ctbp gene in flies results in lost PcG protein recruitment to Polycomb Response Elements (PREs) and lost PcG repression. However, the mechanism of reduced PcG DNA binding in CtBP mutant backgrounds is unknown. We show here that in a Drosophila CtBP mutant background, intergenic transcripts are induced across several PRE sequences and this corresponds to reduced DNA binding by PcG proteins Pleiohomeotic (PHO) and Polycomb (Pc), and reduced trimethylation of histone H3 on lysine 27, a hallmark of PcG repression. Restoration of CtBP levels by expression of a CtBP transgene results in repression of intergenic transcripts, restored PcG binding, and elevated trimethylation of H3 on lysine 27. Our results support a model in which CtBP regulates expression of intergenic transcripts that controls DNA binding by PcG proteins and subsequent histone modifications and transcriptional activity. PMID:20082324

  5. DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

    PubMed Central

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

    2013-01-01

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

  6. DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

    PubMed

    Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

    2013-03-29

    The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

  7. Regulation of ribosomal DNA amplification by the TOR pathway

    PubMed Central

    Jack, Carmen V.; Cruz, Cristina; Hull, Ryan M.; Keller, Markus A.; Ralser, Markus; Houseley, Jonathan

    2015-01-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  8. Structure and organization of the rDNA intergenic spacer in lake trout (Salvelinus namaycush).

    PubMed

    Reed, K M; Phillips, R B

    2000-01-01

    A total-genomic cosmid library was created to isolate complete copies of the rDNA cistron of lake trout (Salvelinus namaycush) in order to study the structure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA-positive clones (average inserts > 25 kb) was recovered by screening the library with a rDNA-specific probe. Positive clones were assayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) and the entire IGS fragment was successfully amplified from 42 clones by PCR. Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restriction mapping of the IGS-PCR products of several clones indicated two regions of extensive length variation surrounding a central region with sequence conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region responsible for this variation. Over 9 kb of DNA sequence was obtained for one clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number of short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequences. These included an 89-bp tandem repeat (alpha repeats), an 82-bp element (beta repeats), a 168-177-bp element (chi repeats), and a 179-201-bp element (delta repeats). Overall nucleotide composition of the IGS was biased towards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to produce homogeneous copies across the genome.

  9. DnaK-facilitated ribosome assembly in Escherichia coli revisited

    PubMed Central

    ALIX, JEAN-HERVÉ; NIERHAUS, KNUD H.

    2003-01-01

    Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30°C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37°C implicates a direct or indirect role for DnaK in this process. PMID:12810912

  10. Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe.

    PubMed

    Cunningham, C O; Collins, C M; Malmberg, G; Mo, T A

    2003-12-29

    The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.

  11. Sequencing Bands of Ribosomal Intergenic Spacer Analysis Fingerprints for Characterization and Microscale Distribution of Soil Bacterium Populations Responding to Mercury Spiking

    PubMed Central

    Ranjard, Lionel; Brothier, Elisabeth; Nazaret, Sylvie

    2000-01-01

    Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to β-proteobacteria (Ralstonia-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking. PMID:11097911

  12. Non-canonical ribosomal DNA segments in the human genome, and nucleoli functioning.

    PubMed

    Kupriyanova, Natalia S; Netchvolodov, Kirill K; Sadova, Anastasia A; Cherepanova, Marina D; Ryskov, Alexei P

    2015-11-10

    Ribosomal DNA (rDNA) in the human genome is represented by tandem repeats of 43 kb nucleotide sequences that form nucleoli organizers (NORs) on each of five pairs of acrocentric chromosomes. RDNA-similar segments of different lengths are also present on (NOR)(-) chromosomes. Many of these segments contain nucleotide substitutions, supplementary microsatellite clusters, and extended deletions. Recently, it was shown that, in addition to ribosome biogenesis, nucleoli exhibit additional functions, such as cell-cycle regulation and response to stresses. In particular, several stress-inducible loci located in the ribosomal intergenic spacer (rIGS) produce stimuli-specific noncoding nucleolus RNAs. By mapping the 5'/3' ends of the rIGS segments scattered throughout (NOR)(-) chromosomes, we discovered that the bonds in the rIGS that were most often susceptible to disruption in the rIGS were adjacent to, or overlapped with stimuli-specific inducible loci. This suggests the interconnection of the two phenomena - nucleoli functioning and the scattering of rDNA-like sequences on (NOR)(-) chromosomes. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

    PubMed Central

    Vafadar-Isfahani, Natasha; Parr, Christina; McMillan, Lara E.; Sanner, Juliane; Yeo, Zhao; Saddington, Stephen; Peacock, Oliver; Cruickshanks, Hazel A.; Meehan, Richard R.; Lund, Jonathan N.

    2017-01-01

    ABSTRACT Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergenic, transpositionally inactive LINE-1 elements and expression of the LINE-1 chimeric transcript (LCT) 13 and LCT14 driven by their antisense promoters (L1-ASP). Our data from DNA modification, expression, and 5′RACE analyses suggest that colorectal cancer methylation in the regions analyzed is not always associated with LCT repression. Consistent with this, in HCT116 colorectal cancer cells lacking DNA methyltransferases DNMT1 or DNMT3B, LCT13 expression decreases, while cells lacking both DNMTs or treated with the DNMT inhibitor 5-azacytidine (5-aza) show no change in LCT13 expression. Interestingly, levels of the H4K20me3 histone modification are inversely associated with LCT13 and LCT14 expression. Moreover, at these LINE-1s, H4K20me3 levels rather than DNA methylation seem to be good predictor of their sensitivity to 5-aza treatment. Therefore, by studying individual LINE-1 promoters we have shown that in some cases these promoters can be active without losing methylation; in addition, we provide evidence that other factors (e.g., H4K20me3 levels) play prominent roles in their regulation. PMID:28300471

  14. An AluI fragment isolated from lake trout (Salvelinus namaycush), maps to the intergenic spacer region of the rDNA cistron.

    PubMed

    Reed, K M; Oakley, T H; Phillips, R B

    1997-02-20

    The relationship between a 217-bp AluI fragment (SnAluI-33c) from lake trout (Salvelinus namaycush) which hybridizes to the nucleolar organizer regions (NORs) and the ribosomal RNA genes was examined by Southern analysis and comparative hybridization. Restriction enzymes with recognition sites mapped in the lake trout rDNA cistron were used to digest genomic DNA into fragments of predetermined size. Comparison of the hybridization pattern of SnAluI-33c with those of two rDNA-specific probes placed this fragment within the intergenic spacer region of the rDNA cistron, approximately 3 kb upstream (5') of the 18S gene. This finding is consistent with in situ hybridization experiments showing hybridization of this fragment to sites of rDNA [Reed, K.M. and Phillips, R.B., Cytogenet. Cell Genet. 70 (1995) 104-107]. Based on cross hybridization and sequence comparisons, homologous sequences are present in other salmonid species.

  15. 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

  16. Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives.

    PubMed

    Fregene, M A; Vargas, J; Ikea, J; Angel, F; Tohme, J; Asiedu, R A; Akoroda, M O; Roca, W M

    1994-11-01

    Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.

  17. Intraspecific Variation in Ribosomal DNA in Populations of the Potato Cyst Nematode Globodera pallida

    PubMed Central

    Blok, V. C.; Malloch, G.; Harrower, B.; Phillips, M. S.; Vrain, T. C.

    1998-01-01

    The relationships among a number of populations of Globodera pallida from Britian, the Netherlands, Germany, Switzerland, and South America were examined using PCR amplification of the ribosomal cistron between the 18S and 28S genes that include the two intergenic spacer regions (ITS1 and ITS2) and the 5.8S gene. Amplifications produced a similar-sized product of 1150 bp from all populations. Digestion of the amplified fragment with a number of restriction enzymes showed differences among the populations. The restriction enzyme RsaI distinguished the most populations. The RFLP patterns revealed by this enzyme were complex and could have arisen from heterogeneity between individuals within populations and from differences between the repeats of an individual. Sequence analysis from six of the populations, together with RFLP analysis of PCR products, shows that there is intraspecific variation in the rDNA of G. pallida. PMID:19274220

  18. Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

    PubMed

    Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

    2017-02-01

    Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome

    PubMed Central

    Robicheau, Brent M.; Susko, Edward; Harrigan, Amye M.

    2017-01-01

    Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an “rDNA-like signal”, representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. PMID:28204512

  20. Zuotin, a ribosome-associated DnaJ molecular chaperone.

    PubMed Central

    Yan, W; Schilke, B; Pfund, C; Walter, W; Kim, S; Craig, E A

    1998-01-01

    Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins. In Saccharomyces cerevisiae, SSB proteins are ribosome-associated Hsp70s which interact with the newly synthesized nascent polypeptide chain. Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ-related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity. Zuo1, which has been shown previously to be a nucleic acid-binding protein, is also a ribosome-associated protein localized predominantly in the cytosol. Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA. We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome. PMID:9707440

  1. Zuotin, a ribosome-associated DnaJ molecular chaperone.

    PubMed

    Yan, W; Schilke, B; Pfund, C; Walter, W; Kim, S; Craig, E A

    1998-08-17

    Correct folding of newly synthesized polypeptides is thought to be facilitated by Hsp70 molecular chaperones in conjunction with DnaJ cohort proteins. In Saccharomyces cerevisiae, SSB proteins are ribosome-associated Hsp70s which interact with the newly synthesized nascent polypeptide chain. Here we report that the phenotypes of an S.cerevisiae strain lacking the DnaJ-related protein Zuotin (Zuo1) are very similar to those of a strain lacking Ssb, including sensitivities to low temperatures, certain protein synthesis inhibitors and high osmolarity. Zuo1, which has been shown previously to be a nucleic acid-binding protein, is also a ribosome-associated protein localized predominantly in the cytosol. Analysis of zuo1 deletion and truncation mutants revealed a positive correlation between the ribosome association of Zuo1 and its ability to bind RNA. We propose that Zuo1 binds to ribosomes, in part, by interaction with ribosomal RNA and that Zuo1 functions with Ssb as a chaperone on the ribosome.

  2. Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

    PubMed

    Zhuo, L; Reed, K M; Phillips, R B

    1995-06-01

    Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

  3. Ribosomal DNA variation, recombination and inheritance in the basidiomycete Trichaptum abietinum: implications for reticulate evolution.

    PubMed

    Kauserud, H; Schumacher, T

    2003-08-01

    Two divergent nuclear ribosomal DNA (nrDNA) types, designated alpha and beta, were found distributed in 11 North European populations of the basidiomycete Trichaptum abietinum. These types differed by a 220 bp indel in the internal transcribed spacer 1 (ITS1) sequence and a number of linked substitutions and small indel motives in the internal transcribed and intergenic spacers (ITS1, ITS2, IGS1 and IGS2). The alpha and beta haplotypes co-occurred in heterozygous somatic individuals (dikaryons) and segregated in a Mendelian fashion in monokaryotic single spore progenies. This result suggests that the haplotypes are encoded in different nuclei of field-collected dikaryons and inherited as a single locus. No meiotic recombinants were observed among the sequenced monokaryons. Population genetic analyses by PCR-RFLP revealed that a low frequency of evolutionary intermediate nrDNA types also existed in natural populations, presumably as a result of meiotic recombination of alpha and beta nrDNA. The existence of divergent nrDNA types in T. abietinum could be a result of a former independent evolution followed by a hybridization event. Phylogenetic analyses of ITS sequences suggest that the sister taxon T. fusco-violaceum has been involved in the evolutionary history of T. abietinum. Sequence polymorphisms observed in the translation elongation factor 1alpha (efa) and glyceraldehyde-3-phosphate dehydrogenase (gpd) genes, did not reveal two well-defined types of these genes. The results are discussed in the light of other evolutionary mechanisms as well.

  4. [Structural organization of 5S ribosomal DNA of Rosa rugosa].

    PubMed

    Tynkevych, Iu O; Volkov, R A

    2014-01-01

    In order to clarify molecular organization of the genomic region encoding 5S rRNA in diploid species Rosa rugosa several 5S rDNA repeated units were cloned and sequenced. Analysis of the obtained sequences revealed that only one length variant of 5S rDNA repeated units, which contains intact promoter elements in the intergenic spacer region (IGS) and appears to be transcriptionally active is present in the genome. Additionally, a limited number of 5S rDNA pseudogenes lacking a portion of coding sequence and the complete IGS was detected. A high level of sequence similarity (from 93.7 to 97.5%) between the IGS of major 5S rDNA variants of East Asian R. rugosa and North American R. nitida was found indicating comparatively recent divergence of these species.

  5. The localization of ribosomal DNA in Sciaridae (Diptera: Nematocera) reassessed.

    PubMed

    Madalena, Christiane Rodriguez Gutierrez; Amabis, José Mariano; Stocker, Ann Jacob; Gorab, Eduardo

    2007-01-01

    The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.

  6. PCR Primers for Metazoan Mitochondrial 12S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Kweskin, Matthew; Knowlton, Nancy

    2012-01-01

    Background Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. Methodology/Principal Findings A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. Conclusions/Significance Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans. PMID:22536450

  7. Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba.

    PubMed

    Maggini, F; Cremonini, R; Zolfino, C; Tucci, G F; D'Ovidio, R; Delre, V; DePace, C; Scarascia Mugnozza, G T; Cionini, P G

    1991-05-01

    Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.

  8. The evolution of ribosomal DNA: Divergent paralogues and phylogenetic implications

    SciTech Connect

    Buckler, E.S. IV; Ippolito, A.; Holtsford, T.P.

    1997-03-01

    Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies. 56 refs., 3 figs., 3 tabs.

  9. Analysis of the genetic diversity of Legionella by sequencing the 23S-5S ribosomal intergenic spacer region: from phylogeny to direct identification of isolates at the species level from clinical specimens.

    PubMed

    Grattard, Florence; Ginevra, Christophe; Riffard, Serge; Ros, Alain; Jarraud, Sophie; Etienne, Jerome; Pozzetto, Bruno

    2006-01-01

    This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.

  10. Ribosomal DNA copy number loss and sequence variation in cancer

    PubMed Central

    Xu, Baoshan; Li, Hua; Perry, John M.; Singh, Vijay Pratap; Yu, Zulin; Zakari, Musinu; Li, Linheng

    2017-01-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments. PMID:28640831

  11. Ribosomal DNA copy number loss and sequence variation in cancer.

    PubMed

    Xu, Baoshan; Li, Hua; Perry, John M; Singh, Vijay Pratap; Unruh, Jay; Yu, Zulin; Zakari, Musinu; McDowell, William; Li, Linheng; Gerton, Jennifer L

    2017-06-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

  12. Human skin color is influenced by an intergenic DNA polymorphism regulating transcription of the nearby BNC2 pigmentation gene.

    PubMed

    Visser, Mijke; Palstra, Robert-Jan; Kayser, Manfred

    2014-11-01

    Single nucleotide polymorphisms (SNPs) found to be statistically significant when associated with human diseases, and other phenotypes are most often located in non-coding regions of the genome. One example is rs10765819 located in the first intron of the BNC2 gene previously associated with (saturation of) human skin color. Here, we demonstrate that a nearby intergenic SNP (rs12350739) in high linkage disequilibrium with rs10756819 is likely the causal DNA variant for the observed BNC2 skin color association. The highly conserved region surrounding rs12350739 functions as an enhancer element regulating BNC2 transcription in human melanocytes, while the activity of this enhancer element depends on the allelic status of rs12350739. When the rs12350739-AA allele is present, the chromatin at the region surrounding rs12350739 is inaccessible and the enhancer element is only slightly active, resulting in low expression of BNC2, corresponding with light skin pigmentation. When the rs12350739-GG allele is present however, the chromatin at the region surrounding rs12350739 is more accessible and the enhancer is active, resulting in a higher expression of BNC2, corresponding with dark skin pigmentation. Overall, we demonstrate the identification of the functional DNA variant that explains the BNC2 skin color association signal, providing another important step towards further understanding human pigmentation genetics beyond statistical association. We thus deliver a clear example of how an intergenic non-coding DNA variant modulates the regulatory potential of the enhancer element it is located within, which in turn results in allele-dependent differential gene expression affecting variation in common human traits. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. [Specific organization of ribosomal DNA arrays in Squamata].

    PubMed

    Voronov, A S; Shibalev, D V; Kupriianova, N S

    2008-11-01

    A first report on structural organization of ribosomal DNA arrays in some members of the order Squamata is presented. The data obtained point to unusually small (for vertebrates) size of the rDNA repetitive unit (approximately, 10 to 15 kb) in the lizard species examined. Analysis of BAC library of Uta stansburiana (Iguania) showed that haploid genome of this lizard contained a single cluster, consisting of about ten rDNA repeats. Determination of the extent of rDNA unit repetition in some other representatives of the order Squamata, using the method of comparative real-time PCR, showed that the number of rDNA units varied from one or several dozens in Iguanina to several hundred repeats in Scincomorpha and Varonoidea. The results are discussed in terms of an ambiguous position of the family Iguania on the evolutionary trees constructed based on morphological and molecular data.

  14. Engineering the ribosomal DNA in a megabase synthetic chromosome.

    PubMed

    Zhang, Weimin; Zhao, Guanghou; Luo, Zhouqing; Lin, Yicong; Wang, Lihui; Guo, Yakun; Wang, Ann; Jiang, Shuangying; Jiang, Qingwen; Gong, Jianhui; Wang, Yun; Hou, Sha; Huang, Jing; Li, Tianyi; Qin, Yiran; Dong, Junkai; Qin, Qin; Zhang, Jiaying; Zou, Xinzhi; He, Xi; Zhao, Li; Xiao, Yibo; Xu, Meng; Cheng, Erchao; Huang, Ning; Zhou, Tong; Shen, Yue; Walker, Roy; Luo, Yisha; Kuang, Zheng; Mitchell, Leslie A; Yang, Kun; Richardson, Sarah M; Wu, Yi; Li, Bing-Zhi; Yuan, Ying-Jin; Yang, Huanming; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Bader, Joel S; Cai, Yizhi; Boeke, Jef D; Dai, Junbiao

    2017-03-10

    We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.

  15. A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

    PubMed Central

    Cahyani, Inswasti; Cridge, Andrew G.; Engelke, David R.; Ganley, Austen R. D.

    2014-01-01

    The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

  16. A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences.

    PubMed

    Kumar, Aundy; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma

    2013-06-01

    Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

  17. Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

    PubMed

    Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

    2017-01-23

    Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental

  18. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression

    PubMed Central

    Ehrlich, Kenneth C.; Paterson, Heather L.; Lacey, Michelle; Ehrlich, Melanie

    2016-01-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1’s super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation. PMID:28018137

  19. DNA Hypomethylation in Intragenic and Intergenic Enhancer Chromatin of Muscle-Specific Genes Usually Correlates with their Expression.

    PubMed

    Ehrlich, Kenneth C; Paterson, Heather L; Lacey, Michelle; Ehrlich, Melanie

    2016-12-01

    Tissue-specific enhancers are critical for gene regulation. In this study, we help elucidate the contribution of muscle-associated differential DNA methylation to the enhancer activity of highly muscle-specific genes. By bioinformatic analysis of 44 muscle-associated genes, we show that preferential gene expression in skeletal muscle (SkM) correlates with SkM-specific intragenic and intergenic enhancer chromatin and overlapping foci of DNA hypomethylation. Some genes, e.g., CASQ1 and FBXO32, displayed broad regions of both SkM- and heart-specific enhancer chromatin but exhibited focal SkM-specific DNA hypomethylation. Half of the genes had SkM-specific super-enhancers. In contrast to simple enhancer/gene-expression correlations, a super-enhancer was associated with the myogenic MYOD1 gene in both SkM and myoblasts even though SkM has < 1 percent as much MYOD1 expression. Local chromatin differences in this super-enhancer probably contribute to the SkM/myoblast differential expression. Transfection assays confirmed the tissue-specificity of the 0.3-kb core enhancer within MYOD1's super-enhancer and demonstrated its repression by methylation of its three CG dinucleotides. Our study suggests that DNA hypomethylation increases enhancer tissue-specificity and that SkM super-enhancers sometimes are poised for physiologically important, rapid up-regulation.

  20. Restless 5S: the re-arrangement(s) and evolution of the nuclear ribosomal DNA in land plants.

    PubMed

    Wicke, Susann; Costa, Andrea; Muñoz, Jesùs; Quandt, Dietmar

    2011-11-01

    Among eukaryotes two types of nuclear ribosomal DNA (nrDNA) organization have been observed. Either all components, i.e. the small ribosomal subunit, 5.8S, large ribosomal subunit, and 5S occur tandemly arranged or the 5S rDNA forms a separate cluster of its own. Generalizations based on data derived from just a few model organisms have led to a superimposition of structural and evolutionary traits to the entire plant kingdom asserting that plants generally possess separate arrays. This study reveals that plant nrDNA organization into separate arrays is not a distinctive feature, but rather assignable almost solely to seed plants. We show that early diverging land plants and presumably streptophyte algae share a co-localization of all rRNA genes within one repeat unit. This raises the possibility that the state of rDNA gene co-localization had occurred in their common ancestor. Separate rDNA arrays were identified for all basal seed plants and water ferns, implying at least two independent 5S rDNA transposition events during land plant evolution. Screening for 5S derived Cassandra transposable elements which might have played a role during the transposition events, indicated that this retrotransposon is absent in early diverging vascular plants including early fern lineages. Thus, Cassandra can be rejected as a primary mechanism for 5S rDNA transposition in water ferns. However, the evolution of Cassandra and other eukaryotic 5S derived elements might have been a side effect of the 5S rDNA cluster formation. Structural analysis of the intergenic spacers of the ribosomal clusters revealed that transposition events partially affect spacer regions and suggests a slightly different transcription regulation of 5S rDNA in early land plants. 5S rDNA upstream regulatory elements are highly divergent or absent from the LSU-5S spacers of most early divergent land plant lineages. Several putative scenarios and mechanisms involved in the concerted relocation of hundreds of 5S

  1. Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions.

    PubMed

    Karpathy, Sandor E; Dasch, Gregory A; Eremeeva, Marina E

    2007-08-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.

  2. Ribosomal DNA polymorphisms in the yeast Geotrichum candidum.

    PubMed

    Alper, Iraz; Frenette, Michel; Labrie, Steve

    2011-12-01

    The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.

  3. Repetitive sequence variation and dynamics in the ribosomal DNA array of Saccharomyces cerevisiae as revealed by whole-genome resequencing

    PubMed Central

    James, Stephen A.; O'Kelly, Michael J.T.; Carter, David M.; Davey, Robert P.; van Oudenaarden, Alexander; Roberts, Ian N.

    2009-01-01

    Ribosomal DNA (rDNA) plays a key role in ribosome biogenesis, encoding genes for the structural RNA components of this important cellular organelle. These genes are vital for efficient functioning of the cellular protein synthesis machinery and as such are highly conserved and normally present in high copy numbers. In the baker's yeast Saccharomyces cerevisiae, there are more than 100 rDNA repeats located at a single locus on chromosome XII. Stability and sequence homogeneity of the rDNA array is essential for function, and this is achieved primarily by the mechanism of gene conversion. Detecting variation within these arrays is extremely problematic due to their large size and repetitive structure. In an attempt to address this, we have analyzed over 35 Mbp of rDNA sequence obtained from whole-genome shotgun sequencing (WGSS) of 34 strains of S. cerevisiae. Contrary to expectation, we find significant rDNA sequence variation exists within individual genomes. Many of the detected polymorphisms are not fully resolved. For this type of sequence variation, we introduce the term partial single nucleotide polymorphism, or pSNP. Comparative analysis of the complete data set reveals that different S. cerevisiae genomes possess different patterns of rDNA polymorphism, with much of the variation located within the rapidly evolving nontranscribed intergenic spacer (IGS) region. Furthermore, we find that strains known to have either structured or mosaic/hybrid genomes can be distinguished from one another based on rDNA pSNP number, indicating that pSNP dynamics may provide a reliable new measure of genome origin and stability. PMID:19141593

  4. Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae).

    PubMed

    Adams, S P; Leitch, I J; Bennett, M D; Chase, M W; Leitch, A R

    2000-11-01

    All Aloe taxa (∼400 species) share a conserved bimodal karyotype with a basic genome of four large and three small submetacentric/acrocentric chromosomes. We investigated the physical organization of 18S-5.8S-26S and 5S ribosomal DNA (rDNA) using fluorescent in situ hybridization (FISH) to 13 Aloe species. The organization was compared with a phylogenetic tree of 28 species (including the 13 used for FISH) constructed by sequence analysis of the internal transcribed spacer (ITS) of 18S-5.8S-26S rDNA. The phylogeny showed little divergence within Aloe, although distinct, well-supported clades were found. FISH analysis of 5S rDNA distribution showed a similar interstitial location on a large chromosome in all species examined. In contrast, the distribution of 18S-5.8S-26S rDNA was variable, with differences in number, location, and size of loci found between species. Nevertheless, within well-supported clades, all species had the same organizational patterns. Thus, despite the striking stability of karyotype structure and location of 5S rDNA, the distribution of 18S-5.8S-26S rDNA is not so constrained and has clearly changed during Aloe speciation.

  5. Coding DNA repeated throughout intergenic regions of the Arabidopsis thaliana genome: Evolutionary footprints of RNA silencing

    USDA-ARS?s Scientific Manuscript database

    Pyknons are non-random sequence patterns significantly repeated throughout non-coding genomic DNA that also appear at least once among genes. They are interesting because they portend an unforeseen connection between coding and non-coding DNA. Pyknons have only been discovered in the human genome,...

  6. MexZ-mediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA.

    PubMed

    Matsuo, Yasuhiro; Eda, Shima; Gotoh, Nobuyuki; Yoshihara, Eisaku; Nakae, Taiji

    2004-09-01

    MexZ is a transcriptional regulator of the mexXY multidrug transporter operon, which confers aminoglycoside resistance on Pseudomonas aeruginosa. Highly purified MexZ showed direct binding with a specific site of the mexZ-mexX intergenic DNA when probed by a gel retardation assay. Both in vitro chemical cross-linking experiments and an in vivo two-hybrid expression system showed that the active form of MexZ, which is capable of binding the intergenic DNA, appeared to be a dimer. These results explain the mechanism by which MexZ represses transcription of the mexXY operon, but do not explain the substrate-induced hyperproduction of MexXY. The presence of inducer antibiotic in the gel-retardation assay mixture failed to detect altered MexZ-probe DNA interaction suggesting the possible involvement of an additional regulator.

  7. Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush).

    PubMed

    Zhuo, L; Sajdak, S L; Phillips, R B

    1994-08-01

    Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5' external spacer region (5' ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5' ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3-6 kb from the 18S. Sequencing of a 609-b segment of the 5' ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5' ETS using the polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Extensive ribosomal DNA genic variation in the columnar cactus Lophocereus.

    PubMed

    Hartmann, S; Nason, J D; Bhattacharya, D

    2001-08-01

    Sequence analysis of the hypervariable internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) is commonly used to gain insights into plant and animal population structure and phylogeny. We characterized ITS1, ITS2, and the 5.8S coding region of 18 senita (Lophocereus) individuals from 12 different populations in Baja as well as from closely related cactus species. Analyses of multiple clones demonstrated extensive paralogy in the senita rDNA gene family. We identified at least two putatively non-recombining rDNA operons in senita as well as multiple paralogous sequences within each operon. Usage of PCR, reverse transcriptase (RT)-PCR, Southern blot, primary sequence analyses of the 18S rDNA gene, and secondary structure analyses of the 5.8S rRNA showed that one of the operons encodes rDNA pseudogenes in a low copy-number (Truncated), whereas the second operon encodes an expressed rRNA (Functional). Surprisingly, we found extensive paralogy not only in the ITS regions but also in the 5.8S coding regions in senita both within and between operons. Phylogenetic analyses suggest that the second rDNA operon originated prior to the divergence of Lophocereus. A significant (p < 0.05) divergence-rate acceleration was found in the Lophocereus 5.8S rDNA coding region in the Functional operon in comparison to Pereskiopsis porteri (Cactaceae) and Portulaca molokiniensis (Portulacaceae) with Silene dioica and Spinacia oleracea as the outgroups.

  9. RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.

    PubMed Central

    Brabant, F; Acheson, N H

    1995-01-01

    RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion. PMID:7769704

  10. DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

    PubMed

    Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

    2016-04-01

    RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

  11. 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.

  12. Effectiveness of Enterobacterial Repetitive Intergenic Consensus PCR and Random Amplified Polymorphic DNA Fingerprinting for Helicobacter pylori Strain Differentiation

    PubMed Central

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

    2006-01-01

    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

  13. DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S-23S rDNA intergenic transcribed spacer (ITS) regions.

    PubMed

    Dingman, Douglas W

    2009-01-01

    Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

  14. Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

    PubMed

    Almamun, Md; Kholod, Olha; Stuckel, Alexei J; Levinson, Benjamin T; Johnson, Nathan T; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H

    2017-01-17

    A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

  15. Mitochondrial swinger replication: DNA replication systematically exchanging nucleotides and short 16S ribosomal DNA swinger inserts.

    PubMed

    Seligmann, Hervé

    2014-11-01

    Assuming systematic exchanges between nucleotides (swinger RNAs) resolves genomic 'parenthood' of some orphan mitochondrial transcripts. Twenty-three different systematic nucleotide exchanges (bijective transformations) exist. Similarities between transcription and replication suggest occurrence of swinger DNA. GenBank searches for swinger DNA matching the 23 swinger versions of human and mouse mitogenomes detect only vertebrate mitochondrial swinger DNA for swinger type AT+CG (from five different studies, 149 sequences) matching three human and mouse mitochondrial genes: 12S and 16S ribosomal RNAs, and cytochrome oxidase subunit I. Exchange A<->T+C<->G conserves self-hybridization properties, putatively explaining swinger biases for rDNA, against protein coding genes. Twenty percent of the regular human mitochondrial 16S rDNA consists of short swinger repeats (from 13 exchanges). Swinger repeats could originate from recombinations between regular and swinger DNA: duplicated mitochondrial genes of the parthenogenetic gecko Heteronotia binoei include fewer short A<->T+C<->G swinger repeats than non-duplicated mitochondrial genomes of that species. Presumably, rare recombinations between female and male mitochondrial genes (and in parthenogenetic situations between duplicated genes), favors reverse-mutations of swinger repeat insertions, probably because most inserts affect negatively ribosomal function. Results show that swinger DNA exists, and indicate that swinger polymerization contributes to the genesis of genetic material and polymorphism.

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

    PubMed Central

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

    2007-01-01

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

  17. Activated levels of rRNA synthesis in fission yeast are driven by an intergenic rDNA region positioned over 2500 nucleotides upstream of the initiation site.

    PubMed Central

    Liu, Z; Zhao, A; Chen, L; Pape, L

    1997-01-01

    RNA polymerase I-catalyzed synthesis of the Schizosaccharomyces pombe approximately 37S pre-rRNAs was shown to be sensitive to regulatory sequences located several kilobases upstream of the initiation site for the rRNA gene. An in vitro transcription system for RNA polymerase I-catalyzed RNA synthesis was established that supports correct and activated transcription from templates bearing a full S. pombe rRNA gene promoter. A 780 bp region starting at -2560 significantly stimulates transcription of ac is-located rDNA promoter and competes with an rDNA promoter in trans, thus displaying some of the activities of rDNA transcriptional enhancers in vitro. Deletion of a 30 bp enhancer-homologous domain in this 780 bp far upstream region blocked its cis-stimulatory effect. The sequence of the S. pombe 3.5 kb intergenic spacer was determined and its organization differs from that of vertebrate, Drosophila, Acanthamoeba and plant intergenic rDNA spacers: it does not contain multiple, imperfect copies of the rRNA gene promoter nor repetitive elements of 140 bp, as are found in vertebrate rDNA enhancers. PMID:9016610

  18. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

    PubMed Central

    Alix, J H; Guérin, M F

    1993-01-01

    To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature. PMID:8105482

  19. Tsukamurella tyrosinosolvens intravascular catheter infection identified using 16S ribosomal DNA sequencing.

    PubMed

    Sheridan, Elizabeth A S; Warwick, Simon; Chan, Anthony; Dall'Antonia, Martino; Koliou, Maria; Sefton, Armine

    2003-03-01

    Cultures of blood from a hemodialysis line repeatedly yielded a gram-positive rod. The organism was identified as Tsukamurella tyrosinosolvens by 16S ribosomal DNA sequencing, and the patient was treated successfully by removal of the line.

  20. Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

    PubMed Central

    Torres, Davi Coe; Lima, João Paulo Matos Santos; Fernandes, Afrânio Gomes; Nunes, Edson Paula; Grangeiro, Thalles Barbosa

    2011-01-01

    Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens. PMID:21734825

  1. The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor.

    PubMed

    Kramer, Gisela; Ramachandiran, Vasanthi; Horowitz, Paul M; Hardesty, Boyd

    2002-07-01

    The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes. We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides. For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated. Truncated forms of rhodanese and E. coli translation initiation factor 3 (IF3) were generated with tandem rare arginine codons in the coding sequence. These codons cause strong translational pausing during coupled transcription/translation in E. coli extracts, generating nascent polypeptides on ribosomes. Protein synthesis in the TF(-) extract was initiated with biotin-Met-tRNA(f). Ribosomes with nascent polypeptides were isolated by interaction of the N-terminal biotin with streptavidin on magnetobeads. These translating ribosomes that lack TF contain the molecular chaperone DnaK in considerably less than stoichiometric amounts.

  2. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.

    PubMed

    Warsen, Adelaide E; Krug, Melissa J; LaFrentz, Stacey; Stanek, Danielle R; Loge, Frank J; Call, Douglas R

    2004-07-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

  3. Phenotypic variability confirmed by nuclear ribosomal DNA suggests a possible natural hybrid zone of Triatoma brasiliensis species complex.

    PubMed

    Costa, Jane; Bargues, Maria Dolores; Neiva, Vanessa Lima; Lawrence, Gena G; Gumiel, Marcia; Oliveira, Genova; Cabello, Pedro; Lima, Marli Maria; Dotson, Ellen; Provance, David William; Almeida, Carlos Eduardo; Mateo, Lucia; Mas-Coma, Santiago; Dujardin, Jean Pierre

    2016-01-01

    Triatoma brasiliensis macromelasoma occurs in Pernambuco state, Brazil, which is situated between the distribution areas of Triatoma brasiliensis brasiliensis (north) and Triatoma juazeirensis (south). T. b. macromelasoma displays greater variations in its chromatic phenotype than either T. b. brasiliensis or T. juazeirensis, and patterns reminiscent of one or the other. Experimental crosses from each of these members of the T. brasiliensis species complex generated fertile offspring suggesting that viable hybrids could be present in nature, despite their significant genetic distances. Considering the geographical position of occurrence of the T. b. macromelasoma (in Pernambuco) it was proposed to be an area capable of supporting natural hybridization between T. b. brasiliensis and T. juazeirensis. Since phenotypic variability is expected, this study investigated the existence of intermediate chromatic phenotypes for T. b. macromelasoma in various locations in areas between the T. b. brasiliensis and T. juazeirensis occurrences. Thirteen different color patterns were for the first time characterized and nine of those displayed intermediate phenotypes. Molecular analysis performed using ribosomal DNA intergenic region, grouped all within the T. brasiliensis complex. The intermediate chromatic phenotypes, molecular analysis and experimental crosses all support the distinction of a zone of hybridization that gave rise to the T. b. macromelasoma through homoploidal evolution.

  4. Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA.

    PubMed Central

    Bertin, J; Sunstrom, N A; Acheson, N H

    1993-01-01

    During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus. Images PMID:8396655

  5. Differential targeting of Tetrahymena ORC to ribosomal DNA and non-rDNA replication origins.

    PubMed

    Donti, Taraka R; Datta, Shibani; Sandoval, Pamela Y; Kapler, Geoffrey M

    2009-02-04

    The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis-acting replication determinant--the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non-rDNA fragment containing two closely associated replicators, ARS1-A (0.8 kb) and ARS1-B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA-independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non-rDNA ARS1 chromosome changed across the cell cycle. In G(2) phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1-A/B. Remarkably, ORC and Mcm6 associated with just the ARS1-A replicator in G(1) phase when pre-replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non-rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.

  6. Morphology and Small-Subunit Ribosomal DNA Sequence of Henneguya Adiposa (Myxosporea) From Ictalurus punctatus (Siluriformes)

    USDA-ARS?s Scientific Manuscript database

    The original description of Henneguya adiposa, a myxozoan parasitizing channel catfish Ictalurus punctatus, is supplemented with new data on spore morphology, including photomicrographs and line drawings, as well as 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence. Elongate, translucent, linear...

  7. Characterization of the ribosomal DNA units in two related Prunus species (P. Cerasifera and P. Spinosa).

    PubMed

    Reynders-Aloisi, S; Grellet, F

    1994-08-01

    The genetic relationships between two Prunus species, involved in rootstock breeding, were examined at the level of the ribosomal RNA genes. Twenty clones of P. cerasifera, a diploid species, and 12 clones of P. spinosa, a tetraploid wild species, were studied. The use of three heterologous ribosomal DNA probes covering different regions of the ribosomal tandem repeats enabled us to construct restriction maps for EcoRI and BamHI. We identified two unit types (unit I and unit II) in P. cerasifera. In P. spinosa, P. cerasifera units were present in addition to a third ribosomal unit type (unit III). These results appeared to confirm previous cytological studies (Salesses 1973) indicating that one of the genomes in P.spinosa has homology with the one from P. cerasifera.

  8. Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA.

    PubMed

    Kane, Nolan; Sveinsson, Saemundur; Dempewolf, Hannes; Yang, Ji Yong; Zhang, Dapeng; Engels, Johannes M M; Cronk, Quentin

    2012-02-01

    To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

  9. Tetrahymena ORC contains a ribosomal RNA fragment that participates in rDNA origin recognition.

    PubMed

    Mohammad, Mohammad M; Donti, Taraka R; Sebastian Yakisich, J; Smith, Aaron G; Kapler, Geoffrey M

    2007-12-12

    The Tetrahymena thermophila ribosomal DNA (rDNA) replicon contains dispersed cis-acting replication determinants, including reiterated type I elements that associate with sequence-specific, single-stranded binding factors, TIF1 through TIF4. Here, we show that TIF4, previously implicated in cell cycle-controlled DNA replication and rDNA gene amplification, is the T. thermophila origin recognition complex (TtORC). We further demonstrate that TtORC contains an integral RNA subunit that participates in rDNA origin recognition. Remarkably, this RNA, designated 26T, spans the terminal 282 nts of 26S ribosomal RNA. 26T RNA exhibits extensive complementarity to the type I element T-rich strand and binds the rDNA origin in vivo. Mutations that disrupt predicted interactions between 26T RNA and its complementary rDNA target change the in vitro binding specificity of ORC and diminish in vivo rDNA origin utilization. These findings reveal a role for ribosomal RNA in chromosome biology and define a new mechanism for targeting ORC to replication initiation sites.

  10. Ribosomal and Mitochondrial DNA Analyses of Xiphinema americanum-Group Populations

    PubMed Central

    Lazarova, Stela S.; Malloch, Gaynor; Oliveira, Claudio M.G.; Hübschen, Judith; Neilson, Roy

    2006-01-01

    The 18S ribosomal DNA (rDNA) and cytochrome oxidase I region of mitochondrial DNA (mtDNA) were sequenced for 24 Xiphinema americanum-group populations sourced from a number of geographically disparate locations. Sequences were subjected to phylogenetic analysis and compared. 18S rDNA strongly suggested that only X. pachtaicum, X. simile (two populations) and a X. americanum s.l. population from Portugal were different from the other 20 populations studied, whereas mtDNA indicated some heterogeneity between populations. Phylogenetically, based on mtDNA, an apparent dichotomy existed amongst X. americanum-group populations from North America and those from Asia, South America and Oceania. Analyses of 18S rDNA and mtDNA sequences underpin the classical taxonomic issues of the X. americanum-group and cast doubt on the degree of speciation within the X. americanum-group. PMID:19259456

  11. Ultra-barcoding in cacao (Theobroma spp.; malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA

    USDA-ARS?s Scientific Manuscript database

    High-throughput next-generation sequencing was used to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an indivi...

  12. Role of messenger RNA-ribosome complex in complementary DNA display.

    PubMed

    Naimuddin, Mohammed; Ohtsuka, Isao; Kitamura, Koichiro; Kudou, Motonori; Kimura, Shinnosuke

    2013-07-15

    In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    PubMed

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  14. Absence of ribosomal DNA amplification in the meroistic (telotrophic) ovary of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae)

    PubMed Central

    1975-01-01

    In the typical meroistic insect ovary, the oocyte nucleus synthesizes little if any RNA. Nurse cells or trophocytes actively synthesize ribosomes which are transported to and accumulated by the oocyte. In the telotrophic ovary a morphological separation exists, the nurse cells being localized at the apical end of each ovariole and communicating with the ooocytes via nutritive cords. In order to determine whether the genes coding for ribosomal RNA (rRNA) are amplified in the telotrophic ovary of the milkweed bug Oncopeltus fasciatus, the percentages of the genome coding for ribosomal RNA in somatic cells, spermatogenic cells, ovarian follicles, and nurse cells were compared. The oocytes and most of the nurse cells of O. fasciatus are uninucleolate. DNA hybridizing with ribosomal RNA is localized in a satellite DNA, the density of which is 1.712 g/cm(-3). The density of main-band DNA is 1.694 g/cm(-3). The ribosomal DNA satellite accounts for approximately 0.2% of the DNA in somatic and gametogenic tissues of both males and females. RNA-DNA hybridization analysis demonstrates that approximately 0.03% of the DNA in somatic tissues, testis, ovarian follicles, and isolated nurse cells hybridizes with ribosomal RNA. The fact that the percentage of DNA hybridizing with rRNA is the same in somatic and in male and female gametogenic tissues indicates that amplification of ribosomal DNA does not occur in nurse cells and that if it occurs in oocytes, it represents less than a 50- fold increase in ribosomal DNA. An increase in total genome DNA accounted by polyploidization appears to provide for increasing the amount of ribosomal DNA in the nurse cells. PMID:1158969

  15. Genome-Wide Anaplasma phagocytophilum AnkA-DNA Interactions Are Enriched in Intergenic Regions and Gene Promoters and Correlate with Infection-Induced Differential Gene Expression

    PubMed Central

    Dumler, J. Stephen; Sinclair, Sara H.; Pappas-Brown, Valeria; Shetty, Amol C.

    2016-01-01

    Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils, and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR)-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: (i) intergenic regions predicted to be MARs; (ii) within predicted lamina-associated domains; and (iii) at promoters ≤ 3000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome “re-organizer.” AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity. PMID:27703927

  16. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    NASA Astrophysics Data System (ADS)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  17. Intergenic and repeat transcription in human, chimpanzee and macaque brains measured by RNA-Seq.

    PubMed

    Xu, Augix Guohua; He, Liu; Li, Zhongshan; Xu, Ying; Li, Mingfeng; Fu, Xing; Yan, Zheng; Yuan, Yuan; Menzel, Corinna; Li, Na; Somel, Mehmet; Hu, Hao; Chen, Wei; Pääbo, Svante; Khaitovich, Philipp

    2010-07-01

    Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq) to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20-28% of non-ribosomal transcripts correspond to annotated exons and 20-23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40-48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20%) represents 3'UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

  18. Intergenic and Repeat Transcription in Human, Chimpanzee and Macaque Brains Measured by RNA-Seq

    PubMed Central

    Xu, Ying; Li, Mingfeng; Fu, Xing; Yan, Zheng; Yuan, Yuan; Menzel, Corinna; Li, Na; Somel, Mehmet; Hu, Hao; Chen, Wei; Pääbo, Svante; Khaitovich, Philipp

    2010-01-01

    Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq) to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20–28% of non-ribosomal transcripts correspond to annotated exons and 20–23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40–48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20%) represents 3′UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits. PMID:20617162

  19. A model for regulation of mammalian ribosomal DNA transcription. Co-ordination of initiation and termination.

    PubMed Central

    Nashimoto, M; Mishima, Y

    1988-01-01

    Based on recent experimental data about transcription initiation and termination, a model for regulation of mammalian ribosomal DNA transcription is developed using a simple kinetic scheme. In this model, the existence of the transition pathway from the terminator to the promoter increases the rate of ribosomal RNA precursor synthesis. In addition to this 'non-transcribed spacer' traverse of RNA polymerase I, the co-ordination of initiation and termination allows a rapid on/off switch transition from the minimum to the maximum rate of ribosomal RNA precursor synthesis. Furthermore, taking account of the participation of two factors in the termination event, we propose a plausible molecular mechanism for the co-ordination of initiation and termination. This co-ordination is emphasized by repetition of the terminator unit. PMID:3223915

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

    PubMed Central

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

    2004-01-01

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

  1. The Intergenerational Impact of Genetic and Psychological Factors on Blood Pressure (InterGEN) Study: Design and Methods for Complex DNA Analysis.

    PubMed

    Taylor, Jacquelyn Y; Wright, Michelle L; Crusto, Cindy A; Sun, Yan V

    2016-10-01

    The Intergenerational Impact of Genetic and Psychological Factors on Blood Pressure (InterGEN) study aims to delineate the independent and interaction effects of genomic (genetic and epigenetic) and psychological-environmental (maternally perceived racial discrimination, mental health, and parenting behavior) factors on blood pressure (BP) among African American mother-child dyads over time. The purpose of this article is to describe the two-step genetic and epigenetic approach that will be executed to explore Gene × Environment interactions on BP using a longitudinal cohort design. Procedure for the single collection of DNA at Time 1 includes the use of the Oragene 500-format saliva sample collection tube, which provides enough DNA for both the Illumina Multi-Ethnic Genotyping and 850K EPIC methylation analyses. BP readings, height, weight, percentage of body fat, and percentage of body water will be measured on all participants every 6 months for 2 years for a total of 4 time points. Genomic data analyses to be completed include multivariate modeling, assessment of population admixture and structure, and extended analyses including Bonferroni correction, false discovery rate methods, Monte Carlo approach, EIGENSTRAT methods, and so on, to determine relationships among both main and interaction effects of genetic, epigenetic, and psychological environmental factors on BP. © The Author(s) 2016.

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

    PubMed Central

    Machida, Ryuji J.; Knowlton, Nancy

    2012-01-01

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

  3. COII/tRNA[sup Lys] intergenic 9-bp deletion and other mtDNA markers clearly reveal that the Tharus (Southern Nepal) have oriental affinities

    SciTech Connect

    Passarino, G.; Semino, O.; Santachiara-Benerecetti, A.S.; Modiano, G. )

    1993-09-01

    The authors searched for the East Asian mtDNA 9-bp deletion in the intergenic COII/tRNA[sup Lys] region in a sample of 107 Tharus (50 from central Terai and 57 from eastern Terai), a population whose anthropological origin has yet to be completely clarified. The deletion, detected by electrophoresis of the PCR-amplified nt 7392-8628 mtDNA fragment after digestion with HaeIII, was found in about 8% of both Tharu groups but was found in none of the 76 Hindus who were examined as a non-Oriental neighboring control population. A complete triplication of the 9-bp unit, the second case so far reported, was also observed in one eastern Tharu. All the mtDNAs with the deletion, and that with the triplication, were further characterized (by PCR amplification of the relevant mTDNA fragments and their digestion with the appropriate enzymes) to locate them in the Ballinger et al. phylogeny of East Asian mtDNA haplotypes. The deletion was found to be associated with four different haplotypes, two of which are reported for the first time. One of the deletions and especially the triplication could be best explained by the assumption of novel length-change events. Ballinger's classification of East Asian mtDNA haplotypes is mainly based on the phenotypes for the DdeI site at nt 10394 and the AluI site at nt 10397. Analysis of the entire Tharu sample revealed that more than 70% of the Tharus have both sites, the association of which has been suggested as an ancient East Asian peculiarity. These results conclusively indicate that the Tharus have a predominantly maternal Oriental ancestry. Moreover, they show at least one and perhaps two further distinct length mutations, and this suggests that the examined region is a hot spot of rearrangements. 21 refs., 5 figs., 6 tabs.

  4. Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

    PubMed Central

    Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

    2015-01-01

    Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

  5. Ribosomal DNA spacer probes for yeast identification: studies in the genus Metschnikowia.

    PubMed

    Henriques, M; Sá-Nogueira, I; Giménez-Jurado, G; van Uden, N

    1991-02-01

    To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7.7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0.65 kb BamHI-HpaI fragment containing non-transcribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclusively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G + C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.

  6. Combined ribosomal DNA and morphological analysis of individual gyrodactylid monogeneans.

    PubMed

    Harris, P D; Cable, J; Tinsley, R C; Lazarus, C M

    1999-04-01

    A method is presented for the isolation and analysis of hamuli, marginal hooks, and bars from individual gyrodactylid monogeneans using scanning electron microscopy (SEM), while simultaneously processing parasites for rDNA analysis using the polymerase chain reaction (PCR). The haptors of ethanol-fixed gyrodactylids were protease digested to liberate hooks for SEM, whereas DNA extracted from the bodies was used for PCR. The method resulted in hooks and hamuli being prepared from more than 90% of Gyrodactylus turnbulli individuals, a significant improvement on previously published digestion-based SEM techniques. PCR on the same parasites was less successful, but sequence data were obtained from 50% of individuals. Amplification of rDNA internal-transcribed spacer regions from individual worms used for SEM gave PCR products consistent with those predicted from our previous sequence analysis. This method allows the correlation of morphology and DNA sequence from the same individual and can be applied to ethanol-fixed material, such as field collected and museum specimens.

  7. Phylogenetic relationships in Nuphar (Nymphaeaceae): evidence from morphology, chloroplast DNA, and nuclear ribosomal DNA.

    PubMed

    Padgett, D J; Les, D H; Crow, G E

    1999-09-01

    The genus Nuphar consists of yellow-flowered waterlilies and is widely distributed in north-temperate bodies of water. Despite regular taxonomic evaluation of these plants, no explicit phylogenetic hypotheses have been proposed for the genus. We investigated phylogenetic relationships in Nuphar using morphology and sequences of the chloroplast gene matK and of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Two major lineages within Nuphar are consistently resolved with the morphological and molecular data sets. One lineage comprises New World taxa and the other represents a primarily Old World lineage. Relationships within the major lineages were poorly resolved by morphology and ITS, yet certain relationships were elucidated by all analyses. Most notable is the strong support for a monophyletic lineage of dwarf taxa and the alliance of the North American N. microphylla with the Eurasian taxa. Minor discordance between the independent cladograms is accounted for by hybridization. The common taxonomic practice of uniting all North American and Eurasian taxa under one species is not supported phylogenetically.

  8. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    PubMed Central

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences. Images PMID:2997749

  9. Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions.

    PubMed

    Gonzalez, Omar Garcia; Assfalg, Robin; Koch, Sylvia; Schelling, Adrian; Meena, Jitendra K; Kraus, Johann; Lechel, Andre; Katz, Sarah-Fee; Benes, Vladimir; Scharffetter-Kochanek, Karin; Kestler, Hans A; Günes, Cagatay; Iben, Sebastian

    2014-08-13

    In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.

  10. The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage

    PubMed Central

    Larsen, Dorthe H; Hari, Flurina; Clapperton, Julie A; Gwerder, Myriam; Gutsche, Katrin; Altmeyer, Matthias; Jungmichel, Stephanie; Toledo, Luis I; Fink, Daniel; Rask, Maj-Britt; Grøfte, Merete; Lukas, Claudia; Nielsen, Michael L; Smerdon, Stephen J; Lukas, Jiri; Stucki, Manuel

    2016-01-01

    Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in-trans signaling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identified TCOF1-Treacle, a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle-dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in-trans in the presence of distant chromosome breaks. PMID:25064736

  11. Horizontal alignment of 5′ -> 3′ intergene distance segment tropy with respect to the gene as the conserved basis for DNA transcription

    PubMed Central

    Sarin, Hemant

    2017-01-01

    Aim: To study the conserved basis for gene expression in comparative cell types at opposite ends of the cell pressuromodulation spectrum, the lymphatic endothelial cell and the blood microvascular capillary endothelial cell. Methods: The mechanism for gene expression is studied in terms of the 5′ -> 3′ direction paired point tropy quotients (prpTQs) and the final 5′ -> 3′ direction episodic sub-episode block sums split-integrated weighted average-averaged gene overexpression tropy quotient (esebssiwaagoTQ). Results: The final 5′ -> 3′ esebssiwaagoTQ classifies an lymphatic endothelial cell overexpressed gene as a supra-pressuromodulated gene (esebssiwaagoTQ ≥ 0.25 < 0.75) every time and classifies a blood microvascular capillary endothelial cell overexpressed gene every time as an infra-pressuromodulated gene (esebssiwaagoTQ < 0.25) (100% sensitivity; 100% specificity). Conclusion: Horizontal alignment of 5′ -> 3′ intergene distance segment tropy wrt the gene is the basis for DNA transcription in the pressuromodulated state. PMID:28344824

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

    PubMed

    Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

    2012-04-17

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

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

    PubMed Central

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

    2012-01-01

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

  14. Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

    NASA Astrophysics Data System (ADS)

    Xu, Jiajie; Jiang, Bo; Chai, Sanming; He, Yuan; Zhu, Jianyi; Shen, Zonggen; Shen, Songdong

    2016-09-01

    Filamentous Bangia, which are distributed extensively throughout the world, have simple and similar morphological characteristics. Scientists can classify these organisms using molecular markers in combination with morphology. We successfully sequenced the complete nuclear ribosomal DNA, approximately 13 kb in length, from a marine Bangia population. We further analyzed the small subunit ribosomal DNA gene (nrSSU) and the internal transcribed spacer (ITS) sequence regions along with nine other marine, and two freshwater Bangia samples from China. Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences (00.003; 0-0.006, respectively) from each other, but high divergences (0.123-0.126; 0.198, respectively) from freshwater samples. An exception is the marine sample collected from Weihai, which shows high divergence from both other marine samples (0.063-0.065; 0.129, respectively) and the freshwater samples (0.097; 0.120, respectively). A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades, with the two marine sample clades containing Bangia spp. from North America, Europe, Asia, and Australia; and one freshwater clade, containing Bangia atropurpurea from North America and China.

  15. Increased sensitivity and specificity of Borrelia burgdorferi 16S ribosomal DNA detection.

    PubMed

    Lee, Sin Hang; Vigliotti, Veronica S; Vigliotti, Jessica S; Jones, William; Pappu, Suri

    2010-04-01

    The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

  16. Development of a Ribosomal DNA ITS2 Marker for the Identification of the Thrips, Scirtothrips dorsalis

    PubMed Central

    Farris, R E; Ruiz-Arce, R; Ciomperlik, M; Vasquez, J D; DeLeón, R

    2010-01-01

    The thrips Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is an invasive pest that poses a significant economical threat to U.S. agriculture and trade. In this study, DNA sequence data and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for S. dorsalis. The DNA sequence variation from the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA (rDNA) was analyzed from various thrips species, including S. dorsalis. A primer set and polymerase chain reaction cycling parameters were designed for the amplification of a single marker fragment of S. dorsalis ITS2 rDNA. Specificity tests performed on ten thrips species, efficacy tests performed on fifteen S. dorsalis populations, and tests on primer sensitivity and robustness all demonstrated the diagnostic utility of this marker. This diagnostic PCR assay provides a quick, simple, and reliable molecular technique to be used in the identification of S. dorsalis. PMID:20578948

  17. Population genetic structure and phylogeographical pattern of a relict tree fern, Alsophila spinulosa (Cyatheaceae), inferred from cpDNA atpB- rbcL intergenic spacers.

    PubMed

    Su, Yingjuan; Wang, Ting; Zheng, Bo; Jiang, Yu; Chen, Guopei; Gu, Hongya

    2004-11-01

    Sequences of chloroplast DNA (cpDNA) atpB- rbcL intergenic spacers of individuals of a tree fern species, Alsophila spinulosa, collected from ten relict populations distributed in the Hainan and Guangdong provinces, and the Guangxi Zhuang region in southern China, were determined. Sequence length varied from 724 bp to 731 bp, showing length polymorphism, and base composition was with high A+T content between 63.17% and 63.95%. Sequences were neutral in terms of evolution (Tajima's criterion D=-1.01899, P>0.10 and Fu and Li's test D*=-1.39008, P>0.10; F*=-1.49775, P>0.10). A total of 19 haplotypes were identified based on nucleotide variation. High levels of haplotype diversity (h=0.744) and nucleotide diversity (Dij=0.01130) were detected in A. spinulosa, probably associated with its long evolutionary history, which has allowed the accumulation of genetic variation within lineages. Both the minimum spanning network and neighbor-joining trees generated for haplotypes demonstrated that current populations of A. spinulosa existing in Hainan, Guangdong, and Guangxi were subdivided into two geographical groups. An analysis of molecular variance indicated that most of the genetic variation (93.49%, P<0.001) was partitioned among regions. Wright's isolation by distance model was not supported across extant populations. Reduced gene flow by the Qiongzhou Strait and inbreeding may result in the geographical subdivision between the Hainan and Guangdong + Guangxi populations (FST=0.95, Nm=0.03). Within each region, the star-like pattern of phylogeography of haplotypes implied a population expansion process during evolutionary history. Gene genealogies together with coalescent theory provided significant information for uncovering phylogeography of A. spinulosa.

  18. Molecular characterization of Fasciola spp. from the endemic area of northern Iran based on nuclear ribosomal DNA sequences.

    PubMed

    Amor, Nabil; Halajian, Ali; Farjallah, Sarra; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine

    2011-07-01

    Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic

  19. Phylogenetic information content of Copepoda ribosomal DNA repeat units: ITS1 and ITS2 impact.

    PubMed

    Zagoskin, Maxim V; Lazareva, Valentina I; Grishanin, Andrey K; Mukha, Dmitry V

    2014-01-01

    The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals.

  20. Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

    PubMed Central

    Zagoskin, Maxim V.; Lazareva, Valentina I.; Grishanin, Andrey K.; Mukha, Dmitry V.

    2014-01-01

    The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals. PMID:25215300

  1. Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

    PubMed Central

    Yoon, Y; Sanchez, J A; Brun, C; Huberman, J A

    1995-01-01

    New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit. PMID:7739533

  2. Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

    PubMed

    Mikaeili, F; Kia, E B; Sharbatkhori, M; Sharifdini, M; Jalalizand, N; Heidari, Z; Zarei, Z; Stensvold, C R; Mirhendi, H

    2013-06-01

    Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels.

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

    PubMed

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

    2001-10-08

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

  4. Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

    PubMed Central

    Angeletti, Silvia; Lorino, Giulia; Gherardi, Giovanni; Battistoni, Fabrizio; De Cesaris, Marina; Dicuonzo, Giordano

    2001-01-01

    For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddlE. faecalis and ddlE. faecium primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative. PMID:11158155

  5. Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability

    PubMed Central

    Krawczyk, Claudia; Dion, Vincent; Schär, Primo; Fritsch, Olivier

    2014-01-01

    Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA. PMID:24574527

  6. Karyotype, chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda).

    PubMed

    Orosová, Martina; Ivica, Králová-Hromadová; Eva, Bazsalovicsová; Marta, Spakulová

    2010-09-01

    Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n=20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.

  7. Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1989-03-01

    Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes.

  8. Population variation in the A chromosome distribution of satellite DNA and ribosomal DNA in the grasshopper Eyprepocnemis plorans.

    PubMed

    Cabrero, J; Perfectti, F; Gómez, R; Camacho, J P M; López-León, M D

    2003-01-01

    The double FISH analysis of two repetitive DNAs (a satellite DNA and ribosomal DNA) in 12 natural populations of the grasshopper Eyprepocnemis plorans collected at the south (Granada and Málaga provinces) and south-east (Albacete and Murcia provinces) of the Iberian Peninsula has shown their wide-spread presence throughout the whole genome as well as extensive variation among populations. Both DNAs are found in most A chromosomes. Regularly, both DNAs occurred in the S11 and X chromosomes, rDNA in the S10 and satDNA in the L2 and M3. No correlation was found between the number of satDNA and rDNA clusters in the A genomes of the 12 populations analysed, and both figures were independent of the presence of B chromosomes. The genomic distribution of both DNAs showed no association with the geographical localization of the populations analysed. Finally, we provide evidence that the supernumerary chromosome segment proximally located on the S11 chromosome is, in most cases, the result of satDNA amplification but, in some cases, it might also derive from amplification of both satDNA and rDNA.

  9. Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

    PubMed Central

    Jakočiūnas, Tadas; Domange Jordö, Marie; Aït Mebarek, Mazhoura; Bünner, Camilla Marie; Verhein-Hansen, Janne; Oddershede, Lene B.; Thon, Geneviève

    2013-01-01

    Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain. PMID:24191010

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

    PubMed

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

    2004-01-01

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

  11. DNA methyltransferase inhibition may limit cancer cell growth by disrupting ribosome biogenesis.

    PubMed

    Moss, Tom

    2011-02-01

    "Mutations" in the pattern of CpG methylation imprinting of the human genome have been correlated with a number of diseases including cancer. In particular, aberrant imprinting of tumor suppressor genes by gain of CpG methylation has been observed in many cancers and thus represents an important alternative pathway to gene "mutation" and tumor progression. Inhibitors of DNA methylation display therapeutic effects in the treatment of certain cancers, and it has been assumed these effects are due to the reversal of "mutant" gene imprinting. However, significant reactivation of imprinted tumor suppressor genes is rarely observed in vivo following treatment with DNA methylation inhibitors. A recent study revealed an unexpected requirement for CpG methylation in the synthesis and assembly of the ribosome, an essential function for cell growth and proliferation. As such, the data provide an unforeseen explanation of the action of DNA methylation inhibitors in restricting cancer cell growth.

  12. Ribosomal DNA haplotype distribution of Bursaphelenchus xylophilus in Kyushu and Okinawa islands, Japan

    PubMed Central

    Nose, Mine; Miyahara, Fumihiko; Ohira, Mineko; Matsunaga, Koji; Tobase, Masashi; Koyama, Takao; Yoshimoto, Kikuo

    2009-01-01

    Ribosomal DNA region sequences (partial 18S, 28S and complete ITS1, 5.8S, and ITS2) of the pinewood nematode (Bursaphelenchus xylophilus) were obtained from DNA extracted directly from wood pieces collected from wilted pine trees throughout the Kyushu and Okinawa islands, Japan. Either a 2569bp or 2573bp sequence was obtained from 88 of 143 samples. Together with the 45 rDNA sequences of pinewood nematode isolates previously reported, there were eight single nucleotide polymorphisms and two indels of two bases. Based on these mutations, nine haplotypes were estimated. The haplotype frequencies differed among regions in Kyushu island (northwest, northeast and center, southeast, and southwest), and the distribution was consistent with the invasion and spreading routes of the pinewood nematode previously estimated from past records of pine wilt and wood importation. There was no significant difference in haplotype frequencies among the collection sites on Okinawa island. PMID:22736814

  13. Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi.

    PubMed

    Desquesnes, Marc; Marc, Desquesnes; Kamyingkird, Ketsarin; Ketsarin, Kamyingkird; Yangtara, Sarawut; Sarawut, Yangtara; Milocco, Cristina; Cristina, Milocco; Ravel, Sophie; Sophie, Ravel; Wang, Ming-Hui; Ming-Hui, Wang; Lun, Zhao-Rong; Zhao-Rong, Lun; Morand, Serge; Serge, Morand; Jittapalapong, Sathaporn; Sathaporn, Jittapalapong

    2011-08-01

    Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1-10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.

  14. 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

    PubMed Central

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

    2016-01-01

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

  15. Structure and inheritance of ribosomal DNA variants in cultivated and wild hop, Humulus lupulus L.

    PubMed

    Pillay, M; Kenny, S T

    1996-08-01

    Genetic variation was assessed among cultivated and wild hop, Humulus lupulus, by restriction fragment length polymorphisms (RFLPs) of the ribosomal RNA genes (rDNA). Two rDNA length variants of 10.3 and 9.3 kbp represented by three phenotypes designated A, B and C were detected with XhoI. Restriction-site mapping showed that hop rDNA is structurally similar to those of most higher plants. A high level of homogeneity existed in rDNA repeat lengths among the diverse hop genotypes. Generally, phenotype A was predominant in wild and cultivated European and Asian genotypes; phenotype B in North American cultivars; while phenotype C was present only in native North American hop, providing a potential molecular marker for the identification of this germ plasm. The rDNA data provided genetic evidence for the separation of native and cultivated American genotypes and supports the hypothesis that North American hop cultivars are of hybrid origin from European and native American genotypes. The segregation of rDNA phenotypes in four F1 families suggests that a single locus with two co-dominant alleles controls genetic variability for rDNA variants in hop.

  16. Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants

    PubMed Central

    Tanner, Michael A.; Goebel, Brett M.; Dojka, Michael A.; Pace, Norman R.

    1998-01-01

    Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella (formerly Zoogloea), Acinetobacter, Stenotrophomonas, Escherichia, Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms. PMID:9687486

  17. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    PubMed Central

    Kvich, Lasse; Eickhardt, Steffen; Omland, Lars H.; Bjarnsholt, Thomas; Moser, Claus

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS. PMID:25854484

  18. Long-term evolution of 5S ribosomal DNA seems to be driven by birth-and-death processes and selection in Ensis razor shells (Mollusca: Bivalvia).

    PubMed

    Vierna, Joaquín; González-Tizón, Ana M; Martínez-Lage, Andrés

    2009-10-01

    A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.

  19. Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum.

    PubMed

    Han, Eun-Heui; Cho, KyeMan; Goo, YoungMin; Kim, ManBae; Shin, Young-Wook; Kim, Yun-Hee; Lee, Shin-Woo

    2016-04-01

    Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

  20. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

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

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

    PubMed

    Ironside, Joseph Edward

    2013-01-01

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

  2. Rpd3- and Spt16-Mediated Nucleosome Assembly and Transcriptional Regulation on Yeast Ribosomal DNA Genes

    PubMed Central

    Johnson, Joseph M.; French, Sarah L.; Osheim, Yvonne N.; Li, Mingguang; Hall, Lindsey; Beyer, Ann L.

    2013-01-01

    Ribosomal DNA (rDNA) genes in eukaryotes are organized into multicopy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, ∼50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study, we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully reactivate polymerase I (Pol I) transcription when cells exit stationary phase. PMID:23689130

  3. Genetic diversity of Azotobacter strains isolated from soils by amplified ribosomal DNA restriction analysis.

    PubMed

    Mazinani, Z; Asgharzadeh, A

    2014-01-01

    Strains of Azotobacter mediate in the nitrogen fixation process by reducing of N2 to ammonia. In this study, 50 strains were isolated from different rhizospheric soil in central Iran, by using soil paste-plate method. These strains were biochemically identified and characterized on differential LG medium based on morphological and physiological properties. Results obtained showed that identified strains were belonging to three species, namely A. chroococcum, A. vinelandii and A. beijernckii. In order to molecular analysis, the 16S rRNA gene was amplified using 27f and 1495r primers and PCR products were subsequently digested with RsaI, HpaII and HhaI. Cluster analysis based on amplified ribosomal DNA restriction analysis were revealed intraspecific polymorphism and differentiated strains into two mains clusters, clusters A and B. Cluster A strains were related to the A. vinelandii, whereas cluster B strains were related to the A. chroococcum and A. beijerinckii. The results show that amplified ribosomal DNA restriction analysis is a powerful and discriminatory tool for the identification of members of the genus Azotobacter.

  4. An unusual case of Streptococcus anginosus group pyomyositis diagnosed using direct 16S ribosomal DNA sequencing.

    PubMed

    Walkty, Andrew; Embil, John M; Nichol, Kim; Karlowsky, James

    2014-01-01

    Bacteria belonging to the Streptococcus anginosus group (Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case of S anginosus group pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to the S anginosus group should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection.

  5. Systematic analysis and evolution of 5S ribosomal DNA in metazoans

    PubMed Central

    Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

    2013-01-01

    Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades. PMID:23838690

  6. Systematic analysis and evolution of 5S ribosomal DNA in metazoans.

    PubMed

    Vierna, J; Wehner, S; Höner zu Siederdissen, C; Martínez-Lage, A; Marz, M

    2013-11-01

    Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

  7. Unveiling cryptic species diversity of flowering plants: successful biological species identification of Asian Mitella using nuclear ribosomal DNA sequences.

    PubMed

    Okuyama, Yudai; Kato, Makoto

    2009-05-16

    Although DNA sequence analysis is becoming a powerful tool for identifying species, it is not easy to assess whether the observed genetic disparity corresponds to reproductive isolation. Here, we compared the efficiency of biological species identification between nuclear ribosomal and chloroplast DNA sequences, focusing on an Asian endemic perennial lineage of Mitella (Asimitellaria; Saxifragaceae). We performed artificial cross experiments for 43 pairs of ten taxonomic species, and examined their F1 hybrid pollen fertility in vitro as a quantitative measure of postzygotic reproductive isolation. A nonlinear, multiple regression analysis indicated that the nuclear ribosomal DNA distances are sufficient to explain the observed pattern of F1 hybrid pollen fertility, and supplementation with chloroplast DNA distance data does not improve the explanatory power. Overall, with the exception of a recently diverged species complex with more than three biological species, nuclear ribosomal DNA sequences successfully circumscribed ten distinct biological species, of which two have not been described (and an additional one has not been regarded as a distinct taxonomic species) to date. We propose that nuclear ribosomal DNA sequences contribute to reliable identification of reproductively isolated and cryptic species of Mitella. More comparable studies for other plant groups are needed to generalize our findings to flowering plants.

  8. Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis.

    PubMed

    Masih, Shet; Arora, Sunil K; Vasishta, Rakesh K

    2011-09-01

    The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmania donovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund's adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1×10(7) L. donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.

  9. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

  10. Cellular regulation of ribosomal DNA transcription:both rat and Xenopus UBF1 stimulate rDNA transcription in 3T3 fibroblasts.

    PubMed

    Hannan, R; Stefanovsky, V; Arino, T; Rothblum, L; Moss, T

    1999-02-15

    A novel RNA polymerase I (RPI) driven reporter gene has been used to investigate the in vivo role of the architectural ribosomal transcription factor UBF in gene activation and species specificity. It is shown that the level of UBF overexpression in NIH3T3 cells leads to a proportionate increase in the activities of both reporter and endogenous ribosomal genes. Further, co-expression of UBF antisense RNA suppresses reporter gene expression. Thus, UBF is limiting for ribosomal transcription in vivo and represents a potential endogenous ribosomal gene regulator. In contrast to some in vitro studies, in vivo, the mammalian and Xenopus forms of UBF1 show an equal ability to activate a mouse RPI promoter. This activity is severely impaired in mutants compromised for either dimerization or DNA binding. Similarly, the natural UBF2 splice variant shows a severely impaired capacity to activate RPI transcription. The data strongly suggest that UBF predominantly regulates ribosomal transcription by binding to and activating the ribosomal genes, but does not eliminate a possible secondary role in titrating ribosomal gene repressors such as Rb. Consistent with the DNA folding ability and cellular abundance of the UBF, we suggest that the protein may regulate a structural transition between the potentially active and active chromatin states.

  11. Molecular detection of bacteria in plant tissues, using universal 16S ribosomal DNA degenerated primers

    PubMed Central

    Tsoktouridis, Georgios; Tsiamis, George; Koutinas, Nikolaos; Mantell, Sinclair

    2014-01-01

    Highly specific, sensitive and rapid tests are required for the detection and identification of covert bacterial contaminations in plant tissue cultures. Current methods available for this purpose are tedious, time consuming, highly error prone, expensive, require advanced technical expertise and are sometimes ineffective. We report here the development of a sensitive polymerase chain reaction (PCR) based method for the rapid detection and identification of bacteria occurring in plant tissue cultures. A total of 121 16S ribosomal DNA (rDNA) coding regions from 14 different groups of bacteria, algae and plants, available in the Gene Bank/European Molecular Biology Laboratory databases, were aligned and several conserved DNA sequences of bacterial origin were identified. From those, five degenerated primers were designed in order to amplify only the bacterial DNA present in mixed plant/bacteria genomic DNA extracts. A known amount of bacterial suspension of either covert Pseudomonas or covert Bacillus were added to in vitro plant leaves and total plant/bacterial DNA extracted using three different methods to determine the lowest number of bacteria required to be present in order to allow their detection. The highest sensitivity of the bacterial cell detection was 2.5 × 106 cells of both Bacillus and Pseudomonas inoculums, using template DNA prepared by the MiniPrep method. Generation of PCR amplification fragments was achieved only for the 16S rDNA bacterial gene by using four combinations of degenerated primers. Successive sequence analysis of these amplified fragments led to the rapid detection and molecular identification of bacteria covertly associated with plants. PMID:26019546

  12. Chromosomal localization of ribosomal and telomeric DNA provides new insights on the evolution of gomphocerinae grasshoppers.

    PubMed

    Jetybayev, I E; Bugrov, A G; Karamysheva, T V; Camacho, J P M; Rubtsov, N B

    2012-01-01

    Chromosome location of ribosomal DNA (rDNA) and telomeric repeats was analysed in mitotic chromosomes of 15 species of Gomphocerinae grasshoppers belonging to the tribes Arcypterini, Gomphocerini, Stenobothrini, and Chrysochraontini. Two types of rDNA distribution were found in the Gomphocerini tribe. Type 1, found in 9 species, was characterized by the presence of rDNA in the short arm of the long biarmed chromosomes 2 and 3 and, in some species, also in the X chromosome. Type 2 was found only in Aeropus sibiricus and Stauroderus scalaris and consisted in the presence of pericentromeric rDNA blocks in all chromosomes. A comparison of rDNA distribution in Gomphocerini species with 2n ♂ = 23, 2n ♂ = 21, and 2n ♂ = 17 suggested the possible involvement of chromosome 6 in the ancestral karyotype (2n ♂ = 23) in 1 of the 3 centric fusions that decreased the chromosome number in these species. In the tribe Stenobothrini, Stenobothrus eurasius carried a single rDNA cluster in the X chromosome, likewise 2 Spanish species previously analysed, but Omocestus viridulus unusually showed a single rDNA cluster in the longest autosome. Telomeric repeats were located primarily on the ends of chromosome arms. In 2 species, however, we observed the presence of interstitial clusters outside telomeric regions. The first one, Aeropus sibiricus, exhibited a polymorphic interstitial site of telomeric repeats in chromosome 6 as a consequence of a paracentric inversion. Most remarkably, Chorthippus jacobsoni showed the presence of telomeric repeats in the pericentric regions of the 3 biarmed chromosome pairs originated by centric fusion, thus suggesting that these rearrangements were not of the Robertsonian type but true centric fusion with a probable generation of dicentric chromosomes.

  13. Copy number variation of ribosomal DNA and Pokey transposons in natural populations of Daphnia

    PubMed Central

    2012-01-01

    Background Despite their ubiquity and high diversity in eukaryotic genomes, DNA transposons are rarely encountered in ribosomal DNA (rDNA). In contrast, R-elements, a diverse group of non-LTR retrotransposons, specifically target rDNA. Pokey is a DNA transposon that targets a specific rDNA site, but also occurs in many other genomic locations, unlike R-elements. However, unlike most DNA transposons, Pokey has been a stable component of Daphnia genomes for over 100 million years. Here we use qPCR to estimate the number of 18S and 28S ribosomal RNA genes and Pokey elements in rDNA (rPokey), as well as other genomic locations (gPokey) in two species of Daphnia. Our goals are to estimate the correlation between (1) the number of 18S and 28S rRNA genes, (2) the number of 28S genes and rPokey, and (3) the number of rPokey and gPokey. In addition, we ask whether Pokey number and distribution in both genomic compartments are affected by differences in life history between D. pulex and D. pulicaria. Results We found differences in 18S and 28S gene number within isolates that are too large to be explained by experimental variation. In general, Pokey number within isolates is modest (< 20), and most are gPokey. There is no correlation between the number of rRNA genes and rPokey, or between rPokey and gPokey. However, we identified three isolates with unusually high numbers of both rPokey and gPokey, which we infer is a consequence of recent transposition. We also detected other rDNA insertions (rInserts) that could be degraded Pokey elements, R- elements or the divergent PokeyB lineage recently detected in the Daphnia genome sequence. Unlike rPokey, rInserts are positively correlated with rRNA genes, suggesting that they are amplified by the same mechanisms that amplify rDNA units even though rPokey is not. Overall, Pokey frequency and distribution are similar in D. pulex and D. pulicaria suggesting that differences in life history have no impact on Pokey. Conclusions The

  14. A group I intron in the nuclear small subunit ribosomal DNA of Gaeumannomyces graminis.

    PubMed

    Fouly, H M; Wilkinson, H T

    2000-05-01

    The length of the small subunit ribosomal DNA (SSU rDNA) differs among isolates of species and varieties of Gaeumannomyces. The sequence of the 3' region of the SSU rDNA revealed 340-, 365-, and 520-bp insertions for G. graminis varieties avenae, tritici, and graminis, respectively. The intron sequences from varities tritici and avenae were similar, except there was an insert of 23 nucleotides at base 328 from the 5' end of the G. g. var. tritici intron. The G. g. var. graminis intron sequences had 92.4% homology compared with the intron sequences of varieties tritici and avenae. In addition, the intron sequence of variety graminis is larger, having an insert of 155 nucleotides at base 365 of the 5' end of the intron. Little variation in the DNA sequences flanking the introns has been detected among the isolates of Gaeumannomyces that either have or lack an intron. Reverse transcriptase PCR (RT-PCR) indicated the absence of the intron in the mature rRNA. The intron sequence had both a conserved sequence and secondary structural elements classifying it as a group I intron.

  15. An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

    PubMed Central

    McGrath, K E; Smothers, J F; Dadd, C A; Madireddi, M T; Gorovsky, M A; Allis, C D

    1997-01-01

    An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo. Images PMID:9017598

  16. Large-scale organization of ribosomal DNA chromatin is regulated by Tip5.

    PubMed

    Zillner, Karina; Filarsky, Michael; Rachow, Katrin; Weinberger, Michael; Längst, Gernot; Németh, Attila

    2013-05-01

    The DNase I accessibility and chromatin organization of genes within the nucleus do correlate to their transcriptional activity. Here, we show that both serum starvation and overexpression of Tip5, a key regulator of ribosomal RNA gene (rDNA) repression, dictate DNase I accessibility, facilitate the association of rDNA with the nuclear matrix and thus regulate large-scale rDNA chromatin organization. Tip5 contains four AT-hooks and a TAM (Tip5/ARBP/MBD) domain, which were proposed to bind matrix-attachment regions (MARs) of the genome. Remarkably, the TAM domain of Tip5 functions as nucleolar localization and nuclear matrix targeting module, whereas AT-hooks do not mediate association with the nuclear matrix, but they are required for nucleolar targeting. These findings suggest a dual role for Tip5's AT-hooks and TAM domain, targeting the nucleolus and anchoring to the nuclear matrix, and suggest a function for Tip5 in the regulation of higher-order rDNA chromatin structure.

  17. Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

    PubMed

    Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

    2006-03-15

    Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

  18. Large-scale organization of ribosomal DNA chromatin is regulated by Tip5

    PubMed Central

    Zillner, Karina; Filarsky, Michael; Rachow, Katrin; Weinberger, Michael; Längst, Gernot; Németh, Attila

    2013-01-01

    The DNase I accessibility and chromatin organization of genes within the nucleus do correlate to their transcriptional activity. Here, we show that both serum starvation and overexpression of Tip5, a key regulator of ribosomal RNA gene (rDNA) repression, dictate DNase I accessibility, facilitate the association of rDNA with the nuclear matrix and thus regulate large-scale rDNA chromatin organization. Tip5 contains four AT-hooks and a TAM (Tip5/ARBP/MBD) domain, which were proposed to bind matrix-attachment regions (MARs) of the genome. Remarkably, the TAM domain of Tip5 functions as nucleolar localization and nuclear matrix targeting module, whereas AT-hooks do not mediate association with the nuclear matrix, but they are required for nucleolar targeting. These findings suggest a dual role for Tip5’s AT-hooks and TAM domain, targeting the nucleolus and anchoring to the nuclear matrix, and suggest a function for Tip5 in the regulation of higher-order rDNA chromatin structure. PMID:23580549

  19. Phylogenetic relationships in Brassicaceae tribe Alysseae inferred from nuclear ribosomal and chloroplast DNA sequence data.

    PubMed

    Rešetnik, Ivana; Satovic, Zlatko; Schneeweiss, Gerald M; Liber, Zlatko

    2013-12-01

    Numerous molecular systematic studies within Brassicaceae have resulted in a strongly improved classification of the family, as morphologically defined units at and above the generic level were often found to poorly reflect phylogenetic relationships. Here, we focus on tribe Alysseae, which despite its size (accounting for about 7% of all species) has only received limited coverage in previous phylogenetic studies. Specifically, we want to test phylogenetic hypotheses implied by current tribal and generic circumscriptions and to put diversification within tribe Alysseae into a temporal context. To this end, sequence data from the nrDNA ITS and two plastid regions (ndhF gene, trnL-F intergenic spacer) were obtained for 176 accessions, representing 16 out of 17 currently recognized genera of the tribe, and were phylogenetically analysed, among others, using a relaxed molecular clock. Due to large discrepancies with respect to published ages of Brassicaceae, age estimates concerning Alysseae are, however, burdened with considerable uncertainty. The tribe is monophyletic and contains four strongly supported major clades and Alyssum homalocarpum, whose relationships among each other remain uncertain due to incongruences between nuclear and plastid DNA markers. The largest genus of the tribe, Alyssum, is not monophyletic and contains, apart from A. homalocarpum, two distinct lineages, corresponding to sections Alyssum, Psilonema, Gamosepalum and to sections Odontarrhena and Meniocus, respectively. Clypeola, whose monophyly is supported only by the plastid data, is very closely related to and possibly nested within the second Alyssum lineage. Species of the genus Fibigia intermingle with those of Alyssoides, Clastopus, Degenia, and Physoptychis, rendering Fibigia polyphyletic. The monotypic genera Leptoplax and Physocardamum are embedded in Bornmuellera.

  20. [Identification of fish species based on ribosomal DNA ITS2 locus].

    PubMed

    Yuan, Wan-An

    2010-04-01

    To prevent illegal fishing and sale, the most difficult problem is identification of marketed fish species, especially the parts that are difficult to be differentiated with morphological method (e.g., larval, eggs, scales, meat, products etc. To assist conservation and management of fishery resources, this paper reported a molecular genetic approach based on ribosomal internal transcribed spacer 2 locus. The method includes two steps: (1) the order general primers were designed according to the conservative nature of 5.8SrRAN and 28SrRNA genes within an order, and the DNA ribosomal internal transcribed spacer 2 locus fragment were then amplified and sequenced. (2) The species-specific ladders and the species-specific primers for each species were designed according to the sequencing results. The map of molecular taxonomy was constructed. This approach employs multiplex PCR that is formatted for fish species identification. We tested 210 single-species samples and 40 mix-species samples from different regions of China. The approach distinguished accurately and sensitively samples from each of the five species. This genetic and molecular approach will be useful for fish conservation, assessment, management and exploitation, strengthen in law enforcement of fishery manager, combat rare and endangered fish smuggling, and prevent commercial fraud and biological invasion by harmful nonnative species.

  1. Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

    PubMed

    Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

    2013-11-01

    Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

  2. Development and Evaluation of a Quality-Controlled Ribosomal Sequence Database for 16S Ribosomal DNA-Based Identification of Staphylococcus Species

    PubMed Central

    Becker, Karsten; Harmsen, Dag; Mellmann, Alexander; Meier, Christian; Schumann, Peter; Peters, Georg; von Eiff, Christof

    2004-01-01

    To establish an improved ribosomal gene sequence database as part of the Ribosomal Differentiation of Microorganisms (RIDOM) project and to overcome the drawbacks of phenotypic identification systems and publicly accessible sequence databases, both strands of the 5′ end of the 16S ribosomal DNA (rDNA) of 81 type and reference strains comprising all validly described staphylococcal (sub)species were sequenced. Assuming a normal distribution for pairwise distances of all unique staphylococcal sequences and choosing a reporting criterion of ≥98.7% similarity for a “distinct species,” a statistical error probability of 1.0% was calculated. To evaluate this database, a 16S rDNA fragment (corresponding to Escherichia coli positions 54 to 510) of 55 clinical Staphylococcus isolates (including those of the small-colony variant phenotype) were sequenced and analyzed by the RIDOM approach. Of these isolates, 54 (98.2%) had a similarity score above the proposed threshold using RIDOM; 48 (87.3%) of the sequences gave a perfect match, whereas 83.6% were found by searching National Center for Biotechnology Information (NCBI) database entries. In contrast to RIDOM, which showed four ambiguities at the species level (mainly concerning Staphylococcus intermedius versus Staphylococcus delphini), the NCBI database search yielded 18 taxon-related ambiguities and showed numerous matches exhibiting redundant or unspecified entries. Comparing molecular results with those of biochemical procedures, ID 32 Staph (bioMérieux, Marcy I'Etoile, France) and VITEK 2 (bioMérieux) failed to identify 13 (23.6%) and 19 (34.5%) isolates, respectively, due to incorrect identification and/or categorization below acceptable values. In contrast to phenotypic methods and the NCBI database, the novel high-quality RIDOM sequence database provides excellent identification of staphylococci, including rarely isolated species and phenotypic variants. PMID:15528685

  3. A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome

    PubMed Central

    Yu, Shoukai; Lemos, Bernardo

    2016-01-01

    Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. PMID:27797956

  4. B-chromosome ribosomal DNA is functional in the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruiz-Estévez, Mercedes; López-León, Ma Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2012-01-01

    B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA) genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B(24) chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B(24) chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them, and not by recruitment of nucleolar materials formed in A chromosome nucleolar organizing regions. Therefore, B-chromosome rDNA in E. plorans is functional since it is actively transcribed to form the nucleolus attached to the B chromosome. This demonstrates that some heterochromatic B chromosomes can harbour functional genes.

  5. B-Chromosome Ribosomal DNA Is Functional in the Grasshopper Eyprepocnemis plorans

    PubMed Central

    Ruiz-Estévez, Mercedes; Cabrero, Josefa; Camacho, Juan Pedro M.

    2012-01-01

    B-chromosomes are frequently argued to be genetically inert elements, but activity for some particular genes has been reported, especially for ribosomal RNA (rRNA) genes whose expression can easily be detected at the cytological level by the visualization of their phenotypic expression, i.e., the nucleolus. The B24 chromosome in the grasshopper Eyprepocnemis plorans frequently shows a nucleolus attached to it during meiotic prophase I. Here we show the presence of rRNA transcripts that unequivocally came from the B24 chromosome. To detect these transcripts, we designed primers specifically anchoring at the ITS-2 region, so that the reverse primer was complementary to the B chromosome DNA sequence including a differential adenine insertion being absent in the ITS2 of A chromosomes. PCR analysis carried out on genomic DNA showed amplification in B-carrying males but not in B-lacking ones. PCR analyses performed on complementary DNA showed amplification in about half of B-carrying males. Joint cytological and molecular analysis performed on 34 B-carrying males showed a close correspondence between the presence of B-specific transcripts and of nucleoli attached to the B chromosome. In addition, the molecular analysis revealed activity of the B chromosome rDNA in 10 out of the 13 B-carrying females analysed. Our results suggest that the nucleoli attached to B chromosomes are actively formed by expression of the rDNA carried by them, and not by recruitment of nucleolar materials formed in A chromosome nucleolar organizing regions. Therefore, B-chromosome rDNA in E. plorans is functional since it is actively transcribed to form the nucleolus attached to the B chromosome. This demonstrates that some heterochromatic B chromosomes can harbour functional genes. PMID:22570730

  6. Species identification of spiny lobster phyllosome larvae via ribosomal DNA analysis.

    PubMed

    Silberman, J D; Walsh, P J

    1992-06-01

    Within the tropical northwestern Atlantic, Panulirus argus, P. guttatus, and P. laevicauda (Palinuridae family), are sympatric. Numerous studies have examined the distribution and abundance of planktonic phyllosome larvae with respect to recruitment of spiny lobsters to the benthic population, but the data are of limited use because larvae of these species cannot yet be distinguished from one another by morphological characteristics. A simple molecular method that unambiguously differentiates adults or larvae of P. argus, P. guttatus, and P. laevicauda is described: a 5' region of 28s ribosomal DNA is amplified in vitro and then cut with a diagnostic restriction enzyme to identify each species. Data are also presented from the application of this method to representative plankton tows.

  7. 18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines.

    PubMed

    Rivera, Windell L; Adao, Davin Edric V

    2009-10-01

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

  8. Phylogenetic analysis of Bambusa (Poaceae: Bambusoideae) based on internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Sun, Ye; Xia, Nianhe; Lin, Rushun

    2005-12-01

    Phylogenetic analyses of Bambusa species were performed using internal transcribed spacer sequences of nuclear ribosomal DNA. The 21 species sampled included members of Bambusa (sensu stricto), Dendrocalamopsis, Dendrocalamus, Guadua, Leleba, and Lingnania. Arundinaria gigantea was used as an outgroup. Using the maximum parsimony method with PAUP*, gaps were treated as missing states or new states. Parsimonious analysis revealed that Dendrocalamus latiflorus was closely related to the members of Dendrocalamopsis. Dendrocalamus membranaceus was a sister species to Dendrocalamus strictus. Three Dendrocalamus species were closely related to and nested in a polyphyletic Bambusa. Bambusa subaequalis was a sister species to B. multiplex, B. emeiensis to B. chungii, B. contracta to B. hainanensis, and B. flexuosa was a sister species to B. sinospinosa, B. tuldoides, B. surrecta, B. intermedia, and B. valida group, which raised doubts about the monophyly of the subgenera Bambusa (sensu stricto), Dendrocalamopsis, Leleba, and Lingnania under the genus Bambusa.

  9. A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.

    PubMed Central

    Kambhampati, S

    1995-01-01

    Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat. PMID:7534409

  10. Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea).

    PubMed

    Warberg, Rikke; Jensen, K Thomas; Frydenberg, Jane

    2005-11-01

    In searching for species-specific DNA sequences of microphallid species (Digenea, Trematoda) we examined the ribosomal internal transcribed spacer regions (ITS) of three closely related species (Levinseniella group) hosted by mud snails (first intermediate host) and marine crustaceans (second intermediate host). In the ITS1 region we found consistent patterns of repeating sequences of 130 bp. Within each main repeat there was a varying number of subrepeats specific for each of the species. All repeats including subrepeats were identified by a similar starting sequence: 5'-CCTGTGG-3'. As this sequence has close resemblance to the chi sequence 5'-GCTGGTGG-3' found in phage lambda we speculate if it serves the same function as a recombination hotspot. Alternatively but less likely, it could be an inactive, mutational relic of a sequence that once served this purpose.

  11. Giant panda ribosomal protein S14: cDNA, genomic sequence cloning, sequence analysis, and overexpression.

    PubMed

    Wu, G-F; Hou, Y-L; Hou, W-R; Song, Y; Zhang, T

    2010-10-13

    RPS14 is a component of the 40S ribosomal subunit encoded by the RPS14 gene and is required for its maturation. The cDNA and the genomic sequence of RPS14 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively; they were both sequenced and analyzed. The length of the cloned cDNA fragment was 492 bp; it contained an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 3421 bp; it contains four exons and three introns. Alignment analysis indicates that the nucleotide sequence shares a high degree of homology with those of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Danio rerio (93.64, 83.37, 92.54, 91.89, 87.28, 84.21, and 84.87%, respectively). Comparison of the deduced amino acid sequences of the giant panda with those of these other species revealed that the RPS14 of giant panda is highly homologous with those of B. taurus, R. norvegicus and D. rerio (85.99, 99.34 and 99.34%, respectively), and is 100% identical with the others. This degree of conservation of RPS14 suggests evolutionary selection. Topology prediction shows that there are two N-glycosylation sites, three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, four N-myristoylation sites, two amidation sites, and one ribosomal protein S11 signature in the RPS14 protein of the giant panda. The RPS14 gene can be readily expressed in Escherichia coli. When it was fused with the N-terminally His-tagged protein, it gave rise to accumulation of an expected 22-kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained can be purified for studies of its function.

  12. Ribosomal DNA, heterochromatin, and correlation with genome size in diploid and polyploid North American endemic sagebrushes (Artemisia, Asteraceae)

    Treesearch

    Sonia Garcia; Teresa Garnatje; Jaume Pellicer; E. Durant McArthur; Sonja Siljak-Yakovlev; Joan Valles

    2009-01-01

    Subgenus Tridentatae (Artemisia, Asteraceae) can be considered a polyploid complex. Both polyploidy and hybridization have been documented in the Tridentatae. Fluorescent in situ hybridization (FISH) and fluorochrome banding were used to detect and analyze ribosomal DNA changes linked to polyploidization in this group by studying four diploidpolyploid species pairs. In...

  13. Molecular phylogenetic analysis of Enterobius vermicularis and development of an 18S ribosomal DNA-targeted diagnostic PCR.

    PubMed

    Zelck, Ulrike E; Bialek, Ralf; Weiss, Michael

    2011-04-01

    We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed.

  14. Molecular Phylogenetic Analysis of Enterobius vermicularis and Development of an 18S Ribosomal DNA-Targeted Diagnostic PCR▿

    PubMed Central

    Zelck, Ulrike E.; Bialek, Ralf; Weiß, Michael

    2011-01-01

    We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed. PMID:21248085

  15. Ribosomal DNA sequence of Nucleospora salmonis Hedrick, Groff and Baxa, 1991 (Microsporea:Enterocytozoonidae): implications for phylogeny and nomenclature.

    PubMed

    Docker, M F; Kent, M L; Hervio, D M; Khattra, J S; Weiss, L M; Cali, A; Devlin, R H

    1997-01-01

    Rules of zoological nomenclature, morphological data, and ribosomal DNA sequence data support the validity of the genus Nucleospora, and its placement in the family Enterocytozoonidae. Although Nucleospora exhibits most of the distinguishing morphological characteristics of the family Enterocytozoonidae Cali and Owen, 1990, the distinctively different hosts (fish and humans, respectively) and sites of development (the nuclei of immature blood cells and the cytoplasm of enterocytes) support the placement of Nucleospora and Enterocytozoon into separate genera. Ribosomal DNA sequence comparisons between Nucleospora salmonis and Enterocytozoon bieneusi showed 19.8% genetic divergence in the large and small subunit regions. Although more inter- and intrageneric comparisons are needed before percent homology of ribosomal DNA can be used as a criterion for the separation of genera, the genetic divergence between the two species is sufficiently large to deter suppression of the genus Nucleospora as a junior synonym of Enterocytozoon. A polymerase chain reaction test for the detection of N. salmonis in chinook salmon (Oncorhynchus tshawytscha), based on N. salmonis-specific ribosomal DNA sequence, is described.

  16. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  17. Identification of Clinical Isolates of Actinomyces Species by Amplified 16S Ribosomal DNA Restriction Analysis

    PubMed Central

    Hall, Val; Talbot, P. R.; Stubbs, S. L.; Duerden, B. I.

    2001-01-01

    Amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA), using enzymes HaeIII and HpaII, was applied to 176 fresh and 299 stored clinical isolates of putative Actinomyces spp. referred to the Anaerobe Reference Unit of the Public Health Laboratory Service for confirmation of identity. Results were compared with ARDRA results obtained previously for reference strains and with conventional phenotypic reactions. Identities of some strains were confirmed by analysis of partial 16S rDNA sequences. Of the 475 isolates, 331 (70%) were clearly assigned to recognized Actinomyces species, including 94 isolates assigned to six recently described species. A further 52 isolates in 12 ARDRA profiles were designated as apparently resembling recognized species, and 44 isolates, in 18 novel profiles, were confirmed as members of genera other than Actinomyces. The identities of 48 isolates in nine profiles remain uncertain, and they may represent novel species of Actinomyces. For the majority of species, phenotypic results, published reactions for the species, and ARDRA profiles concurred. However, of 113 stored isolates originally identified as A. meyeri or resembling A. meyeri by phenotypic tests, only 21 were confirmed as A. meyeri by ARDRA; 63 were reassigned as A. turicensis, 7 as other recognized species, and 22 as unidentified actinomycetes. Analyses of incidence and clinical associations of Actinomyces spp. add to the currently sparse knowledge of some recently described species. PMID:11574572

  18. DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

    PubMed

    Marilley, M; Radebaugh, C A; Geiss, G K; Laybourn, P J; Paule, M R

    2002-08-01

    Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

  19. Evolution of the ITS sequences of ribosomal DNA in Enteromorpha (Chlorophyceae).

    PubMed

    Leskinen, E; Pamilo, P

    1997-01-01

    Ribosomal DNA (rDNA) region, including the 3' end of the 18S rRNA gene, the entire 5.8S rRNA gene, the 5' end of the 28S rRNA gene, and the internal transcribed spacers ITS 1 and ITS 2, of Enteromorpha green algae from the Baltic Sea, were sequenced. The evolution of the Enteromorpha sequences differed from those of other green algae in several important ways. The ITS regions were short and had a high nucleotide bias. The frequency of nucleotides G and C was up to 70% in the ITS sequences, whereas the frequencies were close to 50% in the 5.8S rDNA. Furthermore, the sequence divergence was much higher in ITS 1 than in ITS 2. Two haplotypes, differing only by two nucleotides, were detected in the E. intestinalis/compressa complex. The difference coincides with a morphological differentiation (branching of thalli) and may represent distinct gene pools.

  20. SIRT1 regulates the ribosomal DNA locus: epigenetic candles twinkle longevity in the Christmas tree.

    PubMed

    Salminen, Antero; Kaarniranta, Kai

    2009-01-02

    Ribosomal RNA (rRNA) genes arrange themselves in a tandem pattern in nucleolus and during the transcription of rRNA genes, the elongating nascent rRNA transcripts create a structure called Christmas tree. rRNA genes in the rDNA locus can be either active or silent depending on the epigenetic regulation of the chromatin structure. Yeast Sir2 (silent information regulator 2) protein containing complexes can repress the recombination in the rDNA locus and subsequently extend the replicative lifespan of the budding yeast. The mammalian rDNA locus is also under the epigenetic regulation by protein complexes, such as NoRC (nucleolar remodeling complex) and eNoSC (energy-dependent nucleolar silencing complex), involving histone deacetylases and methyltransferases. SIRT1, a NAD(+)-dependent histone deacetylase, is the key component in the eNoSC complex and hence energetic changes can regulate the activation of eNoSC complex and in this way mediate the epigenetic silencing of rRNA gene expression. The eNoSC complex links SIRT1-induced longevity regulation to the metabolic rate theory of aging.

  1. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing.

    PubMed

    Kruse, Alexandra Y C; Kvich, Lasse; Eickhardt, Steffen; Omland, Lars H; Bjarnsholt, Thomas; Moser, Claus

    2015-06-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii-specific PCR and immunohistochemistry. The patient was later diagnosed with HIV/AIDS. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed Central

    2010-01-01

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

  3. Phylogeography of East Asian Lespedeza buergeri (Fabaceae) based on chloroplast and nuclear ribosomal DNA sequence variations.

    PubMed

    Jin, Dong-Pil; Lee, Jung-Hyun; Xu, Bo; Choi, Byoung-Hee

    2016-09-01

    The dynamic changes in land configuration during the Quaternary that were accompanied by climatic oscillations have significantly influenced the current distribution and genetic structure of warm-temperate forests in East Asia. Although recent surveys have been conducted, the historical migration of forest species via land bridges and, especially, the origins of Korean populations remains conjectural. Here, we reveal the genetic structure of Lespedeza buergeri, a warm-temperate shrub that is disjunctively distributed around the East China Sea (ECS) at China, Korea, and Japan. Two non-coding regions (rpl32-trnL, psbA-trnH) of chloroplast DNA (cpDNA) and the internal transcribed spacer of nuclear ribosomal DNA (nrITS) were analyzed for 188 individuals from 16 populations, which covered almost all of its distribution. The nrITS data demonstrated a genetic structure that followed geographic boundaries. This examination utilized AMOVA, comparisons of genetic differentiation based on haplotype frequency/genetic mutations among haplotypes, and Mantel tests. However, the cpDNA data showed contrasting genetic pattern, implying that this difference was due to a slower mutation rate in cpDNA than in nrITS. These results indicated frequent migration by this species via an ECS land bridge during the early Pleistocene that then tapered gradually toward the late Pleistocene. A genetic isolation between western and eastern Japan coincided with broad consensus that was suggested by the presence of other warm-temperate plants in that country. For Korean populations, high genetic diversity indicated the existence of refugia during the Last Glacial Maximum on the Korean Peninsula. However, their closeness with western Japanese populations at the level of haplotype clade implied that gene flow from western Japanese refugia was possible until post-glacial processing occurred through the Korea/Tsushima Strait land bridge.

  4. The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates.

    PubMed

    Stoeck, T; Przybos, E; Dunthorn, M

    2014-05-01

    Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.

  5. A two-locus DNA sequence database for identifying host-specific pathogens and phylogenetic diversity within the Fusarium oxysporum species complex

    USDA-ARS?s Scientific Manuscript database

    An electronically portable two-locus DNA sequence database, comprising partial sequences of the translation elongation factor gene (EF-1a, 634 bp alignment) and nearly complete sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA, 2220 bp alignment) for 850 isolates spanning the phy...

  6. Variation in Ribosomal DNA among Isolates of the Mycorrhizal Fungus Cenococcum Geophilum FR.

    NASA Astrophysics Data System (ADS)

    Lobuglio, Katherine Frances

    1990-01-01

    Cenococcum geophilum Fr., a cosmopolitan mycorrhizal fungus, is well-known for its extremely wide host and habitat range. The ecological diversity of C. geophilum sharply contrasts its present taxonomic status as a monotypic form -genus. Restriction fragment length polymorphisms (RFLPs) in nuclear ribosomal DNA (rDNA) was used to assess the degree of genetic variation among 72 isolates of C. geophilum. The probe used in this study was the rDNA repeat cloned from C. geophilum isolate A145 (pCG15). Length of the rDNA repeat was approximately 9 kb. The rDNA clone was mapped for 5 restriction endonucleases. Hybridization with cloned Saccharomyces cerevisiae rDNA (pSR118, and pSR125 containing the 18S, and 5.8-25S rRNA genes respectively), and alignment of restriction endonuclease sites conserved in the rDNA genes of other fungi, were used to position the corresponding rDNAs of C. geophilum. Southern hybridizations with EcoRI, HindIII, XhoI, and PstI digested DNAs indicated extensive variation among the C. geophilum isolates, greater than has been previously reported to occur within a fungal species. Most of the rDNA polymorphisms occurred in the IGS region. Restriction endonuclease site and length polymorphisms were also observed in the 5.8S-26S genic regions. Sixteen size categories of length mutations, 6 restriction endonuclease site additions, and 4 restriction endonuclease site deletions were determined using isolate A145 as a reference. The rDNA repeat length among the isolates varied from approximately 8.5 to 10.2 kb. RFLPs were also observed in the mitochondrial (mt) 24S rRNA gene and flanking regions of HindIII digested DNAs of C. geophilum isolates representing both geographically distinct and similar origins. Among the C. geophilum isolates analyzed there were fewer RFLPs in mt-DNA than in nuclear rDNA. EcoRI rDNA phenotypes between C. geophilum and Elaphomyces anthracinus, its proposed teleomorph or sexual state, did not correspond. In addition, the four

  7. Application of the Ribosomal DNA ITS2 Region of Physalis (Solanaceae): DNA Barcoding and Phylogenetic Study.

    PubMed

    Feng, Shangguo; Jiang, Mengying; Shi, Yujun; Jiao, Kaili; Shen, Chenjia; Lu, Jiangjie; Ying, Qicai; Wang, Huizhong

    2016-01-01

    Recently, commercial interest in Physalis species has grown worldwide due to their high nutritional value, edible fruit, and potential medicinal properties. However, many Physalis species have similar shapes and are easily confused, and consequently the phylogenetic relationships between Physalis species are poorly understood. This hinders their safe utilization and genetic resource conservation. In this study, the nuclear ribosomal ITS2 region was used to identify species and phylogenetically examine Physalis. Eighty-six ITS2 regions from 45 Physalis species were analyzed. The ITS2 sequences were aligned using Clustal W and genetic distances were calculated using MEGA V6.0. The results showed that ITS2 regions have significant intra- and inter-specific divergences, obvious barcoding gaps, and higher species discrimination rates (82.2% for both the BLASTA1 and nearest distance methods). In addition, the secondary structure of ITS2 provided another way to differentiate species. Cluster analysis based on ITS2 regions largely concurred with the relationships among Physalis species established by many previous molecular analyses, and showed that most sections of Physalis appear to be polyphyletic. Our results demonstrated that ITS2 can be used as an efficient and powerful marker in the identification and phylogenetic study of Physalis species. The technique provides a scientific basis for the conservation of Physalis plants and for utilization of resources.

  8. Application of the Ribosomal DNA ITS2 Region of Physalis (Solanaceae): DNA Barcoding and Phylogenetic Study

    PubMed Central

    Feng, Shangguo; Jiang, Mengying; Shi, Yujun; Jiao, Kaili; Shen, Chenjia; Lu, Jiangjie; Ying, Qicai; Wang, Huizhong

    2016-01-01

    Recently, commercial interest in Physalis species has grown worldwide due to their high nutritional value, edible fruit, and potential medicinal properties. However, many Physalis species have similar shapes and are easily confused, and consequently the phylogenetic relationships between Physalis species are poorly understood. This hinders their safe utilization and genetic resource conservation. In this study, the nuclear ribosomal ITS2 region was used to identify species and phylogenetically examine Physalis. Eighty-six ITS2 regions from 45 Physalis species were analyzed. The ITS2 sequences were aligned using Clustal W and genetic distances were calculated using MEGA V6.0. The results showed that ITS2 regions have significant intra- and inter-specific divergences, obvious barcoding gaps, and higher species discrimination rates (82.2% for both the BLASTA1 and nearest distance methods). In addition, the secondary structure of ITS2 provided another way to differentiate species. Cluster analysis based on ITS2 regions largely concurred with the relationships among Physalis species established by many previous molecular analyses, and showed that most sections of Physalis appear to be polyphyletic. Our results demonstrated that ITS2 can be used as an efficient and powerful marker in the identification and phylogenetic study of Physalis species. The technique provides a scientific basis for the conservation of Physalis plants and for utilization of resources. PMID:27486467

  9. Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

    PubMed Central

    2017-01-01

    Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy. PMID:28880866

  10. Triatomine vectors of Trypanosoma cruzi: a molecular perspective based on nuclear ribosomal DNA markers.

    PubMed

    Bargues, María Dolores; Marcilla, Antonio; Dujardin, Jean Pierre; Mas-Coma, Santiago

    2002-04-01

    Chagas disease (American trypanosomiasis) is mainly transmitted by blood-sucking bugs of the reduviid subfamily Triatominae (Hemiptera: Prosorrhyncha). Control strategies are directed mainly against these insect vectors, as no vaccine is available and, except in the very early stage of infection, there is no effective chemótherapy. Studies of deoxyribonucleic acid (DNA) will lead to major advances in our knowledge of Triatominae and their relationships to Chagas disease transmission, epidemiology and control. Analyses of complete sequences of nuclear genes coding for ribosomal ribonucleic acid (rRNA) (rRNA genes) and spacers furnish significant information at the levels of higher taxons, genera, species, subspecies, hybrids, varieties and populations of Triatominae. This paper briefly reviews the contributions of studies on the slowly-evolving 18S or small subunit (SSU) rRNA gene and the quickly-evolving second internal transcribed spacer (ITS-2). The whole 18S rRNA gene is a useful marker for supraspecific relationships in Triatominae. ITS-2 is complementary to it, enabling resolution at specific and infraspecific levels. All the evidence suggests that ITS-2 will become the DNA marker of excellence for studies of Triatominae at specific and subspecific levels, as it is in other groups of organisms. Possible applications of data obtained from the study of rRNA and ITS-2 sequences of Triatominae are discussed.

  11. Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture.

    PubMed

    Pitkäranta, M; Meklin, T; Hyvärinen, A; Paulin, L; Auvinen, P; Nevalainen, A; Rintala, H

    2008-01-01

    In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.

  12. Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates.

    PubMed

    Laila, Rawnak; Robin, Arif Hasan Khan; Yang, Kiwoung; Choi, Gyung Ja; Park, Jong-In; Nou, Ill-Sup

    2017-01-04

    Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups.

  13. Detection of Ribosomal DNA Sequence Polymorphisms in the Protist Plasmodiophora brassicae for the Identification of Geographical Isolates

    PubMed Central

    Laila, Rawnak; Robin, Arif Hasan Khan; Yang, Kiwoung; Choi, Gyung Ja; Park, Jong-In; Nou, Ill-Sup

    2017-01-01

    Clubroot is a soil-borne disease caused by the protist Plasmodiophora brassicae (P. brassicae). It is one of the most economically important diseases of Brassica rapa and other cruciferous crops as it can cause remarkable yield reductions. Understanding P. brassicae genetics, and developing efficient molecular markers, is essential for effective detection of harmful races of this pathogen. Samples from 11 Korean field populations of P. brassicae (geographic isolates), collected from nine different locations in South Korea, were used in this study. Genomic DNA was extracted from the clubroot-infected samples to sequence the ribosomal DNA. Primers and probes for P. brassicae were designed using a ribosomal DNA gene sequence from a Japanese strain available in GenBank (accession number AB526843; isolate NGY). The nuclear ribosomal DNA (rDNA) sequence of P. brassicae, comprising 6932 base pairs (bp), was cloned and sequenced and found to include the small subunits (SSUs) and a large subunit (LSU), internal transcribed spacers (ITS1 and ITS2), and a 5.8s. Sequence variation was observed in both the SSU and LSU. Four markers showed useful differences in high-resolution melting analysis to identify nucleotide polymorphisms including single- nucleotide polymorphisms (SNPs), oligonucleotide polymorphisms, and insertions/deletions (InDels). A combination of three markers was able to distinguish the geographical isolates into two groups. PMID:28054984

  14. A unified model of nucleic acid unwinding by the ribosome and the hexameric and monomeric DNA helicases.

    PubMed

    Xie, Ping

    2015-09-07

    DNA helicases are enzymes that use the chemical energy to separate DNA duplex into their single-stranded forms. The ribosome, which catalyzes the translation of messenger RNAs (mRNAs) into proteins, can also unwind mRNA duplex. According to their structures, the DNA helicases can fall broadly into hexameric and monomeric forms. A puzzling issue for the monomeric helicases is that although they have similar structures, in vitro biochemical data showed convincingly that in the monomeric forms some have very weak DNA unwinding activities, some have relatively high unwinding activities while others have high unwinding activities. However, in the dimeric or oligomeric forms all of them have high unwinding activities. In addition, in the monomeric forms all of them can translocate efficiently along the single-stranded DNA (ssDNA). Here, we propose a model of the translocation along the ssDNA and DNA unwinding by the monomeric helicases, providing a consistent explanation of these in vitro experimental data. Moreover, by comparing the present model for the monomeric helicases with the model for the hexameric helicases and that for the ribosome which were proposed before, a unified model of nucleic acid unwinding by the three enzymes is proposed.

  15. Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence.

    PubMed

    Huang, W Y; He, B; Wang, C R; Zhu, X Q

    2004-02-26

    Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere.

  16. Identification of novel fusion genes with 28S ribosomal DNA in hematologic malignancies.

    PubMed

    Kobayashi, Satoru; Taki, Tomohiko; Nagoshi, Hisao; Chinen, Yoshiaki; Yokokawa, Yuichi; Kanegane, Hirokazu; Matsumoto, Yosuke; Kuroda, Junya; Horiike, Shigeo; Nishida, Kazuhiro; Taniwaki, Masafumi

    2014-04-01

    Fusion genes are frequently observed in hematologic malignancies and soft tissue sarcomas, and are usually associated with chromosome abnormalities. Many of these fusion genes create in-frame fusion transcripts that result in the production of fusion proteins, and some of which aid tumorigenesis. These fusion proteins are often associated with disease phenotype and clinical outcome, and act as markers for minimal residual disease and indicators of therapeutic targets. Here, we identified the 28S ribosomal DNA (RN28S1) gene as a novel fusion partner of the B-cell leukemia/lymphoma 11B gene (BCL11B), the immunoglobulin κ variable 3-20 gene (IGKV3-20) and the component of oligomeric Golgi complex 1 gene (COG1) in hematologic malignancies. The RN28S1-BCL11B fusion transcript was identified in a case with mixed-lineage (T/myeloid) acute leukemia having t(6;14)(q25;q32) by cDNA bubble PCR using BCL11B primers; however, the gene fused to BCL11B on 14q32 was not on 6q25. IGKV3-20-RN28S1 and COG1-RN28S1 fusion transcripts were identified in the Burkitt lymphoma cell line HBL-5, and the multiple myeloma cell line KMS-18. RN28S1 would not translate, and the breakpoints in partner genes of RN28S1 were within the coding exons, suggesting that disruption of fusion partners by fusion to RN28S1 is the possible mechanism of tumorigenesis. Although further analysis is needed to elucidate the mechanism(s) through which these RN28S1-related fusions play roles in tumorigenesis, our findings provide important insights into the role of rDNA function in human genomic architecture and tumorigenesis.

  17. Intranuclear Anchoring of Repetitive DNA Sequences

    PubMed Central

    Weipoltshammer, Klara; Schöfer, Christian; Almeder, Marlene; Philimonenko, Vlada V.; Frei, Klemens; Wachtler, Franz; Hozák, Pavel

    1999-01-01

    Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using “physiological” conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton. PMID:10613900

  18. Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.

    PubMed

    Volokhov, Dmitriy V; George, Joseph; Liu, Sue X; Ikonomi, Pranvera; Anderson, Christine; Chizhikov, Vladimir

    2006-08-01

    We have completed sequencing the 16S-23S rRNA intergenic transcribed spacer (ITS) region of most known Mycoplasma , Acholeplasma , Ureaplasma , Mesoplasma , and Spiroplasma species. Analysis of the sequence data revealed a significant interspecies variability and low intraspecies polymorphism of the ITS region among Mollicutes . This finding enabled the application of a combined polymerase chain reaction-microarray technology for identifying Mollicutes species. The microarray included individual species-specific oligonucleotide probes for characterizing human Mollicutes species and other species known to be common cell line contaminants. Evaluation of the microarray was conducted using multiple, previously characterized, Mollicutes species. The microarray analysis of the samples used demonstrated a highly specific assay, which is capable of rapid and accurate discrimination among Mollicutes species.

  19. Prevalence and Dynamics of Ribosomal DNA Micro-heterogeneity Are Linked to Population History in Two Contrasting Yeast Species

    PubMed Central

    James, Stephen A.; West, Claire; Davey, Robert P.; Dicks, Jo; Roberts, Ian N.

    2016-01-01

    Despite the considerable number and taxonomic breadth of past and current genome sequencing projects, many of which necessarily encompass the ribosomal DNA, detailed information on the prevalence and evolutionary significance of sequence variation in this ubiquitous genomic region are severely lacking. Here, we attempt to address this issue in two closely related yet contrasting yeast species, the baker’s yeast Saccharomyces cerevisiae and the wild yeast Saccharomyces paradoxus. By drawing on existing datasets from the Saccharomyces Genome Resequencing Project, we identify a rich seam of ribosomal DNA sequence variation, characterising 1,068 and 970 polymorphisms in 34 S. cerevisiae and 26 S. paradoxus strains respectively. We discover the two species sets exhibit distinct mutational profiles. Furthermore, we show for the first time that unresolved rDNA sequence variation resulting from imperfect concerted evolution of the ribosomal DNA region follows a U-shaped allele frequency distribution in each species, similar to loci that evolve under non-concerted mechanisms but arising through rather different evolutionary processes. Finally, we link differences between the shapes of these allele frequency distributions to the two species’ contrasting population histories. PMID:27345953

  20. Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis.

    PubMed

    Saro, C; Ranilla, M J; Carro, M D

    2012-12-01

    Four ruminally cannulated sheep were used in a crossover design to assess the postprandial changes of fiber-degrading microbes in the solid phase of the rumen of sheep fed 2 high-forage diets. The diets had forage:concentrate ratio of 70:30 (DM basis) and either alfalfa (Medicago sativa) hay (AL) or grass hay (GR) as forage (FOR). Sheep were fed twice daily, and samples from solid rumen digesta were taken at 0, 4, and 8 h after the morning feeding. Postprandial changes of DNA concentrations of all determined microbial populations were similar for the 2 diets. Samples taken at 4 h after feeding had lesser (P < 0.05) concentrations of total bacterial DNA determined with real-time PCR and bacterial diversity and greater (P < 0.05) protozoal DNA concentrations, relative abundance of fungal, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus DNA compared with those taken at 0 and 8 h. No effect (P = 0.41 to 0.76) of FOR was detected either on concentrations of bacterial and protozoal DNA or the relative abundance of the 2 Ruminococcus DNA, but GR diet promoted greater (P < 0.001) relative abundance of F. succinogenes and fungal DNA compared with AL diet. Fibrobacter succinogenes was the most abundant (P < 0.05) of the 3 cellulolytic bacteria for both diets, with no differences (P < 0.05) between the 2 Ruminococcus species. Rumen pH and carboxymethylcellulase, Avicelase, and amylase activities were not affected (P = 0.15 to 0.69) by FOR, but xylanase activity was greater (P = 0.01) for GR diet. The influence of FOR on microbial communities in ruminal solid digesta was more evident in the first hours after feeding than at later times after feeding, which highlights the influence of sampling time when investigating dietary effects on rumen function and microbial populations.

  1. Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments

    PubMed Central

    Kuick, R.; Asakawa, J. I.; Neel, J. V.; Kodaira, M.; Satoh, C.; Thoraval, D.; Gonzalez, I. L.; Hanash, S. M.

    1996-01-01

    We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions. PMID:8878694

  2. Transcription Factor UAF, Expansion and Contraction of Ribosomal DNA (rDNA) Repeats, and RNA Polymerase Switch in Transcription of Yeast rDNA

    PubMed Central

    Oakes, Melanie; Siddiqi, Imran; Vu, Loan; Aris, John; Nomura, Masayasu

    1999-01-01

    Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery. PMID:10567580

  3. Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates.

    PubMed

    Bernier, L; Hamelin, R C; Ouellette, G B

    1994-04-01

    The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety.

  4. Comparison of ribosomal DNA length and restriction site polymorphisms in Gremmeniella and Ascocalyx isolates.

    PubMed Central

    Bernier, L; Hamelin, R C; Ouellette, G B

    1994-01-01

    The small subunit (SSU) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA genes from 27 specimens of the fungal genera Gremmeniella and Ascocalyx were amplified by PCR. Length polymorphisms were observed in the SSU and allowed the differentiation of four groups among the isolates tested: (i) Ascocalyx abietis; (ii) Gremmeniella isolates from Picea spp.; (iii) Gremmeniella isolates from Abies balsamea; and (iv) Gremmeniella isolates from Abies sacchalinensis, Larix spp., and Pinus spp. The amplified ITS was the same length for all Gremmeniella specimens and was 60 bp longer in A. abietis. Phylogenetic analysis of length polymorphisms and of 24 restriction sites in the SSU and ITS showed that Gremmeniella isolates were more related to each other than to the Ascocalyx isolate. Furthermore, seven groups were evident within the genus Gremmeniella. Our results confirm that Gremmeniella and Ascocalyx should be kept as different taxa and suggest that the taxonomy of the former could be revised to consider isolates from Abies balsamea and from Picea spp. to be two different varieties while incorporating Gremmeniella laricina into G. abietina, as a new variety. Images PMID:7912501

  5. Global functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription

    SciTech Connect

    Bergstralh, Daniel T. . E-mail: dan.bergstralh@med.unc.edu; Conti, Brian J.; Moore, Chris B.; Brickey, W. June; Taxman, Debra J.; Ting, Jenny P.-Y.

    2007-01-01

    Analysis of lung cancer response to chemotherapeutic agents showed the accumulation of a Taxol-induced protein that reacted with an anti-phospho-MEK1/2 antibody. Mass spectroscopy identified the protein as nucleophosmin/B23 (NPM), a multifunctional protein with diverse roles: ribosome biosynthesis, p53 regulation, nuclear-cytoplasmic shuttling, and centrosome duplication. Our work demonstrates that following cellular exposure to mitosis-arresting agents, NPM is phosphorylated and its chromatographic property is altered, suggesting changes in function during mitosis. To determine the functional relevance of NPM, its expression in tumor cells was reduced by siRNA. Cells with reduced NPM were treated with Taxol followed by microarray profiling accompanied by gene/protein pathway analyses. These studies demonstrate several expected and unexpected consequences of NPM depletion. The predominant downstream effectors of NPM are genes involved in cell proliferation, cancer, and the cell cycle. In congruence with its role in cancer, NPM is over-expressed in primary malignant lung cancer tissues. We also demonstrate a role for NPM in the expression of genes encoding SET (TAF1{beta}) and the histone methylase SET8. Additionally, we show that NPM is required for a previously unobserved G2/M upregulation of TAF1A, which encodes the rDNA transcription factor TAF{sub I}48. These results demonstrate multi-faceted functions of NPM that can affect cancer cells.

  6. Analysis of the relationship between ribosomal DNA ITS sequences and active components in Rhodiola plants.

    PubMed

    Zhang, D J; Yuan, W T; Li, M T; Zhang, Y H

    2016-12-23

    Rhodiola plants are a valuable resource in traditional Chinese medicine. The objective of this study was to evaluate the correlation between ribosomal DNA internal transcribed spacer (ITS) sequences and the three active components in Rhodiola plants. For this, we determined ITS sequence polymorphisms and the concentrations of active components salidroside, tyrosol, and gallic acid in different Rhodiola species from the Tibetan Plateau. In a total of 23 Rhodiola samples, 16 different haplotypes were defined based on their ITS sequences. Analysis of the active components in these same samples revealed that salidroside was not detected in species with haplotypes H4, H5, or H10, tyrosol was not detected with haplotypes H3, H5, H7, H10, H14, or H15, and gallic acid was detected in with all haplotypes except H14 and H15. In addition, the concentrations of salidroside, tyrosol and gallic acid varied between samples with different haplotypes as well as those with the same haplotype, implying that no significant correlation exists between haplotype and salidroside, tyrosol or gallic acid concentrations. However, a statistically significant positive correlation was observed for among these three active components.

  7. Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences.

    PubMed

    Moller, M; Cronk, Q

    1997-07-01

    Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2007-07-20

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

  10. Phylogenetic position of Gromia oviformis Dujardin inferred from nuclear-encoded small subunit ribosomal DNA.

    PubMed

    Burki, Fabien; Berney, Cédric; Pawlowski, Jan

    2002-09-01

    Gromia oviformis Dujardin is a common marine protist characterised by a large, globular test and filose pseudopodia. First considered a foraminifer, Gromia was later placed within the Filosea and recently included among amoebae of uncertain affinities. In order to clarify the phylogenetic position of this genus, we sequenced the complete small-subunit ribosomal DNA gene of G. oviformis collected at five different geographic localities. The high divergence of obtained sequences suggests that G. oviformis is a species complex composed of several genetically distinct sibling species. Sequence analyses show Gromia to be a member of the Cercozoa, a heterogeneous assemblage which includes filose amoebae, the amoeboflagellate cercomonads, the chlorarachniophytes and the plasmodiophorid plant pathogens. Contrary to traditional classification, Gromia is not closely related to other testate filose amoebae (the Euglyphida), but seems to branch early among the Cercozoa. Our analyses also show a close relationship between the Cercozoa and the Acantharea. Because the Cercozoa are related to the Foraminifera based on other molecular data, we propose that most protists possessing filopodia, reticulopodia and axopodia have a common origin.

  11. Molecular phylogeny of Oncaeidae (Copepoda) using nuclear ribosomal internal transcribed spacer (ITS rDNA)

    PubMed Central

    Di Capua, Iole; Maffucci, Fulvio; Pannone, Raimondo; Mazzocchi, Maria Grazia

    2017-01-01

    Copepods belonging to the Oncaeidae family are commonly and abundantly found in marine zooplankton. In the Mediterranean Sea, forty-seven oncaeid species occur, of which eleven in the Gulf of Naples. In this Gulf, several Oncaea species were morphologically analysed and described at the end of the XIX century by W. Giesbrecht. In the same area, oncaeids are being investigated over seasonal and inter-annual scales at the long-term coastal station LTER-MC. In the present work, we identified six oncaeid species using the nuclear ribosomal internal transcribed spacers (ITS rDNA) and the mitochondrial cytochrome c oxidase subunit I (mtCOI). Phylogenetic analyses based on these two genomic regions validated the sisterhood of the genera Triconia and the Oncaea sensu stricto. ITS1 and ITS2 phylogenies produced incongruent results about the position of Oncaea curta, calling for further investigations on this species. We also characterised the ITS2 region by secondary structure predictions and found that all the sequences analysed presented the distinct eukaryotic hallmarks. A Compensatory Base Change search corroborated the close relationship between O. venusta and O. curta and between O. media and O. venusta already identified by ITS phylogenies. The present results, which stem from the integration of molecular and morphological taxonomy, represent an encouraging step towards an improved knowledge of copepod biodiversity: The two complementary approaches, when applied to long-term copepod monitoring, will also help to better understanding their genetic variations and ecological niches of co-occurring species. PMID:28441395

  12. Phylogenetic relationships of Central European wolf spiders (Araneae: lycosidae) inferred from 12S ribosomal DNA sequences.

    PubMed

    Zehethofer, K; Sturmbauer, C

    1998-12-01

    We have analyzed a sequence dataset of a portion of mitochondrial 12S rRNA gene of the ribosomal small subunit for 27 species of the family Lycosidae (wolf spiders) from Central Europe, belonging to six genera (Alopecosa, Arctosa, Pardosa, Pirata, Trochosa, and Xerolycosa) and four subfamilies (Evippinae, Lycosinae, Pardosinae and Venoniinae). Phylogenetic analyses were performed in two steps and corroborate the monophyly of all the genera analyzed with strong bootstrap support. In the first step focusing on the most ancestral splits the genus Pirata consistently emerged as the most ancestral branch, followed by the two genera Arctosa and Xerolycosa, with conflicting branching order, however. The second step of analysis placed Xerolycosa more ancestral than Arctosa. Arctosa appeared as sister group to the genera Alopecosa, Trochosa, and Pardosa. The palearctic genus Xerolycosa was not yet included in previous studies derived from morphological characters, but its placement based on mtDNA sequences is in good agreement to that according to current diagnostic morphological features. Further, the single representative of the genus Arctosa examined in our study was placed at a more ancestral position than in a previous investigation based on phenotypic characters. The superimposition of the currently used diagnostic phenotypic characters on the DNA-based phylogeny shows that both character sets are widely congruent; only 3 out of 16 phenotypic characters were resolved as homoplasious, suggesting their parallel evolution and/or reversal. Among the three different styles of predation found in the Lycosids, tube builders appear to be the most ancestral from which burrow dwellers descended and from which two groups of vagrant hunters evolved in parallel.

  13. A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

    PubMed

    Yu, Shoukai; Lemos, Bernardo

    2016-12-31

    Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  14. Utility of 16S ribosomal DNA sequencing in the diagnosis of Staphylococcus lugdunensis native valve infective endocarditis: case report and literature review.

    PubMed

    Pada, Surinder; Lye, David C; Leo, Yee Sin; Barkham, Timothy

    2009-11-01

    We report a case of possible infective endocarditis without fever presenting with an acutely ischemic limb with prior antimicrobial therapy preventing identification by culture of a microorganism. 16S ribosomal DNA sequencing led to the identification of Staphylococcus lugdunensis from an embolus removed at surgery and subsequent successful antibiotic treatment. We review the utility of 16S ribosomal DNA sequencing in diagnosing infective endocarditis and other infectious conditions.

  15. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations.

    PubMed

    Callejón, R; Halajian, A; de Rojas, M; Marrugal, A; Guevara, D; Cutillas, C

    2012-05-25

    Comparative morphological, biometrical and molecular studies of Trichuris discolor isolated from Bos taurus from Spain and Iran was carried out. Furthermore, Trichuris ovis isolated from B. taurus and Capra hircus from Spain has been, molecularly, analyzed. Morphological studies revealed clear differences between T. ovis and T. discolor isolated from B. taurus but differences were not observed between populations of T. discolor isolated from different geographical regions. Nevertheless, the molecular studies based on the amplification and sequencing of the internal transcribed spacers 1 and 2 ribosomal DNA and 16S partial gene mitochondrial DNA showed clear differences between both populations of T. discolor from Spain and Iran suggesting two cryptic species. Phylogenetic studies corroborated these data. Thus, phylogenetic trees based on ITS1, ITS2 and 16S partial gene sequences showed that individuals of T. discolor from B. taurus from Iran clustered together and separated, with high bootstrap values, of T. discolor isolated from B. taurus from Spain, while populations of T. ovis from B. taurus and C. hircus from Spain clustered together but separated with high bootstrap values of both populations of T. discolor. Furthermore, a comparative phylogenetic study has been carried out with the ITS1and ITS2 sequences of Trichuris species from different hosts. Three clades were observed: the first clustered all the species of Trichuris parasitizing herbivores (T. discolor, T. ovis, Trichuris leporis and Trichuris skrjabini), the second clustered all the species of Trichuris parasitizing omnivores (Trichuris trichiura and Trichuris suis) and finally, the third clustered species of Trichuris parasitizing carnivores (Trichuris muris, Trichuris arvicolae and Trichuris vulpis). Copyright © 2011 Elsevier B.V. All rights reserved.

  16. A Two-locus DNA Sequence Database for Typing Plant and Human Pathogens Within the Fusarium oxysporum Species Complex

    USDA-ARS?s Scientific Manuscript database

    We constructed a two-locus database, comprising partial translation elongation factor (EF-1alpha) gene sequences and nearly full-length sequences of the nuclear ribosomal intergenic spacer region (IGS rDNA) for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex ...

  17. Identical ribosomal DNA sequence data from Pfiesteria piscicida (Dinophyceae) isolates with different toxicity phenotypes.

    PubMed

    Tengs, Torstein; Bowers, Holly A; Glasgow, Howard B; Burkholder, JoAnn M; Oldach, David W

    2003-09-01

    Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.

  18. Non-random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region.

    PubMed

    Ruiz-Estévez, M; Ruiz-Ruano, F J; Cabrero, J; Bakkali, M; Perfectti, F; López-León, M D; Camacho, J P M

    2015-06-01

    We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA-derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8-28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation-free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated. © 2015 The Royal Entomological Society.

  19. Ribosomal DNA clusters and telomeric (TTAGG)n repeats in blue butterflies (Lepidoptera, Lycaenidae) with low and high chromosome numbers

    PubMed Central

    Vershinina, Alisa O.; Anokhin, Boris A.; Lukhtanov, Vladimir A.

    2015-01-01

    Abstract Ribosomal DNA clusters and telomeric repeats are important parts of eukaryotic genome. However, little is known about their organization and localization in karyotypes of organisms with holocentric chromosomes. Here we present first cytogenetic study of these molecular structures in seven blue butterflies of the genus Polyommatus Latreille, 1804 with low and high chromosome numbers (from n=10 to n=ca.108) using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG)n telomeric probes. FISH with the 18S rDNA probe showed the presence of two different variants of the location of major rDNA clusters in Polyommatus species: with one or two rDNA-carrying chromosomes in haploid karyotype. We discuss evolutionary trends and possible mechanisms of changes in the number of ribosomal clusters. We also demonstrate that Polyommatus species have the classical insect (TTAGG)n telomere organization. This chromosome end protection mechanism probably originated de novo in small chromosomes that evolved via fragmentations. PMID:26140159

  20. Phylogenetic Relationships in Bupleurum (Apiaceae) Based on Nuclear Ribosomal DNA ITS Sequence Data

    PubMed Central

    NEVES, SUSANA S.; WATSON, MARK F.

    2004-01-01

    • Backgroud and Aims The genus Bupleurum has long been recognized as a natural group, but its infrageneric classification is controversial and has not yet been studied in the light of sequence data. • Methods Phylogenetic relationships among 32 species (35 taxa) of the genus Bupleurum were investigated by comparative sequencing of the ITS region of the 18–26S nuclear ribosomal DNA repeat. Exemplar taxa from all currently accepted sections and subsections of the genus were included, along with outgroups from four other early branching Apioideae genera (Anginon, Heteromorpha, Physospermum and Pleurospermum). • Key Results Phylogenies generated by maximum parsimony, maximum likelihood, and neighbour‐joining methods show similar topologies, demonstrating monophyly of Bupleurum and the division of the genus into two major clades. This division is also supported by analysis of the 5.8S coding sequence alone. The first branching clade is formed by all the species of the genus with pinnate‐reticulate veined leaves and B. rigidum with a unique type of leaf venation. The other major clade includes the remaining species studied, all of which have more or less parallel‐veined leaves. • Conclusions These phylogenetic results do not agree with any previous classifications of the genus. Molecular data also suggest that the endemic Macaronesian species B. salicifolium is a neoendemic, as the sequence divergence between the populations in Madeira and Canary Islands, and closer mainland relatives in north‐west Africa is small. All endemic north‐west African taxa are included in a single unresolved but well‐supported clade, and the low nucleotide variation of ITS suggests a recent radiation within this group. The only southern hemisphere species, B. mundii (southern Africa), is shown to be a neoendemic, apparently closely related to B. falcatum, a Eurasian species. PMID:14980972

  1. Use of quantitative 16S ribosomal DNA detection for diagnosis of central vascular catheter-associated bacterial infection.

    PubMed

    Warwick, S; Wilks, M; Hennessy, E; Powell-Tuck, J; Small, M; Sharp, J; Millar, M R

    2004-04-01

    Many central vascular catheters (CVCs) are removed unnecessarily because current diagnostic methods for CVC-associated infection are unreliable. A quantitative PCR assay using primers and probe targeted to bacterial 16S ribosomal DNA was used to measure the levels of bacterial DNA in blood samples drawn through the CVC in a population of patients receiving intravenous nutrition. Bacterial DNA concentrations were raised in 16 of 16 blood samples taken during episodes of probable bacterial CVC-associated infection. Bacterial DNA concentrations were raised in 4 of 29 episodes in which bacterial CVC-associated infection was unlikely. The use of this technique has the potential to substantially reduce the unnecessary removal of CVCs.

  2. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    DOE PAGES

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; ...

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

  3. Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

    SciTech Connect

    Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; Schmidt, Marian L.

    2016-04-29

    Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.

  4. Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH.

    PubMed

    Ksiazczyk, T; Taciak, M; Zwierzykowski, Z

    2010-01-01

    This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploid Festuca pratensis and Lolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploid F. pratensis × L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) with L. perenne genomic DNA as a probe, and F. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. In F. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standard F. pratensis karyotypes. Losses of 45S rDNA loci were more frequent in L. perenne cultivars and intergeneric hybrids. Comparison of the F. pratensis and L. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location in L. perenne. A greater instability of F. pratensis-genome-like and L. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 in F. pratensis and on chromosome 3 in L. perenne are useful markers for these chromosomes in intergeneric Festuca × Lolium hybrids.

  5. Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

    PubMed Central

    Krol, Kamil; Jendrysek, Justyna; Debski, Janusz; Skoneczny, Marek; Kurlandzka, Anna; Kaminska, Joanna; Dadlez, Michal; Skoneczna, Adrianna

    2017-01-01

    Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP). PMID:28212567

  6. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

  7. New Primers for Discovering Fungal Diversity Using Nuclear Large Ribosomal DNA

    PubMed Central

    Gloor, Gregory B.; Lindo, Zoë

    2016-01-01

    Metabarcoding has become an important tool in the discovery of biodiversity, including fungi, which are the second most speciose group of eukaryotes, with diverse and important ecological roles in terrestrial ecosystems. We have designed and tested new PCR primers that target the D1 variable region of nuclear large subunit (LSU) ribosomal DNA; one set that targets the phylum Ascomycota and another that recovers all other fungal phyla. The primers yield amplicons compatible with the Illumina MiSeq platform, which is cost-effective and has a lower error rate than other high throughput sequencing platforms. The new primer set LSU200A-F/LSU476A-R (Ascomycota) yielded 95–98% of reads of target taxa from environmental samples, and primers LSU200-F/LSU481-R (all other fungi) yielded 72–80% of target reads. Both primer sets have fairly low rates of data loss, and together they cover a wide variety of fungal taxa. We compared our results with these primers by amplifying and sequencing a subset of samples using the previously described ITS3_KYO2/ITS4_KYO3 primers, which amplify the internal transcribed spacer 2 (ITS2) of Ascomycota and Basidiomycota. With approximately equivalent read depth, our LSU primers recovered a greater number and phylogenetic diversity of sequences than the ITS2 primers. For instance, ITS3_KYO2/ITS4_KYO3 primers failed to pick up any members of Eurotiales, Mytilinidiales, Pezizales, Saccharomycetales, or Venturiales within Ascomycota, or members of Exobasidiomycetes, Microbotryomycetes, Pucciniomycetes, or Tremellomycetes within Basidiomycota, which were retrieved in good numbers from the same samples by our LSU primers. Among the OTUs recovered using the LSU primers were 127 genera and 28 species that were not obtained using the ITS2 primers, although the ITS2 primers recovered 10 unique genera and 16 species that were not obtained using either of the LSU primers These features identify the new primer sets developed in this study as useful

  8. Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences.

    PubMed

    Sreenivasaprasad, S; Mills, P R; Meehan, B M; Brown, A E

    1996-06-01

    The potential use of the ribosomal DNA internal transcribed spacer (ITS) sequences in understanding the phylogeny and systematics of Colletotrichum species has been evaluated. Sequence data from a limited number of isolates revealed that in Colletotrichum species the ITS 1 region (50.3% variable sites) shows a greater degree of intra- and inter-specific divergence than ITS 2 (12.4% variable sites). Nucleotide sequences of the ITS 1 region from 93 isolates representing 18 Colletotrichum species were determined. Data for 71 of these isolates where molecular and morphological identities concurred were used for phylogenetic analysis. The size of the ITS 1 region varied from 159 to 185 base pairs. Maximum intraspecific divergence was recorded with C. acutatum (5.8%), and C. capsici showed the greatest level of interspecific divergence (8.9-23.3%). Parsimony and distance analyses gave similar tree topologies. The bootstrapped consensus parsimony tree divided the 18 Colletotrichum species into six phylogenetic groups, designated 1-6. These groups, however, are not congruent with species clusterings based on spore shape. For example, the straight cylindrical spored species were represented both in groups 1 and 6; group 6 also included the falcate fusiform spored species C. capsici. The molecular evidence suggests refinement of the species concepts of some of the taxa examined. In group 6, divergence between C. gloeosporioides and C. fuscum (0.6-3.0%) or C. kahawae (0.6-3.0%) or C. fragariae (0.6-4.2%) overlap the divergence (3.6%) within C. gloeosporioides. It is suggested that C. fuscum as well as C. kahawae and C. fragariae fall within the group species C. gloeosporioides. ITS 1 data enabled clear distinction (7.1%) of Colletotrichum isolates from maize and sorghum into C. graminicola and C. sublineolum, respectively (group 2). Species such as C. acutatum, C. coccodes, C. dematium, and C. trichellum can be clearly distinguished based on ITS 1 sequence divergence, but C

  9. Clinostomum complanatum and Clinostomum marginatum (Rudolphi, 1819) (Digenea: Clinostomidae) are separate species based on differences in ribosomal DNA.

    PubMed

    Dzikowski, R; Levy, M G; Poore, M F; Flowers, J R; Paperna, I

    2004-04-01

    Infections by metacercariae of Clinostomum (Leidy, 1856) species adversely affect aquacultured fish and are potentially transmissible to humans. Molecular methodologies are efficient tools, which enable diagnosis of all life-history stages of trematodes in their diverse hosts. The small subunit of ribosomal DNA genes of adults of the Old World Clinostomum complanatum (Rudolphi, 1819) and the New World Clinostomum marginatum (Rudolphi, 1819), obtained from a little egret Egretta garzetta (Linnaeus, 1766) and the great blue heron Ardea herodias (Linnaeus, 1758), respectively, were amplified, sequenced, and aligned. The resulting alignment was used to develop a genetic assay to differentiate between these species.

  10. Genetic differentiation of strongyloides stercoralis from two different climate zones revealed by 18S ribosomal DNA sequence comparison.

    PubMed

    Pakdee, Wallop; Thaenkham, Urusa; Dekumyoy, Paron; Sa-Nguankiat, Surapol; Maipanich, Wanna; Pubampen, Somchit

    2012-11-01

    Over 70 countries in tropical and subtropical zones are endemic areas for Strongyloides stercoralis, with a higher prevalence of the parasite often occurring in tropical regions compared to subtropical ones. In order to explore genetic variations of S. stercoralis form different climate zones, 18S ribosomal DNA of parasite specimens obtained from Thailand were sequenced and compared with those from Japan. The maximum likelihood indicates that S. stercoralis populations from these two different climate zones have genetically diverged. The genetic relationship between S. stercoralis populations is not related to the host species, but rather to moisture and temperature. These factors may directly drive genetic differentiation among isolated populations of S. stercoralis.

  11. Evolutionary site-number changes of ribosomal DNA loci during speciation: complex scenarios of ancestral and more recent polyploid events.

    PubMed

    Rosato, Marcela; Moreno-Saiz, Juan C; Galián, José A; Rosselló, Josep A

    2015-11-16

    Several genome duplications have been identified in the evolution of seed plants, providing unique systems for studying karyological processes promoting diversification and speciation. Knowledge about the number of ribosomal DNA (rDNA) loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. In this work, we aim to elucidate the evolutionary dynamics of karyological and rDNA site-number variation in all known taxa of subtribe Vellinae, showing a complex scenario of ancestral and more recent polyploid events. Specifically, we aim to infer the ancestral chromosome numbers and patterns of chromosome number variation, assess patterns of variation of both 45S and 5S rDNA families, trends in site-number change of rDNA loci within homoploid and polyploid series, and reconstruct the evolutionary history of rDNA site number using a phylogenetic hypothesis as a framework. The best-fitting model of chromosome number evolution with a high likelihood score suggests that the Vellinae core showing x = 17 chromosomes arose by duplication events from a recent x = 8 ancestor. Our survey suggests more complex patterns of polyploid evolution than previously noted for Vellinae. High polyploidization events (6x, 8x) arose independently in the basal clade Vella castrilensis-V. lucentina, where extant diploid species are unknown. Reconstruction of ancestral rDNA states in Vellinae supports the inference that the ancestral number of loci in the subtribe was two for each multigene family, suggesting that an overall tendency towards a net loss of 5S rDNA loci occurred during the splitting of Vellinae ancestors from the remaining Brassiceae lineages. A contrasting pattern for rDNA site change in both paleopolyploid and neopolyploid species was linked to diversification of Vellinae lineages. This suggests dynamic and independent changes in rDNA site number during speciation processes and a

  12. The disparate nature of "intergenic" polyadenylation sites.

    PubMed

    Lopez, Fabrice; Granjeaud, Samuel; Ara, Takeshi; Ghattas, Badih; Gautheret, Daniel

    2006-10-01

    The termination of mature eukaryotic mRNAs occurs at specific polyadenylation sites located downstream from stop codons in the 3'-untranslated region (UTR). An accurate delineation of these sites is essential for the study of 3'-UTR-based gene regulation and for the design of pertinent probes for transcriptome analysis. Although typical poly(A) sites are located between 0 and 2 kb from the stop codon, EST sequence analyses have identified sites located at unexpectedly long ranges (5-10 kb) in a number of genes. Here we perform a complete mapping of EST and full-length cDNA sequences on the mouse and human genome to observe putative poly(A) sites extending beyond annotated 3'-ends and into the intergenic regions. We introduce several quality parameters for poly(A) site prediction and train a classification tree to associate P-values to predicted sites. We observe a higher than background level of high-scoring sites up to 12-15 kb past the stop codon, both in human and mouse. This leads to an estimate of about 5000 human genes having unreported 3'-end extensions and about 3500 novel polyadenylated transcripts lying in present "intergenic" regions. These high-scoring, long-range poly(A) sites corresponding to novel transcripts and gene extensions should be incorporated into current human and mouse gene repositories.

  13. Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

    PubMed Central

    2010-01-01

    Background Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR) led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in the non-coding region of

  14. Group I introns in small subunit ribosomal DNA of several Phaeosphaeria species

    USDA-ARS?s Scientific Manuscript database

    In a study of small subunit ribosomal RNA (SSU-rRNA) gene sequences in Phaeosphaeria species, group I introns were found in 9 of 10 P. avenaria f.sp. avenaria (Paa) isolates, 1 of 2 Phaeosphaeria sp. (P-rye) isolates from Polish rye (Sn48-1), 1 Phaeosphaeria sp. from dallis grass (P-dg) (S-93-48) an...

  15. Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis.

    PubMed

    Benitez, Alvaro; Priest, Jeffrey W; Ehigiator, Humphrey N; McNair, Nina; Mead, Jan R

    2011-11-15

    The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

  16. Molecular phylogenetic study of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships.

    PubMed

    Ro, K E; Keener, C S; McPheron, B A

    1997-10-01

    There are only a small number of molecular markers currently proven to be useful for phylogenetic inference within the flowering plants. We demonstrate that the 5' end of the 26S ribosomal DNA (ca. 1100 bp) is of great value for investigating generic to subfamilial relationships. We analyzed DNA sequences from 31 species of the Ranunculaceae and four species of the Berberidaceae to test phylogenetic relationships within the Ranunculaceae. The inferred phylogeny strongly supports the concept that the Thalictrum chromosome group is not monophyletic, but consists of three independent lineages: (1) Hydrastis, (2) Xanthorhiza and Coptis, and (3) Thalictrum, Aquilegia, and Enemion. Based on comparison with conventional taxonomic characters, we propose a hypothesis that the third group also includes the rest of the Thalictrum chromosome taxa that have a base chromosome number of seven. For the Ranunculus chromosome group, our study suggests several relationships that have not been recognized by conventional systematics. The inferred 26S rDNA topology is compared with results from two previously published molecular data sets: DNA sequences from rbcL, atpB, and 18S rDNA genes and restriction fragment length polymorphism data from chloroplast DNA. The three topologies are highly congruent and agree with karyological characters, but not with fruit type, both of which have often been used for the higher classification of the Ra- nunculaceae.

  17. Overexpression of Ribosomal RNA in the Development of Human Cervical Cancer Is Associated with rDNA Promoter Hypomethylation

    PubMed Central

    Zhou, Hong; Wang, Yapei; Lv, Qiongying; Zhang, Juan; Wang, Qing; Gao, Fei; Hou, Haoli; Zhang, Hao; Zhang, Wei; Li, Lijia

    2016-01-01

    The ribosomal RNA (rRNA) gene encodes rRNA for protein synthesis. Aberrant expression of the rRNA gene has been generally observed in tumor cells and levels of its promoter methylation as an epigenetic regulator affect rRNA gene transcription. The possible relationship between expression and promoter methylation of rDNA has not been examined in human clinical cervical cancer. Here we investigate rRNA gene expression by quantitative real time PCR, and promoter methylation levels by HpaII/MspI digestion and sodium bisulfite sequencing in the development of human cervical cancer. We find that indeed rRNA levels are elevated in most of cervical intraepithelial neoplasia (CIN) specimens as compared with non-cancer tissues. The rDNA promoter region in cervical intraepithelial neoplasia (CIN) tissues reveals significant hypomethylation at cytosines in the context of CpG dinucleotides, accompanied with rDNA chromatin decondensation. Furthermore treatment of HeLa cells with the methylation inhibitor drug 5-aza-2’-deoxycytidine (DAC) demonstrates the negative correlation between the expression of 45S rDNA and the methylation level in the rDNA promoter region. These data suggest that a decrease in rDNA promoter methylation levels can result in an increase of rRNA synthesis in the development of human cervical cancer. PMID:27695092

  18. Study on sequences of ribosomal DNA internal transcribed spacers of clams belonging to the Veneridae family (Mollusca: Bivalvia).

    PubMed

    Cheng, Han-Liang; Xia, De-Quan; Wu, Ting-Ting; Meng, Xue-Ping; Ji, Hong-Ju; Dong, Zhi-Guo

    2006-08-01

    The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.

  19. The IGS-ETS in Bacillus (Insecta Phasmida): molecular characterization and the relevance of sex in ribosomal DNA evolution

    PubMed Central

    2008-01-01

    Background DNA encoding for ribosomal RNA (rDNA) is arranged in tandemly-repeated subunits, each containing ribosomal genes and non-coding spacers. Because tandemly-repeated, rDNA evolves under a balanced influence of selection and "concerted evolution", which homogenizes rDNA variants over the genome (through genomic turnover mechanisms) and the population (through sexuality). Results In this paper we analyzed the IGS-ETS of the automictic parthenogen Bacillus atticus and the bisexual B. grandii, two closely related stick-insect species. Both species share the same IGS-ETS structure and sequence, including a peculiar head-to-tail array of putative transcription enhancers, here named Bag530. Sequence variability of both IGS-ETS and Bag530 evidenced a neat geographic and subspecific clustering in B. grandii, while B. atticus shows a little but evident geographic structure. This was an unexpected result, since the parthenogen B. atticus should lack sequence fixation through sexuality. In B. atticus a new variant might spread in a given geographic area through colonization by an all-female clone, but we cannot discard the hypothesis that B. atticus was actually a bisexual taxon in that area at the time the new variant appeared. Moreover, a gene conversion event between two Bag530 variants of B. grandii benazzii and B. grandii maretimi suggested that rRNA might evolve according to the so-called "library hypothesis" model, through differential amplification of rDNA variants in different taxa. Conclusion On the whole, Bacillus rDNA evolution appears to be under a complex array of interacting mechanisms: homogenization may be achieved through genomic turnover that stabilizes DNA-binding protein interactions but, simultaneously, new sequence variants can be adopted, either by direct appearance of newly mutated repeats, or by competition among repeats, so that both DNA-binding proteins and repeat variants drive each other's evolution. All this, coupled with chromosome

  20. [Phylogenetic relations of Salix L. subg. Salix species (Salicaceae) according to sequencing data of intergenic spacers of chloroplasic genomes and ITS rDNA].

    PubMed

    Barkalov, V Iu; Kozyrenko, M M

    2014-08-01

    A phylogenetic analysis based on a comparison of nucleotide sequences of six regions from cpDNA and ITS rDNA (petN-psbM, trnD-trnT, trnC-petN, psaA-ycf3, petG-trnP, and rpoB-trnC) allowed for elucidating the relationship among species and sections belonging to the Salix subgenus and, more generally, to the Salix genus, as well as revealing the relations of the Chosenia genus. The definition of the subgenera of Pleuradenia (including the Urbanianea genus and the Chosenia genus), Salix (without the Triandrae section), Triandrae, and Longifoliae is essentially consistent with current classification schemes of the Salix genus. The previously defined genera of Chosenia and Toisusu (Urbanianea) are not only joined with the Salix genus but are also closely related between themselves. The Protitea subgenus only corresponds to the American species of the Humboldtianae section (S. humboldtiana, S. amygdaloides, S. gooddingii). The relationship of S. chaenomeloides, which is a nomenclatural type of this subgenus, as well as the relationship of the Wilsonia section, remains unresolved. The Humboldtianae section should be interpreted more narrowly, apparently, separating the Acmophillae and Tetraspermae sections from it. The monotypic American Floridanae section is related to the Salix, Salicaster, Tetraspermae, and Wilsonia.

  1. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  2. Ribosomal DNA transcription in the dorsal raphe nucleus is increased in residual but not in paranoid schizophrenia.

    PubMed

    Krzyżanowska, Marta; Steiner, Johann; Brisch, Ralf; Mawrin, Christian; Busse, Stefan; Braun, Katharina; Jankowski, Zbigniew; Bernstein, Hans-Gert; Bogerts, Bernhard; Gos, Tomasz

    2015-03-01

    The central serotonergic system is implicated in the pathogenesis of schizophrenia, where the imbalance between dopamine, serotonin and glutamate plays a key pathophysiological role. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in schizophrenia patients. The study was carried out on paraffin-embedded brains from 17 (8 paranoid and 9 residual) schizophrenia patients and 28 matched controls without mental disorders. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons was evaluated by the AgNOR silver-staining method. An increased rDNA transcriptional activity was found in schizophrenia patients in the cumulative analysis of all DRN subnuclei (t test, P = 0.02). Further subgroup analysis revealed that it was an effect specific for residual schizophrenia versus paranoid schizophrenia or control groups (ANOVA, P = 0.002). This effect was confounded neither by suicide nor by antipsychotic medication. Our findings suggest that increased activity of rDNA in DRN neurons is a distinct phenomenon in schizophrenia, particularly in residual patients. An activation of the rDNA transcription in DRN neurons may represent a compensatory mechanism to overcome the previously described prefrontal serotonergic hypofunction in this diagnostic subgroup.

  3. Phylogenetic Relationships of Globodera millefolii, G. artemisiae, and Cactodera salina Based on ITS Region of Ribosomal DNA

    PubMed Central

    Ferris, V. R.; Krall, E.; Faghihi, J.; Ferris, J. M.

    1999-01-01

    Globodera millefolii and G. artemisiae are interesting because their type localities (Estonia and Russia, respectively) are geographically distant from those of the potato cyst nematodes and other Globodera species that seem to have originated in the Western world, and because the type host for each is a member of Compositae rather than Solanaceae. Sequence data for ITS1, ITS2, and 5.8S ribosomal DNA (ITS rDNA) for G. millefolii and G. artemisiae were nearly identical to sequence data for Cactodera salina from the rhizosphere of the estuary plant Salicornia bigelovii in Sonora, Mexico. The ITS rDNA sequences of these three species were all about 94% similar to those of two other Cactodera species for which ITS rDNA data were obtained. Phylogenetic analysis indicated that, based on the ITS rDNA data, G. millefolii and G. artemisiae are more closely related phylogenetically to the Cactodera species than to other nominal Globodera species. The molecular data further suggest that the genus Cactodera may comprise two or more morphologically similar but separate groups. PMID:19270922

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

    PubMed Central

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

    2013-01-01

    Abstract Agave Linnaeus, 1753 is endemic of America and is considered one of the most important crops in Mexico due to its key role in the country’s economy. Cytogenetic analysis was carried out in Agave tequilana Weber, 1902 ‘Azul’, Agave cupreata Trelease et Berger, 1915 and Agave angustifolia Haworth, 1812. The analysis showed that in all species the diploid chromosome number was 2n = 60, with bimodal karyotypes composed of five pairs of large chromosomes and 25 pairs of small chromosomes. Furthermore, different karyotypical formulae as well as a secondary constriction in a large chromosome pair were found in all species. Fluorescent in situ hybridization (FISH) was used for physical mapping of 5S and 18S ribosomal DNA (rDNA). All species analyzed showed that 5S rDNA was located in both arms of a small chromosome pair, while 18S rDNA was associated with the secondary constriction of a large chromosome pair. Data of FISH analysis provides new information about the position and number of rDNA loci and helps for detection of hybrids in breeding programs as well as evolutionary studies. PMID:24260700

  5. Intraspecific differentiation of Paramecium novaurelia strains (Ciliophora, Protozoa) inferred from phylogenetic analysis of ribosomal and mitochondrial DNA variation.

    PubMed

    Tarcz, Sebastian

    2013-01-01

    Paramecium novaurelia Beale and Schneller, 1954, was first found in Scotland and is known to occur mainly in Europe, where it is the most common species of the P. aurelia complex. In recent years, two non-European localities have been described: Turkey and the United States of America. This article presents the analysis of intraspecific variability among 25 strains of P. novaurelia with the application of ribosomal and mitochondrial loci (ITS1-5.8S-ITS2, 5' large subunit rDNA (5'LSU rDNA) and cytochrome c oxidase subunit 1 (COI) mtDNA). The mean distance observed for all of the studied P. novaurelia sequence pairs was p=0.008/0.016/0.092 (ITS1-5.8S-ITS2/5'LSU rDNA/COI). Phylogenetic trees (NJ/MP/BI) based on a comparison of all of the analysed sequences show that the studied strains of P. novaurelia form a distinct clade, separate from the P. caudatum outgroup, and are divided into two clusters (A and B) and two branches (C and D). The occurrence of substantial genetic differentiation within P. novaurelia, confirmed by the analysed DNA fragments, indicates a rapid evolution of particular species within the Paramecium genus. Copyright © 2012 Elsevier GmbH. All rights reserved.

  6. A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites.

    PubMed

    Vilas, R; Criscione, C D; Blouin, M S

    2005-12-01

    We examined the relative merits of mitochondrial DNA loci and ribosomal DNA internal transcribed spacers for their use in prospecting for cryptic species of platyhelminth parasites. Sequence divergence at ITS1 and ITS2 was compared with divergence at 2 mtDNA loci (NADH dehydrogenase-1 and cytochrome c oxidase I) between closely related species of trematodes and cestodes. Both spacers accumulated substitutions substantially more slowly than mtDNA, which clearly shows a higher level of divergence among species relative to intra-specific variation. Besides a slow rate of substitution, other caveats that may be encountered when using ITS sequences as a prospecting marker are discussed. In particular, we note recent studies that suggest the existence of substantial levels of intra-individual variation in ITS sequences of flatworms. Because it is likely that closely related species share this phenomenon, it may confound the detection of cryptic species, especially if small sample sizes are studied. Although potential limitations of mtDNA are also recognized, the higher rate of evolution and smaller effective population size of this marker increases the probability of detecting diagnostic characters between cryptic species.

  7. Nuclear Ribosomal DNA Variation and Pathogenic Specialization in Alternaria Fungi Known To Produce Host-Specific Toxins †

    PubMed Central

    Kusaba, Motoaki; Tsuge, Takashi

    1994-01-01

    A total of 99 strains of 11 Alternaria species, including 68 strains of seven fungi known to produce host-specific toxins, were subjected to analysis of restriction fragment length polymorphism (RFLP) in nuclear ribosomal DNA (rDNA). Total DNA was digested with XbaI, and the Southern blots were probed with a nuclear rDNA clone of Alternaria kikuchiana. The hybridization gave 17 different RFLPs from the 99 strains. On the basis of these RFLPs, populations of host-specific toxin-producing fungi could not be differentiated from one another nor from nonpathogenic A. alternata. Each population of the toxin-producing fungi carried rDNA variants. Nine different types, named A1 to A6 and B1 to B3, were detected among the toxin-producing fungi and nonpathogenic A. alternata. All of the populations contained the type A4 variant, and the other rDNA types were also shared by different toxin-producing fungi and A. alternata. In contrast, Alternaria species that are morphologically distinguishable from A. alternata could be differentiated from A. alternata on the basis of the rDNA RFLPs. Polymorphisms in rDNA digested with HaeIII and MspI were also evaluated in 61 Alternaria strains. These restriction enzymes produced 31 variations among all of the samples. The seven toxin-producing fungi and nonpathogenic A. alternata could not be resolved by phylogenetic analysis based on the RFLPs, although they could be differentiated from the other Alternaria species studied. These results provide support for the hypothesis that Alternaria fungi known to produce host-specific toxins are intraspecific variants of A. alternata specialized in pathogenicity. Images PMID:16349367

  8. Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

    PubMed

    Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

    2016-06-01

    Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied

  9. cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

    PubMed

    Abolhassani, Mohsen; Roux, Kenneth H

    2009-06-01

    Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  10. Emergence of Tetracycline Resistance in Helicobacter pylori: Multiple Mutational Changes in 16S Ribosomal DNA and Other Genetic Loci

    PubMed Central

    Dailidiene, Daiva; Bertoli, M. Teresita; Miciuleviciene, Jolanta; Mukhopadhyay, Asish K.; Dailide, Giedrius; Pascasio, Mario Alberto; Kupcinskas, Limas; Berg, Douglas E.

    2002-01-01

    Tetracycline is useful in combination therapies against the gastric pathogen Helicobacter pylori. We found 6 tetracycline-resistant (Tetr) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and obtained the following four results: (i) 5 of 6 Tetr isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16S rRNA (AGA965-967 [Escherichia coli coordinates] changed to gGA, AGc, guA, or gGc [lowercase letters are used to represent the base changes]), whereas the sixth (isolate Ind75) retained AGA965-967; (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles transformed recipient strains to Tetr phenotypes, but transformants containing alleles with single substitutions (gGA and AGc) were less resistant than their Tetr parents; (iii) each of 10 Tetr mutants of reference strain 26695 (in which mutations were induced with metronidazole, a mutagenic anti-H. pylori agent) contained the normal AGA965-967 sequence; and (iv) transformant derivatives of Ind75 and of one of the Tetr 26695 mutants that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either parent strain was resistant. Thus, tetracycline resistance in H. pylori results from an accumulation of changes that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps). We suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple mutations and perhaps also the deleterious effects of such mutations on fitness. Formally equivalent mutations with small but additive effects are postulated to contribute importantly to traits such as host specificity and virulence and to H. pylori's great genetic diversity. PMID:12435699

  11. Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

    PubMed Central

    Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

    2004-01-01

    The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the γ- and β-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). γ-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

  12. Fasciolopsis buski (Digenea: Fasciolidae) from China and India may represent distinct taxa based on mitochondrial and nuclear ribosomal DNA sequences.

    PubMed

    Ma, Jun; Sun, Miao-Miao; He, Jun-Jun; Liu, Guo-Hua; Ai, Lin; Chen, Mu-Xin; Zhu, Xing-Quan

    2017-02-22

    Fasciolopsis buski is a zoonotic intestinal fluke infecting humans and pigs, but it has been seriously neglected. It is yet to know whether there is any genetic diversity among F. buski from different geographical locations, particularly in sequences of nuclear ribosomal DNA (rDNA) and mitochondrial (mt) DNA. Therefore, we determined the sequences of partial 18S, the complete internal transcribed spacer (ITS) rDNA and the complete mt genome of F. buski from China, compared the rDNA and mtDNA sequences with those of isolates from India and Vietnam, and assessed the phylogenetic relationships of this fluke and related fasciolid trematodes based on the mtDNA dataset. The complete mt genome sequence of F. buski from China is 14,833 bp, with 36 genes, including 12 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (rrnL and rrnS). The AT content of F. buski from China is 65.12%. The gene content and arrangement of the F. buski mt genome is similar to that of Fascioloides magna. Genetic distances between isolates of F. buski from China and India were high (28.2% in mtDNA, 13.2% in ITS-1 and 9.8% in ITS-2) and distinctly higher than the interspecific differences between Fasciola hepatica and Fasciola gigantica. The rDNA and mtDNA datasets for F. buski from China (isolate from pigs) and Vietnam (isolates from humans) were identical. The intergeneric differences in amino acid and nucleotide sequences among the genera Fasciolopsis, Fascioloides and Fasciola ranged between 24.64-25.56% and 26.35-28.46%, respectively. Our results indicate that F. buski from China and India may represent distinct taxa, while F. buski in Vietnam and China represent the same species. These findings might have implications for the implementation of appropriate control strategies in different regions. Further studies are needed to decode mtDNA and rDNA sequences of F. buski from various geographical isolates for the better understanding of the species complex of F. buski.

  13. Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum).

    PubMed

    May, C E; Appels, R

    1987-09-01

    Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.

  14. Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

    Treesearch

    M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

    2009-01-01

    The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

  15. Phylogenetic relationship of Perkinsus olseni from the Ebro Delta, Spain, to other Perkinsus species, based on ribosomal DNA sequences.

    PubMed

    Elandaloussi, Laurence; Carrasco, Noèlia; Furones, Dolores; Roque, Ana

    2009-09-23

    The phylogenetic relationship of Perkinsus olseni originating from the Ebro Delta, Spain, to other Perkinsus spp. was investigated using the nontranscribed spacer (NTS) region and the internal transcribed spacer (ITS) region (including ITS1, 5.8S and ITS2) of the ribosomal DNA sequences. These 2 molecular markers (NTS and ITS) were sequenced from prezoosporangia of Perkinsus sp. originating from Manila clam Ruditapes philippinarum from the Ebro Delta. The sequence of the 5.8S ITS region of the ribosomal RNA gene was 100% similar to that of P. olseni. Higher genetic variability was found for the NTS sequence, with 80.7 to 81.8% similarity to P. olseni. The NTS sequence of a P. olseni isolate previously detected in R. decussatus from the same area was also obtained and showed 81% identity with our isolate. Evidence obtained from phylogenetic analysis of the 5.8S ITS and NTS aligned sequences appears to indicate that P. olseni strains group together according to their host rather than their geographic origins within a well-resolved P. olseni clade.

  16. Evolutionary site-number changes of ribosomal DNA loci during speciation: complex scenarios of ancestral and more recent polyploid events

    PubMed Central

    Rosato, Marcela; Moreno-Saiz, Juan C.; Galián, José A.; Rosselló, Josep A.

    2015-01-01

    Several genome duplications have been identified in the evolution of seed plants, providing unique systems for studying karyological processes promoting diversification and speciation. Knowledge about the number of ribosomal DNA (rDNA) loci, together with their chromosomal distribution and structure, provides clues about organismal and molecular evolution at various phylogenetic levels. In this work, we aim to elucidate the evolutionary dynamics of karyological and rDNA site-number variation in all known taxa of subtribe Vellinae, showing a complex scenario of ancestral and more recent polyploid events. Specifically, we aim to infer the ancestral chromosome numbers and patterns of chromosome number variation, assess patterns of variation of both 45S and 5S rDNA families, trends in site-number change of rDNA loci within homoploid and polyploid series, and reconstruct the evolutionary history of rDNA site number using a phylogenetic hypothesis as a framework. The best-fitting model of chromosome number evolution with a high likelihood score suggests that the Vellinae core showing x = 17 chromosomes arose by duplication events from a recent x = 8 ancestor. Our survey suggests more complex patterns of polyploid evolution than previously noted for Vellinae. High polyploidization events (6x, 8x) arose independently in the basal clade Vella castrilensis–V. lucentina, where extant diploid species are unknown. Reconstruction of ancestral rDNA states in Vellinae supports the inference that the ancestral number of loci in the subtribe was two for each multigene family, suggesting that an overall tendency towards a net loss of 5S rDNA loci occurred during the splitting of Vellinae ancestors from the remaining Brassiceae lineages. A contrasting pattern for rDNA site change in both paleopolyploid and neopolyploid species was linked to diversification of Vellinae lineages. This suggests dynamic and independent changes in rDNA site number during speciation processes and a

  17. Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

    PubMed Central

    Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

    2015-01-01

    Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

  18. A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear small-subunit ribosomal DNA.

    PubMed

    Cook, Lyn G; Gullan, Penny J; Trueman, Holly E

    2002-10-01

    Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic.

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

    PubMed

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

    1999-07-01

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

  20. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  1. 16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates

    PubMed Central

    Drancourt, Michel; Bollet, Claude; Carlioz, Antoine; Martelin, Rolland; Gayral, Jean-Pierre; Raoult, Didier

    2000-01-01

    Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides

  2. Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

    PubMed

    Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

    2016-04-15

    Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax

  3. 16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

    PubMed Central

    Magalhães Cruz, Leonardo; Maltempi de Souza, Emanuel; Weber, Olmar Baler; Baldani, José Ivo; Döbereiner, Johanna; de Oliveira Pedrosa, Fábio

    2001-01-01

    Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria. PMID:11319127

  4. Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures.

    PubMed

    Majoros, L; Kardos, G; Belák, A; Maráz, A; Asztalos, L; Csánky, E; Barta, Z; Szabó, B

    2003-11-01

    We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.

  5. 16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril).

    PubMed

    Magalhães Cruz, L; de Souza, E M; Weber, O B; Baldani, J I; Döbereiner, J; Pedrosa, F de O

    2001-05-01

    Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.

  6. Ribosomal PCR and DNA sequencing for detection and identification of bacteria: experience from 6 years of routine analyses of patient samples.

    PubMed

    Jensen, Kristine Helander; Dargis, Rimtas; Christensen, Jens Jørgen; Kemp, Michael

    2014-03-01

    The use of broad range PCR and DNA sequencing of bacterial 16S ribosomal RNA genes for routine diagnostics of bacterial infections was evaluated. Here, the results from more than 2600 analyses during a 6-year period (2003-2009) are presented. Almost half of the samples were from joints and bones, and the second most frequent origin of samples was from the central nervous system. Overall, 26% of all samples were positive for bacterial DNA and bacterial identification was obtained in 80% of the PCR-positive samples by subsequent DNA sequencing. Ambiguous species identification was noticed among non-haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci, may not be precisely identified.

  7. A UV-responsive Internal Ribosome Entry Site Enhances Serine Hydroxymethyltransferase 1 Expression for DNA Damage Repair*

    PubMed Central

    Fox, Jennifer T.; Shin, William K.; Caudill, Marie A.; Stover, Patrick J.

    2009-01-01

    Thymidine nucleotides are required for faithful DNA synthesis and repair, and their de novo biosynthesis is regulated by serine hydroxymethyltransferase 1 (SHMT1). The SHMT1 transcript contains a heavy chain ferritin, heterogeneous nuclear ribonucleoprotein H2, and CUG-binding protein 1-responsive internal ribosome entry site (IRES) that regulates SHMT1 translation. In this study a non-lethal dose of UVC is shown to increase SHMT1 IRES activity and protein levels in four different cell lines. The mechanism for the UV-induced activation of the SHMT1 IRES involves an increase in heavy chain ferritin and heterogeneous nuclear ribonucleoprotein H2 expression and the translocation of CUG-binding protein 1 from the nucleus to the cytoplasm. The UV-induced increase in SHMT1 translation is accompanied by an increase in the small ubiquitin-like modifier-dependent nuclear localization of the de novo thymidylate biosynthesis pathway and a decrease in DNA strand breaks, indicating a role for SHMT1 and nuclear folate metabolism in DNA repair. PMID:19734144

  8. Ribosomal DNA and Plastid Markers Used to Sample Fungal and Plant Communities from Wetland Soils Reveals Complementary Biotas

    PubMed Central

    Porter, Teresita M.; Shokralla, Shadi; Baird, Donald; Golding, G. Brian; Hajibabaei, Mehrdad

    2016-01-01

    Though the use of metagenomic methods to sample below-ground fungal communities is common, the use of similar methods to sample plants from their underground structures is not. In this study we use high throughput sequencing of the ribulose-bisphosphate carboxylase large subunit (rbcL) plastid marker to study the plant community as well as the internal transcribed spacer and large subunit ribosomal DNA (rDNA) markers to investigate the fungal community from two wetland sites. Observed community richness and composition varied by marker. The two rDNA markers detected complementary sets of fungal taxa and total fungal composition clustered according to primer rather than by site. The composition of the most abundant plants, however, clustered according to sites as expected. We suggest that future studies consider using multiple genetic markers, ideally generated from different primer sets, to detect a more taxonomically diverse suite of taxa compared with what can be detected by any single marker alone. Conclusions drawn from the presence of even the most frequently observed taxa should be made with caution without corroborating lines of evidence. PMID:26731732

  9. Dicrocoelium chinensis and Dicrocoelium dendriticum (Trematoda: Digenea) are distinct lancet fluke species based on mitochondrial and nuclear ribosomal DNA sequences.

    PubMed

    Liu, Guo-Hua; Yan, Hong-Bin; Otranto, Domenico; Wang, Xing-Ye; Zhao, Guang-Hui; Jia, Wan-Zhong; Zhu, Xing-Quan

    2014-10-01

    Lancet flukes parasitize the bile ducts and gall bladder of a range of mammals, including humans, causing dicrocoeliosis. In the present study, we sequenced and characterized the complete mitochondrial (mt) genomes as well as the first and second internal transcribed spacers (ITS-1 and ITS-2=ITS) of nuclear ribosomal DNA (rDNA) of two lancet flukes, Dicrocoelium chinensis and D. dendriticum. Sequence comparison of a conserved mt gene and nuclear rDNA sequences among multiple individual lancet flukes revealed substantial nucleotide differences between the species but limited sequence variation within each of them. Phylogenetic analysis of the concatenated amino acid and multiple mt rrnS sequences using Bayesian inference supported the separation of D. chinensis and D. dendriticum into two distinct species-specific clades. Results of the present study support the proposal that D. dendriticum and D. chinensis represent two distinct lancet flukes. While providing the first mt genomes from members of the superfamily Plagiorchioidea, the novel mt markers described herein will be useful for further studies of the diagnosis, epidemiology and systematics of the lancet flukes and other trematodes of human and animal health significance.

  10. Repetitive sequences in the ITS1 region of the ribosomal DNA of Tunga penetrans and other flea species (Insecta, Siphonaptera).

    PubMed

    Gamerschlag, Sara; Mehlhorn, Heinz; Heukelbach, Jörg; Feldmeier, Hermann; D'Haese, Jochen

    2008-01-01

    Different Tunga penetrans isolates from various hosts obtained from South America (Fortaleza. Brazil) have been studied by nucleotide sequence comparison of the first and the second internal transcribed spacer (ITS1, ITS2) of the ribosomal deoxyribonucleic acid (rDNA) and part of the mitochondrial 16S rDNA. Results show no significant host-dependent sequence differences. No indication for intraindividual and intraspecific polymorphisms could be detected. Comparing the ITS1 spacer region of T. penetrans from South America with that from Africa (Togo, Cameroon), distinct length variations have been observed caused by a repetitive sequence motif of 99 bp. The ITS1 from the South American T. penetrans contain two tandemly repeated copies, whereas four of these units are present in the spacer of the African T. penetrans. The absence of homogenization of these units indicates a recent separation of both populations. However, the different number of repetitions together with two base substitutions put the evolutionary distance of only 135 years as postulated for the transfer of T. penetrans from South America to Africa into question. Repetitive sequences could also be detected within the ITS1 rDNA region of other flea species Ctenocephalides felis, Echidnophaga gallinacea, Pulex irritans, Spilopsyllus cuniculi, and Xenopsylla cheopis. The repeat units with lengths from 10 to 99 bp are arranged in pure tandem or interspersed. The repetitive elements observed in the ITS1 of various flea species may serve as a valuable tool for phylogeographic studies.

  11. Ribosomal DNA phylogeny of the major extant arthropod classes and the evolution of myriapods.

    PubMed

    Friedrich, M; Tautz, D

    1995-07-13

    The evolutionary relationships among arthropods are of particular interest because the best-studied model system for ontogenetic pattern formation, the insect Drosophila, is a member of this phylum. Evolutionary inferences about the developmental mechanisms that have led to the various designs of the arthropod body plan depend on a knowledge of the phylogenetic framework of arthropod evolution. Based on morphological evidence, but also on palaeontological consideration, the sister group of the insects is believed to be found among the myriapods. Using nuclear ribosomal gene sequences for constructing a molecular phylogeny, we provide strong evidence that the crustaceans and not the myriapods should be considered to be the sister group of the insects. Moreover, the degree of sequence divergence suggests that the diversification of the myriapods occurred during the Cambrian. Our findings have general implications for the course of land colonization by the different arthropod groups, as well as for the interpretation of primitive and derived features of arthropod morphology.

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

    USDA-ARS?s Scientific Manuscript database

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

  13. Epidemiology of Salmonella typhimurium: ribosomal DNA analysis of strains from human and animal sources.

    PubMed Central

    Nastasi, A.; Mammina, C.; Villafrate, M. R.

    1993-01-01

    Salmonella typhimurium is the most frequently identified serovar of Salmonella in Italy. This serovar is characterized by the widespread dissemination among human and non-human sources of phenotypically and genetically well-differentiated clones. In this study 457 strains of S. typhimurium isolated in Italy in the years 1982-91 from human and animal sources were submitted to characterization by the rDNA fingerprinting technique. Application of this typing method, after digestion of chromosomal DNA with HincII endonuclease, confirmed the greatest genetic differentiation of clones of S. typhimurium, allowing reliable identification of 45 rDNA patterns linked into 9 major clusters. rDNA pattern clusters or ribotypes specific to man were not recognized, whereas some rDNA patterns were characteristically related to ducks, pigeons and pet birds. The ribotyping results for isolates from animal hosts suggest that pig and cattle are the main source of human infection. Images Fig. 1 PMID:8519320

  14. 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.

  15. Effects of permissible maximum-contamination levels of VOC mixture in water on total DNA, antioxidant gene expression, and sequences of ribosomal DNA of Drosophila melanogaster.

    PubMed

    Doganlar, Oguzhan; Doganlar, Zeynep Banu; Tabakcioglu, Kiymet

    2015-10-01

    In this study, we aimed to investigate the mutagenic and carcinogenic potential of a volatile organic compound (VOC) mixture with references to the response of D.melanogaster using selected antioxidant gene expressions, RAPD assay and base-pair change of ribosomal 18S, and the internal transcribed spacer, ITS2 rDNA gene sequences. For this purpose, Drosophila melanogaster Oregon R, reared under controlled conditions on artificial diets, were treated with the mixture of thirteen VOCs, which are commonly found in water in concentrations of 10, 20, 50, and 75 ppb for 1 and 5 days. In the random amplified polymorphic DNA (RAPD) assay, band changes were clearly detected, especially at the 50 and 75 ppb exposure levels, for both treatment periods, and the band profiles exhibited clear differences between the treated and untreated flies with changes in band intensity and the loss/appearance of bands. Quantitative real-time PCR (qRT-PCR) analysis of Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione-synthetase (GS) expressions demonstrated that these markers responded significantly to VOC-induced oxidative stress. Whilst CAT gene expressions increased linearly with increasing concentrations of VOCs and treatment times, the 50- and 75-ppb treatments caused decreases in GS expressions compared to the control at 5 days. Treatment with VOCs at both exposure times, especially in high doses, caused gene mutation of the 18S and the ITS2 ribosomal DNA. According to this research, we thought that the permissible maximum-contamination level of VOCs can cause genotoxic effect especially when mixed.

  16. Fine resolution mapping of double-strand break sites for human ribosomal DNA units.

    PubMed

    Pope, Bernard J; Mahmood, Khalid; Jung, Chol-Hee; Park, Daniel J

    2016-12-01

    DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011) [5]; Blondet et al., 2001 Blondet et al. (2001) [1]). Stults et al. (2009) Stults et al. (2009) [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA restructuring is among the most common chromosomal alterations in adult solid tumours. As such, analysis of rDNA regions is likely to have significant prognostic and predictive value, clinically. Tchurikov et al. (2015a, 2016) Tchurikov et al. (2015a, 2016) [7], [9] have made major advances in this direction, reporting that sites of human genome double-strand breaks (DSBs) occur frequently at sites in rDNA that are tightly linked with active transcription - the authors used a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended sites. They reported the relative frequency of these rDNA DSBs within defined co-ordinate 'windows' of varying size and made these data (as well as the relevant 'raw' sequencing information) available to the public (Tchurikov et al., 2015b). Assay designs targeting rDNA DSB hotspots will benefit greatly from the publication of break sites at greater resolution. Here, we re-analyse public RAFT data and make available rDNA DSB co-ordinates to the single-nucleotide level.

  17. DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

    PubMed Central

    Martin-Laurent, F.; Philippot, L.; Hallet, S.; Chaussod, R.; Germon, J. C.; Soulas, G.; Catroux, G.

    2001-01-01

    The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years. PMID:11319122

  18. Small subunit ribosomal DNA-based phylogenetic analysis of foliar nematodes (Aphelenchoides spp.) and their quantitative detection in complex DNA backgrounds.

    PubMed

    Rybarczyk-Mydłowska, Katarzyna; Mooyman, Paul; van Megen, Hanny; van den Elsen, Sven; Vervoort, Mariëtte; Veenhuizen, Peter; van Doorn, Joop; Dees, Robert; Karssen, Gerrit; Bakker, Jaap; Helder, Johannes

    2012-12-01

    Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.

  19. Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

    SciTech Connect

    Tuskan, Gerald A; Gunter, Lee E; DiFazio, Stephen P

    2009-01-01

    The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

  20. Cytogenetic analysis of Populus trichocarpa--ribosomal DNA, telomere repeat sequence, and marker-selected BACs.

    PubMed

    Islam-Faridi, M N; Nelson, C D; DiFazio, S P; Gunter, L E; Tuskan, G A

    2009-01-01

    The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

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

    PubMed Central

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

    2012-01-01

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

  2. Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability.

    PubMed

    Hiergeist, Andreas; Reischl, Udo; Gessner, Andrè

    2016-08-01

    The composition of human as well as animal microbiota has increasingly gained in interest since metabolites and structural components of endogenous microorganisms fundamentally influence all aspects of host physiology. Since many of the bacteria are still unculturable, molecular techniques such as high-throughput sequencing have dramatically increased our knowledge of microbial communities. The majority of microbiome studies published thus far are based on bacterial 16S ribosomal RNA (rRNA) gene sequencing, so that they can, at least in principle, be compared to determine the role of the microbiome composition for host metabolism and physiology, developmental processes, as well as different diseases. However, differences in DNA preparation and purification, 16S rDNA PCR amplification, sequencing procedures and platforms, as well as bioinformatic analysis and quality control measures may strongly affect the microbiome composition results obtained in different laboratories. To systematically evaluate the comparability of results and identify the most influential methodological factors affecting these differences, identical human stool sample replicates spiked with quantified marker bacteria, and their subsequent DNA sequences were analyzed by nine different centers in an external quality assessment (EQA). While high intra-center reproducibility was observed in repetitive tests, significant inter-center differences of reported microbiota composition were obtained. All steps of the complex analysis workflow significantly influenced microbiome profiles, but the magnitude of variation caused by PCR primers for 16S rDNA amplification was clearly the largest. In order to advance microbiome research to a more standardized and routine medical diagnostic procedure, it is essential to establish uniform standard operating procedures throughout laboratories and to initiate regular proficiency testing.

  3. Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis.

    PubMed

    Alfreider, Albin; Vogt, Carsten; Babel, Wolfgang

    2002-08-01

    A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the

  4. Evolution of Ribosomal DNA (Rdna) Genetic Structure in Colonial Californian Populations of Avena Barbata

    PubMed Central

    Cluster, P. D.; Allard, R. W.

    1995-01-01

    DNA samples from 980 plants of Avena barbata from 48 ecologically diverse sites in California and Oregon were assayed to determine their genotype for two duplicated loci governing rDNA variants. More than 40 different rDNA genotypes were observed among which 5 made up 96% of our sample in environmentally homogeneous sites; predominant genotypes were less frequent and recombinant genotypes were more frequent in environmentally heterogeneous sites. The spatial distribution of each predominant rDNA genotype was nearly an exact overlay on both macro- and microgeographical scales of a distinctive habitat and also of the distribution of an eight-locus morphological-allozyme variant genotype. In all, seven different habitat-genotype combinations (ecotypes) were distinguishable on the basis of their morphological-allozyme-rDNA genotypes. None of these seven genotypes has been found in ancestral Spanish populations; thus the above predominant multilocus genotypes (ecotypes) of the colonial populations evidently evolved subsequent to the recent introduction (within 150-200 generations) of A. barbata to California. The precise associations of specific alleles and genotypes of the morphological allozyme and rDNA loci with different specifiable habitats leads us to the conclusion that natural selection favoring particular multilocus combinations of alleles in different habitats was the main guiding force in shaping the internal genetic structure of local populations as well as the overall adaptive landscape of A. barbata over California and Oregon. PMID:7713443

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

    PubMed Central

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

    2013-01-01

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

  6. Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes.

    PubMed

    Alves-Costa, F A; Wasko, A P; Oliveira, C; Foresti, F; Martins, C

    2006-05-01

    5S rDNA sequences present an intense dynamism and have proved to be valuable as genetic markers to distinguish closed related species and also in the understanding of the evolutionary dynamic of repetitive sequences in the genomes. In order to identify patterns of 5S rDNA organization and their evolution in the genome of fish species, such genomic segment was investigated in the tilapias Oreochromis niloticus and Tilapia rendalli, and in the hybrid O. urolepis hornorum x O. mossambicus. A dual 5S rDNA system was identified in the three analyzed tilapia samples. Although each 5S rDNA class was conserved among the three samples, a distinct 5S rDNA genome organization pattern could be evidenced for each sample. The presence of a dual 5S rDNA system seems to be a general trait among non-related teleost fish orders, suggesting that evolutionary events of duplication have occurred before the divergence of the main groups of teleost fishes.

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

    PubMed

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

    2013-06-01

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

  8. Crosstalk in gene expression: coupling and co-regulation of rDNA transcription, pre-ribosome assembly and pre-rRNA processing.

    PubMed

    Granneman, Sander; Baserga, Susan J

    2005-06-01

    Ribosomes, the large RNPs that translate mRNA into protein in the cytoplasm of eukaryotic cells, are synthesized in a subcompartment of the nucleus, the nucleolus. There, transcription by Pol I yields a pre-rRNA which is modified, cleaved and assembled with ribosomal proteins to make functional ribosomes. Previously, rRNA transcription and pre-rRNA cleavage in eukaryotes were considered to be separable steps in gene expression. However, recent findings suggest that these two steps in gene expression can be concurrent and are co-regulated. Unexpectedly, optimal rDNA transcription requires the presence of a defined subset of components of the pre-rRNA processing machinery.

  9. Analysis of Fungal Flora in Indoor Dust by Ribosomal DNA Sequence Analysis, Quantitative PCR, and Culture▿ †

    PubMed Central

    Pitkäranta, M.; Meklin, T.; Hyvärinen, A.; Paulin, L.; Auvinen, P.; Nevalainen, A.; Rintala, H.

    2008-01-01

    In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed. PMID:17981947

  10. Molecular approach to the phylogenetics of sea spiders (Arthropoda: Pycnogonida) using partial sequences of nuclear ribosomal DNA.

    PubMed

    Arango, Claudia P

    2003-09-01

    The phylogenetic relationships among major evolutionary lineages of the sea spiders (subphylum Pycnogonida) were investigated using partial sequences of nuclear DNA, 18S, and 28S ribosomal genes. Topological differences were obtained with separate analyses of 18S and 28S, and estimates of phylogeny were found to be significantly different between a combined molecular data set (18S and 28S) and a subset of a morphological data matrix analyzed elsewhere. Colossendeidae played a major role in the conflicts; it was closely related to Callipallenidae or Nymphonidae with 18S or 28S, respectively, but related to Ammotheidae according to morphological characters. Austrodecidae was defined as a basal taxon for Pycnogonida by these molecular data. The 18S sequences were surprisingly conserved among pycnogonid taxa, suggesting either an unusual case of slow evolution of the gene, or an unexpected recent divergence of pycnogonid lineages. Notwithstanding difficulties such as non-optimal taxon sampling, this is the first attempt to reconstruct the pycnogonid phylogeny based on DNA. Continued studies of sequences and other characters should increase the reliability of the analyses and our understanding of the phylogenetics of sea spiders.

  11. Evaluation of amplified ribosomal DNA restriction analysis (ARDRA) and species-specific PCR for identification of Bifidobacterium species.

    PubMed

    Krízová, Jana; Spanová, Alena; Rittich, Bohuslav

    2006-01-01

    Molecular biological methods based on genus-specific PCR, species-specific PCR, and amplified ribosomal DNA restriction analysis (ARDRA) of two PCR amplicons (523 and 914bp) using six restriction enzymes were used to differentiate among species of Bifidobacterium. The techniques were established using DNA from 16 type and reference strains of bifidobacteria of 11 species. The discrimination power of 914bp amplicon digestion was higher than that of 523bp amplicon digestion. The 914bp amplicon digestion by six restrictases provided unique patterns for nine species; B. catenulatum and B. pseudocatenulatum were not differentiated yet. The NciI digestion of the 914bp PCR product enabled to discriminate between each of B. animalis, B. lactis, and B. gallicum. The reference strain B. adolescentis CCM 3761 was reclassified as a member of the B. catenulatum/B. pseudocatenulatum group. The above-mentioned methods were applied for the identification of seven strains of Bifidobacterium spp. collected in the Culture Collection of Dairy Microorganisms (CCDM). The strains collected in CCDM were differentiated to the species level. Six strains were identified as B. lactis, one strain as B. adolescentis.

  12. Multiple Orientation-Dependent, Synergistically Interacting, Similar Domains in the Ribosomal DNA Replication Origin of the Fission Yeast, Schizosaccharomyces pombe

    PubMed Central

    Kim, Soo-Mi; Huberman, Joel A.

    1998-01-01

    Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small (∼570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast. PMID:9819416

  13. Phylogenetic Relationships and Genetic Variation in Longidorus and Xiphinema Species (Nematoda: Longidoridae) Using ITS1 Sequences of Nuclear Ribosomal DNA

    PubMed Central

    Ye, Weimin; Szalanski, Allen L.; Robbins, R. T.

    2004-01-01

    Genetic analyses using DNA sequences of nuclear ribosomal DNA ITS1 were conducted to determine the extent of genetic variation within and among Longidorus and Xiphinema species. DNA sequences were obtained from samples collected from Arkansas, California and Australia as well as 4 Xiphinema DNA sequences from GenBank. The sequences of the ITS1 region including the 3' end of the 18S rDNA gene and the 5' end of the 5.8S rDNA gene ranged from 1020 bp to 1244 bp for the 9 Longidorus species, and from 870 bp to 1354 bp for the 7 Xiphinema species. Nucleotide frequencies were: A = 25.5%, C = 21.0%, G = 26.4%, and T = 27.1%. Genetic variation between the two genera had a maximum divergence of 38.6% between X. chambersi and L. crassus. Genetic variation among Xiphinema species ranged from 3.8% between X. diversicaudatum and X. bakeri to 29.9% between X. chambersi and X. italiae. Within Longidorus, genetic variation ranged from 8.9% between L. crassus and L. grandis to 32.4% between L. fragilis and L. diadecturus. Intraspecific genetic variation in X. americanum sensu lato ranged from 0.3% to 1.9%, while genetic variation in L. diadecturus had 0.8% and L. biformis ranged from 0.6% to 10.9%. Identical sequences were obtained between the two populations of L. grandis, and between the two populations of X. bakeri. Phylogenetic analyses based on the ITS1 DNA sequence data were conducted on each genus separately using both maximum parsimony and maximum likelihood analysis. Among the Longidorus taxa, 4 subgroups are supported: L. grandis, L. crassus, and L. elongatus are in one cluster; L. biformis and L. paralongicaudatus are in a second cluster; L. fragilis and L. breviannulatus are in a third cluster; and L. diadecturus is in a fourth cluster. Among the Xiphinema taxa, 3 subgroups are supported: X. americanum with X. chambersi, X. bakeri with X. diversicaudatum, and X. italiae and X. vuittenezi forming a sister group with X. index. The relationships observed in this study

  14. RISSC: a novel database for ribosomal 16S–23S RNA genes spacer regions

    PubMed Central

    García-Martínez, Jesús; Bescós, Ignacio; Rodríguez-Sala, Jesús Javier; Rodríguez-Valera, Francisco

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S–23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documen­tation tool for studies on evolution, identification, typing and strain characterization, among others. PMID:11125084

  15. Identification of the 18S-ribosomal-DNA genotypes of Acanthamoeba isolates from the Philippines.

    PubMed

    Rivera, W L; Adao, D E V

    2008-12-01

    Cyst morphology has been commonly used to identify the free-living amoeba Acanthamoeba to subgenus level. A more accurate and consistent method, based on the sequence analysis of the gene coding for the amoeba's small-subunit ribosomal RNA (Rns), has, however, been developed. There have been no attempts to identify the Acanthamoeba genotypes circulating in the Philippines. In this study, therefore, the ASA.S1 region of the Rns gene from 17 Acanthamoeba isolates, collected from soil, water and contact-lens storage cases in different regions of the Philippines, was sequenced. After the isolates were genotyped, using the BLAST program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. For this, the model-based (GTR + Gamma) neighbour-joining, maximum-likelihood and Bayesian-inference analyses and the non-model-based maximum-parsimony analysis were used. All but two of the isolates were identified as the T5 or T4 genotypes, which are probably common in soil, water and contact-lens cases across the Philippines. The only other genotypes identified were T15 (as a single isolate from a contact-lens case) and T3 (as a single soil isolate).

  16. G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase.

    PubMed

    Wallgren, Marcus; Mohammad, Jani B; Yan, Kok-Phen; Pourbozorgi-Langroudi, Parham; Ebrahimi, Mahsa; Sabouri, Nasim

    2016-07-27

    Certain guanine-rich sequences have an inherent propensity to form G-quadruplex (G4) structures. G4 structures are e.g. involved in telomere protection and gene regulation. However, they also constitute obstacles during replication if they remain unresolved. To overcome these threats to genome integrity, organisms harbor specialized G4 unwinding helicases. In Schizosaccharomyces pombe, one such candidate helicase is Pfh1, an evolutionarily conserved Pif1 homolog. Here, we addressed whether putative G4 sequences in S. pombe can adopt G4 structures and, if so, whether Pfh1 can resolve them. We tested two G4 sequences, derived from S. pombe ribosomal and telomeric DNA regions, and demonstrated that they form inter- and intramolecular G4 structures, respectively. Also, Pfh1 was enriched in vivo at the ribosomal G4 DNA and telomeric sites. The nuclear isoform of Pfh1 (nPfh1) unwound both types of structure, and although the G4-stabilizing compound Phen-DC3 significantly enhanced their stability, nPfh1 still resolved them efficiently. However, stable G4 structures significantly inhibited adenosine triphosphate hydrolysis by nPfh1. Because ribosomal and telomeric DNA contain putative G4 regions conserved from yeasts to humans, our studies support the important role of G4 structure formation in these regions and provide further evidence for a conserved role for Pif1 helicases in resolving G4 structures.

  17. Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas.

    PubMed

    Itoh, Naoki; Oda, Tadashi; Yoshinaga, Tomoyoshi; Ogawa, Kazuo

    2003-03-31

    To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20 degrees C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.

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

    PubMed

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

    2017-04-01

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

  19. Ribosomal DNA in diploid and polyploid Setaria (Poaceae) species: number and distribution.

    PubMed

    Nani, Thaís Furtado; Cenzi, Gisele; Pereira, Daniele Lais; Davide, Lisete Chamma; Techio, Vânia Helena

    2015-01-01

    Setaria Beauvois, 1812 is a genus of economically important forage species, including Setaria italica (Linnaeus, 1753) Beauvois, 1812 and Setaria viridis (Linnaeus, 1753) Beauvois, 1812, closely related species and considered as model systems for studies of C4 plants. However, complications and uncertainties related to taxonomy of other species of the genus are frequent due to the existence of numerous synonyms for the same species or multiple species with the same name, and overlapping of morphological characteristics. Cytogenetic studies in Setaria can be useful for taxonomic and evolutionary studies as well as for applications in breeding. Thus, this study is aimed at locating 45S and 5S rDNA sites through fluorescent in situ hybridization (FISH) in Setaria italica, Setaria viridis and Setaria sphacelata (Schumacher, 1827) Stapf, Hubbard, Moss, 1929 cultivars (cvs.) Narok and Nandi. Setaria italica and Setaria viridis have 18 chromosomes with karyotype formulas 6m + 3sm and 9m, respectively. The location of 45S and 5S rDNA for these species was in different chromosome pairs among the evaluated species. Setaria viridis presented a more symmetrical karyotype, strengthening the ancestral relationship with Setaria italica. Setaria sphacelata cvs. Narok and Nandi have 36 chromosomes, and karyotype formulas 11m+7sm and 16m+2sm, respectively. The 45S rDNA signals for both cultivars were also observed in distinct chromosome pairs; however chromosomes bearing 5S rDNA are conserved. Karyotypic variations found among the studied species are evidence of chromosomal rearrangements.

  20. Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers

    PubMed Central

    2008-01-01

    Background In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. Results In total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda. Conclusion Although ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny. PMID:18366624

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

    PubMed

    Bellavia, Daniele; Dimarco, Eufrosina; Caradonna, Fabio

    2016-04-15

    We previously reported the characterization 5S ribosomal DNA (rDNA) clusters in the common sea urchin Paracentrotus lividus and demonstrated the presence of DNA methylation-dependent silencing of embryo specific 5S rDNA cluster in adult tissue. In this work, we show genetic and epigenetic characterization of 18S-26S rDNA clusters in this specie. The results indicate the presence of three different 18S-26S rDNA clusters with different Non-Transcribed Spacer (NTS) regions that have different chromosomal localizations. Moreover, we show that the two largest clusters are hyper-methylated in the promoter-containing NTS regions in adult tissues, as in the 5S rDNA. These findings demonstrate an analogous epigenetic regulation in small and large rDNA clusters and support the logical synchronism in building ribosomes. In fact, all the ribosomal RNA genes must be synchronously and equally transcribed to perform their unique final product. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Ribosomal DNA gene and phylogenetic relationships of Diplura and lower hexapods.

    PubMed

    Luan, Yunxia; Zhang, Yaping; Yue, Qiaoyun; Pang, Junfeng; Xie, Rongdong; Yin, Wenying

    2003-02-01

    The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNAgene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustacean Artemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.

  3. Ribosomal and mitochondrial DNA analysis of Trichuridae nematodes of carnivores and small mammals.

    PubMed

    Guardone, Lisa; Deplazes, Peter; Macchioni, Fabio; Magi, Marta; Mathis, Alexander

    2013-10-18

    Several species of Trichuridae nematodes can infect dogs, cats and wild mammals. The diagnosis of these infections relies on the microscopic identification of eggs which are characterized by a similar "lemon" shape and polar plugs in all Trichuridae. Thus, morphological diagnosis to species level is challenging. The use of biomolecular diagnostic methods is desirable but very little genetic data are known from Trichuridae of carnivores and small mammals. The aim of this work was to genetically characterize several species of Trichuridae that can affect dogs, cats and wild mammals, as a basis to develop molecular diagnostic tests. Specimens (adult worms or eggs) of Eucoleus aerophilus (syn. Capillaria aerophila), Eucoleus boehmi (syn. Capillaria boehmi), Pearsonema plica (syn. Capillaria plica), Aonchotheca putorii (syn. Capillaria putorii), Calodium hepaticum (syn. Capillaria hepatica), Calodium splenaecum (syn. Capillaria splenaeca) and Trichuris vulpis were obtained from carcasses of red foxes, feces of dogs, the liver of a vole and from the spleen of Crocidura sp. Parts of the small subunit rRNA (18S rRNA) gene and of the mitochondrial cytochrome c oxidase subunit I (cox 1 mtDNA) gene were amplified from the above mentioned nematodes, yielding the first 18S rRNA gene sequences of all the capillariid nematodes and the first cox 1 mtDNA sequences of E. boehmi, P. plica, C. hepaticum, A. putorii and T. vulpis. The 18S rRNA gene is highly conserved among the different species and not suitable as a target for specific diagnostic oligonucleotides. However, these sequences contribute to a better understanding of the complex taxonomic relations among Trichuridae. Indeed, a dendrogram based on the 18S rRNA gene locus supports the latest taxonomic revision. Interspecies divergence was much higher at the cox 1 mtDNA gene locus, rendering it suitable for DNA barcoding and particularly valuable in resolving closely related species. Furthermore, the mitochondrial genetic

  4. Identification of Dermatophyte Species by 28S Ribosomal DNA Sequencing with a Commercial Kit

    PubMed Central

    Ninet, Béatrice; Jan, Isabelle; Bontems, Olympia; Léchenne, Barbara; Jousson, Olivier; Panizzon, Renato; Lew, Daniel; Monod, Michel

    2003-01-01

    We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex. PMID:12574293

  5. Ribosomal DNA in diploid and polyploid Setaria (Poaceae) species: number and distribution

    PubMed Central

    Nani, Thaís Furtado; Cenzi, Gisele; Pereira, Daniele Lais; Davide, Lisete Chamma; Techio, Vânia Helena

    2015-01-01

    Abstract Setaria Beauvois, 1812 is a genus of economically important forage species, including Setaria italica (Linnaeus, 1753) Beauvois, 1812 and Setaria viridis (Linnaeus, 1753) Beauvois, 1812, closely related species and considered as model systems for studies of C4 plants. However, complications and uncertainties related to taxonomy of other species of the genus are frequent due to the existence of numerous synonyms for the same species or multiple species with the same name, and overlapping of morphological characteristics. Cytogenetic studies in Setaria can be useful for taxonomic and evolutionary studies as well as for applications in breeding. Thus, this study is aimed at locating 45S and 5S rDNA sites through fluorescent in situ hybridization (FISH) in Setaria italica, Setaria viridis and Setaria sphacelata (Schumacher, 1827) Stapf, Hubbard, Moss, 1929 cultivars (cvs.) Narok and Nandi. Setaria italica and Setaria viridis have 18 chromosomes with karyotype formulas 6m + 3sm and 9m, respectively. The location of 45S and 5S rDNA for these species was in different chromosome pairs among the evaluated species. Setaria viridis presented a more symmetrical karyotype, strengthening the ancestral relationship with Setaria italica. Setaria sphacelata cvs. Narok and Nandi have 36 chromosomes, and karyotype formulas 11m+7sm and 16m+2sm, respectively. The 45S rDNA signals for both cultivars were also observed in distinct chromosome pairs; however chromosomes bearing 5S rDNA are conserved. Karyotypic variations found among the studied species are evidence of chromosomal rearrangements. PMID:26753080

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

    PubMed

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

    2005-04-01

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

  7. Physical localisation of repetitive DNA sequences in Alstroemeria: karyotyping of two species with species-specific and ribosomal DNA.

    PubMed

    Kamstra, S A; Kuipers, A G; De Jeu, M J; Ramanna, M S; Jacobsen, E

    1997-10-01

    Fluorescence in situ hybridization (FISH) was used to localise two species-specific repetitive DNA sequences, A001-I and D32-13, and two highly conserved 25S and 5S rDNA sequences on the metaphase chromosomes of two species of Alstroemeria. The Chilean species, Alstroemeria aurea (2n = 16), has abundant constitutive heterochromatin, whereas the Brazilian species, Alstroemeria inodora, has hardly any heterochromatin. The A. aurea specific A001-I probe hybridized specifically to the C-band regions on all chromosomes. The FISH patterns on A. inodora chromosomes using species-specific probe D32-13 resembled the C-banding pattern and the A001-I pattern on A. aurea chromosomes. There were notable differences in number and distribution of rDNA sites between the two species. The 25S rDNA probe revealed 16 sites in A. aurea that closely colocalised with A001-I sites and 12 in A. inodora that were predominantly detected in the centromeric regions. FISH karyotypes of the two Alstroemeria species were constructed accordingly, enabling full identification of all individual chromosomes. These FISH karyotypes will be useful for monitoring the chromosomes of both Alstroemeria species in hybrids and backcross derivatives.

  8. Septic arthritis and osteomyelitis in a 10-year-old boy, caused by Fusobacterium nucleatum, diagnosed with PCR/16S ribosomal bacterial DNA amplification

    PubMed Central

    Kroon, Elke; Arents, Niek A; Halbertsma, Feico Jan

    2012-01-01

    A 10-year-old boy presented with an atypical non-febrile septic arthritis/osteomyelitis. He was unresponsive to routine antibiotic treatment with flucloxacillin/gentamicin as the pain and fluid collection increased. Synovial fluid cultures are negative and gram stain remained negative. Only after PCR/16S ribosomal bacterial DNA amplification a Fusobacterium nucleatum could be detected, and antibiotic therapy switched to clindamycin with rapid response. Septic osteomyelitis and arthritis are relatively rare but important infections in children needing prompt treatment, and should be considered when a child complaints about joint or bone pain without prior recent trauma. Skin bacteria are the most prevalent causative organisms, whereas Fusobacteria or other anaerobic, Gram-negative microorganisms are very seldom encountered. If cultures remain negative and the patients responds insufficiently to empiric treatment, PCR/16S ribosomal bacterial DNA amplification can be useful to detect the causative microorganisms. PMID:22605875

  9. Delimitation of Sauropus (Phyllanthaceae) Based on Plastid matK and Nuclear Ribosomal ITS DNA Sequence Data

    PubMed Central

    Pruesapan, Kanchana; Telford, Ian R. H.; Bruhl, Jeremy J.; Draisma, Stefano G. A.; Van Welzen, Peter C.

    2008-01-01

    Background and Aims A recent molecular phylogenetic study showed that Sauropus is deeply embedded within Phyllanthus together with its allies, Breynia and Glochidion. As relationships within Sauropus are still problematic and the relationship with Breynia has long been doubted, more molecular data are needed to test/corroborate such a broad definition of Phyllanthus. This study aims to clarify the status and delimitation of Sauropus and establish its position within Phyllanthaceae. Methods Plastid matK and nuclear ribosomal ITS DNA sequence data for Sauropus and its allies were used to construct phylogenetic trees using maximum parsimony and Bayesian methods. Key Results Within Phyllanthus, Sauropus can be split into the mainly south-east Asian Sauropus sensu stricto (s.s.) plus Breynia and the mainly Australian Sauropus (formerly Synostemon). Sauropus s.s. plus Breynia comprise two distinct clades; one is the combination of Sauropus sections Glochidioidei, Sauropus and Schizanthi and the other is the combination of Sauropus sections Cryptogynium and Hemisauropus and the monophyletic genus Breynia. Conclusions Molecular data indicate that Synostemon should be reinstated at the same level as Sauropus s.s. and that Sauropus s.s. should be united with Breynia under the latter, older name. The molecular data corroborate only two of the five infrageneric groups of Sauropus recognized on the basis of morphological data. PMID:18854375

  10. Delimitation of Sauropus (Phyllanthaceae) based on plastid matK and nuclear ribosomal ITS DNA Sequence data.

    PubMed

    Pruesapan, Kanchana; Telford, Ian R H; Bruhl, Jeremy J; Draisma, Stefano G A; Van Welzen, Peter C

    2008-12-01

    A recent molecular phylogenetic study showed that Sauropus is deeply embedded within Phyllanthus together with its allies, Breynia and Glochidion. As relationships within Sauropus are still problematic and the relationship with Breynia has long been doubted, more molecular data are needed to test/corroborate such a broad definition of Phyllanthus. This study aims to clarify the status and delimitation of Sauropus and establish its position within Phyllanthaceae. Plastid matK and nuclear ribosomal ITS DNA sequence data for Sauropus and its allies were used to construct phylogenetic trees using maximum parsimony and Bayesian methods. Within Phyllanthus, Sauropus can be split into the mainly south-east Asian Sauropus sensu stricto (s.s.) plus Breynia and the mainly Australian Sauropus (formerly Synostemon). Sauropus s.s. plus Breynia comprise two distinct clades; one is the combination of Sauropus sections Glochidioidei, Sauropus and Schizanthi and the other is the combination of Sauropus sections Cryptogynium and Hemisauropus and the monophyletic genus Breynia. Molecular data indicate that Synostemon should be reinstated at the same level as Sauropus s.s. and that Sauropus s.s. should be united with Breynia under the latter, older name. The molecular data corroborate only two of the five infrageneric groups of Sauropus recognized on the basis of morphological data.

  11. Strain Identification of Trichophyton rubrum by Specific Amplification of Subrepeat Elements in the Ribosomal DNA Nontranscribed Spacer

    PubMed Central

    Jackson, Colin J.; Barton, Richard C.; Kelly, Steven L.; Evans, E. Glyn V.

    2000-01-01

    Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed. PMID:11101591

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

    PubMed

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

    2014-07-24

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

  13. Amplified ribosomal DNA restriction analysis of free-living bacteria present in the headbox of a Canadian paper machine.

    PubMed

    Prince, Véronique; Simao-Beaunoir, Anne-Marie; Beaulieu, Carole

    2009-07-01

    The headbox water is the main source of bacterial contamination of paper machines. Identification of these bacterial contaminants could be an asset in developing specific control methods. An amplified ribosomal DNA restriction analysis (ARDRA) was carried out to characterize the bacterial communities associated with the headbox water of a paper machine in a Canadian mill in February and July 2006. Eight bacterial genera were identified as the main colonizers present in the headbox water. The genus Meiothermus appeared to be the dominant bacterial group in the Canadian paper machine. Some variation was observed between the February and July clone libraries. Bacterial genera such as Chelatococcus and Hydrogenophilus were only detected in February or in July, respectively. Furthermore, the proportion of Tepidimonas clones in the libraries was higher in July than in February. The metabolic profile of the February and July communities, determined using Biolog EcoPlates, also suggested that temporal variation occurred within the bacterial populations that colonized the headbox of the paper machine.

  14. Phylogeny and biogeography of Panax L. (the ginseng genus, araliaceae): inferences from ITS sequences of nuclear ribosomal DNA.

    PubMed

    Wen, J; Zimmer, E A

    1996-10-01

    Panax, the ginseng genus, is one of the most medicinally important genera in the Orient and demonstrates a classical eastern Asian and eastern North American disjunct distributional pattern. Sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of the nuclear ribosomal DNA repeat were obtained for the 12 species of Panax to reconstruct phylogenetic relationships. Of the 2 eastern North American species, P. quinquefolius and P. trifolius, P. quinquefolius was suggested to be more closely related to the eastern Asian species in the ITS tree, while P. trifolius was phylogenetically isolated. Monophyly of the three medicinally most important species, P. ginseng, P. notoginseng, and P. quinquefolius, suggested by previous workers, was not supported by the ITS data. A close phylogenetic relationship between Panax and Aralia was supported. Several biogeographical implications were inferred: (1) two divergence events have produced the eastern Asian and eastern North American disjunct distribution in Panax, (2) no intercontinental species pairs are found in Panax; (3) a discrepancy between the sequence divergence pattern and the phylogenetic pattern was observed in Panax, suggesting the need for caution in using sequence divergence data alone in inferring biogeographical patterns; (4) the Himalayas and central and western China are the current centers of diversity of the ginseng genus; and (5) the low ITS sequence divergence and a close relationship among species in that region suggest that rapid evolutionary radiation may have created such a diversity of Panax in the Himalayas and in central and western China.

  15. Phylogenetic relationships of species within the tribe Labiostrongylinea (Nematoda: Cloacinidae) from Australian marsupials based on ribosomal DNA spacer sequence data.

    PubMed

    Chilton, Neil B; Huby-Chilton, Florence; Beveridge, Ian; Smales, Lesley R; Gasser, Robin B; Andrews, Ross H

    2011-12-01

    Parasitic nematodes of the tribe Labiostrongylinea (Family Cloacinidae) occur in the stomachs of a wide variety of potoroid and macropodid marsupials in Australia, Papua Indonesia and Papua New Guinea. The aim of the present study was to infer the evolutionary relationships of the five genera of labiostrongyline nematodes that occur in Australian potoroids and macropodids using sequence data of the nuclear first and second internal transcribed spacers of ribosomal DNA. The phylogenetic analyses resulted in the separation of the Labiostrongylinea into two major groups reflecting coevolution between hosts and parasites. Two nematode species belonging to the genus Potorostrongylus formed a sister group to the remaining species of the Labiostrongylinea. This genus occurs exclusively in potoroid marsupials, which are considered to be basal to the macropodid marsupials. The second major group included species of Labiostrongylus, Labiosimplex, Labiomultiplex and Parazoniolaimus, all of which occur in macropodids. These species formed two distinct clades, one predominating in the host genera Thylogale and Onychogalea, and the second in the genus Macropus, which includes the more recent macropodids. However, there is also evidence of colonisation by both nematode clades of relatively unrelated hosts. In addition, genetic differences among individuals of Lm. eugenii from geographically isolated populations of M. eugenii, and among Ls. longispicularis from different subspecies of M. robustus suggest the existence of sibling species that may have arisen by allopatric speciation. The broad coevolutionary relationship between the labiostrongyline nematodes and their marsupial hosts therefore represents a mixture of potential cospeciation and colonisation events.

  16. Genetic heterogeneity and phylogeny of Trichuris spp. from captive non-human primates based on ribosomal DNA sequence data.

    PubMed

    Cavallero, Serena; De Liberato, Claudio; Friedrich, Klaus G; Di Cave, David; Masella, Valentina; D'Amelio, Stefano; Berrilli, Federica

    2015-08-01

    Nematodes of the genus Trichuris, known as whipworms, are recognized to infect numerous mammalian species including humans and non-human primates. Several Trichuris spp. have been described and species designation/identification is traditionally based on host-affiliation, although cross-infection and hybridization events may complicate species boundaries. The main aims of the present study were to genetically characterize adult Trichuris specimens from captive Japanese macaques (Macaca fuscata) and grivets (Chlorocebus aethiops), using the ribosomal DNA (ITS) as molecular marker and to investigate the phylogeny and the extent of genetic variation also by comparison with data on isolates from other humans, non-human primates and other hosts. The phylogenetic analysis of Trichuris sequences from M. fuscata and C. aethiops provided evidences of distinct clades and subclades thus advocating the existence of additional separated taxa. Neighbor Joining and Bayesian trees suggest that specimens from M. fuscata may be distinct from, but related to Trichuris trichiura, while a close relationship is suggested between the subclade formed by the specimens from C. aethiops and the subclade formed by T. suis. The tendency to associate Trichuris sp. to host species can lead to misleading taxonomic interpretations (i.e. whipworms found in primates are identified as T. trichiura). The results here obtained confirm previous evidences suggesting the existence of Trichuris spp. other than T. trichiura infecting non-human living primates. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Molecular systematics of several cyclophyllid families (Cestoda) based on the analysis of 18S ribosomal DNA gene sequences.

    PubMed

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

    2004-07-01

    The sequences of the 18S small subunit ribosomal DNA of five species of cyclophyllidean cestodes from the families Davaineidae, Anoplocephalidae and Dilepididae were determined. A species of tetrabothridid was also sequenced. These 18S sequences were combined with other available eucestode sequences in GenBank. From the 1,838 sites in the alignment, 375 bp (20%) were excluded from the analysis due to alignment issues inferred by manual inspection. Phylogenetic trees were obtained by maximum parsimony, neighbour-joining distance and maximum likelihood methods. Analyses showed that Cyclophyllidea is monophyletic and separate from Tetrabothrius spp. Lyruterina nigropunctata, which is now included in the family Paruterinidae, is more closely related to davaineids of the genus Raillietina than Pseudidiogenes nana (Davaineidae). P. nana and Choanotaenia infundibulum (Dilepididae) derive from the Davaineidae (or Raillietininae). The two species of Taenia (T. parva and T. pisiformis) formed a monophyletic sister group to the Davaineidae and Anoplocephalidae. The systematics of the Paruterinidae have been problematic and our results suggest a review of this family including other species with paruterine organ located in other families. The position of the Idiogeninae as a subfamily in the Davaineidae is also reviewed. Copyright 2004 Springer-Verlag

  18. Phylogenetics of neotropical Platymiscium (Leguminosae: Dalbergieae): systematics, divergence times, and biogeography inferred from nuclear ribosomal and plastid DNA sequence data.

    PubMed

    Saslis-Lagoudakis, Charilaos; Chase, Mark W; Robinson, Daniel N; Russell, Stephen J; Klitgaard, Bente B

    2008-10-01

    Platymiscium is a neotropical legume genus of forest trees in the Pterocarpus clade of the pantropical "dalbergioid" clade. It comprises 19 species (29 taxa), distributed from Mexico to southern Brazil. This study presents a molecular phylogenetic analysis of Platymiscium and allies inferred from nuclear ribosomal (nrITS) and plastid (trnL, trnL-F and matK) DNA sequence data using parsimony and Bayesian methods. Divergence times are estimated using a Bayesian method assuming a relaxed molecular clock (multidivtime). Within the Pterocarpus clade, new sister relationships are recovered: Pterocarpus + Etaballia, Inocarpus + Tipuana and Paramachaerium + Maraniona. Our results support monophyly of Platymiscium, which is resolved into three major clades, each with distinct geographic ranges and ecological preferences. Diversification in Platymiscium has been driven by habitat fragmentation, invasion of novel geographic regions, and ecological diversification, revealing general patterns of diversification in the neotropics. We hypothesize that Platymiscium arose in dry habitats of South America and radiated northward. The Amazon basin was invaded twice both within the last 5.6 My and Central America twice before the closure of the Isthmus of Panama. Divergence times of the P. pubescens complex, restricted to seasonally dry tropical forests of South America, support pre-Pleistocene divergence in this biome.

  19. Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruíz-Estévez, Mercedes; López-León, M Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2013-09-01

    B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

  20. Diversity of Salmonella Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions

    PubMed Central

    Baudart, Julia; Lemarchand, Karine; Brisabois, Anne; Lebaron, Philippe

    2000-01-01

    Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates. PMID:10742240

  1. Restriction Enzyme Analysis of Ribosomal DNA Shows that Candida inconspicua Clinical Isolates Can Be Misidentified as Candida norvegensis with Traditional Diagnostic Procedures

    PubMed Central

    Majoros, L.; Kardos, G.; Belák, Á.; Maráz, A.; Asztalos, L.; Csánky, E.; Barta, Z.; Szabó, B.

    2003-01-01

    We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua. PMID:14605175

  2. Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species.

    PubMed

    Ma, Jun; He, Jun-Jun; Liu, Guo-Hua; Zhou, Dong-Hui; Liu, Jian-Zhi; Liu, Yi; Zhu, Xing-Quan

    2015-04-02

    Rumen flukes parasitize the rumen and reticulum of ruminants, causing paramphistomiasis. Over the years, there has been considerable debate as to whether Paramphistomum leydeni and Paramphistomum cervi are the same or distant species. In the present study, the complete mitochondrial (mt) genome of P. leydeni was amplified using PCR-based sequencing and compared with that of P. cervi. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) of P. leydeni specimens (n = 6) and P. cervi specimens (n = 8) was amplified and then sequenced. Phylogenetic relationship of the concatenated amino acid sequence data for 12 protein-coding genes of the two rumen flukes and selected members of Trematoda was evaluated using Bayesian inference (BI). The complete mt genome of P. leydeni was 14,050 bp in size. Significant nucleotide difference between the P. leydeni mt genome and that of P. cervi (14.7%) was observed. For genetic divergence in ITS-2, sequence difference between P. leydeni and P. cervi was 3.1%, while no sequence variation was detected within each of them. Phylogenetic analysis indicated that P. leydeni and P. cervi are closely-related but distinct rumen flukes. Results of the present study support the proposal that P. leydeni and P. cervi represent two distinct valid species. The mt genome sequences of P. leydeni provide plentiful resources of mitochondrial markers, which can be combined with nuclear markers, for further comparative studies of the biology of P. leydeni and its congeners from China and other countries.

  3. cDNA Cloning and Overexpression of Acidic Ribosomal Phosphoprotein P1 Gene (RPLP1) from the Giant Panda

    PubMed Central

    Du, Yu-Jie; Luo, Xiao-Yan; Hao, Yan-Zhe; Zhang, Tian; Hou, Wan-Ru

    2007-01-01

    RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function. PMID:18071584

  4. Molecular Systematics and Biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) Based on Nuclear Ribosomal and Plastid DNA Sequences

    PubMed Central

    CHEN, SHENGYUN; XIA, TAO; WANG, YUJIN; LIU, JIANQUAN; CHEN, SHILONG

    2005-01-01

    • Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences. • Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae. • Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0·05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11·4–21·4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0·4 to 6·2 Mya. • Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene. PMID:15994844

  5. cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda.

    PubMed

    Du, Yu-Jie; Luo, Xiao-Yan; Hao, Yan-Zhe; Zhang, Tian; Hou, Wan-Ru

    2007-10-26

    RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

  6. Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata.

    PubMed

    Moreira, David; von der Heyden, Sophie; Bass, David; López-García, Purificación; Chao, Ema; Cavalier-Smith, Thomas

    2007-07-01

    Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of

  7. Comparison of fungi within the Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequences.

    PubMed

    Bryan, G T; Daniels, M J; Osbourn, A E

    1995-02-01

    Four ascomycete species of the genus Gaeumannomyces infect roots of monocotyledons. Gaeumannomyces graminis contains four varieties, var. tritici, var. avenae, var. graminis, and var. maydis. G. graminis varieties tritici, avenae, and graminis have Phialophora-like anamorphs and, together with the other Gaeumannomyces and Phialophora species found on cereal roots, constitute the Gaeumannomyces-Phialophora complex. Relatedness of a number of Gaeumannomyces and Phialophora isolates was assessed by comparison of DNA sequences of the 18S rRNA gene, the 5.8S rRNA gene, and the internal transcribed spacers (ITS). G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolates can be distinguished from each other by nucleotide sequence differences in the ITS regions. The G. graminis var. tritici isolates can be further subdivided into R and N isolates (correlating with ability [R] or inability [N] to infect rye). Phylogenetic analysis of the ITS regions of several oat-infecting G. graminis var. tritici isolates suggests that these isolates are actually more closely related to G. graminis var. avenae. The isolates of Magnaporthe grisea included in the analysis showed a surprising degree of relatedness to members of the Gaeumannomyces-Phialophora complex. G. graminis variety-specific oligonucleotide primers were used in PCRs to amplify DNA from cereal seedlings infected with G. graminis var. tritici or G. graminis var. avenae, and these should be valuable for sensitive detection of pathogenic isolates and for diagnosis of take-all.

  8. 18S ribosomal DNA-based PCR for diagnosis of Trichomonas vaginalis.

    PubMed

    Mayta, H; Gilman, R H; Calderon, M M; Gottlieb, A; Soto, G; Tuero, I; Sanchez, S; Vivar, A

    2000-07-01

    Trichomonas vaginalis remains the most common sexually transmitted parasite in the world and is considered a major risk factor in the transmission of the human immunodeficiency virus. A PCR technique using primers targeting a specific region of the 18S rRNA gene of T. vaginalis was developed. The PCR test was standardized using 15 reference strains, giving a single product of 312 bp in all strains. No amplification was observed when DNA from related organisms or human DNA was used as a target. The test was evaluated on 372 vaginal swab specimens and 361 urine samples from women attending infertility and obstetric clinics at two separate hospitals in Lima, Peru. Compared to T. vaginalis culture, the overall sensitivity and specificity of PCR of vaginal swab samples was 100% and 98%, respectively. The PCR of urine samples was 100% sensitive and 99.7% specific compared to culture of vaginal swab, but the sensitivity drops to 83.3% when compared to PCR of vaginal swabs. All culture-positive samples were found to be positive by PCR in either urine or vaginal secretion. None of the PCR-negative samples were positive by culture. The origin of the amplification was confirmed by digestion of PCR products with HaeIII. This PCR assay, which is easy to perform and has a high sensitivity and specificity, should be useful for routine diagnosis of T. vaginalis infection.

  9. Genetic and Molecular Organization of Ribosomal DNA (Rdna) Variants in Wild and Cultivated Barley

    PubMed Central

    Allard, R. W.; Maroof, MAS.; Zhang, Q.; Jorgensen, R. A.

    1990-01-01

    Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in which each variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locus Rrn2 and the 12 longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus Rrn1. Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuring compound alleles marked by two slvs of Rrn1 or of Rrn2, and the other featuring presence in Rrn1 of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions. PMID:2249766

  10. Arabidopsis MAS2, an Essential Gene That Encodes a Homolog of Animal NF-κ B Activating Protein, Is Involved in 45S Ribosomal DNA Silencing.

    PubMed

    Sánchez-García, Ana Belén; Aguilera, Verónica; Micol-Ponce, Rosa; Jover-Gil, Sara; Ponce, María Rosa

    2015-07-01

    Ribosome biogenesis requires stoichiometric amounts of ribosomal proteins and rRNAs. Synthesis of rRNAs consumes most of the transcriptional activity of eukaryotic cells, but its regulation remains largely unclear in plants. We conducted a screen for ethyl methanesulfonate-induced suppressors of Arabidopsis thaliana ago1-52, a hypomorphic allele of AGO1 (ARGONAUTE1), a key gene in microRNA pathways. We identified nine extragenic suppressors as alleles of MAS2 (MORPHOLOGY OF AGO1-52 SUPPRESSED2). Positional cloning showed that MAS2 encodes the putative ortholog of NKAP (NF-κ B activating protein), a conserved eukaryotic protein involved in transcriptional repression and splicing in animals. The mas2 point mutations behave as informational suppressors of ago1 alleles that cause missplicing. MAS2 is a single-copy gene whose insertional alleles are embryonic lethal. In yeast two-hybrid assays, MAS2 interacted with splicing and ribosome biogenesis proteins, and fluorescence in situ hybridization showed that MAS2 colocalizes with the 45S rDNA at the nucleolar organizer regions (NORs). The artificial microRNA amiR-MAS2 partially repressed MAS2 and caused hypomethylation of 45S rDNA promoters as well as partial NOR decondensation, indicating that MAS2 negatively regulates 45S rDNA expression. Our results thus reveal a key player in the regulation of rRNA synthesis in plants. © 2015 American Society of Plant Biologists. All rights reserved.

  11. Small subunit ribosomal DNA suggests that the xenophyophorean Syringammina corbicula is a foraminiferan.

    PubMed

    Pawlowski, Jan; Holzmann, Maria; Fahrni, Jose; Richardson, Susan L

    2003-01-01

    Xenophyophorea are giant deep-sea rhizopodial protists of enigmatic origins. Although species were described as Foraminifera or sponges in the early literature, the xenophyophoreans are currently classified either as a class of Rhizopoda or an independent phylum. To establish the phylogenetic position of Xenophyophorea, we analysed the small subunit (SSU) rRNA gene sequence of Syringammina corbicula Richardson, a newly described xenophyophorean species from the Cape Verde Plateau. The SSUrDNA analyses showed that S. corbicula is closely related to Rhizammina algaeformis, a tubular deep-sea foraminiferan. Both species branch within a group of monothalamous (single-chambered) Foraminifera, which include also such agglutinated genera as Toxisarcon, Rhabdammina, and Saccammina, and the organic-walled genera Gloiogullmia and Cylindrogullmia. Our results are congruent with observations of similar cytoplasmic organisation in Rhizammina and Syringammina. Thus, the Xenophyophorea appear to be a highly specialised group of deep-sea Foraminifera.

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

    PubMed

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

    2012-03-01

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

  13. Role of DNA damage and repair in the function of eukaryotic genes: radiation-induced single-strand breaks and their rejoining in chromosomal and extrachromosomal ribosomal DNA of Tetrahymena

    SciTech Connect

    Chiu, S.M.; Oleinick, N.L.

    1980-04-01

    The production and rejoining of single-strand breaks (SSB) in chromosomal DNA and extrachromosomal ribosomal DNA (rDNA) were investigated after sublethal doses of ..gamma.. radiation to exponentially growing Tetrahymena. Hydrogen-3-labeled total nuclear DNA isolated from either control or irradiated cells was heat denatured and electrophoresed in agarose gels containing formaldehyde. Ribosomal DNA was identified by hybridization to (/sup 32/P)rRNA after transferring the DNA from the gels to nitrocellulose strips. It was found that (a) approximately 0.68 SSB is produced in each strand of rDNA exposed to 40 krad; (b) greater than 80% of SSB were rejoined within the first 20 min after irradiation in both chromosomal and rDNA; and (c) the rejoining process in both chromosomal and rDNA proceeded in the presence of inhibitors of protein synthesis, RNA synthesis, or oxidative metabolism. While the majority of SSB induced by 40 krad is rejoined within 20 min after irradiation, the resumption of rRNA synthesis does not occur until 30 min thereafter; it is concluded that the restoration of the normal size of the rDNA template is probably necessary but not sufficient for the resumption of rRNA synthesis.

  14. Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation

    PubMed Central

    Kerr, Craig H.

    2016-01-01

    To replicate, all viruses depend entirely on the enslavement of host cell ribosomes for their own advantage. To this end, viruses have evolved a multitude of translational strategies to usurp the ribosome. RNA-based structures known as internal ribosome entry sites (IRESs) are among the most notable mechanisms employed by viruses to seize host ribosomes. In this article, we spotlight the intergenic region IRES from the Dicistroviridae family of viruses and its importance as a model for IRES-dependent translation and in understanding fundamental properties of translation. PMID:27053555

  15. cDNA, genomic sequence cloning and overexpression of ribosomal protein S25 gene (RPS25) from the Giant Panda.

    PubMed

    Hao, Yan-Zhe; Hou, Wan-Ru; Hou, Yi-Ling; Du, Yu-Jie; Zhang, Tian; Peng, Zheng-Song

    2009-11-01

    RPS25 is a component of the 40S small ribosomal subunit encoded by RPS25 gene, which is specific to eukaryotes. Studies in reference to RPS25 gene from animals were handful. The Giant Panda (Ailuropoda melanoleuca), known as a "living fossil", are increasingly concerned by the world community. Studies on RPS25 of the Giant Panda could provide scientific data for inquiring into the hereditary traits of the gene and formulating the protective strategy for the Giant Panda. The cDNA of the RPS25 cloned from Giant Panda is 436 bp in size, containing an open reading frame of 378 bp encoding 125 amino acids. The length of the genomic sequence is 1,992 bp, which was found to possess four exons and three introns. Alignment analysis indicated that the nucleotide sequence of the coding sequence shows a high homology to those of Homo sapiens, Bos taurus, Mus musculus and Rattus norvegicus as determined by Blast analysis, 92.6, 94.4, 89.2 and 91.5%, respectively. Primary structure analysis revealed that the molecular weight of the putative RPS25 protein is 13.7421 kDa with a theoretical pI 10.12. Topology prediction showed there is one N-glycosylation site, one cAMP and cGMP-dependent protein kinase phosphorylation site, two Protein kinase C phosphorylation sites and one Tyrosine kinase phosphorylation site in the RPS25 protein of the Giant Panda. The RPS25 gene was overexpressed in E. coli BL21 and Western Blotting of the RPS25 protein was also done. The results indicated that the RPS25 gene can be really expressed in E. coli and the RPS25 protein fusioned with the N-terminally his-tagged form gave rise to the accumulation of an expected 17.4 kDa polypeptide. The cDNA and the genomic sequence of RPS25 were cloned successfully for the first time from the Giant Panda using RT-PCR technology and Touchdown-PCR, respectively, which were both sequenced and analyzed preliminarily; then the cDNA of the RPS25 gene was overexpressed in E. coli BL21 and immunoblotted, which is the first

  16. Ribosomal DNA (rDNA) identification of the culturable bacterial flora on monetary coinage from 17 currencies.

    PubMed

    Xu, Jiru; Moore, John E; Millar, B Cherie

    2005-03-01

    The aim of the investigation reported in this paper was to identify the bacterial microflora on monetary coinage from 17 countries by employment of polymerase chain reaction (PCR) sequenced-based molecular identification of rDNA from bacterial cultures. Silver, bronze, and other alloy coins (approximately 300 g) from 17 currencies were enriched individually by aerobic culturing in tryptone soya broth for 72 hours at 30 degrees C. Next, 20 microL of broth was inoculated onto Columbia blood agar supplemented with 5 percent volume-pervolume (v/v) defibrinated horse blood for 72 hours at 30 degrees C, and resulting colonies were purified by further subculture, as detailed above, for a further 72 hours. All colonies were identified by initial PCR amplification of a partial region of the 16S rDNA gene locus, which was then sequenced, and the sequence was aligned according to the BLASTn algorithm. Twenty-five isolates were obtained from the coinage; of these, 25 (100 percent) were Gram positive, and the most prevalent genus observed was Bacillus (B. megaterium, B. lentus, B. litoralis, B. subtilis, B. circulans and other Bacillus spp.), which accounted for 10 of 25 isolates (40 percent) and was isolated from 10 of 17 countries (58.8 percent). It was followed in prevalence by Staphylococcus spp. (Staph. aureus, Staph. epidermidis, Staph. hominis, Staph. schleiferi), which accounted for 7 of 25 isolates (28 percent) and were isolated from 7 of 17 countries (41.2 percent). Given the organisms identified in this study, it is not believed that monetary coinage presents any particular risk to public health. The authors support the principles of basic hygiene, however, in terms of proper handwashing and the avoidance of handling money when working with food or dressing wounds and skin lesions, In conclusion, the study demonstrated that money from 17 countries was contaminated by environmental Gram-positive flora, in particular Bacillus spp., and that the universal 16S rDNA

  17. Organellar genome, nuclear ribosomal DNA repeat unit, and microsatellites isolated from a small-scale of 454 GS FLX sequencing on two mosses.

    PubMed

    Liu, Yang; Forrest, Laura L; Bainard, Jillian D; Budke, Jessica M; Goffinet, Bernard

    2013-03-01

    Recent innovations in high-throughput DNA sequencing methodology (next generation sequencing technologies [NGS]) allow for the generation of large amounts of high quality data that may be particularly critical for resolving ambiguous relationships such as those resulting from rapid radiations. Application of NGS technology to bryology is limited to assembling entire nuclear or organellar genomes of selected exemplars of major lineages (e.g., classes). Here we outline how organellar genomes and the entire nuclear ribosomal DNA repeat can be obtained from minimal amounts of moss tissue via small-scale 454 GS FLX sequencing. We sampled two Funariaceae species, Funaria hygrometrica and Entosthodon obtusus, and assembled nearly complete organellar genomes and the whole nuclear ribosomal DNA repeat unit (18S-ITS1-5.8S-ITS2-26S-IGS1-5S-IGS2) for both taxa. Sequence data from these species were compared to sequences from another Funariaceae species, Physcomitrella patens, revealing low overall degrees of divergence of the organellar genomes and nrDNA genes with substitutions spread rather evenly across their length, and high divergence within the external spacers of the nrDNA repeat. Furthermore, we detected numerous microsatellites among the 454 assemblies. This study demonstrates that NGS methodology can be applied to mosses to target large genomic regions and identify microsatellites. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

    PubMed Central

    Cavallero, Serena; Snabel, Viliam; Pacella, Francesca; Perrone, Vitantonio; D'Amelio, Stefano

    2013-01-01

    Background The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the world's most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples. Methodology 151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions. Principal Findings No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum. Conclusions/Significance These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This

  19. 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

  20. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    PubMed Central

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  1. Practical identification of human originated Lactobacillus species by amplified ribosomal DNA restriction analysis (ARDRA) for probiotic use.

    PubMed

    Öztürk, Mehmet; Meterelliyöz, Merve

    2015-08-01

    Probiotics are gaining popularity and increasing the importance of their accurate speciation. Lactobacillus species are commonly used as probiotic strains mostly of clinical importance. Present knowledge indicates that at least 14 Lactobacillus species are associated with the human intestinal tract. Currently, researchers are interested in developing efficient techniques for screening and selecting probiotics bacteria, but unfortunately most of these methods are time-consuming, labor-intensive and costly. The aim of this study is to develop reliable, rapid and accurate method to identify 14 references Lactobacillus species that could have been found in the human alimentary tract by 16S ribosomal DNA restriction analysis. In this study, to develop an effective method for the genotype-based identification of the reference Lactobacillus species, 1.5 kb of 16S rRNA nucleotide sequences of 14 Lactobacillus were collected from the Gene Bank aligned, in silico restricted and analyzed in respect to their 16S-rRNA restriction fragment polymorphism. In silico restriction profiles of 16S-rRNA indicated that FspBI, HinfI and DraI restriction enzymes (RE) are convenient for differentiation of 14 Lactobacillus species in human intestinal tract except Lb. casei and Lb. paracasei. The patterns of our experimental findings obtained from 16S PCR-ARDRA completely confirmed our in silico patterns. The present work demonstrated that 16S PCR-ARDRA method with FspBI, HinfI and DraI RE is a rapid, accurate and reliable method for the identification of Lactobacillus species from human alimentary tract, especially during the identification of large numbers of isolates and any laboratory equipped with a thermo cycler for probiotic use.

  2. Nuclear pores and perinuclear expression sites of var and ribosomal DNA genes correspond to physically distinct regions in Plasmodium falciparum.

    PubMed

    Guizetti, Julien; Martins, Rafael Miyazawa; Guadagnini, Stéphanie; Claes, Aurélie; Scherf, Artur

    2013-05-01

    The human malaria parasite Plasmodium falciparum modifies the erythrocyte it infects by exporting variant proteins to the host cell surface. The var gene family that codes for a large, variant adhesive surface protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) plays a particular role in this process, which is linked to pathogenesis and immune evasion. A single member of this gene family is highly transcribed while the other 59 members remain silenced. Importantly, var gene transcription occurs at a spatially restricted, but yet undefined, perinuclear site that is distinct from repressed var gene clusters. To advance our understanding of monoallelic expression, we investigated whether nuclear pores associate with the var gene expression site. To this end, we studied the nuclear pore organization during the asexual blood stage using a specific antibody directed against a subunit of the nuclear pore, P. falciparum Nup116 (PfNup116). Ring and schizont stage parasites showed highly polarized nuclear pore foci, whereas in trophozoite stage nuclear pores redistributed over the entire nuclear surface. Colocalization studies of var transcripts and anti-PfNup116 antibodies showed clear dissociation between nuclear pores and the var gene expression site in ring stage. Similar results were obtained for another differentially transcribed perinuclear gene family, the ribosomal DNA units. Furthermore, we show that in the poised state, the var gene locus is not physically linked to nuclear pores. Our results indicate that P. falciparum does form compartments of high transcriptional activity at the nuclear periphery which are, unlike the case in yeast, devoid of nuclear pores.

  3. Enterobacterial Repetitive Intergenic Consensus Sequence Repeats in Yersiniae: Genomic Organization and Functional Properties

    PubMed Central

    De Gregorio, Eliana; Silvestro, Giustina; Petrillo, Mauro; Carlomagno, Maria Stella; Di Nocera, Pier Paolo

    2005-01-01

    Genome-wide analyses carried out in silico revealed that the DNA repeats called enterobacterial repetitive intergenic consensus sequences (ERICs), which are present in several Enterobacteriaceae, are overrepresented in yersiniae. From the alignment of DNA regions from the wholly sequenced Yersinia enterocolitica 8081 and Yersinia pestis CO92 strains, we could establish that ERICs are miniature mobile elements whose insertion leads to duplication of the dinucleotide TA. ERICs feature long terminal inverted repeats (TIRs) and can fold as RNA into hairpin structures. The proximity to coding regions suggests that most Y. enterocolitica ERICs are cotranscribed with flanking genes. Elements which either overlap or are located next to stop codons are preferentially inserted in the same (or B) orientation. In contrast, ERICs located far apart from open reading frames are inserted in the opposite (or A) orientation. The expression of genes cotranscribed with A- and B-oriented ERICs has been monitored in vivo. In mRNAs spanning B-oriented ERICs, upstream gene transcripts accumulated at lower levels than downstream gene transcripts. This difference was abolished by treating cells with chloramphenicol. We hypothesize that folding of B-oriented elements is impeded by translating ribosomes. Consequently, upstream RNA degradation is triggered by the unmasking of a site for the RNase E located in the right-hand TIR of ERIC. A-oriented ERICs may act in contrast as upstream RNA stabilizers or may have other functions. The hypothesis that ERICs act as regulatory RNA elements is supported by analyses carried out in Yersinia strains which either lack ERIC sequences or carry alternatively oriented ERICs at specific loci. PMID:16291667

  4. The Ribosome Biogenesis Factor Nol11 Is Required for Optimal rDNA Transcription and Craniofacial Development in Xenopus

    PubMed Central

    Griffin, John N.; Sondalle, Samuel B.; del Viso, Florencia; Baserga, Susan J.; Khokha, Mustafa K.

    2015-01-01

    The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival. PMID:25756904

  5. The ribosome biogenesis factor Nol11 is required for optimal rDNA transcription and craniofacial development in Xenopus.

    PubMed

    Griffin, John N; Sondalle, Samuel B; Del Viso, Florencia; Baserga, Susan J; Khokha, Mustafa K

    2015-03-01

    The production of ribosomes is ubiquitous and fundamental to life. As such, it is surprising that defects in ribosome biogenesis underlie a growing number of symptomatically distinct inherited disorders, collectively called ribosomopathies. We previously determined that the nucleolar protein, NOL11, is essential for optimal pre-rRNA transcription and processing in human tissue culture cells. However, the role of NOL11 in the development of a multicellular organism remains unknown. Here, we reveal a critical function for NOL11 in vertebrate ribosome biogenesis and craniofacial development. Nol11 is strongly expressed in the developing cranial neural crest (CNC) of both amphibians and mammals, and knockdown of Xenopus nol11 results in impaired pre-rRNA transcription and processing, increased apoptosis, and abnormal development of the craniofacial cartilages. Inhibition of p53 rescues this skeletal phenotype, but not the underlying ribosome biogenesis defect, demonstrating an evolutionarily conserved control mechanism through which ribosome-impaired craniofacial cells are removed. Excessive activation of this mechanism impairs craniofacial development. Together, our findings reveal a novel requirement for Nol11 in craniofacial development, present the first frog model of a ribosomopathy, and provide further insight into the clinically important relationship between specific ribosome biogenesis proteins and craniofacial cell survival.

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

    PubMed

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

    2013-03-11

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

  7. The use of large and small subunits of ribosomal DNA in evaluating phylogenetic relationships between species of Cornudiscoides Kulkarni, 1969 (Monogenoidea: Dactylogyridae) from India.

    PubMed

    Verma, J; Agrawal, N; Verma, A K

    2017-03-01

    Two partial regions of ribosomal DNA (28S and 18S) were used to evaluate genetic variations among the species of Cornudiscoides, viz. C. proximus, C. geminus and C. agarwali, all parasites of Mystus vittatus (Bagridae) from River Gomati, Ganges River basin, India. Our findings demonstrated that both the large and small ribosomal subunits are useful for species identification and genetic characterization of parasites, leading to resolution of inter/intra-relationships at generic and specific levels. The secondary structures of all three species for 28S and 18S rRNA genes contained exact pattern matches (EMPs) displaying the high degree of similarity among them. The phylogenetic analyses within the members of Dactylogyridae demonstrated that species of Cornudiscoides cluster together for 28S rRNA and 18S rRNA genes.

  8. Deciphering Poxvirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Cao, Shuai; Martens, Craig A.; Porcella, Stephen F.; Xie, Zhi; Ma, Ming; Shen, Ben

    2015-01-01

    ABSTRACT The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. IMPORTANCE This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological

  9. The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons

    SciTech Connect

    Xiong, Y.; Eickbush, T.H.

    1988-01-01

    Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. The authors present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. The authors therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons.

  10. Intraspecific variation and population structure of the German cockroach, Blattella germanica, revealed with RFLP analysis of the non-transcribed spacer region of ribosomal DNA.

    PubMed

    Mukha, D V; Kagramanova, A S; Lazebnaya, I V; Lazebnyi, O E; Vargo, E L; Schal, C

    2007-06-01

    Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.

  11. cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca).

    PubMed

    Hou, W R; Hou, Y L; Wu, G F; Song, Y; Su, X L; Sun, B; Li, J

    2011-01-01

    The ribosomal protein L9 (RPL9), a component of the large subunit of the ribosome, has an unusual structure, comprising two compact globular domains connected by an α-helix; it interacts with 23 S rRNA. To obtain information about rpL9 of Ailuropoda melanoleuca (the giant panda) we designed primers based on the known mammalian nucleotide sequence. RT-PCR and PCR strategies were employed to isolate cDNA and the rpL9 gene from A. melanoleuca; these were sequenced and analyzed. We overexpressed cDNA of the rpL9 gene in Escherichia coli BL21. The cloned cDNA fragment was 627 bp in length, containing an open reading frame of 579 bp. The deduced protein is composed of 192 amino acids, with an estimated molecular mass of 21.86 kDa and an isoelectric point of 10.36. The length of the genomic sequence is 3807 bp, including six exons and five introns. Based on alignment analysis, rpL9 has high similarity among species; we found 85% agreement of DNA and amino acid sequences with the other species that have been analyzed. Based on topology predictions, there are two N-glycosylation sites, five protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, two tyrosine kinase phosphorylation sites, three N-myristoylation sites, one amidation site, and one ribosomal protein L6 signature 2 in the L9 protein of A. melanoleuca. The rpL9 gene can be readily expressed in E. coli; it fuses with the N-terminal GST-tagged protein, giving rise to the accumulation of an expected 26.51-kDa polypeptide, which is in good agreement with the predicted molecular weight. This expression product could be used for purification and further study of its function.

  12. Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

    PubMed Central

    Elhag, G A; Thomas, F J; McCreery, T P; Bourque, D P

    1992-01-01

    Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco. Images PMID:1542565

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

    PubMed

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

    1996-01-01

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

  14. Application of a clustering-based peak alignment algorithm to analyze various DNA fingerprinting data.

    PubMed

    Ishii, Satoshi; Kadota, Koji; Senoo, Keishi

    2009-09-01

    DNA fingerprinting analysis such as amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic PCR (rep-PCR), ribosomal intergenic spacer analysis (RISA), and denaturing gradient gel electrophoresis (DGGE) are frequently used in various fields of microbiology. The major difficulty in DNA fingerprinting data analysis is the alignment of multiple peak sets. We report here an R program for a clustering-based peak alignment algorithm, and its application to analyze various DNA fingerprinting data, such as ARDRA, rep-PCR, RISA, and DGGE data. The results obtained by our clustering algorithm and by BioNumerics software showed high similarity. Since several R packages have been established to statistically analyze various biological data, the distance matrix obtained by our R program can be used for subsequent statistical analyses, some of which were not previously performed but are useful in DNA fingerprinting studies.

  15. Molecular phylogeny of the butterfly tribe Satyrini (Nymphalidae: Satyrinae) with emphasis on the utility of ribosomal mitochondrial genes 16s rDNA and nuclear 28s rDNA.

    PubMed

    Yang, Mingsheng; Zhang, Yalin

    2015-07-09

    The tribe Satyrini is one of the most diverse groups of butterflies, but no robust phylogenetic hypothesis for this group has been achieved. Two rarely used 16s and 28s ribosomal and another seven protein-coding genes were used to reconstruct the phylogeny of the Satyrini, with further aim to evaluate the informativeness of the ribosomal genes. Our maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) analyses consistently recovered three well-supported clades for the eleven sampled subtribes of Satyrini: clade I includes Eritina and Coenonymphina, being sister to the clade II + clade III; clade II contains Parargina, Mycalesina and Lethina, and the other six subtribes constitute clade III. The placements of the taxonomically unstable Davidina Oberthür and geographically restricted Paroeneis Moore in Satyrina are confirmed for the first time based on molecular evidence. The close relationships of Callerebia Butler, Loxerebia Watkins and Argestina Riley are well-supported. We suggest that Rhaphicera Butler belongs to Lethina. The partitioned Bremer support (PBS) values of MP analysis show that the 16s rDNA contributes well to the nodes representing all the taxa from subtribe to species levels, and the 28s rDNA is informative at the subtribe level. Furthermore, our ML analyses show that the ribosomal genes 16s rDNA and 28s rDNA are informative, because most node support values are lower in the ML tree after the removal of them than that in ML tree constructed based on the full nine-gene dataset. This indicates that some other ribosomal genes should be tentatively used through combining with traditionally used protein-coding genes in further analysis on phylogeny of Satyrini, providing that proper representatives are sampled.

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

    USDA-ARS?s Scientific Manuscript database

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

  17. Differences in ribosomal DNA distribution on A and B chromosomes between eastern and western populations of the grasshopper Eyprepocnemis plorans plorans.

    PubMed

    López-León, M D; Cabrero, J; Dzyubenko, V V; Bugrov, A G; Karamysheva, T V; Rubtsov, N B; Camacho, J P M

    2008-01-01

    Distribution of ribosomal DNA (rDNA) on standard (A) and supernumerary (B) chromosomes of the grasshopper Eyprepocnemis plorans was analysed in specimens collected in Turkey and Armenia, belonging to the E. p. plorans subspecies, and in South Africa, belonging to the E. p. meridionalis subspecies. The latter individuals showed rDNA loci in chromosomes 9 and 11 only, whereas those from Armenia carried it in chromosomes 9 and 11 or else in chromosomes 9-11, depending on the population. The specimens from Turkey carried it in chromosomes 1, 9-11 and X. A comparison of this pattern with those previously observed in populations from Spain, Morocco, and Greece (belonging to E. p. plorans) suggests the existence of two evolutionary patterns in rDNA chromosome location in A chromosomes of this subspecies: eastern populations showing rDNA restricted to the small (9-11) chromosomes (as in E. p. meridionalis and other closely related taxa within the Eyprepocneminae subfamily) and western populations carrying rDNA in most A chromosomes (Spain) or all of them (Morocco). The intermediate pattern discerned in geographically intermediate populations (in Greece and Turkey), with rDNA also being located on the X chromosome, suggests a possible east-west cline. Additional support for east-west differentiation in the rDNA location pattern comes from the analysis of B chromosomes. In eastern populations, including Daghestan, Armenia, Turkey, and Greece, B chromosomes are composed mostly of rDNA, whereas in western populations (Spain and Morocco) they contain roughly similar amounts of rDNA and a 180-bp tandem repeat (satDNA), the latter being scarce in eastern Bs. Copyright 2008 S. Karger AG, Basel.

  18. Seasonal shifts in population structure of Vibrio vulnificus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing.

    PubMed

    Lin, Meilan; Schwarz, John R

    2003-07-01

    Abstract The partial sequence (600 bp) containing the most variable region of Vibrio vulnificus 16S ribosomal DNA (rDNA) was determined for 208 randomly selected V. vulnificus strains isolated from Galveston Bay, TX, USA between June 2000 and June 2001. A comparative analysis of the determined partial 16S rDNA sequences revealed the existence of two different partial 16S rDNA sequences (type A and type B, 1.3% base substitutions) among the 208 V. vulnificus isolates. A higher proportion of 16S rDNA type A strains was isolated in June and July while a considerably higher proportion of type B strains was isolated in September. In addition, after no V. vulnificus strains were detected during the winter months (December-February), only type A strains were isolated during the following months (March-May). The results suggest that the relative abundance of type A and type B V. vulnificus strains in Galveston Bay varies with the season and that the differences between the two 16S rDNA types may affect the viability of these organisms in the natural environment.

  19. Anaerospora hongkongensis gen. nov. sp. nov., a novel genus and species with ribosomal DNA operon heterogeneity isolated from an intravenous drug abuser with pseudobacteremia.

    PubMed

    Woo, Patrick C Y; Teng, Jade L L; Leung, Kit-Wah; Lau, Susanna K P; Woo, Gibson K S; Wong, Amy C Y; Wong, Michelle K M; Yuen, Kwok-Yung

    2005-01-01

    A bacterium was isolated from the blood culture of an intravenous drug abuser with pseudobacteremia. The cells were strictly anaerobic, straight or slightly curved, sporulating, Gram-negative rods. It grew on sheep blood agar as non-hemolytic, pinpoint colonies after 48 hr of incubation at 37 C in an anaerobic environment. It was motile but did not produce catalase or cytochrome oxidase. 16S ribosomal DNA (rDNA) sequencing revealed three different copies of 16S rDNA sequences. More than 90% of the differences among them were due to differences in the lengths of the sequences. Phylogenetically, the bacterium is clustered with Dendrosporobacter, Sporomusa, and Propionispora, the other three genera of anaerobic, sporulating, Gram-negative rods. There were 8.6-11.1% differences between the 16S rDNA sequences of the bacterium and that of D. quercicolus, 4.7-15.1% differences between the 16S rDNA sequences of it and those of S. acidovorans, S. aerivorans, S. malonica, S. ovata, S. paucivorans, S. silvacetica, S. spaeroides, and S. termitida, and 7.6-13.1% differences between the 16S rDNA sequences of it and those of P. hippei and P. vibrioides. The G+C content of the bacterium (mean +/- SD) was 46.8 +/- 3.2%. For these reasons, a new genus and species, Anaerospora hongkongensis gen. nov. sp. nov., is proposed, for which HKU15T is the type strain.

  20. Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects.

    PubMed

    Poczai, Péter; Hyvönen, Jaakko

    2010-04-01

    The nuclear ribosomal locus coding for the large subunit is represented in tandem arrays in the plant genome. These consecutive gene blocks, consisting of several regions, are widely applied in plant phylogenetics. The regions coding for the subunits of the rRNA have the lowest rate of evolution. Also the spacer regions like the internal transcribed spacers (ITS) and external transcribed spacers (ETS) are widely utilized in phylogenetics. The fact, that these regions are present in many copies in the plant genome is an advantage for laboratory practice but might be problem for phylogenetic analysis. Beside routine usage, the rDNA regions provide the great potential to study complex evolutionary mechanisms, such as reticulate events or array duplications. The understanding of these processes is based on the observation that the multiple copies of rDNA regions are homogenized through concerted evolution. This phenomenon results to paralogous copies, which can be misleading when incorporated in phylogenetic analyses. The fact that non-functional copies or pseudogenes can coexist with ortholougues in a single individual certainly makes also the analysis difficult. This article summarizes the information about the structure and utility of the phylogenetically informative spacer regions of the rDNA, namely internal- and external transcribed spacer regions as well as the intergenic spacer (IGS).

  1. Intraspecific diversity of the Cassava green mite Mononychellus progresivus (Acari: Tetranychidae) using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding.

    PubMed

    Navajas, M; Gutierrez, J; Bonato, O; Bolland, H R; Mapangou-Divassa, S

    1994-06-01

    Intraspecific diversity in Cassava Green Mite Mononychellus progresivus Doreste was examined using individuals collected in Benin and the Congo and in Columbia and Brazil. Comparisons were based on mitochondrial and ribosomal DNA sequences and the results of several cross-breeding experiments. Sequence variation was examined in a total of 1139 base pairs (bp) constituting the ITS2 ribosomal DNA (805 bp) and a fragment of the Cytochrome Oxidase I (COI) gene (334 bp). Sequence divergence is low, ranging from 0% to 2.1% for COI and from 0% to 0.4% for ITS2. Inter-strain comparisons have shown that the two African populations appear to be identical. They were similar to the Colombian population while the Brazilian population was clearly different. The data support the hypothesis of a single introduction of the species in the two African populations. Crossing experiments have shown partial hybrid sterility, suggesting a genetic incompatibility consistent with differences detected by sequence data. The results show the usefulness of molecular markers as a tool for determining taxonomic status and dispersion paths in spider mites.

  2. Short report: variation in mitochondrial 12S and 16S ribosomal DNA sequences in natural populations of Triatoma infestans (Hemiptera: Reduviidae).

    PubMed

    García, Beatriz A; Manfredi, Candela; Fichera, Laura; Segura, Elsa L

    2003-06-01

    Mitochondrial DNA sequences of the 12S and 16S ribosomal RNA genes were analyzed in five natural populations of the Chagas' disease vector Triatoma infestans from Argentina. DNA sequence comparisons of 878 basepairs (12S plus 16S) revealed 13 haplotypes. A total of 10 private haplotypes were found in four of the populations analyzed, suggesting low current levels of genetic exchange. The levels of genetic differentiation between the population of Chancaní (Córdoba) and other two of the populations analyzed indicated significant deviation from a pattern of unrestricted gene flow. The haplotypic diversity and the private haplotypes found in the geographically closest localities of Chancaní and El Jardín (La Rioja) suggest that the reduction in the population size by insecticide treatment did not avoid the recovery of the populations apparently from survivors of the same area.

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

    PubMed

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

    2005-04-01

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

  4. Multigene family of ribosomal DNA in Drosophila melanogaster reveals contrasting patterns of homogenization for IGS and ITS spacer regions. A possible mechanism to resolve this paradox.

    PubMed Central

    Polanco, C; González, A I; de la Fuente; Dover, G A

    1998-01-01

    The multigene family of rDNA in Drosophila reveals high levels of within-species homogeneity and between-species diversity. This pattern of mutation distribution is known as concerted evolution and is considered to be due to a variety of genomic mechanisms of turnover (e.g., unequal crossing over and gene conversion) that underpin the process of molecular drive. The dynamics of spread of mutant repeats through a gene family, and ultimately through a sexual population, depends on the differences in rates of turnover within and between chromosomes. Our extensive molecular analysis of the intergenic spacer (IGS) and internal transcribed spacer (ITS) spacer regions within repetitive rDNA units, drawn from the same individuals in 10 natural populations of Drosophila melanogaster collected along a latitudinal cline on the east coast of Australia, indicates a relatively fast rate of X-Y and X-X interchromosomal exchanges of IGS length variants in agreement with a multilineage model of homogenization. In contrast, an X chromosome-restricted 24-bp deletion in the ITS spacers is indicative of the absence of X-Y chromosome exchanges for this region that is part of the same repetitive rDNA units. Hence, a single lineage model of homogenization, coupled to drift and/or selection, seems to be responsible for ITS concerted evolution. A single-stranded exchange mechanism is proposed to resolve this paradox, based on the role of the IGS region in meiotic pairing between X and Y chromosomes in D. melanogaster. PMID:9584100

  5. Ribosomal DNA location in the scarab beetle Thorectes intermedius (Costa) (Coleoptera: Geotrupidae) using banding and fluorescent in-situ hybridization.

    PubMed

    Vitturi, R; Colomba, M S; Barbieri, R; Zunino, M

    1999-01-01

    Mitotic metaphase chromosomes of the scarab beetle Thorectes intermedius (Costa) (Coleoptera Scarabaeoidea: Geotrupidae) were analyzed using various banding methods and fluorescent in-situ hybridization (FISH) with a ribosomal probe. The results obtained indicate that silver and CMA3 staining are unable to localize the chromosome sites of nucleolar organizer regions (NORs). Such an inadequacy is a consequence of the extensive silver and CMA3 stainability of both constitutive heterochromatin and heterochromatin associated to the NORs.

  6. Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus).

    PubMed

    Harada, Tetsuya; Kawai, Takao; Jinnai, Michio; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko; Kumeda, Yuko

    2012-09-01

    Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10(1) spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations.

  7. Molecular Evolution of Aromatic Polyketides and Comparative Sequence Analysis of Polyketide Ketosynthase and 16S Ribosomal DNA Genes from Various Streptomyces Species

    PubMed Central

    Metsä-Ketelä, Mikko; Halo, Laura; Munukka, Eveliina; Hakala, Juha; Mäntsälä, Pekka; Ylihonko, Kristiina

    2002-01-01

    A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the γ-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes. PMID:12200302

  8. Transcription Termination Factor reb1p Causes Two Replication Fork Barriers at Its Cognate Sites in Fission Yeast Ribosomal DNA In Vivo

    PubMed Central

    Sánchez-Gorostiaga, Alicia; López-Estraño, Carlos; Krimer, Dora B.; Schvartzman, Jorge B.; Hernández, Pablo

    2004-01-01

    Polar replication fork barriers (RFBs) near the 3′ end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3′ to 5′ order. The transcription termination protein reb1 and its two binding sites, present at the 3′ end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist. PMID:14673172

  9. Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

    PubMed Central

    Edgcomb, Virginia P.; McDonald, John H.; Devereux, Richard; Smith, David W.

    1999-01-01

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 107 to 2.5 × 109 (average, 1.1 × 109 ± 5.2 × 108) cells g of sediment−1. In September, numbers of SRB detected ranged from 5.4 × 108 to 7.3 × 109 (average, 2.5 × 109 ± 1.5 × 109) cells g of sediment−1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments. PMID:10103245

  10. Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections

    PubMed Central

    Ferrer, Consuelo; Colom, Francisca; Frasés, Susana; Mulet, Emilia; Abad, José L.; Alió, Jorge L.

    2001-01-01

    The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples. PMID:11474006

  11. Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

    PubMed Central

    Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

    2015-01-01

    Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

  12. Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription.

    PubMed

    Yamamoto, K; Koga, A; Yamamoto, M; Nishi, Y; Tamura, T; Nogi, Y; Muramatsu, M

    2000-03-01

    Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins directly bound to the core promoter region of murine rDNA. With these techniques, we identified a 70 kDa protein (p70) in the flow-through fraction of a phosphocellulose column (TFIA-fraction). Interestingly, the binding of p70 to the rDNA core promoter was observed only in the presence of the SL1-containing fraction. The probable human orthologue of p70 was also detected in HeLa cells. Consistent with the observation that p70 bound to the core promoter only in the presence of the TFIA- and SL1-fractions, alteration of DNase I footprint pattern over the core promoter element was demonstrated by cooperative action of the TFIA- and SL1-fractions. A reconstituted in vitro transcription assay with further purified p70 indicated that p70 was required for accurate initiation of rDNA transcription. These results indicate that the p70 identified recently by the current DNA-binding experiments represents a novel transcription factor in rDNA transcription.

  13. Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA.

    PubMed

    Zhu, X Q; Podolska, M; Liu, J S; Yu, H Q; Chen, H H; Lin, Z X; Luo, C B; Song, H Q; Lin, R Q

    2007-11-01

    Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecum osculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.

  14. Evaluation of immunogenicity and protective efficacy of a plasmid DNA vaccine encoding ribosomal protein L9 of Brucella abortus in BALB/c mice.

    PubMed

    Jain, Shikha; Afley, Prachiti; Dohre, Sudhir K; Saxena, Nandita; Kumar, Subodh

    2014-07-31

    Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans and existing animal vaccines have limitations. We have previously described the ribosomal protein L9 to have the vaccine potential. In this study, L9 based DNA vaccine (pVaxL9) was generated and evaluated in mouse model. Intramuscular immunisation of pVaxL9 was able to elicit the anti-L9 IgG antibody response of both IgG1 and IgG2a isotypes when compared with PBS and pVax immunised control animals. Heightened antibody response was observed in mice groups immunised with pVaxL9 priming and recombinant L9 boosting (PB) and where pDNA immunisation was carried out by in vivo electroporation (EP). The vaccine groups proliferated splenocytes and released Th1 type cytokines e.g. IFN-γ, TNF-α, IL-2. Further, flow cytometric analysis revealed that IFN-γ was released by both by CD4+ and CD8+ T cells particularly in PB and EP groups when compared with mice immunised with empty control vector. The L9 based pDNA vaccine was able to confer significant protection in mice against challenge with virulent B. abortus with PB and EP groups offering better protection. Taken together, it can be concluded that L9 based DNA vaccine is immunogenic and confer protection in mouse model.

  15. Patterns of evolution in Discula fungi and the origin of dogwood anthracnose in North America, studied using arbitrarily amplified and ribosomal DNA.

    PubMed

    Caetano-Anollés, G; Trigiano, R N; Windham, M T

    2001-07-01

    The anthracnose epidemic caused by exotic filamentous fungi of the genus Discula threatens the future of the prized flowering (Cornus florida L.) and Pacific (C. nuttalli Aud.) dogwoods in North America. A cross-section of fungi that cause anthracnose in broadleaf temperate trees was characterized using DNA amplification fingerprinting, sequence and secondary structure analysis of the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), and compatibility of hyphal anastomosis. ITS-inferred phylogenies rejected the null hypothesis of only one fungal lineage, by defining four monophyletic and well differentiated groups, corresponding to Discula sp., D. quercina, D. umbrinella and D. destructiva, with the last two species sharing a common and recent ancestor. In turn, they showed that the dogwood pathogen, D. destructiva, did not evolve directly from an indigenous population related to Discula sp. In this study, rDNA spacers that are generally considered important for protein synthesis but are selectively neutral, appeared functionally constrained and subject to selective sequence diversification. Results confirmed the high variability of D. umbrinella and the remarkable homogeneity and exotic nature of D. destructiva at the genetic level, clarified the taxonomy and phylogeny of Discula, and provided clues as to the origin and diversification of dogwood anthracnose-causing fungi.

  16. Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

    NASA Astrophysics Data System (ADS)

    Su, Lei; Zhang, Qianqian; Gong, Jun

    2017-07-01

    Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

  17. Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors.

    PubMed

    Osterman, Ilya A; Komarova, Ekaterina S; Shiryaev, Dmitry I; Korniltsev, Ilya A; Khven, Irina M; Lukyanov, Dmitry A; Tashlitsky, Vadim N; Serebryakova, Marina V; Efremenkova, Olga V; Ivanenkov, Yan A; Bogdanov, Alexey A; Sergiev, Petr V; Dontsova, Olga A

    2016-12-01

    In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta'-subunit of RNA polymerase.

    PubMed Central

    Cozens, A L; Walker, J E

    1986-01-01

    The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III. PMID:3530249

  19. Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species, little divergence

    PubMed Central

    Turner, Barbara; Paun, Ovidiu; Munzinger, Jérôme; Chase, Mark W.; Samuel, Rosabelle

    2016-01-01

    Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species. Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices. Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species. Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with

  20. The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice.

    PubMed

    Iborra, Salvador; Soto, Manuel; Carrión, Javier; Nieto, Ana; Fernández, Edgar; Alonso, Carlos; Requena, Jose M

    2003-11-01

    In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-gamma) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4(+) and CD8(+) T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-gamma. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

  1. Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells

    SciTech Connect

    Egyhazi, E.; Pigon, A. ); Chang, Jinhong; Ghaffari, S.H.; Dreesen, T.D.; Wellman, S.E.; Case, S.T.; Olson, M.O.J. )

    1988-10-01

    Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with {sup 32}P-labeled RNA precursors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of {sup 32}P incorporation into 38 S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of {sup 32}P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of {sup 32}P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

  2. Use of Amplified Ribosomal DNA Restriction Analysis for Identification of Ralstonia and Pandoraea Species: Interest in Determination of the Respiratory Bacterial Flora in Patients with Cystic Fibrosis

    PubMed Central

    Segonds, Christine; Paute, Sandrine; Chabanon, Gérard

    2003-01-01

    The recovery of Ralstonia and Pandoraea species from respiratory tract cultures of patients with cystic fibrosis has recently been reported. These species are difficult to identify, and especially to differentiate from Burkholderia cepacia complex organisms, with classical methods. The discriminatory power of amplified ribosomal DNA restriction analysis (ARDRA) within the two genera was assessed by comparing the restriction profiles of reference strains of each species by using a panel of six enzymes already proven suitable for the identification of Burkholderia species. ARDRA provided differentiation of all the Ralstonia species tested and of Pandoraea norimbergensis. Pandoraea species P. pnomenusa, P. sputorum, P. pulmonicola, and P. apista were not discriminated to the species level. This method allowed the identification of five clinical isolates recovered from French cystic fibrosis patients as Ralstonia mannitolilytica. PMID:12843108

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

    PubMed

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

    2013-02-28

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

  4. A paraphyly of the genus Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea) inferred from internal transcribed spacer-2 and 18S ribosomal DNA sequences.

    PubMed

    Skeríková, Andrea; Hypsa, Václav; Scholz, Tomás

    2004-06-01

    Phylogenetic relationships between Bothriocephalus species from freshwater and marine teleosts from different geographical regions were studied using internal transcribed spacer-2 and partial 18S ribosomal DNA. The analyses revealed a paraphyly of Bothriocephalus with respect to the genera Polyonchobothrium, Anantrum, and Clestobothrium. The freshwater species Bothriocephalus claviceps, B. acheilognathi, and Bothriocephalus sp. from Dorosoma petenense formed a well-supported monophyletic cluster, with Polyonchobothrium at its base. In contrast, the type species, B. scorpii, clustered within a distinct lineage formed by a heterogeneous assemblage of marine species, Clestobothrium crassiceps and Anantrum tortum, and the freshwater species B. cf. japonicus. This shows that the current morphology-based classification is unlikely to reflect the phylogenetic relationships within this group and will require a thorough revision.

  5. Non-monophyly of most supraspecific taxa of calcareous sponges (Porifera, Calcarea) revealed by increased taxon sampling and partitioned Bayesian analysis of ribosomal DNA.

    PubMed

    Dohrmann, Martin; Voigt, Oliver; Erpenbeck, Dirk; Wörheide, Gert

    2006-09-01

    Calcareous sponges (Porifera, Calcarea) play an important role for our understanding of early metazoan evolution, since several molecular studies suggested their closer relationship to Eumetazoa than to the other two sponge 'classes,' Demospongiae and Hexactinellida. The division of Calcarea into the subtaxa Calcinea and Calcaronea is well established by now, but their internal relationships remain largely unresolved. Here, we estimate phylogenetic relationships within Calcarea in a Bayesian framework, using full-length 18S and partial 28S ribosomal DNA sequences. Both genes were analyzed separately and in combination and were further partitioned by stem and loop regions, the former being modelled to take non-independence of paired sites into account. By substantially increasing taxon sampling, we show that most of the traditionally recognized supraspecific taxa within Calcinea and Calcaronea are not monophyletic, challenging the existing classification system, while monophyly of Calcinea and Calcaronea is again highly supported.

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

    PubMed Central

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

    2004-01-01

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

  7. Molecular Analysis of the Lance Nematode, Hoplolaimus spp., Using the First Internal Transcribed Spacer and the D1-D3 Expansion Segments of 28S Ribosomal DNA1

    PubMed Central

    Bae, CH; Szalanski, AL; Robbins, RT

    2008-01-01

    DNA sequence analyses of the nuclear ribosomal ITS1 region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene were conducted to characterize the genetic variation of six amphimictic Hoplolaimus species, including H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus and H. seinhorsti. PCR amplifications of the combined D1-D3 expansion segments and the ITS1 region each yielded one distinct amplicon. In the D1-D3 region, there was no nucleotide sequence variation between populations of H. columbus, H. magnistylus, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, whereas the ITS1 sequences had nucleotide variation among species. We detected conserved ITS1 regions located at the 3’ and 5’ end of ITS1 and also in the middle of the ITS1 among Hoplolaimus species. These regions were compared with sequences of distantly related Heterodera and Globedera. PCR-RFLP and sequence analysis of ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus and Hoplolaimus sp. 1. Maximum likelihood and maximum parsimony analysis using the combined ITS1 and D1-D3 expansion segment sequences always produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species (H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution. PMID:19440260

  8. Characterisation of Lymnaea cubensis, L. viatrix and L. neotropica n. sp., the main vectors of Fasciola hepatica in Latin America, by analysis of their ribosomal and mitochondrial DNA.

    PubMed

    Bargues, M D; Artigas, P; Mera Y Sierra, R L; Pointier, J P; Mas-Coma, S

    2007-10-01

    Although, in the endemic areas throughout the world, human fascioliasis presents varying patterns in its epidemiology, the species of lymnaeid snail that act as intermediate hosts and vectors are always crucial in the transmission of the causative parasites. Species in the Galba/Fossaria group of snails, such as Lymnaea cubensis, L. viatrix var. A ventricosa, L. viatrix var. B elongata and Galba truncatula, appear to be frequently involved in the transmission of Fasciola hepatica in Central and South America, although specific classification within this morphologically and anatomically confusing group is often very difficult. To explore the potential use of molecular analyses in the identification of vector snails, regions of the ribosomal DNA - the small subunit (18S) gene and internal transcribed spacers (ITS-2 and ITS-1) - and of the mitochondrial DNA - the cytochrome c oxidase subunit I (COI) - of wild-caught lymnaeid snails of L. cubensis, L. viatrix var. A ventricosa, L. viatrix var. B elongata and G. truncatula have been sequenced. The samples of the Latin American species included specimens from the respective type localities. The genetic distances observed and the results of phylogenetic analyses demonstrate that two different species exist within L. viatrix. Lymnaea neotropica n. sp. (=L. viatrix var. B elongata) is here proposed for specimens from Lima, Peru, and is differentiated from L. viatrix (=L. viatrix var. A ventricosa), L. cubensis and G. truncatula. The data collected on the 18S ribosomal-RNA gene indicate that the snails investigated may cover more than one supraspecific taxon. The ITS-2, ITS-1 and COI nucleotide sequences are clearly useful markers for the differentiation of these morpho-anatomically similar lymnaeid species. The numerous microsatellite repeats found within ITS-2 are potential tools for differentiation at population level.

  9. cDNA cloning, overexpression, purification and pharmacologic evaluation for anticancer activity of ribosomal protein L23A gene (RPL23A) from the Giant Panda.

    PubMed

    Sun, Bing; Hou, Yi-Ling; Hou, Wan-Ru; Zhang, Si-Nan; Ding, Xiang; Su, Xiu-Lan

    2012-01-01

    RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A) gene of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF) of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 μg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids orientation for

  10. Phylogenetic relationships among phrynosomatid lizards as inferred from mitochondrial ribosomal DNA sequences: substitutional bias and information content of transitions relative to transversions.

    PubMed

    Reeder, T W

    1995-06-01

    The phylogenetic relationships among 40 species, representing all genera, within the North American lizard family Phrynosomatidae were inferred from mitochondrial ribosomal RNA gene sequences. Cladistic analysis of the DNA sequence data (779 bp; 162 informative characters) supported the monophyly of the sand lizards (Callisaurus, Cophosaurus, Holbrookia, and Uma), Petrosaurus, Phrynosoma, Urosaurus, and Uta. All the species of Sceloporus, except S. variabilis and S. chrysostictus, formed a clade. Except for a sand lizard + Phrynosoma clade, the intergeneric relationships inferred from the mtDNA were largely incongruent with recent cladistic analyses based on morphology. Sceloporus group monophyly was not supported, with Petrosaurus being a member of a clade containing Sator, Sceloporus, and Urosaurus, to the exclusion of Uta. The phylogenetic placement of Uta was ambiguous. The substitutional bias in the phrynosomatid mitochondrial rDNA sequences was examined, as well as the phylogenetic information content of transitions relative to transversions. There appeared to be a lower transition bias than observed in other vertebrate sequences, with some classes of transversions occurring as frequently as G <-> A transitions. Transitions were no less informative for phylogeny reconstruction than transversions. Therefore, transitions should not be down-weighted in phylogenetic analysis, as is often done.

  11. DIVERSITY OF BAT-ASSOCIATED LEPTOSPIRA IN THE PERUVIAN AMAZON INFERRED BY BAYESIAN PHYLOGENETIC ANALYSIS OF 16S RIBOSOMAL DNA SEQUENCES

    PubMed Central

    MATTHIAS, MICHAEL A.; DÍAZ, M. MÓNICA; CAMPOS, KALINA J.; CALDERON, MARITZA; WILLIG, MICHAEL R.; PACHECO, VICTOR; GOTUZZO, EDUARDO; GILMAN, ROBERT H.; VINETZ, JOSEPH M.

    2008-01-01

    The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans. PMID:16282313

  12. Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures.

    PubMed

    Takishita, Kiyotaka; Yubuki, Naoji; Kakizoe, Natsuki; Inagaki, Yuji; Maruyama, Tadashi

    2007-07-01

    Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the "hidden" microeukaryotic diversity in the environment examined.

  13. Morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence distinctions between two species Platygyra (Cnidaria: Scleractinia) from Hong Kong [corrected].

    PubMed

    Lam, Katherine; Morton, Brian

    2003-01-01

    Two sympatric species of Platygyra have been identified from Hong Kong waters: i.e., P. sinensis and P. pini. The former has been further subdivided into 4 morphotypes based on colony growth form as follows: classic, encrusting, hillocky, and long-valley. Taxonomic confusion raised by overlapping morphological variations and frequent sympatric occurrences, however, has posed problems in relation to Platygyra ecology and population dynamics. This study attempted to differentiate Platygyra pini and morphotypes of P. sinensis by both morphological and ITS1, 5.8S, and partial ITS2 ribosomal DNA sequence analysis. Morphological data based on 9 skeletal characters were subjected to multivariate analysis. No clear groupings were obtained using a multidimensional scaling plot. Most parsimony analysis was conducted using either the rDNA data set including ITS1, 5.8S, and partial ITS2 or the ITS1 region only. Maximum parsimony (MP) and neighbor-joining (NJ) trees obtained from both data sets, clustered samples of P. sinensis and P. pini into 2 clades. The interspecific Kimura 2-parameter sequence divergence value (k2) obtained by the former rDNA data set was 14.275 +/- 0.507%, which is greater than the intraspecific values (1.239 +/- 1.147% for P. sinensis and 0.469 +/- 0.364% for P. pini), indicating that this marker of ITS1, 5.8S, and ITS2 contains substantially high levels of inherent diversity and is useful in resolving the problematic taxonomy of Platygyra.

  14. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

    PubMed

    Wang, Yong; Qian, Pei-Yuan

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

  15. Molecular characterization of Hysterothylacium aduncum (Nematoda: Raphidascaridae) from different fish caught off the Tunisian coast based on nuclear ribosomal DNA sequences.

    PubMed

    Amor, Nabil; Farjallah, Sarra; Merella, Paolo; Said, Khaled; Ben Slimane, Badreddine

    2011-11-01

    Larval forms of the genus Hysterothylacium have been previously reported in teleost fish from the North African coasts of central Mediterranean Sea by morphological analysis. In the present study, samples identified morphologically as Hysterothylacium aduncum (n = 62), from Merluccius merluccius, Trachurus mediterraneus and Pagellus erythrinus from different geographical locations of the Tunisian coasts, were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the sequences obtained with those available in public gene databases confirmed that all the samples from the Tunisian coasts belong to a single species, namely H. aduncum. All specimens from the Tunisian coasts showed one indel in position 787 in ITS-2 sequences not reported by any of the previously published sequences from the Atlantic and Pacific Oceans, Adriatic Sea (Mediterranean Sea) and the East Greenland Sea, suggesting the existence of a population-specific pattern exhibiting a low differentiation of this parasite in this area. This is the first molecular characterization of H. aduncum from the Tunisian coasts using ITS rDNA sequences which allows the definition of genetic markers for their unequivocal identification, and provides further biological data on these nematodes in marine fish off the Tunisian coasts, improving the picture of the occurrence of these taxa in the North African coasts of central Mediterranean Sea.

  16. Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment.

    PubMed

    Tsuge, Kenji; Matsui, Kuniko; Itaya, Mitsuhiro

    2007-05-10

    Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6kb), and sfp (1.0kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40kb.

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

    PubMed

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

    2009-09-01

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

  18. Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

    PubMed

    Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

    2014-04-01

    Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

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

    PubMed

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

    1997-05-01

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

  20. The ribosomal transcription units of Haplorchis pumilio and H. taichui and the use of 28S rDNA sequences for phylogenetic identification of common heterophyids in Vietnam.

    PubMed

    Le, Thanh Hoa; Nguyen, Khue Thi; Nguyen, Nga Thi Bich; Doan, Huong Thi Thanh; Dung, Do Trung; Blair, David

    2017-01-09

    Heterophyidiasis is now a major public health threat in many tropical countries. Species in the trematode family Heterophyidae infecting humans include Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. For molecular phylogenetic and systematic studies on trematodes, we need more prospective markers for taxonomic identification and classification. This study provides near-complete ribosomal transcription units (rTU) from Haplorchis pumilio and H. taichui and demonstrates the use of 28S rDNA sequences for identification and phylogenetic analysis. The near-complete ribosomal transcription units (rTU), consisting of 18S, ITS1, 5.8S, ITS2 and 28S rRNA genes and spacers, from H. pumilio and H. taichui from human hosts in Vietnam, were determined and annotated. Sequence analysis revealed tandem repetitive elements in ITS1 in H. pumilio and in ITS2 in H. taichui. A phylogenetic tree inferred from 28S rDNA sequences of 40 trematode strains/species, including 14 Vietnamese heterophyid individuals, clearly confirmed the status of each of the Vietnamese species: Centrocestus formosanus, Haplorchis pumilio, H. taichui, H. yokogawai, Procerovum varium and Stellantchasmus falcatus. However, the family Heterophyidae was clearly not monophyletic, with some genera apparently allied with other families within the superfamily Opisthorchioidea (i.e. Cryptogonimidae and Opisthorchiidae). These families and their constituent genera require substantial re-evaluation using a combination of morphological and molecular data. Our new molecular data will assist in such studies. The 28S rDNA sequences are conserved among individuals within a species but varied between genera. Based on analysis of 40 28S rDNA sequences representing 19 species in the superfamily Opisthorchioidea and an outgroup taxon (Alaria alata, family Diplostomidae), six common human pathogenic heterophyids were identified and clearly resolved. The

  1. Characterization of the Dominant and Rare Members of a Young Hawaiian Soil Bacterial Community with Small-Subunit Ribosomal DNA Amplified from DNA Fractionated on the Basis of Its Guanine and Cytosine Composition

    PubMed Central

    Nüsslein, Klaus; Tiedje, James M.

    1998-01-01

    The small-subunit ribosomal DNA (rDNA) diversity was found to be very high in a Hawaiian soil community that might be expected to have lower diversity than the communities in continental soils because the Hawaiian soil is geographically isolated and only 200 years old, is subjected to a constant climate, and harbors low plant diversity. Since an underlying community structure could not be revealed by analyzing the total eubacterial rDNA, we first fractionated the DNA on the basis of guanine-plus-cytosine (G+C) content by using bis-benzimidazole and equilibrium centrifugation and then analyzed the bacterial rDNA amplified from a fraction with a high biomass (63% G+C fraction) and a fraction with a low biomass (35% G+C fraction). The rDNA clone libraries were screened by amplified rDNA restriction analysis to determine phylotype distribution. The dominant biomass reflected by the 63% G+C fraction contained several dominant phylotypes, while the community members that were less successful (35% G+C fraction) did not show dominance but there was a very high diversity of phylotypes. Nucleotide sequence analysis revealed taxa belonging to the groups expected for the G+C contents used. The dominant phylotypes in the 63% G+C fraction were members of the Pseudomonas, Rhizobium-Agrobacterium, and Rhodospirillum assemblages, while all of the clones sequenced from the 35% G+C fraction were affiliated with several Clostridium assemblages. The two-step rDNA analysis used here uncovered more diversity than can be detected by direct rDNA analysis of total community DNA. The G+C separation step is also a way to detect some of the less dominant organisms in a community. PMID:9546163

  2. Mitochondrial ribosomes in cancer.

    PubMed

    Kim, Hyun-Jung; Maiti, Priyanka; Barrientos, Antoni

    2017-04-23

    Mitochondria play fundamental roles in the regulation of life and death of eukaryotic cells. They mediate aerobic energy conversion through the oxidative phosphorylation (OXPHOS) system, and harbor and control the intrinsic pathway of apoptosis. As a descendant of a bacterial endosymbiont, mitochondria retain a vestige of their original genome (mtDNA), and its corresponding full gene expression machinery. Proteins encoded in the mtDNA, all components of the multimeric OXPHOS enzymes, are synthesized in specialized mitochondrial ribosomes (mitoribosomes). Mitoribosomes are therefore essential in the regulation of cellular respiration. Additionally, an increasing body of literature has been reporting an alternative role for several mitochondrial ribosomal proteins as apoptosis-inducing factors. No surprisingly, the expression of genes encoding for mitoribosomal proteins, mitoribosome assembly factors and mitochondrial translation factors is modified in numerous cancers, a trait that has been linked to tumorigenesis and metastasis. In this article, we will review the current knowledge regarding the dual function of mitoribosome components in protein synthesis and apoptosis and their association with cancer susceptibility and development. We will also highlight recent developments in targeting mitochondrial ribosomes for the treatment of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

    PubMed Central

    Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

    2001-01-01

    The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

  4. Sequence variability in internal transcribed spacers of nuclear ribosomal DNA among isolates of the oxyurid nematodes Syphacia obvelata and Aspiculuris tetraptera from mice reared in laboratories in China.

    PubMed

    Qiu, J H; Lou, Y; Zhang, Y; Chang, Q C; Liu, Z X; Duan, H; Guo, D H; Gao, D Z; Yue, D M; Wang, C R

    2016-01-01

    This study examined sequence variability in internal transcribed spacers (ITS) of nuclear ribosomal DNA among Syphacia obvelata and Aspiculuris tetraptera isolates from laboratory mice from different geographical locations in China. ITS1, 5.8S and ITS2 rDNA were amplified separately from adult S. obvelata and A. tetraptera individuals by polymerase chain reaction (PCR), and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of ITS1, 5.8S and ITS2 rDNA from both nematodes were 314 bp and 456 bp, 157 bp, and 273 bp and 419 bp, respectively. The intraspecific sequence variations in S. obvelata ITS1 were 0-0.3%. For A. tetraptera they were 0-0.7% in ITS1 and 0-1.0% in ITS2. However, the interspecific sequence differences among members of the infraorder Oxyuridomorpha were significantly higher, being 54.0-65.5% for ITS1 and 55.3-64.1% for ITS2. Phylogenetic analysis based on the combined partial sequences of ITS1 and ITS2 using three inference methods - Bayesian inference, maximum likelihood and maximum parsimony - revealed that all the S. obvelata and A. tetraptera samples formed independent monophyletic groups. Syphacia obvelata was closer to Syphacia muris than to A. tetraptera, consistent with morphological classification. These results demonstrate that ITS1 and ITS2 rDNA sequences are useful markers for population genetic studies of oxyurid nematodes.

  5. A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

    PubMed Central

    Emond, E; Fliss, I; Pandian, S

    1993-01-01

    cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate. Images PMID:8368854

  6. 16S ribosomal DNA sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Köhrer, Karl; Gougoula, Christina; Sager, Martin

    2014-10-01

    Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥ 99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥ 97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥ 95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.

  7. The DNA virus white spot syndrome virus uses an internal ribosome entry site for translation of the highly expressed nonstructural protein ICP35.

    PubMed

    Kang, Shih-Ting; Wang, Han-Ching; Yang, Yi-Ting; Kou, Guang-Hsiung; Lo, Chu-Fang

    2013-12-01

    Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (∼300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5' untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the α subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.

  8. Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae).

    PubMed

    Vesgueiro, Fabiana Tavares; Demari-Silva, Bruna; Malafronte, Rosely dos Santos; Sallum, Maria Anice Mureb; Marrelli, Mauro Toledo

    2011-02-01

    Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.

  9. The first determination of Trichuris sp. from roe deer by amplification and sequenation of the ITS1-5.8S-ITS2 segment of ribosomal DNA.

    PubMed

    Salaba, O; Rylková, K; Vadlejch, J; Petrtýl, M; Scháňková, S; Brožová, A; Jankovská, I; Jebavý, L; Langrová, I

    2013-03-01

    Trichuris nematodes were isolated from roe deer (Capreolus capreolus). At first, nematodes were determined using morphological and biometrical methods. Subsequently genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from ribosomal DNA (RNA) was amplified and sequenced using PCR techniques. With u sing morphological and biometrical methods, female nematodes were identified as Trichuris globulosa, and the only male was identified as Trichuris ovis. The females were classified into four morphotypes. However, analysis of the internal transcribed spacers (ITS1-5.8S-ITS2) of specimens did not confirm this classification. Moreover, the female individuals morphologically determined as T. globulosa were molecularly identified as Trichuris discolor. In the case of the only male molecular analysis match the result of the molecular identification. Furthermore, a comparative phylogenetic study was carried out with the ITS1 and ITS2 sequences of the Trichuris species from various hosts. A comparison of biometric information from T. discolor individuals from this study was also conducted.

  10. Ribosomal tag pyrosequencing of DNA and RNA from benthic coral reef microbiota: community spatial structure, rare members and nitrogen-cycling guilds.

    PubMed

    Gaidos, Eric; Rusch, Antje; Ilardo, Melissa

    2011-05-01

    Ribosomal tag libraries based on DNA and RNA in coral reef sediment from Hawaii show the microbial community to be dominated by the bacterial phyla Proteobacteria, Firmicutes and Actinobacteria, the archaeal order Nitrosopumilales and the uncultivated divisions Marine Group III (Euryarchaeota) and Marine Benthic Group C (Crenarchaeota). Operational taxonomic units (OTUs) number in the high thousands, and richness varies with site, presence or absence of porewater sulfide (sediment depth), and nucleotide pool. Rank abundance curves of DNA-based libraries, but not RNA-based libraries, possess a tail of low abundance taxa, supporting the existence of an inactive 'rare' biosphere. While bacterial libraries from two oxic samples differ markedly, those from two anoxic (sulfidic) samples are similar. The four dominant bacterial OTUs are members of genera that include pathogens, but are found in marine environments, and include facultative anaerobes, i.e. dissimilatory nitrate reducers and denitrifiers. This may explain their abundance in both oxic and anoxic samples. A numerous archaeon is closely related to the lithoautotrophic ammonia oxidizer Nitrosopumilus maritimus. Known bacterial ammonia oxidizers are essentially absent, but bacterial nitrite oxidizers are abundant. Although other studies of this reef found evidence for anaerobic ammonia oxidizers, primer bias rendered that clade invisible to this study. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  11. Nucleotide sequence of Marchantia polymorpha chloroplast DNA: a region possibly encoding three tRNAs and three proteins including a homologue of E. coli ribosomal protein S14.

    PubMed Central

    Umesono, K; Inokuchi, H; Ohyama, K; Ozeki, H

    1984-01-01

    The nucleotide sequence of a region of Marchantia polymorpha chloroplast DNA was determined. On this DNA sequence (3.38kb), three open reading frames (ORFs) and three putative tRNA genes were detected in the following order: -ORF701-tRNASer(UGA)-ORF702-tRNAGly(GCC)-initiator tRNAMet(CAU)-ORF703-. The ORF703 is composed of 100 codons in which those for lysine (15%) and arginine (11%) are abundant, and could be accounted for as a counterpart of E. coli ribosomal protein S14 since they share 45% homology in the amino acid sequences. The ORF701 appears to code for a membrane protein, showing a periodic appearance of seven clusters of hydrophobic amino acids. Although the mechanisms remain unknown, the ORF701 causes a streptomycin-sensitive phenotype in resistant mutants of E. coli. The ORFs and tRNA genes are separated from each other by extremely AT-rich spacers containing sequences of dyad symmetry. The third letter positions of the codons in the ORFs are also rich in A and T residues. PMID:6393057

  12. Molecular phylogeny and genetic variation in the genus Lilium native to China based on the internal transcribed spacer sequences of nuclear ribosomal DNA.

    PubMed

    Du, Yun-peng; He, Heng-bin; Wang, Zhong-xuan; Li, Shuang; Wei, Chi; Yuan, Xiao-na; Cui, Qi; Jia, Gui-xia

    2014-03-01

    We present a comprehensive phylogeny derived from nuclear ribosomal DNA (nrDNA) for 214 samples representing 98 species and five varieties, including 44 species and five varieties native to China. Our collection of 25 species and five varieties (44 samples) covering all five sections of the genus (Comber) distributed in China also were included in the internal transcribed spacer (ITS) database. This study incorporates previous research with an emphasis on Chinese species, including the controversial subsection, Sinomartagon 5c Comber. In the phylogenetic tree obtained by maximum parsimony (PAUP) and maximum likelihood (RAxML) analyses, the samples were divided into four major groups. Our results suggest that the subsection (subsect.) 5c Comber should be classified into the true subsect. 5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum I, subsect. Lophophorum II, and subsect. Lophophorum III). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, we report that L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion 6a. Our results support Comber's subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.

  13. Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

    PubMed Central

    Rogers, G. B.; Hart, C. A.; Mason, J. R.; Hughes, M.; Walshaw, M. J.; Bruce, K. D.

    2003-01-01

    The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients. PMID:12904354

  14. Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles

    PubMed Central

    Woo, Patrick C. Y.; Ng, Kenneth H. L.; Lau, Susanna K. P.; Yip, Kam-tong; Fung, Ami M. Y.; Leung, Kit-wah; Tam, Dorothy M. W.; Que, Tak-lun; Yuen, Kwok-yung

    2003-01-01

    Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S r

  15. Characterization of Trichuris trichiura from humans and T. suis from pigs in China using internal transcribed spacers of nuclear ribosomal DNA.

    PubMed

    Liu, G H; Zhou, W; Nisbet, A J; Xu, M J; Zhou, D H; Zhao, G H; Wang, S K; Song, H Q; Lin, R Q; Zhu, X Q

    2014-03-01

    Trichuris trichiura and Trichuris suis parasitize (at the adult stage) the caeca of humans and pigs, respectively, causing trichuriasis. Despite these parasites being of human and animal health significance, causing considerable socio-economic losses globally, little is known of the molecular characteristics of T. trichiura and T. suis from China. In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of T. trichiura and T. suis from China were amplified by polymerase chain reaction (PCR), the representative amplicons were cloned and sequenced, and sequence variation in the ITS rDNA was examined. The ITS rDNA sequences for the T. trichiura and T. suis samples were 1222-1267 bp and 1339-1353 bp in length, respectively. Sequence analysis revealed that the ITS-1, 5.8S and ITS-2 rDNAs of both whipworms were 600-627 bp and 655-661 bp, 154 bp, and 468-486 bp and 530-538 bp in size, respectively. Sequence variation in ITS rDNA within and among T. trichiura and T. suis was examined. Excluding nucleotide variations in the simple sequence repeats, the intra-species sequence variation in the ITS-1 was 0.2-1.7% within T. trichiura, and 0-1.5% within T. suis. For ITS-2 rDNA, the intra-species sequence variation was 0-1.3% within T. trichiura and 0.2-1.7% within T. suis. The inter-species sequence differences between the two whipworms were 60.7-65.3% for ITS-1 and 59.3-61.5% for ITS-2. These results demonstrated that the ITS rDNA sequences provide additional genetic markers for the characterization and differentiation of the two whipworms. These data should be useful for studying the epidemiology and population genetics of T. trichiura and T. suis, as well as for the diagnosis of trichuriasis in humans and pigs.

  16. Evolution of the assassin's arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea).

    PubMed

    Zhang, Junxia; Gordon, Eric R L; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R; Johnson, Kevin P; Meier, Rudolf; Weirauch, Christiane

    2016-02-26

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators.

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

    PubMed

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

    2016-02-01

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

  18. Evolution of the assassin’s arms: insights from a phylogeny of combined transcriptomic and ribosomal DNA data (Heteroptera: Reduvioidea)

    PubMed Central

    Zhang, Junxia; Gordon, Eric R. L.; Forthman, Michael; Hwang, Wei Song; Walden, Kim; Swanson, Daniel R.; Johnson, Kevin P.; Meier, Rudolf; Weirauch, Christiane

    2016-01-01

    Assassin bugs (Reduvioidea) are one of the most diverse (>7,000 spp.) lineages of predatory animals and have evolved an astounding diversity of raptorial leg modifications for handling prey. The evolution of these modifications is not well understood due to the lack of a robust phylogeny, especially at deeper nodes. We here utilize refined data from transcriptomes (370 loci) to stabilize the backbone phylogeny of Reduvioidea, revealing the position of major clades (e.g., the Chagas disease vectors Triatominae). Analyses combining transcriptomic and Sanger-sequencing datasets result in the first well-resolved phylogeny of Reduvioidea. Despite amounts of missing data, the transcriptomic loci resolve deeper nodes while the targeted ribosomal genes anchor taxa at shallower nodes, both with high support. This phylogeny reveals patterns of raptorial leg evolution across major leg types. Hairy attachment structures (fossula spongiosa), present in the ancestor of Reduvioidea, were lost multiple times within the clade. In contrast to prior hypotheses, this loss is not directly correlated with the evolution of alternative raptorial leg types. Our results suggest that prey type, predatory behavior, salivary toxicity, and morphological adaptations pose intricate and interrelated factors influencing the evolution of this diverse group of predators. PMID:26916580

  19. Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism.

    PubMed

    Kengne, P; Antonio-Nkondjio, C; Awono-Ambene, H P; Simard, F; Awolola, T S; Fontenille, D

    2007-06-01

    Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

  20. Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).

    PubMed

    Tarcz, Sebastian; Potekhin, Alexey; Rautian, Maria; Przyboś, Ewa

    2012-05-01

    This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2011-01-01

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

  2. Molecular systematics of Gagea and Lloydia (Liliaceae; Liliales): implications of analyses of nuclear ribosomal and plastid DNA sequences for infrageneric classification

    PubMed Central

    Zarrei, M.; Wilkin, P.; Fay, M. F.; Ingrouille, M. J.; Zarre, S.; Chase, M. W.

    2009-01-01

    Background and Aims Gagea is a Eurasian genus of petaloid monocots, with a few species in North Africa, comprising between 70 and approximately 275 species depending on the author. Lloydia (thought to be the closest relative of Gagea) consists of 12–20 species that have a mostly eastern Asian distribution. Delimitation of these genera and their subdivisions are unresolved questions in Liliaceae taxonomy. The objective of this study is to evaluate generic and infrageneric circumscription of Gagea and Lloydia using DNA sequence data. Methods A phylogenetic study of Gagea and Lloydia (Liliaceae) was conducted using sequences of nuclear ribosomal internal transcribed spacer (ITS) and plastid (rpl16 intron, trnL intron, trnL-F spacer, matK and the psbA-trnH spacer) DNA regions. This included 149 accessions (seven as outgroups), with multiple accessions of some taxa; 552 sequences were included, of which 393 were generated as part of this research. Key Results A close relationship of Gagea and Lloydia was confirmed in analyses using different datasets, but neither Gagea nor Lloydia forms a monophyletic group as currently circumscribed; however, the ITS and plastid analyses did not produce congruent results for the placement of Lloydia relative to the major groups within Gagea. Gagea accessions formed five moderately to strongly supported clades in all trees, with most Lloydia taxa positioned at the basal nodes; in the strict consensus trees from the combined data a basal polytomy occurs. There is limited congruence between the classical, morphology-derived infrageneric taxonomy in Gagea (including Lloydia) and clades in the present phylogenetic analyses. Conclusions The analyses support monophyly of Gagea/Lloydia collectively, and they clearly comprise a single lineage, as some previous authors have hypothesized. The results provide the basis for a new classification of Gagea that has support from some morphological features. Incongruence between plastid and nuclear

  3. Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

    PubMed Central

    Schroeder, Jill M.; Booton, Gregory C.; Hay, John; Niszl, Ingrid A.; Seal, David V.; Markus, Miles B.; Fuerst, Paul A.; Byers, Thomas J.

    2001-01-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK. PMID:11326011

  4. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

    PubMed

    Schroeder, J M; Booton, G C; Hay, J; Niszl, I A; Seal, D V; Markus, M B; Fuerst, P A; Byers, T J

    2001-05-01

    This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

  5. Using the small subunit of nuclear ribosomal DNA to reveal the phylogenetic position of the plerocercoid larvae of Spirometra tapeworms.

    PubMed

    Zhang, Xi; Duan, Jiang Yang; Wang, Zhong Quan; Jiang, Peng; Liu, Ruo Dan; Cui, Jing

    2017-04-01

    Although medically important, the systematics of Spirometra and the taxonomic position of S. erinaceieuropaei remain unclear. In this study, the 18S rDNA gene of S. erinaceieuropaei sparganum from naturally infected frogs caught in 14 geographical locations of China was sequenced. In addition, all available 18S sequences of the family Diphyllobothriidae in the Genbank database were included to reconstruct the phylogeny of diphyllobothriid tapeworms. The secondary structure model of the 18S rDNA was also predicated to further explore the sequence variation. Phylogenetic analyses were performed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. The intraspecific divergences of 18S rDNA in Chinese sparganum isolates ranged from 0.0 to 0.4%. Regions of V2, V4 and V7 were the most variable regions in the secondary structure of 18S rDNA. With the exception of genera Duthiersia and Probothriocephalus, other genera (i.e., Adenocephalus, Diphyllobothrium, Diplogonoporus, Duthiersia, Schistocephalus and Spirometra) selected in the Diphyllobothriidae shared similar topologies of V2, V4 and V7 structures. The topology of generated phylogenetic trees revealed close relationships among Adenocephalus, Digramma, Diphyllobothrium, Diplogonoporus, Ligula, Sparganum and Spirometra. The exact phylogenetic position of Spirometra species should be further analyzed with more sampling and more useful molecular markers.

  6. Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea

    PubMed Central

    Kong, Hyun-Hee; Shin, Ji-Yeol; Yu, Hak-Sun; Kim, Jin; Hahn, Tae-Won; Hahn, Young-Ho; Chung, Dong-Il

    2002-01-01</