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Sample records for 5s rrna assay

  1. Compilation of 5S rRNA and 5S rRNA gene sequences

    PubMed Central

    Specht, Thomas; Wolters, Jörn; Erdmann, Volker A.

    1990-01-01

    The BERLIN RNA DATABANK as of Dezember 31, 1989, contains a total of 667 sequences of 5S rRNAs or their genes, which is an increase of 114 new sequence entries over the last compilation (1). It covers sequences from 44 archaebacteria, 267 eubacteria, 20 plastids, 6 mitochondria, 319 eukaryotes and 11 eukaryotic pseudogenes. The hardcopy shows only the list (Table 1) of those organisms whose sequences have been determined. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information. PMID:1692116

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

    NASA Technical Reports Server (NTRS)

    Yang, Y.; Fox, G. E.

    1996-01-01

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

  3. The 5S rRNA and the rRNA intergenic spacer of the two varieties of Cryptococcus neoformans.

    PubMed

    Fan, M; Chen, L C; Ragan, M A; Gutell, R R; Warner, J R; Currie, B P; Casadevall, A

    1995-01-01

    The intergenic spacers (IGS) separating the 23S-like and 16S-like rDNAs of the two varieties of the human pathogenic fungus Cryptococcus neoformans were amplified, cloned and sequenced. The C. neoformans var. neoformans IGS was 2421 nt with 5S rRNA at positions 1228-1345 3' of the 23S-like rRNA. The C. neoformans var. gattii IGS was 2480 nt with 5S rRNA at positions 1268-1385 3' of the 23S-like rRNA. For both varieties the 5S rDNA genes were in the same orientation as the 16S-5.8-23S genes and encode a 118 nt molecule of identical sequence. Phylogenetic comparison of C. neoformans 5S rDNA with that of other fungi placed this fungus in close relationship with other basidiomycetes including Tremella mesenterica, Bullera alba, and Cryptococcus laurentii. A secondary structure model for the deduced 5S rRNA was constructed by comparative sequence analysis. Polymerase chain reaction-amplified IGS of 12 C. neoformans var. neoformans strains revealed extensive size variation ranging from 100 to 300 nt. Size variation between strains in the length of the IGS may be useful for distinguishing strains. Structurally, the IGS were characterized by the presence of occasional short direct GC-rich 19-nt repeats. Overall IGS sequence identity between the C. neoformans varieties was only 78.5%, in sharp contrast to the identical or nearly identical sequences for the rDNA genes, and suggests rapid evolution for IGS sequences.

  4. A yeast transcription system for the 5S rRNA gene.

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin. Images PMID:7145700

  5. Crystallization of engineered Thermus flavus 5S rRNA under earth and microgravity conditions.

    PubMed

    Lorenz, S; Perbandt, M; Lippmann, C; Moore, K; DeLucas, L J; Betzel, C; Erdmann, V A

    2000-04-01

    Thermus flavus 5S rRNA with a molecular weight of about 40 kDa was modified at the 5' and 3' ends. Crystals were obtained under earth and microgravity conditions. The best crystals were obtained during NASA space mission STS 94. For the first time, it was possible to collect a complete data set from 5S rRNA crystals to 7.8 A resolution and to assign the space group as R32, with unit-cell parameters a = b = 110.3, c = 387.6 A, alpha = beta = 90, gamma = 120 degrees.

  6. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The

  7. The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

    PubMed

    Onofrillo, Carmine; Galbiati, Alice; Montanaro, Lorenzo; Derenzini, Massimo

    2017-01-17

    Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

  8. Gene arrangement and sequence of the 5S rRNA in Filobasidiella neoformans (Cryptococcus neoformans) as a phylogenetic indicator.

    PubMed

    Kwon-Chung, K J; Chang, Y C

    1994-04-01

    We cloned the 5S rRNA gene and determined its organization in the four genes encoding rRNAs in a ribosomal DNA repeat unit of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans. The 5S rRNA gene contained 118 nucleotides and was located 1 kb upstream from the 18S rRNA gene within the 8.6-kb fragment of the ribosomal DNA repeat unit. The sequence of the 5S rRNA gene from F. neoformans was more similar to the sequence of the 5S rRNA gene from Tremella mesenterica than to the sequences of the 5S rRNA genes from Filobasidium species. The arrangement of the rRNA genes in F. neoformans closely resembles the arrangement of the rRNA genes in mushrooms such as Schizophyllum commune, Agaricus bisporus, and Coprinus cinereus in that the 5S rRNA-coding region not only is located within the repeat unit that encodes the other rRNAs but also is transcribed in the same direction as the other rRNA genes. This is the first description of the arrangement of rRNA genes in a species belonging to the Heterobasidiomycetes.

  9. Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

    PubMed Central

    Condon, C; Brechemier-Baey, D; Beltchev, B; Grunberg-Manago, M; Putzer, H

    2001-01-01

    Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored. PMID:11233981

  10. The 5S rRNA-histone repeat in the crustacean Artemia: structure, polymorphism and variation of the 5S rRNA segment in different populations.

    PubMed Central

    Cruces, J; Díaz-Guerra, M; Gil, I; Renart, J

    1989-01-01

    5S rRNA genes are linked to the histone genes in the 13 populations of the crustacean Artemia that we have studied. In all cases, two types of repeat units are found. Southern blot analysis of all populations shows that they can be grouped into three classes: a) American bisexuals; b) Eurasian bisexuals, and c) parthenogenetic organisms (all from Eurasia). Restriction analysis of a bisexual population from San Francisco Bay shows that the two repeat units are of 9.0 and 8.5 kb (with minor heterogeneities of restriction sites). In parthenogenetic organisms, the two repeat units are of approximately 12 kb. Sequencing data from the region of the 5S rRNA from the San Francisco Bay population, shows that in both types of units, the single 5S rRNA gene (315 bp in length), is located 430 bp downstream the 3' regulatory sequences of the H2A gene, the last gene in the histone cluster. We have isolated three clones that contain 5S rRNA sequences. Two of them (one from an American bisexual and the other from a parthenogenetic population) contain histone and 5S rRNA genes, both with the same transcriptional polarity. The third clone, lacking histone genes, is likely to be an orphon derived from the parthenogenetic population. Images PMID:2570403

  11. Analysis of a 5S rRNA gene cloned from Euplotes eurstomus

    SciTech Connect

    Roberson, A.E.; Wolffe, A.; Olins, D.E.

    1987-05-01

    The macronucleus of the hypotrichous ciliated protozoan Euplotes eurystomus lends itself to the study of eukaryotic gene and chromatin structure because native macronuclear DNA exists as linear, gene-sized fragments between 400 and 20,000 bp in length. The macronuclear chromatin, while arranged in a typical nucleosomal structure, is freely soluble in low ionic strength buffers without treatment by nucleases. Thus, specific genes may be enriched as native, intact chromatin molecules. The 5S rRNA gene from Euplotes has been cloned to facilitate investigation of 5S gene-chromatin following characterization of the gene at the DNA level. It has been demonstrated that the gene, while in circular or linear form, can be transcribed in vitro by a Xenopus oocyte nuclear extract. The transcript generated in vitro is 120 nucleotides in length and is synthesized by RNA polymerase III. Anti-Xenopus TFIIIA antibodies recognize a Euplotes macronuclear chromatin-associated protein which is approx. 80 KD in size. It has been established that the sequence of the telomere flanking the 5S gene in Euplotes eurystomus is the same telomeric sequence published for Euplotes aediculatus.

  12. An unusual 5S rRNA, from Sulfolobus acidocaldarius, and its implications for a general 5S rRNA structure.

    PubMed Central

    Stahl, D A; Luehrsen, K R; Woese, C R; Pace, N R

    1981-01-01

    The nucleotide sequence of the 5S ribosomal RNA of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was determined. The high degree of evident secondary structure in the molecule has implications for the common higher order structure of other 5S rRNAs, both bacterial and eukaryotic. Images PMID:6273825

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

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1985-01-01

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

  14. Evolution of green plants as deduced from 5S rRNA sequences.

    PubMed

    Hori, H; Lim, B L; Osawa, S

    1985-02-01

    We have constructed a phylogenic tree for green plants by comparing 5S rRNA sequences. The tree suggests that the emergence of most of the uni- and multicellular green algae such as Chlamydomonas, Spirogyra, Ulva, and Chlorella occurred in the early stage of green plant evolution. The branching point of Nitella is a little earlier than that of land plants and much later than that of the above green algae, supporting the view that Nitella-like green algae may be the direct precursor to land plants. The Bryophyta and the Pteridophyta separated from each other after emergence of the Spermatophyta. The result is consistent with the view that the Bryophyta evolved from ferns by degeneration. In the Pteridophyta, Psilotum (whisk fern) separated first, and a little later Lycopodium (club moss) separated from the ancestor common to Equisetum (horsetail) and Dryopteris (fern). This order is in accordance with the classical view. During the Spermatophyta evolution, the gymnosperms (Cycas, Ginkgo, and Metasequoia have been studied here) and the angiosperms (flowering plants) separated, and this was followed by the separation of Metasequoia and Cycas (cycad)/Ginkgo (maidenhair tree) on one branch and various flowering plants on the other.

  15. Evolution of green plants as deduced from 5S rRNA sequences

    PubMed Central

    Hori, Hiroshi; Lim, Byung-Lak; Osawa, Syozo

    1985-01-01

    We have constructed a phylogenic tree for green plants by comparing 5S rRNA sequences. The tree suggests that the emergence of most of the uni- and multicellular green algae such as Chlamydomonas, Spirogyra, Ulva, and Chlorella occurred in the early stage of green plant evolution. The branching point of Nitella is a little earlier than that of land plants and much later than that of the above green algae, supporting the view that Nitella-like green algae may be the direct precursor to land plants. The Bryophyta and the Pteridophyta separated from each other after emergence of the Spermatophyta. The result is consistent with the view that the Bryophyta evolved from ferns by degeneration. In the Pteridophyta, Psilotum (whisk fern) separated first, and a little later Lycopodium (club moss) separated from the ancestor common to Equisetum (horsetail) and Dryopteris (fern). This order is in accordance with the classical view. During the Spermatophyta evolution, the gymnosperms (Cycas, Ginkgo, and Metasequoia have been studied here) and the angiosperms (flowering plants) separated, and this was followed by the separation of Metasequoia and Cycas (cycad)/Ginkgo (maidenhair tree) on one branch and various flowering plants on the other. PMID:16593540

  16. Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons

    PubMed Central

    Pagano, Johanna F.B.; Ensink, Wim A.; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Zhu, Kongju; Spaink, Herman P.; Girard, Geneviève; Rauwerda, Han; Jonker, Martijs J.; Dekker, Rob J.

    2017-01-01

    5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci. PMID:28003516

  17. Tomato (Solanum lycopersicum) variety discrimination and hybridization analysis based on the 5S rRNA region.

    PubMed

    Sun, Yan-Lin; Kang, Ho-Min; Kim, Young-Sik; Baek, Jun-Pill; Zheng, Shi-Lin; Xiang, Jin-Jun; Hong, Soon-Kwan

    2014-05-04

    The tomato (Solanum lycopersicum) is a major vegetable crop worldwide. To satisfy popular demand, more than 500 tomato varieties have been bred. However, a clear variety identification has not been found. Thorough understanding of the phylogenetic relationship and hybridization information of tomato varieties is very important for further variety breeding. Thus, in this study, we collected 26 tomato varieties and attempted to distinguish them based on the 5S rRNA region, which is widely used in the determination of phylogenetic relations. Sequence analysis of the 5S rRNA region suggested that a large number of nucleotide variations exist among tomato varieties. These variable nucleotide sites were also informative regarding hybridization. Chromas sequencing of Yellow Mountain View and Seuwiteuking varieties indicated three and one variable nucleotide sites in the non-transcribed spacer (NTS) of the 5S rRNA region showing hybridization, respectively. Based on a phylogenetic tree constructed using the 5S rRNA sequences, we observed that 16 tomato varieties were divided into three groups at 95% similarity. Rubiking and Sseommeoking, Lang Selection Procedure and Seuwiteuking, and Acorn Gold and Yellow Mountain View exhibited very high identity with their partners. This work will aid variety authentication and provides a basis for further tomato variety breeding.

  18. Small Ubiquitin-like Modifier (SUMO)-mediated Repression of the Xenopus Oocyte 5 S rRNA Genes*

    PubMed Central

    Malik, Mariam Q.; Bertke, Michelle M.; Huber, Paul W.

    2014-01-01

    The 5 S rRNA gene-specific transcription factor IIIA (TFIIIA) interacts with the small ubiquitin-like modifier (SUMO) E3 ligase PIAS2b and with one of its targets, the transcriptional corepressor, XCtBP. PIAS2b is restricted to the cytoplasm of Xenopus oocytes but relocates to the nucleus immediately after fertilization. Following the midblastula transition, PIAS2b and XCtBP are present on oocyte-type, but not somatic-type, 5 S rRNA genes up through the neurula stage, as is a limiting amount of TFIIIA. Histone H3 methylation, coincident with the binding of XCtBP, also occurs exclusively on the oocyte-type genes. Immunohistochemical staining of embryos confirms the occupancy of a subset of the oocyte-type genes by TFIIIA that become positioned at the nuclear periphery shortly after the midblastula transition. Inhibition of SUMOylation activity relieves repression of oocyte-type 5 S rRNA genes and is correlated with a decrease in methylation of H3K9 and H3K27 and disruption of subnuclear localization. These results reveal a novel function for TFIIIA as a negative regulator that recruits histone modification activity through the CtBP repressor complex exclusively to the oocyte-type 5 S rRNA genes, leading to their terminal repression. PMID:25368327

  19. Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus.

    PubMed

    Dimarco, Eufrosina; Cascone, Eleonora; Bellavia, Daniele; Caradonna, Fabio

    2012-10-15

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants.

  20. Evolution of multicellular animals as deduced from 5S rRNA sequences: a possible early emergence of the Mesozoa.

    PubMed

    Ohama, T; Kumazaki, T; Hori, H; Osawa, S

    1984-06-25

    The nucleotide sequences of 5S rRNA from a mesozoan Dicyema misakiense and three metazoan species, i.e., an acorn-worm Saccoglossus kowalevskii, a moss-animal Bugula neritina, and an octopus Octopus vulgaris have been determined. A phylogenic tree of multicellular animals has been constructed from 73 5S rRNA sequences available at present including those from the above four sequences. The tree suggests that the mesozoan is the most ancient multicellular animal identified so far, its emergence time being almost the same as that of flagellated or ciliated protozoans. The branching points of planarians and nematodes are a little later than that of the mesozoan but are clearly earlier than other metazoan groups including sponges and jellyfishes. Many metazoan groups seem to have diverged within a relatively short period.

  1. In vivo analyses of the internal control region in the 5S rRNA gene from Saccharomyces cerevisiae.

    PubMed

    Lee, Y; Erkine, A M; Van Ryk, D I; Nazar, R N

    1995-02-25

    The internal control region of the Saccharomyces cerevisiae 5S rRNA gene has been characterized in vivo by genomic DNase I footprinting and by mutational analyses using base substitutions, deletions or insertions. A high copy shuttle vector was used to efficiently express mutant 5S rRNA genes in vivo and isotope labelling kinetics were used to distinguish impeded gene expression from nascent RNA degradation. In contrast to mutational studies in reconstituted systems, the analyses describe promoter elements which closely resemble the three distinct sequence elements that have been observed in Xenopus laevis 5S rRNA. The results indicate a more highly conserved structure than previously reported with reconstituted systems and suggest that the saturated conditions which are used in reconstitution studies mask sequence dependence which may be physiologically significant. Footprint analyses support the extended region of protein interaction which has recently been observed in some reconstituted systems, but mutational analyses indicate that these interactions are not sequence specific. Periodicity in the footprint provides further detail regarding the in vivo topology of the interacting protein.

  2. Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

    PubMed

    Barman, Anindya Sundar; Singh, Mamta; Singh, Rajeev Kumar; Lal, Kuldeep Kumar

    2016-12-01

    In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120 bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.

  3. The nucleotide sequence of Beneckea harveyi 5S rRNA. [bioluminescent marine bacterium

    NASA Technical Reports Server (NTRS)

    Luehrsen, K. R.; Fox, G. E.

    1981-01-01

    The primary sequence of the 5S ribosomal RNA isolated from the free-living bioluminescent marine bacterium Beneckea harveyi is reported and discussed in regard to indications of phylogenetic relationships with the bacteria Escherichia coli and Photobacterium phosphoreum. Sequences were determined for oligonucleotide products generated by digestion with ribonuclease T1, pancreatic ribonuclease and ribonuclease T2. The presence of heterogeneity is indicated for two sites. The B. harveyi sequence can be arranged into the same four helix secondary structures as E. coli and other prokaryotic 5S rRNAs. Examination of the 5S-RNS sequences of the three bacteria indicates that B. harveyi and P. phosphoreum are specifically related and share a common ancestor which diverged from an ancestor of E. coli at a somewhat earlier time, consistent with previous studies.

  4. Authentication of Saussurea lappa, an endangered medicinal material, by ITS DNA and 5S rRNA sequencing.

    PubMed

    Chen, Feng; Chan, Ho-Yin Edwin; Wong, Ka-Lok; Wang, Jun; Yu, Man-Tang; But, Paul Pui-Hay; Shaw, Pang-Chui

    2008-06-01

    Wild SAUSSUREA LAPPA in the family Asteraceae is a highly endangered plant. On the other hand, the dried root of cultivated S. LAPPA (Radix Aucklandia, Muxiang) is a popular medicinal material for treating various gastrointestinal diseases. In the market, several medicinal plants including VLADIMIRIA BERARDIOIDEA, V. SOULIEI, V. SOULIEI var. MIRABILIS, INULA HELENIUM and I. RACEMOSA in the family Asteraceae and ARISTOLOCHIA DEBILIS in the family Aristolochiaceae have the trade name of Muxiang. To manage the concerned medicinal material, we investigated if the ITS and 5S rRNA intergenic spacers are effective for discriminating S. LAPPA from its substitutes and adulterants. Sequencing results showed that the similarities of ITS-1, ITS-2 and 5S rRNA intergenic spacers among S. LAPPA and related species were 56.3 - 97.8 %, 58.5 - 97.0 %, and 26.4 - 77.9 %, respectively. The intraspecific variation was much lower. There are also several unique changes in the S. LAPPA sequences that may be used as differentiation markers.

  5. 5S rRNA and accompanying proteins in gonads: powerful markers to identify sex and reproductive endocrine disruption in fish.

    PubMed

    Diaz de Cerio, Oihane; Rojo-Bartolomé, Iratxe; Bizarro, Cristina; Ortiz-Zarragoitia, Maren; Cancio, Ibon

    2012-07-17

    In anuran ovaries, 5S rDNA is regulated transcriptionally by transcription factor IIIA (TFIIIA), which upon transcription, binds 5S rRNA, forming 7S RNP. 5S rRNA can be stockpiled also in the form of 42S RNP bound to 42sp43. The aim of the present study was to assess the differential transcriptional regulation of 5S rRNA and associated proteins in thicklip gray mullet (Chelon labrosus) gonads. Up to 75% of the total RNA from mullet ovaries was 5S rRNA. qPCR quantification of 5S rRNA expression, in gonads of histologically sexed individuals from different geographical areas, successfully sexed animals. All males had expression levels that were orders of magnitude below expression levels in females, throughout an annual reproductive cycle, with the exception of two individuals: one in November and one in December. Moreover, intersex mullets from a polluted harbor had expression levels between both sexes. TFIIIA and 42sp43 were also very active transcriptionally in gonads of female and intersex mullets, in comparison to males. Nucleocytoplasmatic transport is important in this context and we also analyzed transcriptional levels of importins-α1, -α2, and -β2 and different exportins. Importin-αs behaved similarly to 5S rRNA. Thus, 5S rRNA and associated proteins constitute very powerful molecular markers of sex and effects of xenosterogens in fish gonads, with potential technological applications in the analysis of fish stock dynamics and reproduction as well as in environmental health assessment.

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

    PubMed

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

    2006-05-01

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

  7. Role of Histone H1 as an Architectural Determinant of Chromatin Structure and as a Specific Repressor of Transcription on Xenopus Oocyte 5S rRNA Genes

    PubMed Central

    Sera, Takashi; Wolffe, Alan P.

    1998-01-01

    We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5′ end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3′ end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes. PMID:9632749

  8. Natural Microbial Community Compositions Compared by a Back-Propagating Neural Network and Cluster Analysis of 5S rRNA

    PubMed Central

    Noble, P. A.; Bidle, K. D.; Fletcher, M.

    1997-01-01

    The community compositions of free-living and particle-associated bacteria in the Chesapeake Bay estuary were analyzed by comparing banding patterns of stable low-molecular-weight RNA (SLMW RNA) which include 5S rRNA and tRNA molecules. By analyzing images of autoradiographs of SLMW RNAs on polyacrylamide gels, band intensities of 5S rRNA were converted to binary format for transmission to a back-propagating neural network (NN). The NN was trained to relate binary input to sample stations, collection times, positions in the water column, and sample types (e.g., particle-associated versus free-living communities). Dendrograms produced by using Euclidean distance and average and Ward's linkage methods on data of three independently trained NNs yielded the following results. (i) Community compositions of Chesapeake Bay water samples varied both seasonally and spatially. (ii) Although there was no difference in the compositions of free-living and particle-associated bacteria in the summer, these community types differed significantly in the winter. (iii) In the summer, most bay samples had a common 121-nucleotide 5S rRNA molecule. Although this band occurred in the top water of midbay samples, it did not occur in particle-associated communities of bottom-water samples. (iv) Regardless of the season, midbay samples had the greatest variety of 5S rRNA sizes. The utility of NNs for interpreting complex banding patterns in electrophoresis gels was demonstrated. PMID:16535593

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

    PubMed Central

    Garcia, S; Kovařík, A

    2013-01-01

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

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

    PubMed

    Garcia, S; Kovařík, A

    2013-07-01

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

  11. An assessment of the phylogenetic relationship among sugarcane and related taxa based on the nucleotide sequence of 5S rRNA intergenic spacers.

    PubMed

    Pan, Y B; Burner, D M; Legendre, B L

    2000-01-01

    5S rRNA intergenic spacers were amplified from two elite sugarcane (Saccharum hybrids) cultivars and their related taxa by polymerase chain reaction (PCR) with 5S rDNA consensus primers. Resulting PCR products were uniform in length from each accession but exhibited some degree of length variation among the sugarcane accessions and related taxa. These PCR products did not always cross hybridize in Southern blot hybridization experiments. These PCR products were cloned into a commercial plasmid vector PCR 2.1 and sequenced. Direct sequencing of cloned PCR products revealed spacer length of 231-237 bp for S. officinarum, 233-237 for sugarcane cultivars, 228-238 bp for S. spontaneum, 239-252 bp for S. giganteum, 385-410 bp for Erianthus spp., 226-230 bp for Miscanthus sinensis Zebra, 206-207 bp for M. sinensis IMP 3057, 207-209 bp for Sorghum bicolor, and 247-249 bp for Zea mays. Nucleotide sequence polymorphism were found at both the segment and single nucleotide level. A consensus sequence for each taxon was obtained by Align X. Multiple sequences were aligned and phylogenetic trees constructed using Align X. CLUSTAL and DNAMAN programs. In general, accessions of the following taxa tended to group together to form distinct clusters: S. giganteum, Erianthus spp., M. sinensis, S. bicolor, and Z. mays. However, the two S. officinarum clones and two sugarcane cultivars did not form distinct clusters but interrelated within the S. spontaneum cluster. The disclosure of these 5S rRNA intergenic spacer sequences will facilitate marker-assisted breeding in sugarcane.

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

    PubMed

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

    2016-02-01

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

  13. Cytogenetic characterization by in situ hybridization techniques and molecular analysis of 5S rRNA genes of the European hazelnut (Corylus avellana).

    PubMed

    Falistocco, E; Marconi, G

    2013-03-01

    The European hazelnut (Corylus avellana L.) is widespread in Europe, where it has been cultivated for centuries. Despite progress in genetics, most of the cytogenetic aspects of this species have been overlooked. The aim of this study was to fill in this gap and obtain basic information on the chromosome structure of this species. Karyomorphological analysis confirmed the chromosome number 2n = 22 and showed that, despite their apparent uniformity, the chromosomes could be separated into three groups of different size: large (L), medium (M), and small (S). As a first step towards the physical mapping of the hazelnut chromosomes, we applied FISH to localize the position of rRNA genes (rDNA). The sites of 45S and 5S rDNA enabled us to identify two chromosome pairs belonging, respectively, to the L and S groups. The self-GISH procedure revealed that repetitive DNA is concentrated in the pericentromeric regions of the chromosomes, as with other species with rather small genomes. The analysis of 5S rDNA repeats offered additional information on the hazelnut genome by obtaining the whole sequence of the transcribed region so far unpublished. The overall results constitute a substantial advance in hazelnut cytogenetics. Further investigation of other species of Corylus could be an effective approach to understanding the phylogenesis of the genus and resolving taxonomic problems.

  14. Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.

    EPA Science Inventory

    Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

  15. Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

    PubMed Central

    Suzuki, Marcelino T.; Taylor, Lance T.; DeLong, Edward F.

    2000-01-01

    Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. PMID:11055900

  16. Chromosomal mapping of H3 histone and 5S rRNA genes in eight species of Astyanax (Pisces, Characiformes) with different diploid numbers: syntenic conservation of repetitive genes.

    PubMed

    Piscor, Diovani; Parise-Maltempi, Patricia Pasquali

    2016-03-01

    The genus Astyanax is widely distributed from the southern United States to northern Patagonia, Argentina. While cytogenetic studies have been performed for this genus, little is known about the histone gene families. The aim of this study was to examine the chromosomal relationships among the different species of Astyanax. The chromosomal locations of the 5S rRNA and H3 histone genes were determined in A. abramis, A. asuncionensis, A. altiparanae, A. bockmanni, A. eigenmanniorum, A. mexicanus (all 2n = 50), A. fasciatus (2n = 46), and A. schubarti (2n = 36). All eight species exhibited H3 histone clusters on two chromosome pairs. In six species (A. abramis, A. asuncionensis, A. altiparanae, A. bockmanni, A. eigenmanniorum, and A. fasciatus), syntenic clusters of H3 histone and 5S rDNA were observed on metacentric (m) or submetacentric (sm) chromosomes. In seven species, clusters of 5S rDNA sequences were located on one or two chromosome pairs. In A. mexicanus, 5S rDNA clusters were located on four chromosome pairs. This study demonstrates that H3 histone clusters are conserved on two chromosome pairs in the genus Astyanax, and specific chromosomal features may contribute to the genomic organization of the H3 histone and 5S rRNA genes.

  17. Chromatin endogenous cleavage and psoralen crosslinking assays to analyze rRNA gene chromatin in vivo.

    PubMed

    Griesenbeck, Joachim; Wittner, Manuel; Charton, Romain; Conconi, Antonio

    2012-01-01

    In eukaryotes, multiple copies of ribosomal RNA (rRNA) genes co-exist in two different chromatin states: actively transcribed (nucleosome depleted) chromatin, and nontranscribed (nucleosomal) chromatin. The presence of two rRNA gene populations compromises the interpretation of analyses obtained by the standard biochemical methods that are used to study chromatin structure (e.g., nuclease digestion and chromatin immunoprecipitation). Here, we provide a protocol to investigate the specific association of proteins with the two rRNA gene chromatin populations in vivo, using Saccharomyces cerevisiae as a model eukaryote.

  18. Helicobacter pylori specific nested PCR assay for the detection of 23S rRNA mutation associated with clarithromycin resistance

    PubMed Central

    Maeda, S; Yoshida, H; Ogura, K; Kanai, F; Shiratori, Y; Omata, M

    1998-01-01

    Background—Clarithromycin is one of the most important antibiotics for Helicobacter pylori eradication. However, 5-10% of strains are reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin. 
Aims—To establish a polymerase chain reaction (PCR) system which amplifies a segment of the 23S rRNA gene containing the mutation points with primers specific for H pylori, so that H pylori infection and the mutation associated with clarithromycin resistance can be examined simultaneously. 
Methods—To detect H pylori infection and the mutation simultaneously, primers specific for the H pylori 23S rRNA gene were designed based on sequence conservation among H pylori strains and sequence specificity as compared with other bacteria. DNA from 57 cultured strains and from 39 gastric juice samples was amplified in the seminested 23S rRNA PCR. Clinical applicability was evaluated in 85patients. 
Results—DNA samples from 57 cultured strains were all amplified. The novel assay and the urease A PCR agreed in 37/39 gastric juice samples with no false positives. The assay did not amplify the DNA of bacteria other than H pylori. Eight of 85 samples had the mutation before treatment. In clarithromycin based treatment, eradication was achieved in 2/5 (40%) with the mutation and 29/34 (85%) without the mutation. 
Conclusion—The assay using gastric juice is quick (within 12 hours) and non-invasive (endoscopy not required), enabling rapid initiation of appropriate antibiotic treatment. 

 Keywords: Helicobacter pylori; eradication; clarithromycin; resistance; point mutation PMID:9863474

  19. How Mg(2+) ion and water network affect the stability and structure of non-Watson-Crick base pairs in E. coli loop E of 5S rRNA: a molecular dynamics and reference interaction site model (RISM) study.

    PubMed

    Shanker, Sudhanshu; Bandyopadhyay, Pradipta

    2016-08-02

    The non-Watson-Crick (non-WC) base pairs of Escherichia coli loop E of 5S rRNA are stabilized by Mg(2+) ions through water-mediated interaction. It is important to know the synergic role of Mg(2+) and the water network surrounding Mg(2+) in stabilizing the non-WC base pairs of RNA. For this purpose, free energy change of the system is calculated using molecular dynamics (MD) simulation as Mg(2+) is pulled from RNA, which causes disturbance of the water network. It was found that Mg(2+) remains hexahydrated unless it is close to or far from RNA. In the pentahydrated form, Mg(2+) interacts directly with RNA. Water network has been identified by two complimentary methods; MD followed by a density-based clustering algorithm and three-dimensional-reference interaction site model. These two methods gave similar results. Identification of water network around Mg(2+) and non-WC base pairs gives a clue to the strong effect of water network on the stability of this RNA. Based on sequence analysis of all Eubacteria 5s rRNA, we propose that hexahydrated Mg(2+) is an integral part of this RNA and geometry of base pairs surrounding it adjust to accommodate the [Formula: see text]. Overall the findings from this work can help in understanding the basis of the complex structure and stability of RNA with non-WC base pairs.

  20. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    EPA Science Inventory

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

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

    PubMed Central

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

    2006-01-01

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

  2. Rapid Estimation of Numbers of Fecal Bacteroidetes by Use of a Quantitative PCR Assay for 16S rRNA Genes

    PubMed Central

    Dick, Linda K.; Field, Katharine G.

    2004-01-01

    Assessment of health risk associated with fecal pollution requires a reliable fecal indicator and a rapid quantification method. We report the development of a Taq nuclease assay for enumeration of 16S rRNA genes of Bacteroidetes. Sensitivity and correlation with standard fecal indicators provide experimental evidence for application of the assay in monitoring fecal pollution. PMID:15345463

  3. Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

    PubMed Central

    Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

    2009-01-01

    Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

  4. Development of quantitative PCR assays targeting the 16S rRNA genes of Enterococcus spp. and their application to the identification of enterococcus species in environmental samples.

    PubMed

    Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan; Santo Domingo, Jorge W

    2013-01-01

    The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

  5. Escherichia coli and Enterococcus spp. in rainwater tank samples: comparison of culture-based methods and 23S rRNA gene quantitative PCR assays.

    PubMed

    Ahmed, W; Richardson, K; Sidhu, J P S; Toze, S

    2012-10-16

    In this study, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichia coli and Enterococcus spp. in water samples collected from rainwater tanks in Southeast Queensland, Australia. Among the 50 rainwater tank samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively. Similarly, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively. The mean E. coli (2.49 ± 0.85) log(10) and Enterococcus spp. (2.72 ± 0.32) log(10) numbers as measured by qPCR assays were significantly (P < 0001) different than E. coli (0.91 ± 0.80) log(10) and Enterococcus spp. (1.86 ± 0.60) log(10) numbers as measured by culture-based method. Weak but significant correlations were observed between both EPA Method 1603 and the E. coli qPCR assay (r = 0.47, P = 0.0009), and EPA Method 1600 and the Enterococcus spp. qPCR assay (r = 0.42, P = 0.002). Good qualitative agreement was found between the culture-based method and the Enterococcus spp. qPCR assay in terms of detecting fecal pollution in water samples from the studied rainwater tanks. More research studies, however, are needed to shed some light on the discrepancies associated with the culture-based methods and qPCR assays for measuring fecal indicator bacteria.

  6. Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

    PubMed Central

    Ryu, Hodon; Griffith, John F.; Khan, Izhar U. H.; Hill, Stephen; Edge, Thomas A.; Toledo-Hernandez, Carlos; Gonzalez-Nieves, Joel

    2012-01-01

    Two novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targeting Streptococcus spp. (gull3) and a hydrolysis TaqMan assay targeting Catellicoccus marimammalium (gull4). The objectives of this study were to compare the host specificity of a previous C. marimammalium qPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n = 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n = 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n = 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters. PMID:22226950

  7. A loop-mediated isothermal amplification (LAMP) assay targeting 16S rRNA gene for rapid detection of Anaplasma phagocytophilum infection in sheep and goats.

    PubMed

    Ning, Changshen; Wang, Jinhong; Zhang, Yan; Wang, Xiaoxing; Cui, Yanyan; Yan, Yaqun; Wang, Rongjun; Jian, Fuchun; Zhang, Longxian

    2017-01-24

    Anaplasma phagocytophilum is a zoonotic pathogen and the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever in various kinds of animals. In the present study, a loop-mediated isothermal amplification (LAMP) assay for rapid detection of A. phagocytophilum was developed using primers specific to 16S rRNA gene of this organism. The LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min. The optimal reaction conditions, under which no cross-reaction was observed with other closely related tick borne parasites (Anaplasma bovis, Anaplasma ovis, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum) was established. The assay exhibited much higher sensitivity when compared with conventional PCR (1 copy vs 1000 copies). To evaluate the applicability of the LAMP assay, 94 sheep field blood samples were analyzed for A. phagocytophilum infection using LAMP, nested PCR and conventional PCR assay at the same time. A positive LAMP result was obtained from 53 of the 94 samples (56.4%), while only 12 (12.8%) and 3 (3.2%) were tested positive by nested PCR and conventional PCR, respectively. In conclusion, this LAMP assay is a specific, sensitive, and rapid method for the detection of A. phagocytophilum in sheep.

  8. Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay

    PubMed Central

    Watzinger, Peter; Hartmann, Johannes David; Kötter, Peter; Entian, Karl-Dieter

    2016-01-01

    Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae’s 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes. PMID:28033325

  9. Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer.

    PubMed

    Saito, Keiko; Drgon, Tomás; Robledo, José A F; Krupatkina, Danara N; Vasta, Gerardo R

    2002-11-01

    Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

  10. 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.

  11. Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.

    PubMed

    Pingle, Maneesh R; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-06-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity.

  12. Use of 18S rRNA gene-based PCR assay for diagnosis of acanthamoeba keratitis in non-contact lens wearers in India.

    PubMed

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K

    2003-07-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the "gold standard," the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.

  13. Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis in Non-Contact Lens Wearers in India

    PubMed Central

    Pasricha, Gunisha; Sharma, Savitri; Garg, Prashant; Aggarwal, Ramesh K.

    2003-01-01

    Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the “gold standard,” the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting. PMID:12843065

  14. Three cases of cat scratch disease diagnosed by indirect immunofluorescence antibody assay and/or polymerase chain reaction of 16S rRNA gene of Bartonella henselae.

    PubMed

    Maruyama, S; Kabeya, H; Nogami, S; Sakai, H; Suzuki, J; Suzuki, H; Sugita, H; Katsube, Y

    2000-12-01

    Three suspected cases of cat scratch disease were diagnosed by indirect immunofluorescence antibody assay and/or polymerase chain reaction. Patient 1 was a 10-year-old female who presented swelling of the right axillary [corrected] lymph nodes with pain and fever. She kept a kitten, and many scratches were observed on her both legs and dorsum manus. Antibody titers against Bartonella (B.) henselae were 1:32 for IgM 3 weeks after the onset of the symptoms and 1:64 for IgG 8 weeks after the onset. The DNA for 16S rRNA type I of B. henselae was detected from the blood sample obtained 3 weeks after the onset of symptoms by polymerase chain reaction for the first time in Japan. Patient 2 was a 22-year-old female veterinary student with a cat scratch at the bottom of her neck by a male kitten. She developed a papule at the scratch, slight fever, and neck pain. Although both Bartonella-specific IgG and IgM antibodies were negative before the scratch, the IgG antibody titer rose to 1:512 14 weeks after the onset. B. henselae was isolated from the kitten and its DNA found to be for 16S rRNA type I by PCR. Patient 3 was a 23-year-old female veterinary student with a cat scratch on her left forearm. A small reddish papule developed on the scratch, and she experienced swelling of the left axillary [corrected] lymph node and pain. Both the IgG and IgM antibodies against B. henselae were negative before the cat scratch, and the IgG titer rose significantly to 1:128 and 1:1,024 in 2 and 5 weeks, respectively, after the onset of the symptoms.

  15. Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment.

    PubMed

    Zhang, Huan; Lin, Senjie

    2005-11-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and 18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5'-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells x ml(-1)), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).

  16. Development of a cob-18S rRNA Gene Real-Time PCR Assay for Quantifying Pfiesteria shumwayae in the Natural Environment†

    PubMed Central

    Zhang, Huan; Lin, Senjie

    2005-01-01

    Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5′-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml−1), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay). PMID:16269741

  17. Is wheat mitochondrial 5S ribosomal RNA prokaryotic in nature?

    PubMed Central

    Gray, M W; Spencer, D F

    1981-01-01

    Küntzel et al. (1981) (Nucleic Acids Res. 9, 1451-1461) recently concluded that the sequence of wheat mitochondrial 5S rRNA is significantly more related to prokaryotic than to eukaryotic 5S rRNA sequences, and displays an especially high affinity to that of the thermophilic Gram-negative bacterium, Thermus aquaticus. However, the sequence on which this conclusion was based, although attributed to us, differs in several places from the one determined by us. We show here that the correct sequence (Spencer, D.F., Bonen, L. and Gray, M.W. (1981) Biochemistry, in press) does not support the conclusions of Küntzel et al. about potential secondary structure in wheat mitochondrial 5S rRNA and its phylogenetic significance. We further show that when the wheat mitochondrial 5S rRNA sequence is matched against published alignments for E. coli, T. aquaticus, and wheat cytosol 5S rRNAs, the mitochondrial sequence shows no greater homology to the T. aquaticus sequence than to the E. coli sequence, and only slightly more homology to these two sequences than to wheat cytosol 5S rRNA. This analysis confirms our original view (Biochemistry, in press) that wheat mitochondrial 5S rRNA is neither obviously prokaryotic nor eukaryotic in nature, but shows characteristics of both classes of 5S rRNA, as well as some unique features. PMID:7024917

  18. Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063.

    PubMed

    Suzuki, Yu; Seto, Junji; Shimotai, Yoshitaka; Ikeda, Tatsuya; Yahagi, Kazue; Mizuta, Katsumi; Matsuzaki, Yoko; Hongo, Seiji

    2016-12-01

    The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

  19. Characterization of Streptomyces venezuelae ATCC 10595 rRNA gene clusters and cloning of rrnA.

    PubMed Central

    La Farina, M; Stira, S; Mancuso, R; Grisanti, C

    1996-01-01

    Streptomyces venezuelae ATCC 10595 harbors seven rRNA gene clusters which can be distinguished by BglII digestion. The three rRNA genes present in each set are closely linked with the general structure 16S-23S-5S. We cloned rrnA and sequenced the 16S-23S spacer region and the region downstream of the 5S rRNA gene. No tRNA gene was found in these regions. PMID:8631730

  20. Application of a 16S rRNA PCR-high-resolution melt analysis assay for rapid detection of Salmonella Bacteremia.

    PubMed

    Jeng, Kevin; Yang, Samuel; Won, Helen; Gaydos, Charlotte A; Hsieh, Yu-Hsiang; Kecojevic, Alex; Carroll, Karen C; Hardick, Justin; Rothman, Richard E

    2012-03-01

    Current culture and phenotypic protocols for diagnosing Salmonella infections can be time-consuming. Here, we describe the application of a 16S rRNA PCR coupled to high-resolution melt analysis (HRMA) for species and serotype identification within 6 h of blood sample collection from a patient with Salmonella enterica serotype Enteritidis bacteremia.

  1. Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

    PubMed Central

    Layton, Alice; McKay, Larry; Williams, Dan; Garrett, Victoria; Gentry, Randall; Sayler, Gary

    2006-01-01

    Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds. PMID:16751534

  2. Development of real-time PCR assays for detection of the Streptococcus milleri group from cystic fibrosis clinical specimens by targeting the cpn60 and 16S rRNA genes.

    PubMed

    Olson, A B; Sibley, C D; Schmidt, L; Wilcox, M A; Surette, M G; Corbett, C R

    2010-04-01

    Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

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

    PubMed

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

    2016-06-01

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

  4. 5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

    NASA Technical Reports Server (NTRS)

    Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

    1989-01-01

    The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

  5. Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay.

    PubMed

    Samarasinghe, Bimba; Johnson, Johanna; Ryan, Una

    2008-04-01

    The ITS1 sequences for C. suis, C. belli, C. rivolta, C. felis, and C. ohioensis-like oocysts were determined and a diagnostic PCR-RFLP assay specific for Cystosisopora species was developed. Phylogenetic analysis of ITS1 sequences of Cystosisopora species along with ITS1 sequences for Toxoplasma, Neospora, Sarcocystis and Eimeria spp. using distance, minimum evolution and parsimony-based methods confirmed previous studies, which suggested that the genus Cystoisospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae.

  6. Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites.

    PubMed

    Kawahara, Fumiya; Zhang, Guohong; Mingala, Claro N; Tamura, Yu; Koiwa, Masateru; Onuma, Misao; Nunoya, Tetsuo

    2010-11-24

    At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

  7. The 5S ribosomal RNAs of Paracoccus denitrificans and Prochloron

    NASA Technical Reports Server (NTRS)

    Mackay, R. M.; Salgado, D.; Bonen, L.; Doolittle, W. F.; Stackebrandt, E.

    1982-01-01

    The nucleotide sequences of the 5S rRNAs of Paracoccus denitrificans and Prochloron sp. are presented, along with the demonstrated phylogenetic relationships of P. denitrificans with purple nonsulfur bacteria, and of Prochloron with cyanobacteria. Structural findings include the following: (1) helix II in both models is much shorter than in other eubacteria, (2) a base-pair has been deleted from helix IV of P. denitrificans 5S, and (3) Prochloron 5S has the potential to form four base-pairs between residues. Also covered are the differences between pairs of sequences in P. denitrificans, Prochloron, wheat mitochondion, spinach chloroplast, and nine diverse eubacteria. Findings include the observation that Prochloron 5S rRNA is much more similar to the 5S of the cyanobacterium Anacystis nidulans (25 percent difference) than either are to any of the other nine eubacterial 5S rRNAs.

  8. Next-Generation Sequencing Combined with Specific PCR Assays To Determine the Bacterial 16S rRNA Gene Profiles of Middle Ear Fluid Collected from Children with Acute Otitis Media

    PubMed Central

    Kramna, Lenka; Oikarinen, Sami; Sipilä, Markku; Rautiainen, Markus; Aittoniemi, Janne; Laranne, Jussi; Hyöty, Heikki; Cinek, Ondrej

    2017-01-01

    ABSTRACT The aim of the study was to analyze the bacteriome of acute otitis media with a novel modification of next-generation sequencing techniques. Outpatient children with acute otitis media were enrolled in the study, and middle ear fluids were collected during 90 episodes from 79 subjects aged 5 to 42 months (median age, 19 months). The bacteriome profiles of middle ear fluid samples were determined by a nested-PCR amplification of the 16S rRNA gene (V4 region), followed by mass sequencing. The profiling results were compared to the results of specific PCR assays targeting selected prevalent pathogens. Bacteriome profiling using nested amplification of low-volume samples was aided by a bioinformatic subtraction of signal contaminants from the recombinant polymerase, achieving a sensitivity slightly lower than that of specific PCR detection. Streptococcus pneumoniae was detected in 28 (31%) samples, Haemophilus influenzae in 24 (27%), Moraxella catarrhalis in 18 (20%), Staphylococcus spp. in 21 (23%), Turicella otitidis in 5 (5.6%), Alloiococcus otitidis in 3 (3.3%), and other bacteria in 14 (16%) using bacteriome profiling. S. pneumoniae was the dominant pathogen in 14 (16%) samples, H. influenzae in 15 (17%), M. catarrhalis in 5 (5.6%), T. otitidis in 2, and Staphylococcus auricularis in 2. Weaker signals of Prevotella melaninogenica, Veillonella dispar, and Veillonella montpellierensis were noted in several samples. Fourteen samples (16%) were not explainable by bacterial pathogens; novel causative agents were not detected. In conclusion, unbiased bacteriome profiling helped in depicting the true mutual quantitative ratios of ear bacteria, but at present, its complicated protocol impedes its routine clinical use. IMPORTANCE Although S. pneumoniae, H. influenzae, and M. catarrhalis have been long established as the most important pathogens in acute otitis media using culture and specific PCR assays, the knowledge of their mutual quantitative relations

  9. Sequence characterization of 5S ribosomal RNA from eight gram positive procaryotes

    NASA Technical Reports Server (NTRS)

    Woese, C. R.; Luehrsen, K. R.; Pribula, C. D.; Fox, G. E.

    1976-01-01

    Complete nucleotide sequences are presented for 5S rRNA from Bacillus subtilis, B. firmus, B. pasteurii, B. brevis, Lactobacillus brevis, and Streptococcus faecalis, and 5S rRNA oligonucleotide catalogs and partial sequence data are given for B. cereus and Sporosarcina ureae. These data demonstrate a striking consistency of 5S rRNA primary and secondary structure within a given bacterial grouping. An exception is B. brevis, in which the 5S rRNA sequence varies significantly from that of other bacilli in the tuned helix and the procaryotic loop. The localization of these variations suggests that B. brevis occupies an ecological niche that selects such changes. It is noted that this organism produces antibiotics which affect ribosome function.

  10. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli

    PubMed Central

    Takada, Hiraku; Shimada, Tomohiro; Dey, Debashish; Quyyum, M. Zuhaib; Nakano, Masahiro; Ishiguro, Akira; Yoshida, Hideji; Yamamoto, Kaneyoshi; Sen, Ranjan

    2016-01-01

    Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3’ proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed

  11. 5S RRNA GENE DELETIONS CAUSE AN UNEXPECTEDLY HIGH FITNESS LOSS IN ESCHERICHIA COLI. (R825354)

    EPA Science Inventory

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

  12. Microbial identification by immunohybridization assay of artificial RNA labels

    NASA Technical Reports Server (NTRS)

    Kourentzi, Katerina D.; Fox, George E.; Willson, Richard C.

    2002-01-01

    Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

  13. 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

  14. 5S rRNA-recognition module of CTC family proteins and its evolution.

    PubMed

    Korobeinikova, A V; Gongadze, G M; Korepanov, A P; Eliseev, B D; Bazhenova, M V; Garber, M B

    2008-02-01

    The effects of amino acid replacements in the RNA-binding sites of homologous ribosomal proteins TL5 and L25 (members of the CTC family) on ability of these proteins to form stable complexes with ribosomal 5S RNA were studied. It was shown that even three simultaneous replacements of non-conserved amino acid residues by alanine in the RNA-binding site of TL5 did not result in noticeable decrease in stability of the TL5-5S rRNA complex. However, any replacement among five conserved residues in the RNA-binding site of TL5, as well as of L25 resulted in serious destabilization or complete impossibility of complex formation. These five residues form an RNA-recognition module in TL5 and L25. These residues are strictly conserved in proteins of the CTC family. However, there are several cases of natural replacements of these residues in TL5 and L25 homologs in Bacilli and Cyanobacteria, which are accompanied by certain changes in the CTC-binding site of 5S rRNAs of the corresponding organisms. CTC proteins and specific fragments of 5S rRNA of Enterococcus faecalis and Nostoc sp. were isolated, and their ability to form specific complexes was tested. It was found that these proteins formed specific complexes only with 5S rRNA of the same organism. This is an example of coevolution of the structures of two interacting macromolecules.

  15. The primary structure of oocyte and somatic 5S rRNAs from the loach Misgurnus fossilis.

    PubMed Central

    Mashkova, T D; Serenkova, T I; Mazo, A M; Avdonina, T A; Timofeyeva MYa; Kisselev, L L

    1981-01-01

    Somatic and oocyte 5S rRNAs from the liver and unfertilized eggs of the loach (Misgurnus fossilis have been sequenced and found to differ in six nucleotides. All the substitutions are confined to the 5'-half of the molecules; 4 of them are pyrimidine-pyrimidine substitutions, and 2 are purine-pyrimidine ones. Considerable differences, both in the position and the character of substitutions, have been established when these 5S rRNAs were compared with somatic and oocyte 5S rRNAs from Xenopus borealis and Xenopus laevis. Among the known primary structures, somatic 5S rRNA of M. fossilis is most similar to trout 5S rRNA. Images PMID:7197777

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

    PubMed Central

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

    2015-01-01

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

  17. Arabidopsis chloroplast mini-ribonuclease III participates in rRNA maturation and intron recycling.

    PubMed

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

    2015-03-01

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

  18. Evolutionary dynamics of rRNA gene clusters in cichlid fish

    PubMed Central

    2012-01-01

    Background Among multigene families, ribosomal RNA (rRNA) genes are the most frequently studied and have been explored as cytogenetic markers to study the evolutionary history of karyotypes among animals and plants. In this report, we applied cytogenetic and genomic methods to investigate the organization of rRNA genes among cichlid fishes. Cichlids are a group of fishes that are of increasing scientific interest due to their rapid and convergent adaptive radiation, which has led to extensive ecological diversity. Results The present paper reports the cytogenetic mapping of the 5S rRNA genes from 18 South American, 22 African and one Asian species and the 18S rRNA genes from 3 African species. The data obtained were comparatively analyzed with previously published information related to the mapping of rRNA genes in cichlids. The number of 5S rRNA clusters per diploid genome ranged from 2 to 15, with the most common pattern being the presence of 2 chromosomes bearing a 5S rDNA cluster. Regarding 18S rDNA mapping, the number of sites ranged from 2 to 6, with the most common pattern being the presence of 2 sites per diploid genome. Furthermore, searching the Oreochromis niloticus genome database led to the identification of a total of 59 copies of 5S rRNA and 38 copies of 18S rRNA genes that were distributed in several genomic scaffolds. The rRNA genes were frequently flanked by transposable elements (TEs) and spread throughout the genome, complementing the FISH analysis that detect only clustered copies of rRNA genes. Conclusions The organization of rRNA gene clusters seems to reflect their intense and particular evolutionary pathway and not the evolutionary history of the associated taxa. The possible role of TEs as one source of rRNA gene movement, that could generates the spreading of ribosomal clusters/copies, is discussed. The present paper reinforces the notion that the integration of cytogenetic data and genomic analysis provides a more complete picture for

  19. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

    PubMed Central

    Valach, Matus; Burger, Gertraud; Gray, Michael W.; Lang, B. Franz

    2014-01-01

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. PMID:25429974

  20. Exceptionally high and diverse mutation rates in insects small rRNA.

    PubMed

    Feng, Y X; Krupp, G; Gross, J H

    1985-10-01

    The nucleotide sequence of 5S rRNA from the posterior silk gland of the silk worm Philosamia cynthia ricini has been determined. The comparison with other insect 5S rRNAs revealed an exceptionally conserved secondary structure, in spite of an extremely high mutation rate: Thirteen nucleotides are different in Philosamia and Drosophila 5S rRNA, but all substitutions are either compensatory or occur in loops or introduce G:U base pairs. The rates of base substitution per site per year of several insect species (diptera and lepidoptera) 5S and 5.8S rRNAs are compared with those occurring in vertebrate rRNAs. In the latter cases the rates are remarkably constant, whereas their value is not only about twofold higher in insect rRNAs, but is found to be extremely large in the 5S rRNA of the silkworm Bombyx mori. These data demonstrate that phylogenetic conclusions derived from small rRNA sequence comparisons are only of limited value.

  1. Abundant 5S rRNA-Like Transcripts Encoded by the Mitochondrial Genome in Amoebozoa ▿ †

    PubMed Central

    Bullerwell, Charles E.; Burger, Gertraud; Gott, Jonatha M.; Kourennaia, Olga; Schnare, Murray N.; Gray, Michael W.

    2010-01-01

    5S rRNAs are ubiquitous components of prokaryotic, chloroplast, and eukaryotic cytosolic ribosomes but are apparently absent from mitochondrial ribosomes (mitoribosomes) of many eukaryotic groups including animals and fungi. Nevertheless, a clearly identifiable, mitochondrion-encoded 5S rRNA is present in Acanthamoeba castellanii, a member of Amoebozoa. During a search for additional mitochondrial 5S rRNAs, we detected small abundant RNAs in other members of Amoebozoa, namely, in the lobose amoeba Hartmannella vermiformis and in the myxomycete slime mold Physarum polycephalum. These RNAs are encoded by mitochondrial DNA (mtDNA), cosediment with mitoribosomes in glycerol gradients, and can be folded into a secondary structure similar to that of bona fide 5S rRNAs. Further, in the mtDNA of another slime mold, Didymium nigripes, we identified a region that in sequence, potential secondary structure, and genomic location is similar to the corresponding region encoding the Physarum small RNA. A mtDNA-encoded small RNA previously identified in Dictyostelium discoideum is here shown to share several characteristics with known 5S rRNAs. Again, we detected genes encoding potential homologs of this RNA in the mtDNA of three other species of the genus Dictyostelium as well as in a related genus, Polysphondylium. Taken together, our results indicate a widespread occurrence of small, abundant, mtDNA-encoded RNAs with 5S rRNA-like structures that are associated with the mitoribosome in various amoebozoan taxa. Our working hypothesis is that these novel small abundant RNAs represent radically divergent mitochondrial 5S rRNA homologs. We posit that currently unrecognized 5S-like RNAs may exist in other mitochondrial systems in which a conventional 5S rRNA cannot be identified. PMID:20304999

  2. 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.

  3. Coordinate regulation of ribosomal component synthesis in Acanthamoeba castellanii: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity.

    PubMed Central

    Matthews, J L; Zwick, M G; Paule, M R

    1995-01-01

    Transcription of large rRNA precursor and 5S RNA were examined during encystment of Acanthamoeba castellanii. Both transcription units are down regulated almost coordinately during this process, though 5S RNA transcription is not as completely shut down as rRNA transcription. The protein components necessary for transcription of 5S RNA and tRNA were determined, and fractions containing transcription factors comparable to TFIIIA, TFIIIB, and TFIIIC, as well as RNA polymerase III and a 3'-end processing activity, were identified. Regulation of 5S RNA transcription could be recapitulated in vitro, and the activities of the required components were compared. In contrast to regulation of precursor rRNA, there is no apparent change during encystment in the activity of the polymerase dedicated to 5S RNA expression. Similarly, the transcriptional and promoter-binding activities of TFIIIC are not altered in parallel with 5S RNA regulation. TFIIIB transcriptional activity is unaltered in encysting cells. In contrast, both the transcriptional and DNA-binding activities of TFIIIA are strongly reduced in nuclear extracts from transcriptionally inactive cells. These results were analyzed in terms of mechanisms for coordinate regulation of rRNA and 5S RNA expression. PMID:7760828

  4. Intraindividual and interspecies variation in the 5S rDNA of coregonid fish.

    PubMed

    Sajdak, S L; Reed, K M; Phillips, R B

    1998-06-01

    This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa.

  5. The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

    PubMed Central

    2011-01-01

    Background Ribosomal 5S genes are well known for the critical role they play in ribosome folding and functionality. These genes are thought to evolve in a concerted fashion, with high rates of homogenization of gene copies. However, the majority of previous analyses regarding the evolutionary process of rDNA repeats were conducted in invertebrates and plants. Studies have also been conducted on vertebrates, but these analyses were usually restricted to the 18S, 5.8S and 28S rRNA genes. The recent identification of divergent 5S rRNA gene paralogs in the genomes of elasmobranches and teleost fishes indicate that the eukaryotic 5S rRNA gene family has a more complex genomic organization than previously thought. The availability of new sequence data from lower vertebrates such as teleosts and elasmobranches enables an enhanced evolutionary characterization of 5S rDNA among vertebrates. Results We identified two variant classes of 5S rDNA sequences in the genomes of Potamotrygonidae stingrays, similar to the genomes of other vertebrates. One class of 5S rRNA genes was shared only by elasmobranches. A broad comparative survey among 100 vertebrate species suggests that the 5S rRNA gene variants in fishes originated from rounds of genome duplication. These variants were then maintained or eliminated by birth-and-death mechanisms, under intense purifying selection. Clustered multiple copies of 5S rDNA variants could have arisen due to unequal crossing over mechanisms. Simultaneously, the distinct genome clusters were independently homogenized, resulting in the maintenance of clusters of highly similar repeats through concerted evolution. Conclusions We believe that 5S rDNA molecular evolution in fish genomes is driven by a mixed mechanism that integrates birth-and-death and concerted evolution. PMID:21627815

  6. Structure and organization of rRNA operons in the region of the replication origin of the Bacillus subtilis chromosome.

    PubMed Central

    Ogasawara, N; Moriya, S; Yoshikawa, H

    1983-01-01

    Structure and organization of two complete ribosomal RNA (rRNA) gene sets, rrnO and rrnA, were determined for the first time in Bacillus subtilis. They are located at the region of the replication origin of the chromosome. Each set constitutes a single operon of: two tandem promoters - leader sequence - 16S rRNA gene - Ile-tRNA gene - Ala-tRNA gene - 23S rRNA gene - 5S rRNA gene - termination signal. The first promoter (P1) of rrnO differs from that of rrnA in sequence and function. P1 of rrnO was used very little for transcription either in vivo or in vitro while P1 was predominantly used in rrnA. A putative transcript of the entire operon was determined and constructed into a secondary structure. Analysis of in vivo transcripts by S1 mapping revealed primary processing sites at the loop and stem structure of 16S rRNA in rrnO and rrnA. A unique sequence in the leader region of rrnO can be formed into a highly complexed secondary structure and affects processing of mature 16S rRNA. The sequences of the two spacer tRNA genes are highly conserved between B. subtilis and Escherichia coli. Images PMID:6312418

  7. Chicken rRNA Gene Cluster Structure

    PubMed Central

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

    2016-01-01

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

  8. Sensitive high-performance liquid chromatographic method for the determination of 5-S-cysteinyldopamine, 5-S-cysteinyl-3,4-dihydroxyphenylacetic acid and 5-S-cysteinyl-3,4-dihydroxyphenylalanine.

    PubMed

    Fornstedt-Wallin, B; Bergh, I

    1995-01-06

    A new HPLC method for the determination of 5-S-cysteinylcatechols has been developed. The alumina adsorbed fraction of the supernatant of brain homogenate was injected onto a reversed-phase column and a citrate-phosphate buffer containing 1-nonyl sulphate was used as mobile phase (pH 2.1). Two dual-series working electrodes of a thin-layer cell were operating together, joined by a special coupler. The assay allows determination of the 5-S-cysteinylcatechols in the striatum, limbic system and mesencephalon of one guinea pig. Recoveries of the three 5-S-cysteinylcatechols were 59-76%, whereas the limit of quantitation was 0.04-0.10 pmol. The coefficient of variation was less than 0.76-1.10% and linearity was found up to a concentration of 500 pmol. By adding ascorbic acid to the samples, artifacts resulting in HPLC peaks were either reduced in size or deleted.

  9. DNA-methylation dependent regulation of embryo-specific 5S ribosomal DNA cluster transcription in adult tissues of sea urchin Paracentrotus lividus.

    PubMed

    Bellavia, Daniele; Dimarco, Eufrosina; Naselli, Flores; Caradonna, Fabio

    2013-10-01

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism. Using single strand conformation polymorphism (SSCP) analysis, we demonstrate the existence of two classes of 5S rRNA, one which is embryo-specific and encoded by the smallest (700 bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950 bp). We also demonstrate that the embryo-specific class of 5S rRNA is expressed in oocytes and embryonic stages and is silenced in adult tissue and that this phenomenon appears to be due exclusively to DNA methylation, as indicated by sensitivity to 5-azacytidine, unlike Xenopus where this mechanism is necessary but not sufficient to maintain the silenced status.

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

    PubMed Central

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

    2016-01-01

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

  11. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  12. Multiplex-polymerase chain reaction assay for the authentication of the mackerel Scomber colias in commercial canned products.

    PubMed

    Infante, Carlos; Manchado, Manuel

    2006-01-01

    A multiplex-polymerase chain reaction (PCR) system was developed for the authentication of the mackerel Scomber colias in commercial canned products. This novel method consists of an S. colias-specific fragment [159 base pairs (bp)] located in the nontranscribed spacer (NTS) sequence, and a Scomber genus-specific PCR product in the 5S rRNA gene (196-201 bp) as a positive amplification control. The system was assayed using 18 different canned products labeled as S. colias. A positive identification was made in all but one sample, revealing this methodology as a potential molecular tool for direct application in the authentication of S. colias canned products.

  13. Mouse nucleolin binds to 4.5S RNAH, a small noncoding RNA

    SciTech Connect

    Hirose, Yutaka Harada, Fumio

    2008-01-04

    4.5S RNAH is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAH is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAH-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAHin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAH-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAH recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAH was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.

  14. The rRNA evolution and procaryotic phylogeny

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1986-01-01

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

  15. Higher-order structure of rRNA

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  16. 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

  17. Ultraviolet damage and nucleosome folding of the 5S ribosomal RNA gene.

    SciTech Connect

    Liu, X; Mann, David B.; Suquet, C; Springer, David L. ); Smerdon, Michael J.

    2000-01-25

    The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.

  18. Role of Escherichia coli YbeY, a highly conserved protein, in rRNA processing

    PubMed Central

    Davies, Bryan W.; Köhrer, Caroline; Jacob, Asha I.; Simmons, Lyle A.; Zhu, Jianyu; Aleman, Lourdes M.; RajBhandary, Uttam L.; Walker, Graham C.

    2010-01-01

    The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homolog, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′- and 3′-ends of 16S rRNA as well as maturation of the 5′-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria. PMID:20807199

  19. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

    PubMed

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

    2015-08-01

    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  20. Sequence homologies between eukaryotic 5.8S rRNA and the 5' end of prokaryotic 23S rRNa: evidences for a common evolutionary origin.

    PubMed Central

    Jacq, B

    1981-01-01

    The question of the evolutionary origin of eukaryotic 5.8S rRNA was re-examined after the recent publication of the E. coli 23S rRNA sequence (26,40). A region of the 23S RNA located at its 5' end was found to be approximately 50% homologous to four different eukaryotic 5.8S rRNAs. A computer comparison analysis indicates that no other region of the E. coli ribosomal transcription unit (greater than 5 000 nucleotides in length) shares a comparable homology with 5.8S rRNA. Homology between the 5' end of e. coli 23S and four different eukaryotic 5.8S rRNAs falls within the same range as that between E. coli 5S RNA from the same four eukaryotic species. All these data strongly suggest that the 5' end of prokaryotic 23S rRNA and eukaryotic 5.8S RNA have a common evolutionary origin. Secondary structure models are proposed for the 5' region of E. coli 23S RNA. Images PMID:7024907

  1. High-Resolution Infrared Spectroscopy of Carbon-Sulfur Chains: II. C_5S and SC_5S

    NASA Astrophysics Data System (ADS)

    Thorwirth, Sven; Salomon, Thomas; Dudek, John B.

    2016-06-01

    Unbiased high-resolution infrared survey scans of the ablation products from carbon-sulfur targets in the 2100 to 2150 cm-1 regime reveal two bands previously not observed in the gas phase. On the basis of comparison against laboratory matrix-isolation work and new high-level quantum-chemical calculations these bands are attributed to the linear C_5S and SC_5S clusters. While polar C_5S was studied earlier using Fourier-transform microwave techniques, the present work marks the first gas-phase spectroscopic detection of SC_5S. H. Wang, J. Szczepanski, P. Brucat, and M. Vala 2005, Int. J. Quant. Chem. 102, 795 Y. Kasai, K. Obi, Y. Ohshima, Y. Hirahara, Y. Endo, K. Kawaguchi, and A. Murakami 1993, ApJ 410, L45 V. D. Gordon, M. C. McCarthy, A. J. Apponi, and P. Thaddeus 2001, ApJS 134, 311

  2. Enterococcus faecium PBP5-S/R, the Missing Link between PBP5-S and PBP5-R

    PubMed Central

    Pietta, Ester; Montealegre, Maria Camila; Roh, Jung Hyeob; Cocconcelli, Pier Sandro

    2014-01-01

    During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs. PMID:25182648

  3. Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

    PubMed

    Pietta, Ester; Montealegre, Maria Camila; Roh, Jung Hyeob; Cocconcelli, Pier Sandro; Murray, Barbara E

    2014-11-01

    During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs.

  4. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

    PubMed Central

    Taylor, D E; Ge, Z; Purych, D; Lo, T; Hiratsuka, K

    1997-01-01

    In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer. PMID:9420030

  5. Analyzing Digital Library Initiatives: 5S Theory Perspective

    ERIC Educational Resources Information Center

    Isah, Abdulmumin; Mutshewa, Athulang; Serema, Batlang; Kenosi, Lekoko

    2015-01-01

    This article traces the historical development of Digital Libraries (DLs), examines some DL initiatives in developed and developing countries and uses 5S Theory as a lens for analyzing the focused DLs. The analysis shows that present-day systems, in both developed and developing nations, are essentially content and user centric, with low level…

  6. Phylogenetic Analysis of Bacteroidales 16S rRNA Genes Unveils Sequences Specific to Diverse Swine Fecal Sources

    EPA Science Inventory

    Two of the currently available methods to assess swine fecal pollution (Bac1 and PF163) target Bacteroidales 16S rRNA genes. However, these assays have been shown to exhibit poor host-specificity and low detection limits in environmental waters, in part due to the limited number...

  7. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to th...

  8. Karyotypic diversification in Mytilus mussels (Bivalvia: Mytilidae) inferred from chromosomal mapping of rRNA and histone gene clusters

    PubMed Central

    2014-01-01

    Background Mussels of the genus Mytilus present morphologically similar karyotypes that are presumably conserved. The absence of chromosome painting probes in bivalves makes difficult verifying this hypothesis. In this context, we comparatively mapped ribosomal RNA and histone gene families on the chromosomes of Mytilus edulis, M. galloprovincialis, M. trossulus and M. californianus by fluorescent in situ hybridization (FISH). Results Major rRNA, core and linker histone gene clusters mapped to different chromosome pairs in the four taxa. In contrast, minor rRNA gene clusters showed a different behavior. In all Mytilus two of the 5S rDNA clusters mapped to the same chromosome pair and one of them showed overlapping signals with those corresponding to one of the histone H1 gene clusters. The overlapping signals on mitotic chromosomes became a pattern of alternate 5S rRNA and linker histone gene signals on extended chromatin fibers. Additionally, M. trossulus showed minor and major rDNA clusters on the same chromosome pair. Conclusion The results obtained suggest that at least some of the chromosomes bearing these sequences are orthologous and that chromosomal mapping of rRNA and histone gene clusters could be a good tool to help deciphering some of the many unsolved questions in the systematic classification of Mytilidae. PMID:25023072

  9. Complete rRNA sequence, arrangement of tandem repeated units and phylogeny of Nosema fumiferanae from spruce budworm, Choristoneura fumiferana (Clemens).

    PubMed

    Kyei-Poku, George; Gauthier, Debbie; van-Frankenhuyzen, Kees

    2012-01-01

    We provide molecular systematics of a microporidian species, Nosema fumiferanae, one of the most common natural enemies of spruce budworm, Choristoneura fumiferana. The uncharacterized flanking region upstream of the large subunit (LSU) rRNA and the complete rRNA cistron of N. fumiferanae was 4,769 bp long. The organization of the rRNA gene was 5'-LSU rRNA-ITS-SSU rRNA-IGS-5S-3' and corresponded primarily to most insect (i.e. lepidopteran) Nosema species identified and classified to date. Phylogenetic analysis based on the complete rRNA cistron indicated that N. fumiferanae is closely related to Nosema plutellae and is correctly assigned to the "true" Nosema group. Suggestions were provided on a criterion to delineate the "true" Nosema from other microsporidian species.

  10. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  11. Description of an Unusual Neisseria meningitidis Isolate Containing and Expressing Neisseria gonorrhoeae-Specific 16S rRNA Gene Sequences

    PubMed Central

    Skvoretz, Rhonda; Montgomery-Fullerton, Megan; Jonas, Vivian; Brentano, Steve

    2013-01-01

    An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination. PMID:23863567

  12. RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms.

    PubMed

    Wachino, Jun-Ichi; Shibayama, Keigo; Kimura, Kouji; Yamane, Kunikazu; Suzuki, Satowa; Arakawa, Yoshichika

    2010-10-01

    Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance.

  13. rRNA transcription rate in Escherichia coli.

    PubMed Central

    Gotta, S L; Miller, O L; French, S L

    1991-01-01

    The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s. Images FIG. 1 PMID:1717439

  14. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  15. 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

  16. rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

    PubMed

    Wulff, N A; Eveillard, S; Foissac, X; Ayres, A J; Bové, J-M

    2009-08-01

    Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

  17. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis

    PubMed Central

    El Gawhary, Somaia; El-Anany, Mervat; Ali, Doaa; El Gameel, El Qassem

    2016-01-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen. PMID:26494728

  18. The Role of 16S rRNA Gene Sequencing in Confirmation of Suspected Neonatal Sepsis.

    PubMed

    El Gawhary, Somaia; El-Anany, Mervat; Hassan, Reem; Ali, Doaa; El Gameel, El Qassem

    2016-02-01

    Different molecular assays for the detection of bacterial DNA in the peripheral blood represented a diagnostic tool for neonatal sepsis. We targeted to evaluate the role of 16S rRNA gene sequencing to screen for bacteremia to confirm suspected neonatal sepsis (NS) and compare with risk factors and septic screen testing. Sixty-two neonates with suspected NS were enrolled. White blood cells count, I/T ratio, C-reactive protein, blood culture and 16S rRNA sequencing were performed. Blood culture was positive in 26% of cases, and PCR was positive in 26% of cases. Evaluation of PCR for the diagnosis of NS showed sensitivity 62.5%, specificity 86.9%, PPV 62.5%, NPV 86.9% and accuracy of 79.7%. 16S rRNA PCR increased the sensitivity of detecting bacterial DNA in newborns with signs of sepsis from 26 to 35.4%, and its use can be limited to cases with the most significant risk factors and positive septic screen.

  19. Structure and organization of the rrnD operon of 'Brevibacterium lactofermentum': analysis of the 16S rRNA gene.

    PubMed

    Amador, E; Castro, J M; Correia, A; Martín, J F

    1999-04-01

    Five rRNA operons (rrn) were found by hybridization in the genome of 'Brevibacterium lactofermentum' ATCC 13869 and Corynebacterium glutamicum ATCC 13032. 'B. lactofermentum' DSM 20412 differed from the other corynebacteria tested in showing six hybridizing BamHI bands. Two of the rrn operons (rrnD and rrnE) were located in a single cosmid. Sequencing of the rrnD operon showed that it contains a complete 16S rRNA-23S RNA-5S rRNA gene cluster. Phylogenetic studies using the complete 16S rRNA sequence showed that 'B. lactofermentum' is closely related to several species of the genus Corynebacterium but only distantly related to the type species Brevibacterium linens and the authors suggest that it should be reclassified as Corynebacterium lactofermentum. The 5' end of mature 16S rRNA was identified by primer extension. Sequence elements similar to those of mycobacteria implicated in transcription antitermination (Boxes A, B, C) and in processing of the pre-rRNA to 16S rRNA were identified. An open reading frame encoding an rpoD-like sigma factor (named SigC) different from the previously reported SigA and SigB proteins was found upstream of rrnD in the opposite orientation. Both rpoD and sigC seem to be expressed from a bidirectional promoter region.

  20. Synthesis and bioactivity profile of 5-s-lipoylhydroxytyrosol-based multidefense antioxidants with a sizeable (poly)sulfide chain.

    PubMed

    Panzella, Lucia; Verotta, Luisella; Goya, Luis; Ramos, Sonia; Martín, María Angeles; Bravo, Laura; Napolitano, Alessandra; d'Ischia, Marco

    2013-02-27

    Novel polyfunctionalized antioxidants, 5-S-lipoylhydroxytyrosol (1) and its disulfide 2, trisulfide 3, and tetrasulfide 4, were prepared from tyrosol and dihydrolipoic acid in the presence, when appropriate, of sulfur. Compound 1 exhibited significant activity in the ferric reducing/antioxidant power (FRAP) assay (1.60 Trolox equiv), whereas polysulfides 2-4 were more efficient in the DPPH reduction assay (88-93% reduction vs 68% by Trolox). At 10 μM concentration, all compounds 1-4 proved to be efficient hydroxyl radical scavengers (56-69% inhibition) in a Fenton reaction assay. When administered to human HepG2 cells, 1-4 proved to be nontoxic and exhibited marked protective effects against reactive oxygen species (ROS) generation (60-84% inhibition at 1 μM concentration) and cell damage induced by 400 μM tert-butylhydroperoxide. All compounds 1-4 exhibited overall greater antioxidant activity than hydroxytyrosol.

  1. Essential role of conserved DUF177A protein in plastid 23S rRNA accumulation and plant embryogenesis

    PubMed Central

    Yang, Jiani; Suzuki, Masaharu; McCarty, Donald R.

    2016-01-01

    DUF177 proteins are nearly universally conserved in bacteria and plants except the Chlorophyceae algae. Thus far, duf177 mutants in bacteria have not established a function. In contrast, duf177a mutants have embryo lethal phenotypes in maize and Arabidopsis. In maize inbred W22, duf177a mutant embryos arrest at an early transition stage, whereas the block is suppressed in the B73 inbred background, conditioning an albino seedling phenotype. Background-dependent embryo lethal phenotypes are characteristic of maize plastid gene expression mutants. Consistent with the plastid gene expression hypothesis, quantitative real-time PCR revealed a significant reduction of 23S rRNA in an Escherichia coli duf177 knockout. Plastid 23S rRNA contents of duf177a mutant tissues were also markedly reduced compared with the wild-type, whereas plastid 16S, 5S, and 4.5S rRNA contents were less affected, indicating that DUF177 is specifically required for accumulation of prokaryote-type 23S rRNA. An AtDUF177A–green fluorescent protein (GFP) transgene controlled by the native AtDUF177A promoter fully complemented the Arabidopsis atduf177a mutant. Transient expression of AtDUF177A–GFP in Nicotiana benthamiana leaves showed that the protein was localized in chloroplasts. The essential role of DUF177A in chloroplast–ribosome formation is reminiscent of IOJAP, another highly conserved ribosome-associated protein, suggesting that key mechanisms controlling ribosome formation in plastids evolved from non-essential pathways for regulation of the prokaryotic ribosome. PMID:27574185

  2. 500-MHz proton NMR evidence for two solution structures of the common arm base-paired segment of wheat germ 5S ribosomal RNA

    SciTech Connect

    Wu, Jiejun; Marshall, A.G. )

    1990-02-20

    The base-pair protons of the common arm duplex fragment of wheat germ (Triticum aestivum) ribosomal 5S RNA have been identified and assigned by means of 500-MHz proton NMR spectroscopy. The two previously reported extra base pairs within the fragment are now explained by the presence of two distinct solution structures of the common arm fragment (and its corresponding base-paired segment in intact 5S rRNA). The present conclusions are supported by one- and two-dimensional proton homonuclear Overhauser enhancements in H{sub 2}O and by temperature variation and Mg{sup 2+} titration of the downfield {sup 1}H NMR spectrum. The difference between the two conformers is most likely due to difference in helical tightness. Some additional amino proton resonances have also been assigned.

  3. Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

    PubMed

    Dorsch, M; Lane, D; Stackebrandt, E

    1992-01-01

    The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

  4. Molecular identification of adulteration in mutton based on mitochondrial 16S rRNA gene.

    PubMed

    Xu, Jia; Zhao, Wei; Zhu, Mengru; Wen, Yuanju; Xie, Tao; He, Xiaoqian; Zhang, Yongfeng; Cao, Suizhong; Niu, Lili; Zhang, Hongping; Zhong, Tao

    2016-01-01

    The aim of this study is to set up a protocol for identification of the adulteration in mutton based on mitochondrial 16S rRNA gene. The multiplex polymerase chain reaction (multi-PCR) assay was carried out to trace the impure DNA in mutton. A universal primer pair yielded an approximate 610 bp fragment in mutton, pork, duck, chicken, horse and cat meats. The amplicons of multi-PCR assay represented the species-specific products, which could be discriminated by the size ranging from 106 bp to 532 bp. Subsequently, the authentication of each fragment was also confirmed by sequencing. Random analyses of adulterants with various meats yielded the identical results to their components, showing the suitability of the multi-PCR assay for tracing of adulterant meats with high-accuracy and precision. This assay was sensitive to detect the species-specific DNA in different proportional mixtures of mutton and duck/pork (9.1%-90.9%). In conclusion, this multi-PCR assay successfully discriminated the double-, triple-, quadruple-, and quintuple-mixtures containing variant counterparts. This method will be particularly useful in the detection of mutton adulteration in processed foods further.

  5. Measurement of rRNA Variations in Natural Communities of Microorganisms on the Southeastern U.S. Continental Shelf †

    PubMed Central

    Kramer, Jonathan G.; Singleton, Fred L.

    1993-01-01

    The development of a clear understanding of the physiology of marine prokaryotes is complicated by the difficulties inherent in resolving the activity of various components of natural microbial communities. Application of appropriate molecular biological techniques offers a means of overcoming some of these problems. In this regard, we have used direct probing of bulk RNA purified from selective size fractions to examine variations in the rRNA content of heterotrophic communities and Synechococcus populations on the southeastern U.S. continental shelf. Heterotrophic communities in natural seawater cultures amended with selected substrates were examined. Synechococcus populations were isolated from the water column by differential filtration. The total cellular rRNA content of the target populations was assayed by probing RNA purified from these samples with an oligonucleotide complementing a universally conserved region in the eubacterial 16S rRNA (heterotrophs) or with a 1.5-kbp fragment encoding the Synechococcus sp. strain WH 7803 16S rRNA (cyanobacteria). The analyses revealed that heterotrophic bacteria responded to the addition of glucose and trace nutrients after a 6-h lag period. However, no response was detected after amino acids were added. The cellular rRNA content increased 48-fold before dropping to a value 20 times that detected before nutrients were added. Variations in the rRNA content from Synechococcus spp. followed a distinct diel pattern imposed by the phasing of cell division within the irradiance cycle. The results indicate that careful application of these appropriate molecular biological techniques can be of great use in discerning basic physiological characteristics of selected natural populations and the mechanisms which regulate growth at the subcellular level. Images PMID:16349009

  6. Hexosaminidase assays.

    PubMed

    Wendeler, Michaela; Sandhoff, Konrad

    2009-11-01

    beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

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

    PubMed

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

    2003-01-01

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

  8. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment.

    PubMed

    LaFrentz, B R; Waldbieser, G C; Welch, T J; Shoemaker, C A

    2014-07-01

    Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.

  9. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals

    PubMed Central

    Kakumanu, Madhavi L.; Ponnusamy, Loganathan; Sutton, Haley T.; Meshnick, Steven R.; Nicholson, William L.

    2016-01-01

    A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia-specific PCR targets (ompA, gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “Candidatus Rickettsia amblyommii,” R. montanensis, R. felis, and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii. PMID:26818674

  10. Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

    PubMed

    Kakumanu, Madhavi L; Ponnusamy, Loganathan; Sutton, Haley T; Meshnick, Steven R; Nicholson, William L; Apperson, Charles S

    2016-04-01

    A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.

  11. Control of rRNA transcription in Escherichia coli.

    PubMed Central

    Condon, C; Squires, C; Squires, C L

    1995-01-01

    The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response. PMID:8531889

  12. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    PubMed

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

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

    PubMed Central

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

    1994-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  15. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

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

    PubMed Central

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

    2016-01-01

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

  17. A backward view from 16S rRNA to archaea to the universal tree of life to progenotes: reminiscences of Carl Woese.

    PubMed

    Garrett, Roger A

    2014-01-01

    I first became aware of Carl Woese in the mid-1970s when he and George Fox criticized a few of the 16S rRNA oligonucleotide sequences emerging from Strasbourg in the 10-12 y RNA sequencing project of the first 16S rRNA from Escherichia coli, some of which we were using for assembling RNA binding sites of ribosomal proteins. When I realized that they were attempting to sequence 16S rRNAs from a range of bacteria to classify them phylogenetically, I seriously questioned their sanity. Not because of the goal, which was admirable, but because of the sheer technical difficulty, and slowness, of sequencing large RNA molecules using the original Sanger RNA sequencing method, not to mention the health hazards of regularly preparing rRNA using 20-30 mCi [ (32)P]. My view changed radically, however, with their subsequent prediction of 5S rRNA secondary structures using a phylogenetic approach. Previously, the molecular biology community had been competing to generate the maximum numbers of base pairs in the model RNA molecule E. coli 5S RNA when Fox and Woese introduced the concept of compensatory base changes based on phylogeny for defining secondary structure and applied it to 5S RNA, they found evidence for only about 50% base pairing. This approach had previously been used for tRNA secondary structure predictions but its more general significance had never been acknowledged. Carl subsequently persuaded Harry Noller to apply the same method to predicting secondary structures of the large rRNAs.

  18. Real-Time PCR and Sequencing Assays for Rapid Detection and Identification of Avian Schistosomes in Environmental Samples

    PubMed Central

    Mull, Bonnie J.; Brant, Sara V.; Loker, Eric S.; Collinson, Jeremy; Secor, W. Evan; Hill, Vincent R.

    2015-01-01

    Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification. PMID:25862226

  19. Real-time PCR and sequencing assays for rapid detection and identification of avian schistosomes in environmental samples.

    PubMed

    Jothikumar, Narayanan; Mull, Bonnie J; Brant, Sara V; Loker, Eric S; Collinson, Jeremy; Secor, W Evan; Hill, Vincent R

    2015-06-15

    Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed. A PCR assay was also developed to amplify a 28S rDNA region for subsequent sequencing to identify schistosomes. When applied to surface water samples seeded with Schistosoma mansoni cercariae, the 18S rDNA TaqMan assay enabled detection at a level of 5 S. mansoni cercariae in 100 liters of lake water. The 18S rDNA TaqMan and 28S rDNA PCR sequencing assays were also applied to 100-liter water samples collected from lakes in Nebraska and Wisconsin where there were reported dermatitis outbreaks. Avian schistosome DNA was detected in 11 of 34 lake water samples using the TaqMan assay. Further 28S rDNA sequence analysis of positive samples confirmed the presence of avian schistosome DNA and provided a preliminary identification of the avian schistosomes in 10 of the 11 samples. These data indicate that the broadly schistosome-reactive TaqMan assay can be effective for rapid screening of large-volume water samples for detection of avian schistosomes, thereby facilitating timely response actions to mitigate or prevent dermatitis outbreaks. Additionally, samples positive by the 18S rDNA TaqMan assay can be further assayed using the 28S rDNA sequencing assay to both confirm the presence of schistosomes and contribute to their identification.

  20. An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria.

    PubMed

    Asai, T; Zaporojets, D; Squires, C; Squires, C L

    1999-03-02

    Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.

  1. Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids.

    PubMed Central

    Miller, C A; Patterson, W L; Johnson, P K; Swartzell, C T; Wogoman, F; Albarella, J P; Carrico, R J

    1988-01-01

    A novel nucleic acid hybridization assay with a DNA probe immobilized on 1.25-micron-diameter latex particles was developed. Hybridization of the immobilized probe DNA with sample rRNA was complete in 10 to 15 min. Alkaline phosphatase-labeled anti-DNA-RNA was allowed to bind to the DNA-RNA hybrids on the latex particles. Then the latex was collected on a small glass fiber filter pad, and bound alkaline phosphatase was quantitated by reflectance rate measurement. The method detected a broad range of bacterial species and had a detection limit of 500 cells per assay. The assay was used to screen urine samples for bacteriuria and had a sensitivity of 96.2% compared with conventional culture at a decision level of greater than or equal to 10(4) CFU/ml. The hybridization method could have broad application to the detection of bacteria and viruses. PMID:2457597

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

    PubMed Central

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

    2012-01-01

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

  3. Identification of Erwinia stewartii by a ligase chain reaction assay.

    PubMed Central

    Wilson, W J; Wiedmann, M; Dillard, H R; Batt, C A

    1994-01-01

    A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material. Images PMID:7509585

  4. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  5. 5-S-GAD, a novel radical scavenging compound, prevents lens opacity development.

    PubMed

    Akiyama, Nobuko; Umeda, Izumi O; Sogo, Shunji; Nishigori, Hideo; Tsujimoto, Masafumi; Natori, Shunji

    2009-02-15

    The ability of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD)-a novel catechol derivative isolated from an insect as an antibacterial substance-to scavenge free radicals and prevent cataract progression was examined. 5-S-GAD scavenged 1,1-diphenylpicrylhydrazyl (DPPH) and superoxide anions (O(2)(*)(-)), and inhibited lipid peroxidation. It also significantly inhibited the onset of glucocorticoid-induced lens opacification in chick embryos. These effects of 5-S-GAD were stronger than those of N-acetylcarnosine and TEMPOL, which are reported to be effective radical scavengers in the prevention of cataract progression. 5-S-GAD clearly delayed the maturation of cataracts induced by diamide in cultured lenses of rats. Daily instillation of 5-S-GAD retarded the development of lens opacity in galactose-fed rats. Biochemical analysis of the lenses revealed that 20-kDa proteins, presumably consisting of alpha-crystallin, were the most susceptible to oxidative stress, which leads to the carbonylation of the side chains of these proteins. alpha-Crystallin carbonylation induced by diamide or galactose was notably inhibited by 5-S-GAD in a dose-dependent manner. Our results show that 5-S-GAD prevents acute lens opacification in these short-term experimental models, possibly in part by virtue of its antioxidative property, and 5-S-GAD is expected to have long-term pharmaceutical effects.

  6. Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species.

    PubMed

    Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

    2016-08-01

    A PCR-based method targeting single-nucleotide polymorphisms (SNPs) in the 16S rRNA gene was developed for differential identification of Legionella pneumophila and non-Legionella pneumophila. Based on the bioinformatics analysis for 176 Legionella 16S rRNA gene fragments of 56 different Legionella species, a set of SNPs, A(628)C(629) was found to be highly specific to L. pneumophila strains. A multiplex assay was designed that was able to distinguish sites with limited sequence heterogeneity between L. pneumophila and non-L. pneumophila in the targeted 16S rRNA gene. The assay amplified a 261-bp amplicon for Legionella spp. and a set of 203- and 97-bp amplicons only specific to L. pneumophila species. Among 49 ATCC strains and 284 Legionella isolates from environmental water and clinical samples, 100 % of L. pneumophila and non-L. pneumophila strains were correctly identified and differentiated by this assay. The assay presents a more rapid, sensitive and alternative method to the currently available PCR-sequencing detection and differentiation method.

  7. Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.

    PubMed

    Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde

    2008-12-01

    The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

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

    PubMed

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

    2013-01-16

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

  9. The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion

    PubMed Central

    Blyn, Lawrence B.; Risen, Lisa M.; Griffey, Richard H.; Draper, David E.

    2000-01-01

    Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the ‘GTPase center’ of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg2+, L11 and thiostrepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg2+, L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure. PMID:10734197

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

  12. Use of quantitative 16S rRNA PCR to determine bacterial load does not augment conventional cerebrospinal fluid (CSF) cultures among children undergoing treatment for CSF shunt infection☆,☆☆

    PubMed Central

    Simon, Tamara D.; Van Yserloo, Brian; Nelson, Kevin; Gillespie, David; Jensen, Randy; McAllister, James P.; Riva-Cambrin, Jay; Stockmann, Chris; Daly, Judy A.; Blaschke, Anne J.

    2013-01-01

    The aim of this study was to develop a quantitative 16S rRNA assay for determination of bacterial nucleic acid load in cerebrospinal fluid (CSF) shunt infection and to compare quantitative 16S rRNA polymerase chain reaction (PCR) findings to those of conventional bacterial culture in patients treated for CSF shunt infection. We developed a quantitative 16S rRNA PCR assay that detected bacterial load across a range of 2.5 × 109 down to 2.5 × 104 16S copies/mL CSF under experimental conditions for numerous Gram-positive and Gram-negative organisms. However, when applied to archived CSF samples from 25 shunt infection episodes, correlations between positive bacterial culture and 16S rRNA levels were seen in only half of infections, and 16S rRNA levels dropped precipitously after an initial peak on the first day of sample collection. Bacterial load measured using 16S rRNA PCR does not provide sufficient information beyond bacterial culture to inform CSF shunt infection treatment. PMID:23953744

  13. 8 CFR 1236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants... OF ALIENS ORDERED REMOVED Detention of Aliens Prior to Order of Removal § 1236.4 Removal of S-5, S-6, and S-7 nonimmigrants. (a) Condition of classification. As a condition of classification and...

  14. The 5S lean method as a tool of industrial management performances

    NASA Astrophysics Data System (ADS)

    Filip, F. C.; Marascu-Klein, V.

    2015-11-01

    Implementing the 5S (seiri, seiton, seiso, seiketsu, and shitsuke) method is carried out through a significant study whose purpose to analyse and deployment the management performance in order to emphasize the problems and working mistakes, reducing waste (stationary and waiting times), flow transparency, storage areas by properly marking and labelling, establishing standards work (everyone knows exactly where are the necessary things), safety and ergonomic working places (the health of all employees). The study describes the impact of the 5S lean method implemented to storing, cleaning, developing and sustaining a production working place from an industrial company. In order to check and sustain the 5S process, it is needed to use an internal audit, called “5S audit”. Implementing the 5S methodology requires organization and safety of the working process, properly marking and labelling of the working place, and audits to establish the work in progress and to maintain the improved activities.

  15. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function

    PubMed Central

    Nemoto, Naoki; Udagawa, Tsuyoshi; Chowdhury, Wasimul; Kitabatake, Makoto; Shin, Byung-shik; Hiraishi, Hiroyuki; Wang, Suzhi; Singh, Chingakham Ranjit; Brown, Susan J.; Ohno, Mutsuhito; Asano, Katsura

    2013-01-01

    In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation. PMID:26824023

  17. Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency.

    PubMed

    Graziadei, Andrea; Masiewicz, Pawel; Lapinaite, Audrone; Carlomagno, Teresa

    2016-05-01

    RNA modifications confer complexity to the 4-nucleotide polymer; nevertheless, their exact function is mostly unknown. rRNA 2'-O-ribose methylation concentrates to ribosome functional sites and is important for ribosome biogenesis. The methyl group is transferred to rRNA by the box C/D RNPs: The rRNA sequence to be methylated is recognized by a complementary sequence on the guide RNA, which is part of the enzyme. In contrast to their eukaryotic homologs, archaeal box C/D enzymes can be assembled in vitro and are used to study the mechanism of 2'-O-ribose methylation. In Archaea, each guide RNA directs methylation to two distinct rRNA sequences, posing the question whether this dual architecture of the enzyme has a regulatory role. Here we use methylation assays and low-resolution structural analysis with small-angle X-ray scattering to study the methylation reaction guided by the sR26 guide RNA fromPyrococcus furiosus We find that the methylation efficacy at sites D and D' differ substantially, with substrate D' turning over more efficiently than substrate D. This observation correlates well with structural data: The scattering profile of the box C/D RNP half-loaded with substrate D' is similar to that of the holo complex, which has the highest activity. Unexpectedly, the guide RNA secondary structure is not responsible for the functional difference at the D and D' sites. Instead, this difference is recapitulated by the nature of the first base pair of the guide-substrate duplex. We suggest that substrate turnover may occur through a zip mechanism that initiates at the 5'-end of the product.

  18. The specific isolation of complete 5S rDNA units from chromosome 1A of hexaploid, tetraploid, and diploid wheat species using PCR with head-to-head oriented primers.

    PubMed

    Van Campenhout, S; Stappen, J V; Volckaert, G

    2001-08-01

    The presence of 5S rDNA units on chromosome 1A of Triticum aestivum was shown by the development of a specific PCR test, using head-to-head oriented primers. This primer set allowed the amplification of complete 5S DNA units and was used to isolate SS-Rrna-A1 sequences from polyploid and diploid wheat species. Multiple-alignment and parsimony analyses of the 132 sequences divided the sequences into four types. The isolates from T. aestivum and the tetraploid species (T. dicoccoides, T. dicoccum, T durum, T. araraticum, and T timopheevi) were all of one type, which was shown to be closely related to the type mainly characteristic for T. urartu. The other two types were isolated exclusively from the diploid species T. monococcum, T aegilopoides, T. thaoudar, and T. sinskajae and the hexaploid species T. zhukovski. Triticum monococcum was the only species for which representatives of each of the four sequence types were found to be present. Further, we discuss the possible multicluster arrangement of the 5S-Rrna-A1 array.

  19. Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

    PubMed Central

    Lucchini, R; Sogo, J M

    1992-01-01

    The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer. Images PMID:1406621

  20. Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

    PubMed Central

    Whitfeld, P R; Leaver, C J; Bottomley, W; Atchison, B

    1978-01-01

    A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves. Images Fig. 8. PMID:743229

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

    PubMed

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

    2011-07-01

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

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

    PubMed

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

    2008-10-28

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

  3. 104. JOB NO. 1347F, SHEET 5S 1927, ASSEMBLY BUILDING; FORD ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    104. JOB NO. 1347-F, SHEET 5S 1927, ASSEMBLY BUILDING; FORD MOTOR COMPANY; LONGITUDINAL SECTION AND TRUSS DETAILS - Ford Motor Company Long Beach Assembly Plant, Assembly Building, 700 Henry Ford Avenue, Long Beach, Los Angeles County, CA

  4. Hyperfine splitting of B mesons and Bs production at the Υ(5S)

    NASA Astrophysics Data System (ADS)

    Lee-Franzini, J.; Heintz, U.; Lovelock, D. M. J.; Narain, M.; Schamberger, R. D.; Willins, J.; Yanagisawa, C.; Franzini, P.; Tuts, P. M.

    1990-12-01

    Using the Columbia University-Stony Brook (CUSB-II) detector we have studied the inclusive photon spectrum from 2.9×104 Υ(5S) decays. We observe a strong signal due to B*-->Bγ decays. From this we obtain (i) the average B*-B mass difference, 46.7+/-0.4 MeV, (ii) the photon yield per Υ(5S) decay, <γ/Υ(5S)>=1.09+/-0.06, and (iii) the average velocity of the B*'s, <β>=0.156+/-0.010, for a mix of nonstrange (B) and strange (Bs) B* mesons from Υ(5S) decays. From the shape of the photon line, we find that both B and Bs mesons are produced with nearly equal values for the hyperfine splitting of the B and Bs meson systems.

  5. [Comparative study of single strand conformation polymorphism of 4.5S RNA gene in enterobacteria].

    PubMed

    Huang, Y; Gong, L; Zhang, L; Li, S; Zhu, S

    1994-04-01

    A recently developed technique, non-isotopic single strand conformation polymorphism analysis (PCR-SSCP), was applied to study the conserved feature of 4.5S RNA gene in enterobacteria. The 4.5S RNA gene was amplified by the polymerase chain reaction, using the template DNA extracted respectively from five strains of Escherichia coli and three strains of different genera in Enterobacteriaceae, i.e. Proteus vulgaris, Serratia marcescens and Enterobacter aerogenes. The PCR products were then carried out SSCP analysis. The experimental results showed that there seemed to be no detectable differences in the size and single strand conformation of 4.5S RNA genes from above strains, except the negative strand conformation of Enterobacter aerogenes. Thus it can be seen that the secondary structures of 4.5S RNA gene in enterobacteria are quite conservative.

  6. [Implementation of "5S" methodology in laboratory safety and its effect on employee satisfaction].

    PubMed

    Dogan, Yavuz; Ozkutuk, Aydan; Dogan, Ozlem

    2014-04-01

    Health institutions use the accreditation process to achieve improvement across the organization and management of the health care system. An ISO 15189 quality and efficiency standard is the recommended standard for medical laboratories qualification. The "safety and accommodation conditions" of this standard covers the requirement to improve working conditions and maintain the necessary safety precautions. The most inevitable precaution for ensuring a safe environment is the creation of a clean and orderly environment to maintain a potentially safe surroundings. In this context, the 5S application which is a superior improvement tool that has been used by the industry, includes some advantages such as encouraging employees to participate in and to help increase the productivity. The main target of this study was to implement 5S methods in a clinical laboratory of a university hospital for evaluating its effect on employees' satisfaction, and correction of non-compliance in terms of the working environment. To start with, first, 5S education was given to management and employees. Secondly, a 5S team was formed and then the main steps of 5S (Seiri: Sort, Seiton: Set in order, Seiso: Shine, Seiketsu: Standardize, and Shitsuke: Systematize) were implemented for a duration of 3 months. A five-point likert scale questionnaire was used in order to determine and assess the impact of 5S on employees' satisfaction considering the areas such as facilitating the job, the job satisfaction, setting up a safe environment, and the effect of participation in management. Questionnaire form was given to 114 employees who actively worked during the 5S implementation period, and the data obtained from 63 (52.3%) participants (16 male, 47 female) were evaluated. The reliability of the questionnaire's Cronbach's alpha value was determined as 0.858 (p< 0.001). After the implementation of 5S it was observed and determined that facilitating the job and setting up a safe environment created

  7. Absolute frequency measurement of rubidium 5S-7S two-photon transitions.

    PubMed

    Morzyński, Piotr; Wcisło, Piotr; Ablewski, Piotr; Gartman, Rafał; Gawlik, Wojciech; Masłowski, Piotr; Nagórny, Bartłomiej; Ozimek, Filip; Radzewicz, Czesław; Witkowski, Marcin; Ciuryło, Roman; Zawada, Michał

    2013-11-15

    We report the absolute frequency measurements of rubidium 5S-7S two-photon transitions with a cw laser digitally locked to an atomic transition and referenced to an optical frequency comb. The narrow, two-photon transition, 5S-7S (760 nm), insensitive to first-order in a magnetic field, is a promising candidate for frequency reference. The performed tests yielded more accurate transition frequencies than previously reported.

  8. 8 CFR 236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants... of Aliens Prior to Order of Removal § 236.4 Removal of S-5, S-6, and S-7 nonimmigrants. (a) Condition... section 101(a)(15)(S) of the Act, nonimmigrants in S classification must have executed Form I-854, Part...

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

    PubMed Central

    McDonald, James E.; Larsen, Niels; Pennington, Andrea; Connolly, John; Wallis, Corrin; Rooks, David J.; Hall, Neil; McCarthy, Alan J.; Allison, Heather E.

    2016-01-01

    PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by ‘universal’ primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology. PMID:27276347

  10. One-stage surgery through posterior approach-for L5-S1 spondyloptosis

    PubMed Central

    Suslu, Hikmet Turan; Celikoglu, Erhan; Borekcı, Ali; Hıcdonmez, Tufan; Suslu, Hüsnü

    2011-01-01

    Grade 5 spondylolisthesis or spondyloptosis is a rare condition. Generally, the surgical management of spondyloptosis includes multi-staged procedures instead of one-staged procedures. One-stage treatment for spondyloptosis is very rare. A 15-year-old girl with L5-S1 spondyloptosis was admitted with severe low back pain. There was no history of trauma. The patient underwent L5 laminectomy, L5-S1 discectomy, resection of sacral dome, reduction, L3-L4-L5-S1 pedicular screw fixation, and interbody-posterolateral fusion through the posterior approach. The reduction was maintained with bilateral L5-S1 discectomy, resection of the sacral dome, and transpedicular instrumentation from L3 to S1. In this particular case, one-staged approach was adequate for the treatment of L5-S1 spondyloptosis. One-staged surgery using the posterior approach may be adequate for the treatment of L5-S1 spondyloptosis while avoiding the risks inherent in anterior approaches. PMID:23125496

  11. Evaluation of 16S rRNA Gene PCR Sensitivity and Specificity for Diagnosis of Prosthetic Joint Infection: a Prospective Multicenter Cross-Sectional Study

    PubMed Central

    Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-01-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥1 positive sample for strict pathogens and ≥2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

  12. Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

    PubMed

    Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

    2014-10-01

    There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis.

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

    PubMed Central

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

    2016-01-01

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

  14. Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements

    SciTech Connect

    Kim, J.H.; Marshall, A.G. )

    1990-01-23

    Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G{sub 80}{center dot}C{sub 92}{center dot}G{sub 81}{center dot}C{sub 91}-G{sub 82}{center dot}C{sub 90}-A{sub 83}{center dot}U{sub 89}-C{sub 84}{center dot}G{sub 88} and three unpaired U's in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G{sub 21}{center dot}C{sub 58}-A{sub 20}{center dot}U{sub 59}-G{sub 19}{center dot}C{sub 60}-A{sub 18}{center dot}U{sub 61} in helix II, U{sub 32}{center dot}A{sub 46}-G{sub 31}{center dot}C{sub 47}-C{sub 30}{center dot}G{sub 48}-C{sub 29}{center dot}G{sub 49} in helix III, and G{sub 4}{center dot}C{sub 112}-G{sub 5}{center dot}C{sub 111}-U{sub 6}{center dot}G{sub 110} in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.

  15. Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines.

    PubMed

    Liu, Zhan-Lin; Zhang, Daming; Wang, Xiao-Quan; Ma, Xiao-Fei; Wang, Xiao-Ru

    2003-01-01

    Patterns of intragenomic and interspecific variation of 5S rDNA in Pinus (Pinaceae) were studied by cloning and sequencing multiple 5S rDNA repeats from individual trees. Five pines, from both subgenera, Pinus and Strobus, were selected. The 5S rDNA repeat in pines has a conserved 120-base pair (bp) transcribed region and an intergenic spacer region of variable length (382-608 bp). The evolutionary rate in the spacer region is three- to sevenfold higher than in the genic region. We found substantial sequence divergence between the two subgenera. Intragenomic sequence heterogeneity was high for all species, and more than 86% of the clones within each individual were unique. The 5S gene tree revealed that different 5S repeats within individuals are polyphyletic, indicating that their ancestral divergence preceded the speciation events. The degrees of interspecific and intragenomic divergence among diploxylon pines are similar. The observed sequence patterns suggest that concerted evolution has been acting after the diversification of the two subgenera but very weak after the speciation of the four diploxylon pines. Sequence patterns in P. densata are consistent with hybrid origin. It had higher intragenomic diversity and maintained polymorphic copies of the parental types in addition to new and recombinant types unique to the hybrid.

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

    PubMed

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

    2016-08-01

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

  17. Species identification through mitochondrial rRNA genetic analysis

    PubMed Central

    Yang, Li; Tan, Zongqing; Wang, Daren; Xue, Ling; Guan, Min-xin; Huang, Taosheng; Li, Ronghua

    2014-01-01

    Inter-species and intraspecific variations in mitochondrial DNA (mtDNA) were observed in a bioinformatics analysis of the mitochondrial genomic sequences of 11 animal species. Some highly conserved regions were identified in the mitochondrial 12S and 16S ribosomal RNA (rRNA) genes of these species. To test whether these sequences are universally conserved, primers were designed to target the conserved regions of these two genes and were used to amplify DNA from 21 animal tissues, including two of unknown origin. By sequencing these PCR amplicons and aligning the sequences to a database of non-redundant nucleotide sequences, it was confirmed that these amplicons aligned specifically to mtDNA sequences from the expected species of origin. This molecular technique, when combined with bioinformatics, provides a reliable method for the taxonomic classification of animal tissues. PMID:24522485

  18. B semileptonic decays at the Υ(4S) and the Υ(5S)

    NASA Astrophysics Data System (ADS)

    Yanagisawa, C.; Heintz, U.; Lee-Franzini, J.; Lovelock, D. M. J.; Narain, M.; Schamberger, R. D.; Willins, J.; Franzini, P.; Tuts, P. M.

    1991-05-01

    B-meson semileptonic decay spectra have been obtained at the Υ(4S) and at the Υ(5S) at the Cornell Electron Storage Ring with the Columbia University-Stony Brook detector. The branching ratio for B-->eνX at the Υ(4S) is found to be (10.0+/-0.5)%. The electron spectrum of B-->eνX at the Υ(5S) is observed for the first time and the average branching ratio of B,Bs-->eνX is consistent with that for B's from Υ(4S) decays. The shape of the electron spectrum at the Υ(5S) indicates production of B mesons which are heavier than nonstrange B's, presumably strange B's.

  19. Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract.

    PubMed Central

    Reynolds, W F; Bloomer, L S; Gottesfeld, J M

    1983-01-01

    A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography. Images PMID:6866764

  20. Characterization of the L4-L5-S1 motion segment using the stepwise reduction method.

    PubMed

    Jaramillo, Héctor Enrique; Puttlitz, Christian M; McGilvray, Kirk; García, José J

    2016-05-03

    The two aims of this study were to generate data for a more accurate calibration of finite element models including the L5-S1 segment, and to find mechanical differences between the L4-L5 and L5-S1 segments. Then, the range of motion (ROM) and facet forces for the L4-S1 segment were measured using the stepwise reduction method. This consists of sequentially testing and reducing each segment in nine stages by cutting the ligaments, facet capsules, and removing the nucleus. Five L4-S1 human segments (median: 65 years, range: 53-84 years, SD=11.0 years) were loaded under a maximum pure moment of 8Nm. The ROM was measured using stereo-photogrammetry via tracking of three markers and the facet contact forces (CF) were measured using a Tekscan system. The ROM for the L4-L5 segment and all stages showed good agreement with published data. The major differences in ROM between the L4-L5 and L5-S1 segments were found for lateral bending and all stages, for which the L4-L5 ROM was about 1.5-3 times higher than that of the L5-S1 segment, consistent with L5-S1 facet CF about 1.3 to 4 times higher than those measured for the L4-L5 segment. For the other movements and few stages, the L4-L5 ROM was significantly lower that of the L5-S1 segment. ROM and CF provide important baseline data for more accurate calibration of FE models and to understand the role that their structures play in lower lumbar spine mechanics.

  1. Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs

    NASA Technical Reports Server (NTRS)

    Delihas, N.; Fox, G. E.

    1987-01-01

    In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.

  2. Xenopus U3 snoRNA GAC-Box A′ and Box A Sequences Play Distinct Functional Roles in rRNA Processing

    PubMed Central

    Borovjagin, Anton V.; Gerbi, Susan A.

    2001-01-01

    Mutations in the 5′ portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3′ region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5′ and 3′ ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A′ region is necessary for site 1 cleavage, and the 3′ end of box A′ and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5′ and 3′ ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage. PMID:11509664

  3. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  4. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  5. Relationship between 5S and 20S forms of malate synthase in maturing cottonseeds

    SciTech Connect

    Turley, R.B.; Trelease, R.N.

    1987-04-01

    Malate synthase (MS) activity appears and increases during seed maturation persists during desiccation, then increases again following germination. Because different modes of synthesis and organelle import of MS may occur in maturing and germinated seeds, a comparative study was performed. A comparison of immunoprecipitations from in-vivo-labeled seeds (/sup 35/S-met) and in-vitro translations of Poly A+ RNA (wheat germ) showed no detectable differences in subunit mol wt. When MS activity first appears (42 DPA) only the cytosolic 5S form is found in rate-zonal gradients (5-25% sucrose). At 48 DPA, O d, and 48 h germinated seeds both the 5S and glyoxysomal 20S forms are present, with the 20S becoming more prevalent. Western blots of SDS-PAGE gels showed that no other form(s) of MS (inactive) are present in rate-zonal fractions. Calculations of radiospecific activity (per MS activity) of 5S and 20S forms radiolabeled in vivo (/sup 35/S-met) at various time periods provided further convincing evidence that there is a 5S precursor to 20S product relationship during both seed maturation and seedling growth.

  6. Nucleotide sequences of 5S rRNAs from four jellyfishes.

    PubMed

    Hori, H; Ohama, T; Kumazaki, T; Osawa, S

    1982-11-25

    The nucleotide sequences of 5S rRNAs from four jellyfishes, Spirocodon saltatrix, Nemopsis dofleini, Aurelia aurita and Chrysaora quinquecirrha have been determined. The sequences are highly similar to each other. A fairly high similarity was also found between these jellyfishes and a sea anemone, Anthopleura japonica.

  7. USE OF INTERSPECIES CORRELATION ESTIMATIONS TO PREDICT HC5'S BASED ON MINIMAL DATA

    EPA Science Inventory

    Dyer, S., S. Belanger, J. Chaney, D. Versteeg and F. Mayer. In press. Use of Interspecies Correlation Estimations to Predict HC5's Based on Minimal Data (Abstract). To be presented at the SETAC Fourth World Congress, 14-18 November 2004, Portland, OR. 1 p. (ERL,GB R1013).

  8. 5. S U.S. HIGHWAY 34 AND EAST (ILLINOIS) APPROACH TO ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. S U.S. HIGHWAY 34 AND EAST (ILLINOIS) APPROACH TO BRIDGE WITH EAST BRIDGE HOUSE IN RIGHT FOREGROUND. VIEW TO WEST. - MacArthur Bridge, Spanning Mississippi River on Highway 34 between IA & IL, Burlington, Des Moines County, IA

  9. Photodissociation dynamics of superexcited O2: Dissociation channels O(5S) vs. O(3S)

    NASA Astrophysics Data System (ADS)

    Zhou, Yiyong; Meng, Qingnan; Mo, Yuxiang

    2014-07-01

    The photodissociation dynamics of O2, O2 + hυ → O(3P) + O(2p3(4S)3s, 3S/5S), has been studied by combining the XUV laser pump / UV laser probe and velocity map imaging methods in the photon energy range 14.64-15.20 eV. The fragment yield spectra of O(3S) and O(5S) and their velocity map images have been recorded using the state-selective (1+1) REMPI method to detect the fragments. The fragment yield spectra show resolved fine structure that arises from the predissociated Rydberg states I, I' and I″ (3ΠΩ = 0,1,2). The branching ratios between the two decay channels have been measured by one-photon ionization of the fragments O(3S) and O(5S) simultaneously. It is surprising to find that the dissociation cross sections for the production of O(5S) are larger than, or comparable to, those of O(3S) for the I and I' states, while the cross sections for the production of O(5S) are smaller than those of O(3S) for the I″ state. All fragments O(5S) arise from perpendicular transitions, which provides direct experimental evidence about the symmetry assignments of the states I, I' and I″ excited in this energy region. Although most of the fragments O(3S) arise from perpendicular transitions, some of them are from parallel transitions. Based on the calculated ab initio potential energy curves, we propose that the neutral dissociation into O(3P) + O(3S) occurs mainly via the interaction of the Rydberg states I, I', and I″ with the vibrational continuum of the diabatic 83Πu state (1π _u^{ - 1} (a^4 {Π}_u {)3}sσ _g ,^3 Π_u), while the neutral dissociation into O(3P) + O(5S) occurs mainly via the interaction of Rydberg states I, I', and I″ with the diabatic 73Πu (1π _g^{ - 1} (X^2 {Π}_g {)3}p{σ }_u ,^3 Π_u).

  10. Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp

    EPA Science Inventory

    Chicken feces are vectors of human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a pre...

  11. Development and evaluation of 16S rRNA gene targeting Enterococcus genus- and species-specific assays

    EPA Science Inventory

    Enterococci have been widely used as indicators of fecal pollution in recreational waters. Most studies enumerate enterococci using culture-based techniques that are time consuming and do not provide information on the identity of enterococci species within a given sample. Althou...

  12. Evaluation of Borrelia real time PCR DNA targeting OspA, FlaB and 5S-23S IGS and Borrelia 16S rRNA RT-qPCR.

    PubMed

    de Leeuw, Bertie H C G M; Maraha, Boulos; Hollemans, Leonie; Sprong, Hein; Brandenburg, Afke H; Westenend, Pieter J; Kusters, Johannes G

    2014-12-01

    Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

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

    PubMed

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

    2016-05-15

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

  14. Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.

    PubMed

    Clifford, Robert J; Milillo, Michael; Prestwood, Jackson; Quintero, Reyes; Zurawski, Daniel V; Kwak, Yoon I; Waterman, Paige E; Lesho, Emil P; Mc Gann, Patrick

    2012-01-01

    Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.

  15. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  16. Wheat germ 5S ribosomal RNA common arm fragment conformations observed by sup 1 H and sup 31 P nuclear magnetic resonance spectroscopy

    SciTech Connect

    Wu, Jiejun; Marshall, A.G. )

    1990-02-20

    The nonexchangeable protons of the common arm fragment of wheat germ (Triticum aestivum) ribosomal 5S RNA have been observed by means of high-resolution 500-MHz {sup 1}H NMR spectroscopy in D{sub 2}O solution. Although NMR studies on the exchangeable protons support the presence of two distinct solution structures of the common arm fragment (and of the same base-paired segment in intact 5S rRNA), only a single conformation is manifested in the {sup 1}H NMR behavior of all of the H6 and H5 pyrimidine and most of the H8/H2 purine protons under the same salt conditions. The nonexchangeable protons near the base-paired helix have been assigned by a sequential strategy. Conformational features such as the presence of a cytidine-uridine (C{center dot}U) pair at the loop-helix junction and base stacking into the hairpin loop are evaluated from nuclear Overhauser enhancement spectroscopy (NOESY) data. Double-quantum filtered correlation spectroscopy (DQF-COSY) experiments show that most of the 26 riboses are in the C3{prime}-endo conformation. Finally, backbone conformational changes induced by Mg{sup 2+} and heating have been monitored by {sup 31}P NMR spectroscopy. The results show that the common arm RNA segment can assume two conformations which produce distinguishably different NMR environments at the base-pair hydrogen-bond imino protons but not at nonexchangeable base or ribose proton or backbone phosphate sites.

  17. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  18. Polarizability of 5s25p(2P12) atomic indium

    NASA Astrophysics Data System (ADS)

    Guella, T. P.; Miller, Thomas M.; Bederson, B.; Stockdale, J. A. D.; Jaduszliwer, B.

    1984-06-01

    We have measured the static electric dipole polarizability of ground state 115 49In5s25p(2P12) with a small (~9%) admixture of metastable 5s25p(2P32). Three different methods were used: (a) E-H gradient balance, (b) comparison of deflection in an inhomogeneous electric field with an alkalimetal-atom "standard," and (c) a deflection analysis using a computer program with no adjustable parameters except the polarizability itself. All methods agree to within 11%. Our weighted final result is (10.18+/-1.20)×10-24 cm3. This is in very close agreement to a recent computation by Liberman and Zangwill, using fully relativistic wave functions and including electron correlation.

  19. Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa.

    PubMed

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

    2011-12-15

    Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in South Africa, it is compulsory to test and certify them disease free prior to translocation. A T. parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers, although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing studies have revealed a small number of sequence differences within the 18S rRNA gene in both species but the extent of this sequence variation is unknown. The aim of the current study was to sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot (RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area. T. parva and Theileria sp. (buffalo) were identified in 42% and 28%, respectively, of 252 samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with previously published T. parva sequences from GenBank while 10 sequences grouped with a previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with known pathogenic Theileria species. Our phylogenetic analyses confirm the

  20. Revised bond valence parameters for the P+5/S-2 ion pair

    NASA Astrophysics Data System (ADS)

    Sidey, V.; Shteyfan, A.

    2017-04-01

    The physically reasonable bond valence parameters, r0=2.125 Å and b=0.37 Å, have been derived for the P+5/S-2 ion pair from a representative set of accurately determined low-symmetry thiophosphate structures. These parameters can be recommended for bond valence analysis of thiophosphates as a replacement for the (r0; b) sets previously reported for the same ions.

  1. A new RNA-RNA crosslinking reagent and its application to ribosomal 5S RNA.

    PubMed Central

    Wagner, R; Garrett, R A

    1978-01-01

    The synthesis of a new RNA specific bifunctional crosslinking reagent, 1.4-phenyl-diglyoxal, is described which reacts exclusively with guanosines. The properties of the crosslinked products enabled us to develop a straightforward method for identifying the reacted nucleotides. Results obtained with ribosomal 5S RNA of Escherichia coli demonstrate the formation of an intramolecular crosslink between guanosine-2 and guanosine-112 in the stem region. Images PMID:724507

  2. Nqrs Data for C10H9MnO5S (Subst. No. 1226)

    NASA Astrophysics Data System (ADS)

    Chihara, H.; Nakamura, N.

    This document is part of Subvolume A `Substances Containing Ag … C10H15' of Volume 48 `Nuclear Quadrupole Resonance Spectroscopy Data' of Landolt-Börnstein - Group III `Condensed Matter'. It contains an extract of Section `3.2 Data tables' of the Chapter `3 Nuclear quadrupole resonance data' providing the NQRS data for C10H9MnO5S (Subst. No. 1226)

  3. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  4. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

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

    PubMed Central

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

    2014-01-01

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

  6. rRNA promoter activity in the fast-growing bacterium Vibrio natriegens.

    PubMed

    Aiyar, Sarah E; Gaal, Tamas; Gourse, Richard L

    2002-03-01

    The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.

  7. Two spatially separated phases in semiconducting Rb0.8Fe1.5S2

    DOE PAGES

    Wang, Meng; Tian, Wei; Valdivia, P.; ...

    2014-09-26

    We report neutron scattering and transport measurements on semiconducting Rb0.8Fe1.5S2, a compound isostructural and isoelectronic to the well-studied A0.8FeySe2(A = K, Rb, Cs, Tl/K) superconducting systems. Both resistivity and DC susceptibility measurements reveal a magnetic phase transition at T = 275 K. Neutron diffraction studies show that the 275 K transition originates from a phase with rhombic iron vacancy order which exhibits an in-plane stripe antiferromagnetic ordering below 275 K. In addition, the stripe antiferromagnetic phase interdigitates mesoscopically with an ubiquitous phase with √5 x√5 iron vacancy order. This phase has a magnetic transition at TN = 425 K andmore » an iron vacancy order-disorder transition at TS = 600 K. These two different structural phases are closely similar to those observed in the isomorphous Se materials. Based on the close similarities of the in-plane antiferromagnetic structures, moments sizes, and ordering temperatures in semiconducting Rb0.8Fe1.5S2 and K0.81Fe1.58Se2, we argue that the in-plane antiferromagnetic order arises from strong coupling between local moments. Superconductivity, previously observed in the A0.8FeySe2₋ zSz system, is absent in A0.8Fe1.5S2, which has a semiconducting ground state. We discuss the implied relationship between stripe and block antiferromagnetism and superconductivity in these materials as well as a strategy for further investigation.« less

  8. Identification of Group B Streptococci Using 16S rRNA, cfb, scpB, and atr Genes in Pregnant Women by PCR.

    PubMed

    Mousavi, Seyed Masoud; Hosseini, Seyed Mostafa; Mashouf, Rasoul Yousefi; Arabestani, Mohammad Reza

    2016-12-01

    Streptococcus agalactiae is acommensalorganism, but it may cause infection in susceptible hosts. The aim of this study was to evaluate PCR assay compared with conventional culture method for direct detection of Streptococcus agalactiae. Total of 203 paired low vaginal swabs were collected from women at 35-37 weeks of pregnancy from June 2013 through February 2014 for detection of Streptococcus agalactiae using PCR assay targeting 16S rRNA, cfb, scpB, and atr genes and culture method following broth enrichment. The results were recorded and evaluated for determining of sensitivity, specificity, positive and negative predictive values of PCR assaycompared with culture method. Prevalence of Streptococcus agalactiae was determined as 7.39% (n=15) using culture method; 19.70% (n=40) by PCR targeting 16S rRNA gene; 18.23% (n=37) by targeting atr gene; 17.24% (n=35) by cfb gene; and 8.87% (n=18) by scpB gene. Generally, a total of 49 specimens were considered true positive (27 samples by PCR assay using the four genes in sum, 4 samples only by atr gene PCR, 3 samples only by cfb gene PCR, 2 samples only by culture method, and 13 samples by PCR assay and culture method in common) and prevalence of Streptococcus agalactiae determined 24.14% in Hamadan. The current data demonstrated that performing only culture method for detecting GBS from pregnant women leads to missed false negative carrier individuals. Thus, it is recommended that both the PCR assay and conventional culture method to be performed in order to detect Streptococcus agalactiae.

  9. Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms

    PubMed Central

    Bavykin, Sergei G.; Lysov, Yuri P.; Zakhariev, Vladimir; Kelly, John J.; Jackman, Joany; Stahl, David A.; Cherni, Alexey

    2004-01-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group. PMID:15297521

  10. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group.

    SciTech Connect

    Bayvkin, S. G.; Lysov, Y. P.; Zakhariev, V.; Kelly, J. J.; Jackman, J.; Stahl, D. A.; Cherni, A.; Engelhardt Inst. of Molecular Biology; Loyola Univ.; Johns Hopkins Univ.; Univ. of Washington

    2004-08-01

    In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

  11. Sequence variation and methylation of the flax 5S RNA genes.

    PubMed Central

    Goldsbrough, P B; Ellis, T H; Lomonossoff, G P

    1982-01-01

    The complete sequence of the flax 5S DNA repeat is presented. Length heterogeneity is the consequence of the presence or absence of a single direct repeat and the majority of single base changes are transition mutations. No sequence variation has been found in the coding sequence. The extent of methylation of cytosines has been measured at one location in the gene and one in the spacer. The relationship between the observed sequence heterogeneity and the level of methylation is discussed in the context of the operation of a correction mechanism. Images PMID:6290983

  12. Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis.

    PubMed

    Coelho, Marcia Reed Rodrigues; von der Weid, Irene; Zahner, Viviane; Seldin, Lucy

    2003-05-28

    Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

  13. Refinement of the spinal muscular atrophy locus to the interval between D5S435 and MAP1B

    SciTech Connect

    Soares, V.M.; Brzustowicz, L.M.; Kleyn, P.W.; Knowles, J.A.; Palmer, D.A.; Asokan, S.; Penchaszadeh, G.K.; Gilliam, T.C. ); Munsat, T.L. )

    1993-02-01

    The childhood-onset SMA locus has been mapped to chromosome 5q13, in a region bounded by the proximal locus, D5S6, and the closely linked distal loci, D5S112 and MAP1B. We now describe a highly polymorphic, tightly linked microsatellite marker (D5S435) that is very likely the closet proximal marker to the SMA locus. Multipoint linkage analysis firmly establishes the following order of markers at 5q13; centromere-D5S76-D5S6-D5S435-MAP1B/D5S112-D5S39-telomere. The data indicate that SMA resides in an approximately 0.7-cM (range 01.-2.1) region between D5S435 and MAP1B. This finding reduces by approximately fourfold the genetic region that most likely harbors the SMA locus and will facilitate the physical mapping and cloning of the disease gene region. 24 refs., 3 figs., 1 tab.

  14. Identification of dual false indirect exclusions on the D5S818 and FGA loci.

    PubMed

    Jiang, Wenxiao; Kline, Margaret; Hu, Peter; Wang, Yue

    2011-01-01

    Here, we present a case in which the result of a maternity test was obscured due to two false indirect exclusions that occurred in two out of 15 genetic loci through the use of the AmpFlSTR Identifiler PCR Amplification kit (Applied Biosystems, Foster City, CA). The Identifiler kit failed to amplify allele 11 of the D5S818 system on the child and failed to capture the existence of allele 13 on the FGA system on both mother and child. The situation was remedied through use of the PowerPlex 16 PCR Amplification Kit (Promega, Madison, WI) which used different primers with a different allele range than that of the Identifiler kit. Maternity was confirmed through sequencing and it was found that the failure of the Identifiler kit to amplify allele 11 on the D5S818 system was the result of an incompatibility to the primer-binding site due to a mutation that changed a guanine (G) into a thymine (T) 55 base pairs (bp) downstream of the repeat. The inability of the Identifiler kit to pick up allele 13 of the FGA system was due to the out-of-range location of the allele. Indirect exclusions can be misleading if they are not fully investigated since allele range as well as primer-binding affinity are two confounders that must be addressed to ensure accuracy of the test results.

  15. iPhone 4s and iPhone 5s Imaging of the Eye

    PubMed Central

    Jalil, Maaz; Ferenczy, Sandor R.; Shields, Carol L.

    2017-01-01

    Background/Aims To evaluate the technical feasibility of a consumer-grade cellular iPhone camera as an ocular imaging device compared to existing ophthalmic imaging equipment for documentation purposes. Methods A comparison of iPhone 4s and 5s images was made with external facial images (macrophotography) using Nikon cameras, slit-lamp images (microphotography) using Zeiss photo slit-lamp camera, and fundus images (fundus photography) using RetCam II. Results In an analysis of six consecutive patients with ophthalmic conditions, both iPhones achieved documentation of external findings (macrophotography) using standard camera modality, tap to focus, and built-in flash. Both iPhones achieved documentation of anterior segment findings (microphotography) during slit-lamp examination through oculars. Both iPhones achieved fundus imaging using standard video modality with continuous iPhone illumination through an ophthalmic lens. Comparison to standard ophthalmic cameras, macrophotography and microphotography were excellent. In comparison to RetCam fundus photography, iPhone fundus photography revealed smaller field and was technically more difficult to obtain, but the quality was nearly similar to RetCam. Conclusions iPhone versions 4s and 5s can provide excellent ophthalmic macrophotography and microphotography and adequate fundus photography. We believe that iPhone imaging could be most useful in settings where expensive, complicated, and cumbersome imaging equipment is unavailable. PMID:28275604

  16. Magnetization reversal phenomena in (Cr0.70Ti0.30)5S6

    NASA Astrophysics Data System (ADS)

    Hashimoto, Satoshi; Matsuda, Yuji; Sato, Tetsuya; Anzai, Shuichiro

    2005-12-01

    Magnetization reversal phenomena (MRP) along magnetic order-order transitions have recently been reported on impurity-doped magnetic systems. Because imperfect long-range magnetic order exists in these systems, it is expected that a systematic investigation of MRP will give physical information on thermomagnetic processes of magnetic systems in the range from the micro- to nanoscales. As a typical order-order transition (a state doubly modulated by helical and canting orders to a collinear ferrimagnetic state) has been known to occur on Cr5S6 at a transition temperature Tt, we investigate the magnetizations of (Cr0.70Ti0.30)5S6 on heating and cooling runs in various magnetic fields. At 20Oe, the field-cooled magnetization just below the Curie temperature has a positive sign; the sign turns negative below the compensation temperature TCM (first step) and finally returns to positive below Tt (second step). The first-step MRP observed in this system is explained by the potential barriers resulting from anisotropy energy when the preferred direction of collinear ferrimagnetic moment reverses. The proposed mechanism for second-step MRP is the pinning effect caused by the impurity atoms (Ti) in the helical long-range-order chains. Comparing other examples of MRPs, we discuss the roles of local impurity centers in the thermomagnetic process in magnetic order-order transitions.

  17. Measurements of exclusive Bs0 decays at the Υ(5S) resonance

    NASA Astrophysics Data System (ADS)

    Drutskoy, A.; Abe, K.; Adachi, I.; Aihara, H.; Anipko, D.; Bakich, A. M.; Barberio, E.; Bedny, I.; Bitenc, U.; Bizjak, I.; Blyth, S.; Bondar, A.; Bračko, M.; Browder, T. E.; Chang, M.-C.; Chang, P.; Chao, Y.; Chen, A.; Chen, K.-F.; Chen, W. T.; Cheon, B. G.; Chistov, R.; Choi, Y.; Dalseno, J.; Danilov, M.; Dash, M.; Dragic, J.; Eidelman, S.; Fratina, S.; Gabyshev, N.; Golob, B.; Ha, H.; Haba, J.; Hara, T.; Hayashii, H.; Hazumi, M.; Heffernan, D.; Hoshi, Y.; Hou, W.-S.; Hsiung, Y. B.; Ikado, K.; Inami, K.; Ishikawa, A.; Ishino, H.; Itoh, R.; Iwasaki, M.; Iwasaki, Y.; Kang, J. H.; Kapusta, P.; Kawai, H.; Kawasaki, T.; Kim, H. J.; Kim, H. O.; Kim, Y. J.; Kinoshita, K.; Korpar, S.; Križan, P.; Krokovny, P.; Kulasiri, R.; Kumar, R.; Kuo, C. C.; Kuzmin, A.; Kwon, Y.-J.; Lee, M. J.; Limosani, A.; Lin, S.-W.; Liventsev, D.; MacNaughton, J.; Majumder, G.; Matsumoto, T.; McOnie, S.; Mitaroff, W.; Miyabayashi, K.; Miyata, H.; Miyazaki, Y.; Moloney, G. R.; Nakano, E.; Nakao, M.; Natkaniec, Z.; Nishida, S.; Ogawa, S.; Ohshima, T.; Okuno, S.; Olsen, S. L.; Onuki, Y.; Ozaki, H.; Pakhlov, P.; Pakhlova, G.; Pestotnik, R.; Piilonen, L. E.; Sakai, Y.; Satoyama, N.; Schietinger, T.; Schneider, O.; Schümann, J.; Schwartz, A. J.; Seidl, R.; Senyo, K.; Sevior, M. E.; Shapkin, M.; Shibuya, H.; Singh, J. B.; Somov, A.; Soni, N.; Stanič, S.; Starič, M.; Stoeck, H.; Sumisawa, K.; Sumiyoshi, T.; Suzuki, S.; Takasaki, F.; Tamai, K.; Tanaka, M.; Taylor, G. N.; Teramoto, Y.; Tian, X. C.; Tikhomirov, I.; Uehara, S.; Ueno, K.; Unno, Y.; Uno, S.; Ushiroda, Y.; Usov, Y.; Varner, G.; Villa, S.; Vinokurova, A.; Wang, C. H.; Watanabe, Y.; Wicht, J.; Yabsley, B. D.; Yamaguchi, A.; Yamashita, Y.; Yamauchi, M.; Zhilich, V.; Zhulanov, V.; Zupanc, A.

    2007-07-01

    Several exclusive Bs0 decays are studied using a 1.86fb-1 data sample collected at the Υ(5S) resonance with the Belle detector at the KEKB asymmetric energy e+e- collider. In the Bs0→Ds-π+ decay mode we find 10 Bs0 candidates and measure the corresponding branching fraction. Combining the Bs0→Ds(*)-π+, Bs0→Ds(*)-ρ+, Bs0→J/ψϕ, and Bs0→J/ψη decay modes, a significant Bs0 signal is observed. The ratio σ(e+e-→Bs*B¯s*)/σ(e+e-→Bs(*)B¯s(*))=(93-9+7±1)% is obtained at the Υ(5S) energy, indicating that Bs0 meson production proceeds predominantly through the creation of Bs*B¯s* pairs. The Bs0 and Bs* meson masses are measured to be M(Bs0)=(5370±1±3)MeV/c2 and M(Bs*)=(5418±1±3)MeV/c2. Upper limits on the Bs0→γγ, Bs0→ϕγ, Bs0→K+K-, and Bs0→Ds(*)+Ds(*)- branching fractions are also reported.

  18. Superconducting H5S2 phase in sulfur-hydrogen system under high-pressure

    PubMed Central

    Ishikawa, Takahiro; Nakanishi, Akitaka; Shimizu, Katsuya; Katayama-Yoshida, Hiroshi; Oda, Tatsuki; Suzuki, Naoshi

    2016-01-01

    Recently, hydrogen sulfide was experimentally found to show the high superconducting critical temperature (Tc) under high-pressure. The superconducting Tc shows 30–70 K in pressure range of 100–170 GPa (low-Tc phase) and increases to 203 K, which sets a record for the highest Tc in all materials, for the samples annealed by heating it to room temperature at pressures above 150 GPa (high-Tc phase). Here we present a solid H5S2 phase predicted as the low-Tc phase by the application of the genetic algorithm technique for crystal structure searching and first-principles calculations to sulfur-hydrogen system under high-pressure. The H5S2 phase is thermodynamically stabilized at 110 GPa, in which asymmetric hydrogen bonds are formed between H2S and H3S molecules. Calculated Tc values show 50–70 K in pressure range of 100–150 GPa within the harmonic approximation, which can reproduce the experimentally observed low-Tc phase. These findings give a new aspect of the excellent superconductivity in compressed sulfur-hydrogen system. PMID:26983593

  19. Superconducting H5S2 phase in sulfur-hydrogen system under high-pressure

    NASA Astrophysics Data System (ADS)

    Ishikawa, Takahiro; Nakanishi, Akitaka; Shimizu, Katsuya; Katayama-Yoshida, Hiroshi; Oda, Tatsuki; Suzuki, Naoshi

    2016-03-01

    Recently, hydrogen sulfide was experimentally found to show the high superconducting critical temperature (Tc) under high-pressure. The superconducting Tc shows 30–70 K in pressure range of 100–170 GPa (low-Tc phase) and increases to 203 K, which sets a record for the highest Tc in all materials, for the samples annealed by heating it to room temperature at pressures above 150 GPa (high-Tc phase). Here we present a solid H5S2 phase predicted as the low-Tc phase by the application of the genetic algorithm technique for crystal structure searching and first-principles calculations to sulfur-hydrogen system under high-pressure. The H5S2 phase is thermodynamically stabilized at 110 GPa, in which asymmetric hydrogen bonds are formed between H2S and H3S molecules. Calculated Tc values show 50–70 K in pressure range of 100–150 GPa within the harmonic approximation, which can reproduce the experimentally observed low-Tc phase. These findings give a new aspect of the excellent superconductivity in compressed sulfur-hydrogen system.

  20. Nucleolus-like bodies of fully-grown mouse oocytes contain key nucleolar proteins but are impoverished for rRNA.

    PubMed

    Shishova, Kseniya V; Lavrentyeva, Elena A; Dobrucki, Jurek W; Zatsepina, Olga V

    2015-01-15

    It is well known that fully-grown mammalian oocytes, rather than typical nucleoli, contain prominent but structurally homogenous bodies called "nucleolus-like bodies" (NLBs). NLBs accumulate a vast amount of material, but their biochemical composition and functions remain uncertain. To clarify the composition of the NLB material in mouse GV oocytes, we devised an assay to detect internal oocyte proteins with fluorescein-5-isothiocyanate (FITC) and applied the fluorescent RNA-binding dye acridine orange to examine whether NLBs contain RNA. Our results unequivocally show that, similarly to typical nucleoli, proteins and RNA are major constituents of transcriptionally active (or non-surrounded) NLBs as well as of transcriptionally silent (or surrounded) NLBs. We also show, by exposing fixed oocytes to a mild proteinase K treatment, that the NLB mass in oocytes of both types contains nucleolar proteins that are involved in all major steps of ribosome biogenesis, including rDNA transcription (UBF), early rRNA processing (fibrillarin), and late rRNA processing (NPM1/nucleophosmin/B23, nucleolin/C23), but none of the nuclear proteins tested, including SC35, NOBOX, topoisomerase II beta, HP1α, and H3. The ribosomal RPL26 protein was detected within the NLBs of NSN-type oocytes but is virtually absent from NLBs of SN-type oocytes. Taking into account that the major class of nucleolar RNA is ribosomal RNA (rRNA), we applied fluorescence in situ hybridization with oligonucleotide probes targeting 18S and 28S rRNAs. The results show that, in contrast to active nucleoli, NLBs of fully-grown oocytes are impoverished for the rRNAs, which is consistent with the absence of transcribed ribosomal genes in the NLB mass. Overall, the results of this study suggest that NLBs of fully-grown mammalian oocytes serve for storing major nucleolar proteins but not rRNA.

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

    PubMed Central

    Herzog, Michel; Maroteaux, Luc

    1986-01-01

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

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

    PubMed

    Herzog, M; Maroteaux, L

    1986-11-01

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

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

    PubMed

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

    1996-12-01

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

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

    PubMed

    Coleman, Annette W

    2015-03-01

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

  5. Processing pathway of Escherichia coli 16S precursor rRNA.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1989-01-01

    Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA. Images PMID:2646597

  6. Complete nucleotide sequence of the 23S rRNA gene of the Cyanobacterium, Anacystis nidulans.

    PubMed Central

    Douglas, S E; Doolittle, W F

    1984-01-01

    The nucleotide sequence of the Anacystis nidulans 23S rRNA gene, including the 5'- and 3'-flanking regions has been determined. The gene is 2876 nucleotides long and shows higher primary sequence homology to the 23S rRNAs of plastids (84.5%) than to that of E. coli (79%). The predicted rRNA transcript also shares many secondary structural features with those of plastids, reinforcing the endosymbiont hypothesis for the origin of these organelles. PMID:6326060

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

    ERIC Educational Resources Information Center

    Guan, Min-Xin

    2005-01-01

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

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

    PubMed

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

    2017-03-01

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

  9. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  10. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  11. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  12. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

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

    PubMed Central

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

    2008-01-01

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

  14. A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

    PubMed Central

    Liles, Mark R.; Manske, Brian F.; Bintrim, Scott B.; Handelsman, Jo; Goodman, Robert M.

    2003-01-01

    We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division. PMID:12732537

  15. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  16. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  17. Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I

    SciTech Connect

    Kownin, P.; Bateman, E.; Paule, M.R.

    1988-02-01

    Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.

  18. Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

    PubMed Central

    2010-01-01

    Background Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples. PMID:20205957

  19. Structural Insights into the Methylation of C1402 in 16S rRNA by Methyltransferase RsmI

    PubMed Central

    Liu, Guangfeng; Wang, Li; Wang, Jian; Gao, Zengqiang; Dong, Yuhui; Zhang, Linbo; Gong, Yong

    2016-01-01

    RsmI and RsmH are conserved S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) that are responsible for the 2′-O-methylation and N4-methylation of C1402 in bacterial 16S rRNA, respectively. Methylation of m4Cm1402 plays a role in fine-tuning the shape and functions of the P-site to increase the decoding fidelity, and was recently found to contribute to the virulence of Staphylococcus aureus in host animals. Here we report the 2.20-Å crystal structure of homodimeric RsmI from Escherichia coli in complex with the cofactor AdoMet. RsmI consists of an N-terminal putative RNA-binding domain (NTD) and a C-terminal catalytic domain (CTD) with a Rossmann-like fold, and belongs to the class III MTase family. AdoMet is specifically bound into a negatively charged deep pocket formed by both domains by making extensive contacts. Structure-based mutagenesis and isothermal titration calorimetry (ITC) assays revealed Asp100 and Ala124 are vital for AdoMet-binding. Although the overall fold of RsmI shows remarkable similarities to the characterized MTases involved in vitamin B12 biosynthesis, it exhibits a distinct charge distribution especially around the AdoMet-binding pocket because of different substrate specificity. The docking model of RsmI-AdoMet-RNA ternary complex suggested a possible base-flipping mechanism of the substrate RNA that has been observed in several known RNA MTases. Our structural and biochemical studies provide novel insights into the catalytic mechanism of C1402 methylation in 16S rRNA. PMID:27711192

  20. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  1. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  2. Electric-dipole 5s - 5p Transitions in Promethiumlike Ions

    SciTech Connect

    Vilkas, M J; Ishikawa, Y; Trabert, E

    2008-02-29

    The 5s-5p electric-dipole resonance transitions in highly ionized promethiumlike ions have been studied applying relativistic multi-reference Moeller-Plesset second-order perturbation theory. The transition wavelengths are determined to within 0.2 {angstrom} in the more highly charged ions, where the level degeneracies are small. For somewhat lighter ions a very large reference space was used in order to account for the many degeneracies. In order to calculate transition probabilities and lifetimes, correlation corrections have been added to the transition operator in the next order. The contributions from the higher orders of the theory, that is, frequency-dependent Breit correction, Lamb shift, and mass shifts, have been estimated. The results are used to re-assess spectroscopic data from beam-foil, electron beam ion trap, and tokamak observations.

  3. Solution-Based Processing of the Phase-Change Material KSb5S8

    SciTech Connect

    Mitzi,D.; Raoux, S.; Schrott, A.; Copel, M.; Kellock, A.; Jordan-Sweet, J.

    2006-01-01

    A hydrazine-based process for solution-depositing phase-change materials (PCMs) is demonstrated, using KSb{sub 5}S{sub 8} (KSS) as an example. The process involves dissolving the elemental metals and chalcogen in hydrazine at room temperature and spin-coating the solution onto a substrate, followed by a short low-temperature (T {<=} 250 C) anneal. The spin-coated KSS films, which range in thickness from 10 to 90 nm, are examined using variable temperature X-ray diffraction, medium energy ion scattering (MEIS), Rutherford backscattering spectroscopy (RBS), and scanning electron microscopy (SEM). The spin-coated KSS films exhibit a reversible amorphous-crystalline transition with a relatively high crystallization temperature ({approx}280 C). Selected other chalcogenide-based PCMs are also expected to be suitable for thin-film deposition using this approach.

  4. Donor-acceptor pair recombination in AgIn5S8 single crystals

    NASA Astrophysics Data System (ADS)

    Gasanly, N. M.; Serpengüzel, A.; Aydinli, A.; Gürlü, O.; Yilmaz, I.

    1999-03-01

    Photoluminescence (PL) spectra of AgIn5S8 single crystals were investigated in the 1.44-1.91 eV energy region and in the 10-170 K temperature range. The PL band was observed to be centered at 1.65 eV at 10 K and an excitation intensity of 0.97 W cm-2. The redshift of this band with increasing temperature and with decreasing excitation intensity was observed. To explain the observed PL behavior, we propose that the emission is due to radiative recombination of a donor-acceptor pair, with an electron occupying a donor level located at 0.06 eV below the conduction band, and a hole occupying an acceptor level located at 0.32 eV above the valence band.

  5. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site.

    PubMed

    Calviño, Fabiola R; Kharde, Satyavati; Ori, Alessandro; Hendricks, Astrid; Wild, Klemens; Kressler, Dieter; Bange, Gert; Hurt, Ed; Beck, Martin; Sinning, Irmgard

    2015-04-07

    During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1-RpL5-N-RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1-RpL5-RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP.

  6. Classification of 5-S Epileptic EEG Recordings Using Distribution Entropy and Sample Entropy.

    PubMed

    Li, Peng; Karmakar, Chandan; Yan, Chang; Palaniswami, Marimuthu; Liu, Changchun

    2016-01-01

    Epilepsy is an electrophysiological disorder of the brain, the hallmark of which is recurrent and unprovoked seizures. Electroencephalogram (EEG) measures electrical activity of the brain that is commonly applied as a non-invasive technique for seizure detection. Although a vast number of publications have been published on intelligent algorithms to classify interictal and ictal EEG, it remains an open question whether they can be detected using short-length EEG recordings. In this study, we proposed three protocols to select 5 s EEG segment for classifying interictal and ictal EEG from normal. We used the publicly-accessible Bonn database, which consists of normal, interical, and ictal EEG signals with a length of 4097 sampling points (23.6 s) per record. In this study, we selected three segments of 868 points (5 s) length from each recordings and evaluated results for each of them separately. The well-studied irregularity measure-sample entropy (SampEn)-and a more recently proposed complexity measure-distribution entropy (DistEn)-were used as classification features. A total of 20 combinations of input parameters m and τ for the calculation of SampEn and DistEn were selected for compatibility. Results showed that SampEn was undefined for half of the used combinations of input parameters and indicated a large intra-class variance. Moreover, DistEn performed robustly for short-length EEG data indicating relative independence from input parameters and small intra-class fluctuations. In addition, it showed acceptable performance for all three classification problems (interictal EEG from normal, ictal EEG from normal, and ictal EEG from interictal) compared to SampEn, which showed better results only for distinguishing normal EEG from interictal and ictal. Both SampEn and DistEn showed good reproducibility and consistency, as evidenced by the independence of results on analysing protocol.

  7. Measuring B{sub s} width difference at the {Upsilon}(5s) using quantum entanglement

    SciTech Connect

    Atwood, David; Soni, Amarjit

    2010-08-01

    About 90% of B{sub s}B{sub s} pairs produced at the {Upsilon}(5s) resonance are initially B{sub s}{sup *}B{sub s}{sup *} pairs which decay radiatively to B{sub s}B{sub s}. This implies that the B{sub s}B{sub s} pair will then be in an eigenstate of charge conjugation (i.e. C=-1) and therefore in an entangled state. This allows for a determination of {Delta}{Gamma}{sub s}/{Gamma}{sub s} and the CP phase using a number of possible correlations between the decays of the two B{sub s} mesons. In particular, we consider the time integrated correlation, the time ordering asymmetry, and the time ordering-charge asymmetry, which in addition to time ordering distinguishes B{sub s} from B{sub s}, for various combinations of final states. With the statistics of about O(10{sup 7}-10{sup 8}) {Upsilon}(5s) events available at B factories, we find that the time ordering asymmetry between suitably defined hadronic and flavor specific (tagging) decays offers a promising method for determining the width difference. The corresponding time ordering-charge asymmetry can also bound the mixing phase. Similar observables involving exclusive decays are also considered. At the super B factories with O(50) times greater luminosity time ordering and time ordering-charge asymmetries between inclusive and exclusive modes may also provide additional bounds on the phases in those decays.

  8. Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

    USGS Publications Warehouse

    Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

    2007-01-01

    We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

  9. Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

    PubMed

    Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

    2015-10-01

    The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.

  10. Rapid identification of Penicillium marneffei by PCR-based detection of specific sequences on the rRNA gene.

    PubMed

    Vanittanakom, Nongnuch; Vanittanakom, Pramote; Hay, Roderick J

    2002-05-01

    An emerging pathogenic dimorphic fungus, Penicillium marneffei, is one of the major causes of morbidity in patients with human immunodeficiency virus infection in Southeast Asia. A PCR-hybridization assay has been developed to identify this pathogen. This study describes the use of single and nested PCR methods for the rapid identification of P. marneffei. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA genes of P. marneffei. The outer primers (RRF1 and RRH1) were fungus specific. The inner primers (Pm1 and Pm2) were specific for P. marneffei and were used in nested or single PCR. The specific fragment of approximately 400-bp was amplified from both mold and yeast forms of 13 P. marneffei human isolates, 12 bamboo rat isolates, and 1 soil isolate, but not from other fungi, bacteria, and human DNA. The amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The sensitivities of the single PCR and nested PCR were 1.0 pg/microl and 1.8 fg/microl, respectively. The assay is useful for rapid identification of P. marneffei cultures. Very young culture of P. marneffei (2-day-old filamentous colony, 2 mm in diameter) could be performed by this assay. The species was identified within 7 h (single PCR) or 10 h (nested PCR), compared to 4 to 7 days for confirmation of dimorphism. The application of these PCR methods for early diagnosis of the disease needs to be studied further.

  11. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  12. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  13. Uncultivated microbial eukaryotic diversity: a method to link ssu rRNA gene sequences with morphology.

    PubMed

    Hirst, Marissa B; Kita, Kelley N; Dawson, Scott C

    2011-01-01

    Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages

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

    PubMed

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

    2016-01-01

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

  15. Improved solid-phase extraction and liquid chromatography with electrochemical detection of urinary catecholamines and 5-S-L-cysteinyl-L-dopa.

    PubMed

    Huang, T H; Wall, J; Kabra, P

    1988-10-28

    We describe a rapid, precise, accurate liquid chromatographic procedure for determining urinary catecholamines and 5-S-L-cysteinyl-L-dopa. The catecholamines (norepinephrine, epinephrine, and dopamine) and 5-S-L-cysteinyl-L-dopa are extracted from 1.0 ml of urine together with internal standards, by using a Bond-Elut strong cation-exchange (SCX) and an affinity phenylboronic acid (PBA) extraction column in series. The eluate obtained from PBA column is then chromatographed on a reversed-phase C18 column with a mobile phase containing pentane- and heptanesulfonate as ion-pair reagents. The detection is achieved with an amperometric detector set at an oxidation potential of +0.55 V. The chromatography is complete is less than 8 min for catecholamines and less than 5 min for cysteinyldopa. The method can measure less than 2 micrograms/l for catecholamines and 5 micrograms/l for cysteinyldopa. Analytical recoveries of catecholamines and cysteinyldopa added to urine pool ranged from 90-107%. Between run coefficient of variation ranged from 4.7 to 8%. None of the drugs and catecholamines metabolites tested interfered with the assay.

  16. 5S ribosomal RNA genes in six species of Mediterranean grey mullets: genomic organization and phylogenetic inference.

    PubMed

    Gornung, Ekaterina; Colangelo, Paolo; Annesi, Flavia

    2007-09-01

    This paper describes a study of the 5S ribosomal RNA genes (5S rDNA) in a group of 6 species belonging to 4 genera of Mugilidae. In these 6 species, the relatively short 5S rDNA repeat units, generated by PCR and ranging in size from 219 to 257 bp, show a high level of intragenomic homogeneity of both coding and spacer regions (NTS-I). Phylogenetic reconstructions based on this data set highlight the greater phylogenetic and genetic diversity of Mugil cephalus and Oedalechilus labeo compared with the genera Liza and Chelon. Comparative sequence analysis revealed significant conservation of the short 5S rDNA repeat units across Chelon and Liza. Moreover, a second size class of 5S rDNA repeat units, ranging from roughly 800 to 1100 bp, was produced in the Liza and Chelon samples. Only short 5S rDNA repeat units were found in M. cephalus and O. labeo. The sequences of the long 5S rDNA repeat units, obtained in Chelon labrosus and Liza ramada, differ owing to the presence of 2 large insertion/deletions (indels) in the spacers (NTS-II) and show considerable sequence identity with NTS-I spacers. Interspecific sequence variation of NTS-II spacers, excluding the indels, is low. Southern-blot hybridization patterns suggest an intermixed arrangement of short and long repeat units within a single chromosome locus.

  17. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  18. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  19. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  20. Cysteine-dependent 5-S-cysteinyldopa formation and its regulation by glutathione in normal epidermal melanocytes.

    PubMed

    Benathan, M; Labidi, F

    1996-10-01

    Recent evidence suggests that the melanogenesis intermediate 5-S-cysteinyldopa (5-S-CD) could display antioxidative activity. In the present study, the synthesis of 5-S-CD was examined in human epidermal melanocytes isolated from dark skin type VI (MT) and from white skin type III (GT). The MT melanocytes showed the higher melanin content and dopa oxidase activity. In addition, they produced eumelanin as shown by their ultrastructure, and the solubility and UV/visible absorption of the isolated pigment. Both MT and GT cells showed high levels of 5-S-CD (5.5-6.9 nmol/mg protein). 5-S-CD was also detected in culture supernatants from MT cells; the secretion rate was estimated to be 2.5 nmol/mg protein per 24 h. The role of cysteine and glutathione in 5-S-CD formation was investigated by exposing the melanocytes to the gamma-glutamylcysteine synthetase inhibitor L-buthionine sulfoximine (BSO). A strong reduction in glutathione levels (4-8% of the untreated controls) associated with an increase in cysteine levels (152-154%) was observed. In addition, BSO induced a moderate increase in the cellular levels of 5-S-CD (114-129%) and a decrease in dopa oxidase activity (75-83%). Our results indicate that the direct addition of cysteine to dopaquinone is the main source of 5-S-CD in human epidermal melanocytes. It is proposed that the synthesis of 5-S-CD is a mechanism regulating dopaquinone levels during pigment formation and/or a defence mechanism against oxidative stress.

  1. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  2. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  3. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R; Benett, William J; Coleman, Matthew A; Pearson, Francesca S; Nasarabadi, Shanavaz L

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  4. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  5. Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).

    PubMed

    Kishida, Naohiro; Miyata, Ryo; Furuta, Atsushi; Izumiyama, Shinji; Tsuneda, Satoshi; Sekiguchi, Yuji; Noda, Naohiro; Akiba, Michihiro

    2012-01-01

    We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.

  6. Regulation of gene expression via retrotransposon insertions and the noncoding RNA 4.5S RNAH.

    PubMed

    Ishida, Kentaro; Miyauchi, Kenjyo; Kimura, Yuko; Mito, Mari; Okada, Shunpei; Suzuki, Tsutomu; Nakagawa, Shinichi

    2015-11-01

    Short interspersed elements (SINEs) comprise a significant portion of mammalian genomes and regulate gene expression through a variety of mechanisms. Here, we show that Myodonta clade-specific 4.5S RNAH (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of reporter gene that contains the antisense insertion of SINE B1 via nuclear retention. The depletion of endogenous 4.5SH with antisense oligonucleotides neutralizes the nuclear retention and changes the subcellular distribution of the reporter transcripts containing the antisense SINE B1 insertion. Importantly, endogenous transcripts with antisense SINE B1 were increased in the cytoplasm after knockdown of 4.5SH, leading to a decrease in cellular growth. We propose a tentative hypothesis that the amplification of the 4.5SH cluster in specific rodent species might delineate their evolutionary direction via the regulation of genes containing the antisense insertion of SINE B1.

  7. Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes).

    PubMed

    Noleto, Rafael Bueno; Vicari, Marcelo Ricardo; Cipriano, Roger Raupp; Artoni, Roberto Ferreira; Cestari, Marta Margarete

    2007-06-01

    Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA3-positive. Giemsa karyotypes of tetraodontid species presents 2n=46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n=50 chromosomes (4m+18sm+12st+16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.

  8. Design and performance of a 1 MW-5 s high temperature superconductor magnetic energy storage system

    NASA Astrophysics Data System (ADS)

    Morandi, Antonio; Gholizad, Babak; Fabbri, Massimo

    2016-01-01

    The feasibility of a 1 MW-5 s superconducting magnetic energy storage (SMES) system based on state-of-the-art high-temperature superconductor (HTS) materials is investigated in detail. Both YBCO coated conductors and MgB2 are considered. A procedure for the electromagnetic design of the coil is introduced and the final layout is arrived at and compared for the two materials. The choice of the inductance of the coil is carried out as part of the design procedure. Both low-field (3 T) and high-field (8 T) designs are considered for the YBCO. AC losses during a complete charge/discharge cycle at full power are estimated and the cooling power needed for continuous operation is derived. The power conditioning system and control algorithms needed to carry out various operations are discussed in detail. Performances of the SMES system during voltage sag compensation, load leveling and power factor correction are investigated by means of numerical simulation.

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

    PubMed

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

    2016-05-01

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

  10. Unstable Inheritance of 45S rRNA Genes in Arabidopsis thaliana

    PubMed Central

    Rabanal, Fernando A.; Nizhynska, Viktoria; Mandáková, Terezie; Novikova, Polina Yu.; Lysak, Martin A.; Mott, Richard; Nordborg, Magnus

    2017-01-01

    The considerable genome size variation in Arabidopsis thaliana has been shown largely to be due to copy number variation (CNV) in 45S ribosomal RNA (rRNA) genes. Surprisingly, attempts to map this variation by means of genome-wide association studies (GWAS) failed to identify either of the two likely sources, namely the nucleolus organizer regions (NORs). Instead, GWAS implicated a trans-acting locus, as if rRNA gene CNV was a phenotype rather than a genotype. To explain these results, we investigated the inheritance and stability of rRNA gene copy number using the variety of genetic resources available in A. thaliana — F2 crosses, recombinant inbred lines, the multiparent advanced-generation inter-cross population, and mutation accumulation lines. Our results clearly show that rRNA gene CNV can be mapped to the NORs themselves, with both loci contributing equally to the variation. However, NOR size is unstably inherited, and dramatic copy number changes are visible already within tens of generations, which explains why it is not possible to map the NORs using GWAS. We did not find any evidence of trans-acting loci in crosses, which is also expected since changes due to such loci would take very many generations to manifest themselves. rRNA gene copy number is thus an interesting example of “missing heritability”—a trait that is heritable in pedigrees, but not in the general population. PMID:28188182

  11. Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

    PubMed

    Tongaonkar, Prasad; French, Sarah L; Oakes, Melanie L; Vu, Loan; Schneider, David A; Beyer, Ann L; Nomura, Masayasu

    2005-07-19

    Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Deep sequencing of subseafloor eukaryotic rRNA reveals active Fungi across marine subsurface provinces.

    PubMed

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

    2013-01-01

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

  15. Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences.

    PubMed Central

    Krych, M; Sirdeshmukh, R; Gourse, R; Schlessinger, D

    1987-01-01

    To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed. Images PMID:2445728

  16. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  17. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  18. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  19. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

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

    PubMed

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

    2016-03-01

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

  1. Infective Arthritis: Bacterial 23S rRNA Gene Sequencing as a Supplementary Diagnostic Method

    PubMed Central

    Moser, Claus; Andresen, Keld; Kjerulf, Anne; Salamon, Suheil; Kemp, Michael; Christensen, Jens Jørgen

    2008-01-01

    Consecutively collected synovial fluids were examined for presence of bacterial DNA (a 700-bp fragment of the bacterial 23S rRNA gene) followed by DNA sequencing of amplicons, and by conventional bacteriological methods. One or more microorganisms were identified in 22 of the 227 synovial fluids (9,7%) originating from 17 patients. Sixteen of the patients had clinical signs of arthritis. For 11 patients molecular and conventional bacterial examinations were in agreement. Staphylococcus aureus, Streptococcus dysgalactiae subspecies equisimilis and Streptococcus pneumoniae, were detected in synovial fluids from 6, 2 and 2 patients, respectively. In 3 patients only 23S rRNA analysis was positive; 2 synovial fluids contained S. dysgalactiae subspecies equisimilis and 1 S. pneumoniae). The present study indicates a significant contribution by PCR with subsequent DNA sequencing of the 23S rRNA gene analysis in recognizing and identification of microorganisms from synovial fluids. PMID:19088916

  2. Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage.

    PubMed

    Zhang, Qiao; Shalaby, Nevine A; Buszczak, Michael

    2014-01-17

    Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

  3. Sequence organization of the Acanthamoeba rRNA intergenic spacer: identification of transcriptional enhancers.

    PubMed Central

    Yang, Q; Zwick, M G; Paule, M R

    1994-01-01

    The primary sequence of the entire 2330 bp intergenic spacer of the A.castellanii ribosomal RNA gene was determined. Repeated sequence elements averaging 140 bp were identified and found to bind a protein required for optimum initiation at the core promoter. These repeated elements were shown to stimulate rRNA transcription by RNA polymerase I in vitro. The repeats inhibited transcription when placed in trans, and stimulated transcription when in cis, in either orientation, but only when upstream of the core promoter. Thus, these repeated elements have characteristics similar to polymerase I enhancers found in higher eukaryotes. The number of rRNA repeats in Acanthamoeba cells was determined to be 24 per haploid genome, the lowest number so far identified in any eukaryote. However, because Acanthamoeba is polyploid, each cell contains approximately 600 rRNA genes. Images PMID:7984432

  4. Specific binding of tRNAMet to 23S rRNA of Escherichia coli.

    PubMed Central

    Dahlberg, J E; Kintner, C; Lund, E

    1978-01-01

    tRNAMetf binds to 23S rRNA of Escherichia coli, forming a complex with a melting temperature of 75 degrees (in 0.6 M NaCl). The regions within the RNAs that bind to each other have been isolated and their nucleotide sequences have been determined. The interacting region in tRNAMetf is 17 nucleotides long, extending from G5 in the acceptor stem to D21 (D = 5.6-dihydrouridine) in the D loop. The sequence in 23S rRNA is complementary to that sequence except for an extra Up in the middle and allowing a Gp.D base pair. We propose that association of these two sequences may play a role in initiation of protein synthesis by tRNAMetf. In addition, part of this sequence in 23S rRNA may also stabilize tRNA binding to the ribosome during elongation of nascent polypeptides. Images PMID:349554

  5. Ergometric and metabolic adaptation to a 5-s sprint training programme.

    PubMed

    Linossier, M T; Denis, C; Dormois, D; Geyssant, A; Lacour, J R

    1993-01-01

    The effects of 7 weeks of sprint training (repeated 5-s all-out sprints) on maximal power output (Wv,max) determined during a force-velocity test and a 30-s Wingate test (Wpeak) were studied in ten students [22 (SD 2) years] exercising on a cycle ergometer. Before and after training, muscle biopsies were taken from vastus lateralis muscle at rest for the ten subjects and immediately after a training session for five of them. Sprint training induced an improvement both in peak performances by 25% (Wv,max and Wpeak) and in the 30-s total work by 16%. Before sprint training, the velocity reached with no load (v0) was related to the resting muscle phosphocreatine (PCr) stores (r = 0.87, P < 0.001). The training-induced changes in v0 were observed only when these PCr stores were lowest. This pointed to a possible limiting role of low PCr concentrations in the ability to reach a high velocity. The improvement in performances was linked to an increase in the energy production from anaerobic glycolysis. This result was suggested in muscle by the increase in lactate production measured after a training session associated with the 20% higher activity of both phosphofructokinase and lactate dehydrogenase. The sprint training also increased the proportion of slow twitch fibres closely related to the decrease in fast twitch b fibres. This result would appear to demonstrate an appropriate adaptive reaction following high-intensity intermittent training for the slow twitch fibres which exhibit a greater oxidative capacity.

  6. Network analysis provides insights into evolution of 5S rDNA arrays in Triticum and Aegilops.

    PubMed Central

    Allaby, R G; Brown, T A

    2001-01-01

    We have used network analysis to study gene sequences of the Triticum and Aegilops 5S rDNA arrays, as well as the spacers of the 5S-DNA-A1 and 5S-DNA-2 loci. Network analysis describes relationships between 5S rDNA sequences in a more realistic fashion than conventional tree building because it makes fewer assumptions about the direction of evolution, the extent of sexual isolation, and the pattern of ancestry and descent. The networks show that the 5S rDNA sequences of Triticum and Aegilops species are related in a reticulate manner around principal nodal sequences. The spacer networks have multiple principal nodes of considerable antiquity but the gene network has just one principal node, corresponding to the correct gene sequence. The networks enable orthologous groups of spacer sequences to be identified. When orthologs are compared it is seen that the patterns of intra- and interspecific diversity are similar for both genes and spacers. We propose that 5S rDNA arrays combine sequence conservation with a large store of mutant variations, the number of correct gene copies within an array being the result of neutral processes that act on gene and spacer regions together. PMID:11238418

  7. Conserved 5' flank homologies in dipteran 5S RNA genes that would function on 'A' form DNA.

    PubMed Central

    Rubacha, A; Sumner, W; Richter, L; Beckingham, K

    1984-01-01

    We have sequenced the 480 base pair (bp) repeating unit of the 5S RNA genes of the Dipteran fly Calliphora erythrocephala and compared this sequence to the three known 5S RNA gene sequences from the Dipteran Genus Drosophila (1,2). A striking series of five perfectly conserved homologies identically positioned within the 5' flanks of all four Dipteran 5S RNA coding regions has thus been identified. The spacing (12-13 bp) between all of these homologies is typical of A form rather than B form DNA. Given that the eukaryotic 5S RNA gene specific initiation factor TFIIIA (3) is a DNA unwinding protein (4), a role for these Dipteran 5' flank homologies in initiation site selection on 5S RNA genes transiently unwound for transcription is suggested. One of the Dipteran homology blocks is highly conserved in sequence and position in all but one of the eukaryotic 5S RNA gene sequences known to date (17/18 genes). Its sequence (consensus: TATAAG) and position (average center: -26 bp) are highly reminiscent of the polymerase II gene 'TATA' box (5). PMID:6209610

  8. Phylogenetic origins of the plant mitochondrion based on a comparative analysis of 5S ribosomal RNA sequences

    NASA Technical Reports Server (NTRS)

    Villanueva, E.; Delihas, N.; Luehrsen, K. R.; Fox, G. E.; Gibson, J.

    1985-01-01

    The complete nucleotide sequences of 5S ribosomal RNAs from Rhodocyclus gelatinosa, Rhodobacter sphaeroides, and Pseudomonas cepacia were determined. Comparisons of these 5S RNA sequences show that rather than being phylogenetically related to one another, the two photosynthetic bacterial 5S RNAs share more sequence and signature homology with the RNAs of two nonphotosynthetic strains. Rhodobacter sphaeroides is specifically related to Paracoccus denitrificans and Rc. gelatinosa is related to Ps. cepacia. These results support earlier 16S ribosomal RNA studies and add two important groups to the 5S RNA data base. Unique 5S RNA structural features previously found in P. denitrificans are present also in the 5S RNA of Rb. sphaeroides; these provide the basis for subdivisional signatures. The immediate consequence of obtaining these new sequences is that it is possible to clarify the phylogenetic origins of the plant mitochondrion. In particular, a close phylogenetic relationship is found between the plant mitochondria and members of the alpha subdivision of the purple photosynthetic bacteria, namely, Rb. sphaeroides, P. denitrificans, and Rhodospirillum rubrum.

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

    PubMed

    Wu, Shu; Xiong, Jie; Yu, Yuhe

    2015-01-01

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

  10. 16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

    PubMed Central

    Auchtung, Thomas A.; Takacs-Vesbach, Cristina D.; Cavanaugh, Colleen M.

    2006-01-01

    The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity. PMID:16820509

  11. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

    PubMed Central

    Melançon, P; Tapprich, W E; Brakier-Gingras, L

    1992-01-01

    Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes. PMID:1281147

  12. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  13. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  14. Aerodynamic and engineering design of a 1.5 s high quality microgravity drop tower facility

    NASA Astrophysics Data System (ADS)

    Belser, Valentin; Breuninger, Jakob; Reilly, Matthew; Laufer, René; Dropmann, Michael; Herdrich, Georg; Hyde, Truell; Röser, Hans-Peter; Fasoulas, Stefanos

    2016-12-01

    Microgravity experiments are essential for research in space science, biology, fluid mechanics, combustion, and material sciences. One way to conduct microgravity experiments on Earth is by using drop tower facilities. These facilities combine a high quality of microgravity, adequate payload masses and have the advantage of virtually unlimited repeatability under same experimental conditions, at a low cost. In a collaboration between the Institute of Space Systems (IRS) at the University of Stuttgart and Baylor University (BU) in Waco, Texas, a new drop tower is currently under development at the Center for Astrophysics, Space Physics and Engineering Research (CASPER). The design parameters of the drop tower ask for at least 1.5 s in free fall duration while providing a quality of at least 10-5 g. Previously, this quality has only been achieved in vacuum drop tower facilities where the capsule experiences virtually zero aerodynamic drag during its free fall. Since this design comes at high costs, a different drop tower design concept, which does not require an evacuated drop shaft, was chosen. It features a dual-capsule system in which the experiment capsule is shielded from aerodynamic forces by surrounding it with a drag shield during the drop. As no other dual-capsule drop tower has been able to achieve a quality as good as or better than 10-5 g previous work optimized the design with an aerodynamic perspective by using computational fluid dynamics (CFD) simulations to determine the ideal shape and size of the outer capsule and to specify the aerodynamically crucial dimensions for the overall system. Experiments later demonstrated that the required quality of microgravity can be met with the proposed design. The main focus of this paper is the mechanical realization of the capsule as well as the development and layout of the surrounding components, such as the release mechanism, the deceleration device and the drop shaft. Because the drop tower facility is a

  15. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  16. Transcription of the Drosophila melanogaster 5S RNA gene requires an upstream promoter and four intragenic sequence elements

    SciTech Connect

    Sharp, S.J.; Garcia, A.D.

    1988-03-01

    Linker-scanning (LS) mutations were constructed spanning the length of the Drosophila melanogaster 5S RNA gene. In vitro transcription analysis of the LS 5S DNAs revealed five transcription control regions. One control region essential for the transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants of this upstream promoter region were located between coordinates -39 and -26 (-30 region), but important sequences extended to the transcription start site at position 1. Since mutations in the upstream promoter did not alter the ability of 5S DNA to sequester transcription factors into a stable transcription complex, it appears that this control region involved the interaction of RNA polymerase III. Active 5S DNA transcription additionally required the four intragenic control regions (ICRs) located between coordinates 3 and 18 (ICR I), 37 and 44 (ICR II), 48 and 61 (ICR III), and 78 and 98 (ICR IV). LS mutations in each ICR decreased the ability of 5S DNA to sequester transcription factors. ICR III, ICR IV, and the spacer sequence between were similar in sequence and position to the determinant elements of the multipartite ICR of Xenopus 5S DNA. The importance of ICR III and ICR IV in transcription initiation and in sequestering transcription factors suggests the presence of an activity in D. melanogaster similar to transcription factor TFIIIA of Xenopus laevis and HeLa cells. Transcription initiation of Drosophila 5S DNA was not eliminated by LS mutations in the spacer region even though these mutations reduced the ability of the TFIIIA-like activity to bind.

  17. Chromosomal mapping of rRNA genes, core histone genes and telomeric sequences in Brachidontes puniceus and Brachidontes rodriguezi (Bivalvia, Mytilidae)

    PubMed Central

    2010-01-01

    Background Chromosome rearrangements are an important part of the speciation process in many taxa. The study of chromosome evolution in bivalves is hampered by the absence of clear chromosomal banding patterns and the similarity in both chromosome size and morphology. For this reason, obtaining good chromosome markers is essential for reliable karyotypic comparisons. To begin this task, the chromosomes of the mussels Brachidontes puniceus and B. rodriguezi were studied by means of fluorochrome staining and fluorescent in situ hybridization (FISH). Results Brachidontes puniceus and B. rodriguezi both have 2n = 32 chromosomes but differing karyotype composition. Vertebrate-type telomeric sequences appear at both ends of every single chromosome. B. puniceus presents a single terminal major rRNA gene cluster on a chromosome pair while B. rodriguezi shows two. Both mussels present two 5S rDNA and two core histone gene clusters intercalary located on the long arms of two chromosome pairs. Double and triple-FISH experiments demonstrated that one of the 5S rDNA and one of the major rDNA clusters appear on the same chromosome pair in B. rodriguezi but not in B. puniceus. On the other hand, the second 5S rDNA cluster is located in one of the chromosome pairs also bearing one of the core histone gene clusters in the two mussel species. Conclusion Knowledge of the chromosomal distribution of these sequences in the two species of Brachidontes is a first step in the understanding of the role of chromosome changes on bivalve evolution. PMID:21143946

  18. [16S rRNA gene sequence analysis for bacterial identification in the clinical laboratory].

    PubMed

    Matsumoto, Takehisa; Sugano, Mitsutoshi

    2013-12-01

    The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. For many years, sequencing of the 16S ribosomal RNA (rRNA) gene has served as an important tool for determining phylogenetic relationships between bacteria. The features of this molecular target that make it a useful phylogenetic tool also make it useful for bacterial detection and identification in the clinical laboratory. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, and can lead to the recognition of novel pathogens and noncultured bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques is impossible or difficult. However, there are some cases in which 16S rRNA gene sequence analysis can not differentiate closely related bacteria such as Shigella spp. and Escherichia coli at the species level. Thus, it is important to understand the advantages and disadvantages of 16S rRNA gene sequence analysis.

  19. 16S rRNA Phylogeny of Sponge-Associated Cyanobacteria

    PubMed Central

    Steindler, Laura; Huchon, Dorothée; Avni, Adi; Ilan, Micha

    2005-01-01

    Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales. PMID:16000832

  20. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  1. Molecular diagnosis of Actinomadura madurae infection by 16S rRNA deep sequencing.

    PubMed

    Salipante, Stephen J; Sengupta, Dhruba J; Hoogestraat, Daniel R; Cummings, Lisa A; Bryant, Bronwyn H; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W; Chau, Mimosa; Barbee, Lindley A; Rosenthal, Christopher; Cookson, Brad T; Hoffman, Noah G

    2013-12-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms.

  2. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra.

    PubMed

    Ota-Tsuzuki, C; Brunheira, A T P; Mayer, M P A

    2008-10-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes.

  3. Methyltransferase Erm(37) slips on rRNA to confer atypical resistance in Mycobacterium tuberculosis.

    PubMed

    Madsen, Christian Toft; Jakobsen, Lene; Buriánková, Karolina; Doucet-Populaire, Florence; Pernodet, Jean-Luc; Douthwaite, Stephen

    2005-11-25

    Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

  4. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  5. Detecting 16S rRNA Methyltransferases in Enterobacteriaceae by Use of Arbekacin

    PubMed Central

    Chahine, Sarah; Okafor, Darius; Ong, Ana C.; Maybank, Rosslyn; Kwak, Yoon I.; Wilson, Kerry; Zapor, Michael; Lesho, Emil; Hinkle, Mary

    2015-01-01

    16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test. PMID:26537447

  6. Modified nucleotides m(2)G966/m(5)C967 of Escherichia coli 16S rRNA are required for attenuation of tryptophan operon.

    PubMed

    Prokhorova, Irina V; Osterman, Ilya A; Burakovsky, Dmitry E; Serebryakova, Marina V; Galyamina, Maria A; Pobeguts, Olga V; Altukhov, Ilya; Kovalchuk, Sergey; Alexeev, Dmitry G; Govorun, Vadim M; Bogdanov, Alexey A; Sergiev, Petr V; Dontsova, Olga A

    2013-11-18

    Ribosomes contain a number of modifications in rRNA, the function of which is unclear. Here we show--using proteomic analysis and dual fluorescence reporter in vivo assays--that m(2)G966 and m(5)C967 in 16S rRNA of Escherichia coli ribosomes are necessary for correct attenuation of tryptophan (trp) operon. Expression of trp operon is upregulated in the strain where RsmD and RsmB methyltransferases were deleted, which results in the lack of m(2)G966 and m(5)C967 modifications. The upregulation requires the trpL attenuator, but is independent of the promotor of trp operon, ribosome binding site of the trpE gene, which follows trp attenuator and even Trp codons in the trpL sequence. Suboptimal translation initiation efficiency in the rsmB/rsmD knockout strain is likely to cause a delay in translation relative to transcription which causes misregulation of attenuation control of trp operon.

  7. 23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

    PubMed

    Bae, Hee-Sung; Hou, Aixin

    2013-02-01

    Little is known about the impacts of fecal polluted urban runoff inputs on the structure of enterococci communities in estuarine waters. This study employed a 23S rRNA gene-based polymerase chain reaction (PCR) assay with newly designed genus-specific primers, Ent127F-Ent907R, to determine the possible impacts of Hurricane Katrina floodwaters via the 17th Street Canal discharge on the community structure of enterococci in Lake Pontchartrain. A total of 94 phylotypes were identified through the restriction fragment length polymorphism (RFLP) screening of 494 clones while only 8 phylotypes occurred among 88 cultivated isolates. Sequence analyses of representative phylotypes and their temporal and spatial distribution in the lake and the canal indicated the Katrina floodwater input introduced a large portion of Enterococcus flavescens, Enterococcus casseliflavus, and Enterococcus dispar into the lake; typical fecal groups Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii were detected primarily in the floodwater-impacted waters. This study provides a global picture of enterococci in estuarine waters impacted by Hurricane Katrina-derived urban runoff. It also demonstrates the culture-independent PCR approach using 23S rRNA gene as a molecular marker could be a good alternative in ecological studies of enterococci in natural environments to overcome the limitation of conventional cultivation methods.

  8. Comparative analysis of 16S rRNA and amoA genes from archaea selected with organic and inorganic amendments in enrichment culture.

    PubMed

    Xu, Mouzhong; Schnorr, Jon; Keibler, Brandon; Simon, Holly M

    2012-04-01

    We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the "root" clade, we detected no corresponding amoA gene. The amoA-containing archaea were present in media with either organic or inorganic amendments, whereas archaea representing the root clade were present only when organic amendment was used. Analysis of amoA gene abundance and expression, together with nitrification-coupled growth assays, indicated potential growth by autotrophic ammonia oxidation for members of two group 1.1b clades. Increased abundance of one of these clades, however, also occurred upon the addition of organic amendment. Finally, although amoA-containing group 1.1a archaea were present in enrichments, we detected neither expression of amoA genes nor evidence for nitrification-coupled growth of these organisms. These data support a model of a diverse metabolic community in mesophilic soil archaea that is just beginning to be characterized.

  9. Source bioaerosol concentration and rRNA gene-based identification of microorganisms aerosolized at a flood irrigation wastewater reuse site.

    PubMed

    Paez-Rubio, Tania; Viau, Emily; Romero-Hernandez, Socorro; Peccia, Jordan

    2005-02-01

    Reuse of partially treated domestic wastewater for agricultural irrigation is a growing practice in arid regions throughout the world. A field sampling campaign to determine bioaerosol concentration, culturability, and identity at various wind speeds was conducted at a flooded wastewater irrigation site in Mexicali, Baja California, Mexico. Direct fluorescent microscopy measurements for total microorganisms, culture-based assays for heterotrophs and gram-negative enteric bacteria, and small-subunit rRNA gene-based cloning were used for microbial characterizations of aerosols and effluent wastewater samples. Bioaerosol results were divided into two wind speed regimens: (i) below 1.9 m/s, average speed 0.5 m/s, and (ii) above 1.9 m/s, average speed 4.5 m/s. Average air-borne concentration of total microorganisms, culturable heterotrophs, and gram-negative enteric bacteria were, respectively, 1.1, 4.2, and 6.2 orders of magnitude greater during the high-wind-speed regimen. Small-subunit rRNA gene clone libraries processed from samples from air and the irrigation effluent wastewater during a high-wind sampling event indicate that the majority of air clone sequences were more than 98% similar to clone sequences retrieved from the effluent wastewater sample. Overall results indicate that wind is a potential aerosolization mechanism of viable wastewater microorganisms at flood irrigation sites.

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

    PubMed

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

    2016-01-30

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

  11. Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

    PubMed Central

    Bommakanti, Ananth S.; Lindahl, Lasse; Zengel, Janice M.

    2008-01-01

    The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both Gram-positive and Gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes. PMID:18218702

  12. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

    PubMed Central

    Tatti, Enrico; McKew, Boyd A.; Whitby, Corrine; Smith, Cindy J.

    2016-01-01

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included. PMID:27341629

  13. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    PubMed

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  14. Modified nucleotides m2G966/m5C967 of Escherichia coli 16S rRNA are required for attenuation of tryptophan operon

    NASA Astrophysics Data System (ADS)

    Prokhorova, Irina V.; Osterman, Ilya A.; Burakovsky, Dmitry E.; Serebryakova, Marina V.; Galyamina, Maria A.; Pobeguts, Olga V.; Altukhov, Ilya; Kovalchuk, Sergey; Alexeev, Dmitry G.; Govorun, Vadim M.; Bogdanov, Alexey A.; Sergiev, Petr V.; Dontsova, Olga A.

    2013-11-01

    Ribosomes contain a number of modifications in rRNA, the function of which is unclear. Here we show - using proteomic analysis and dual fluorescence reporter in vivo assays - that m2G966 and m5C967 in 16S rRNA of Escherichia coli ribosomes are necessary for correct attenuation of tryptophan (trp) operon. Expression of trp operon is upregulated in the strain where RsmD and RsmB methyltransferases were deleted, which results in the lack of m2G966 and m5C967 modifications. The upregulation requires the trpL attenuator, but is independent of the promotor of trp operon, ribosome binding site of the trpE gene, which follows trp attenuator and even Trp codons in the trpL sequence. Suboptimal translation initiation efficiency in the rsmB/rsmD knockout strain is likely to cause a delay in translation relative to transcription which causes misregulation of attenuation control of trp operon.

  15. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  16. Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis

    PubMed Central

    Floyd, Brice E; Morriss, Stephanie C; MacIntosh, Gustavo C; Bassham, Diane C

    2015-01-01

    Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2–2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2–2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2–2, atg9–4, atg5–1, rns2–2 atg9–4, and rns2–2 atg5–1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2–2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2–2 atg5–1 double mutant but not by an rns2–2 atg9–4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole. PMID:26735434

  17. Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis.

    PubMed

    Floyd, Brice E; Morriss, Stephanie C; MacIntosh, Gustavo C; Bassham, Diane C

    2015-01-01

    Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.

  18. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

    PubMed Central

    Schmidt, T M; DeLong, E F; Pace, N R

    1991-01-01

    The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms. Images PMID:2066334

  19. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  20. Growth cone collapse assay.

    PubMed

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  1. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  2. Radiative lifetime of the 5S2 metastable state of N/+/. [with interpretation of auroral emission spectrum

    NASA Technical Reports Server (NTRS)

    Knight, R. D.

    1982-01-01

    The radiative lifetime of metastable N(+)(5S2) has been measured to be 4.2 + or - 0.6 msec, a value generally in agreement with theory, by direct monitoring of the spontaneous emission from approximately 1,000,000 ions stored in a radio-frequency ion trap. Additional measurements were made of the quenching rate coefficient (2.5 x 10 to the -9th cu cm/sec) and the production cross section (greater than or equal to 10 to the -18th sq cm) of N(+)(5S2) in N2. This work supports the interpretation of the 2145-A feature in the spectrum of aurorae as being due to N(+)(5S2) emission.

  3. Electron microscopic study of crystals of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex.

    PubMed

    Brown, R S; Ferguson, C; Kingswell, A; Winkler, F K; Leonard, K R

    1988-06-01

    A novel method has been developed to grow crystals of the Xenopus laevis transcription factor IIIA-5S RNA complex directly on grids for examination by electron microscopy. Microcrystals were examined in negative stain and in thin sections to reveal a hexagonal lattice with unit-cell dimensions a = b = 87.1 +/- 4.4 A and c = 143.8 +/- 12.7 A. Optical diffraction patterns from micrographs were obtained about the major crystal axes extending to about 40-A resolution. A packing scheme is proposed for which there are three or six transcription factor IIIA-5S RNA complexes in the unit cell related by 3(1) symmetry along the long cell axis. This would require that the 5S RNA molecules are arranged end-to-end, with the terminal loops of adjacent molecules overlapping.

  4. D5S2500 is an ambiguously characterized STR: Identification and description of forensic microsatellites in the genomics age.

    PubMed

    Phillips, C; Parson, W; Amigo, J; King, J L; Coble, M D; Steffen, C R; Vallone, P M; Gettings, K B; Butler, J M; Budowle, B

    2016-07-01

    In the process of establishing short tandem repeat (STR) sequence variant nomenclature guidelines in anticipation of expanded forensic multiplexes for massively parallel sequencing (MPS), it was discovered that the STR D5S2500 has multiple positions and genomic characteristics reported. This ambiguity is because the marker named D5S2500 consists of two different microsatellites forming separate components in the capillary electrophoresis multiplexes of Qiagen's HDplex (Hilden, Germany) and AGCU ScienTech's non-CODIS STR 21plex (Wuxi, Jiangsu, China). This study outlines the genomic details used to identify each microsatellite and reveals the D5S2500 marker in HDplex has the correctly assigned STR name, while the D5S2500 marker in the AGCU 21plex, closely positioned a further 1643 nucleotides in the human reference sequence, is an unnamed microsatellite. The fact that the D5S2500 marker has existed as two distinct STR loci undetected for almost ten years, even with reported discordant genotypes for the standard control DNA, underlines the need for careful scrutiny of the genomic properties of forensic STRs, as they become adapted for sequence analysis with MPS systems. We make the recommendation that precise chromosome location data must be reported for any forensic marker under development but not in common use, so that the genomic characteristics of the locus are validated to the same level of accuracy as its allelic variation and forensic performance. To clearly differentiate each microsatellite, we propose the name D5S2800 be used to identify the Chromosome-5 STR in the AGCU 21plex.

  5. Rapid Identification of Yersinia enterocolitica in Blood by the 5′ Nuclease PCR Assay

    PubMed Central

    Sen, Keya

    2000-01-01

    Yersinia enterocolitica accounts for 50% of the clinical sepsis episodes caused by the transfusion of contaminated red blood cells. A 5′ nuclease TaqMan PCR assay was developed to detect Y. enterocolitica in blood. Primers and a probe based on the nucleotide sequence of the 16S rRNA gene from Y. enterocolitica were designed. Whole-blood samples were spiked with various numbers of Y. enterocolitica cells, and total chromosomal DNA was extracted. When the TaqMan PCR assay was performed, as few as six bacteria spiked in 200 μl of blood could be detected. The assay was specific and did not detect other Yersinia species. The TaqMan assay is easy to perform, takes 2 h, and has the potential for use in the rapid detection of Y. enterocolitica contamination in stored blood units. PMID:10790127

  6. Impact of the Japanese 5S management method on patients’ and caretakers’ satisfaction: a quasi-experimental study in Senegal

    PubMed Central

    Kanamori, Shogo; Castro, Marcia C.; Sow, Seydou; Matsuno, Rui; Cissokho, Alioune; Jimba, Masamine

    2016-01-01

    Background The 5S method is a lean management tool for workplace organization, with 5S being an abbreviation for five Japanese words that translate to English as Sort, Set in Order, Shine, Standardize, and Sustain. In Senegal, the 5S intervention program was implemented in 10 health centers in two regions between 2011 and 2014. Objective To identify the impact of the 5S intervention program on the satisfaction of clients (patients and caretakers) who visited the health centers. Design A standardized 5S intervention protocol was implemented in the health centers using a quasi-experimental separate pre-post samples design (four intervention and three control health facilities). A questionnaire with 10 five-point Likert items was used to measure client satisfaction. Linear regression analysis was conducted to identify the intervention's effect on the client satisfaction scores, represented by an equally weighted average of the 10 Likert items (Cronbach's alpha=0.83). Additional regression analyses were conducted to identify the intervention's effect on the scores of each Likert item. Results Backward stepwise linear regression (n=1,928) indicated a statistically significant effect of the 5S intervention, represented by an increase of 0.19 points in the client satisfaction scores in the intervention group, 6 to 8 months after the intervention (p=0.014). Additional regression analyses showed significant score increases of 0.44 (p=0.002), 0.14 (p=0.002), 0.06 (p=0.019), and 0.17 (p=0.044) points on four items, which, respectively were healthcare staff members’ communication, explanations about illnesses or cases, and consultation duration, and clients’ overall satisfaction. Conclusions The 5S has the potential to improve client satisfaction at resource-poor health facilities and could therefore be recommended as a strategic option for improving the quality of healthcare service in low- and middle-income countries. To explore more effective intervention modalities

  7. Features of laser-induced luminescence and photoconductivity of layered Cu3In5S9 crystals

    NASA Astrophysics Data System (ADS)

    Guseinov, A. G.; Kyazym-zade, A. G.; Salmanov, V. M.; Mamedov, R. M.; Salmanova, A. A.; Gasanova, L. G.; Mahammadov, A. Z.

    2016-12-01

    Luminescence and photoconductivity of layered Cu3In5S9 crystals at high levels of optical excitation are studied experimentally. A pulsed nanosecond Nd:YAG laser with built-in second and third harmonic generators to generate 1064-, 532-, and 355-nm radiation is used as a light source. It is found that the photoluminescence spectra exhibit two emission bands due to zone-acceptor level and impurity donor-impurity acceptor transitions. It is shown that the photoconductivity in Cu3In5S9 is monopolar. The waveform of the photoconductivity consists of fast and slow components associated with two channels of recombination.

  8. Singly modified amikacin and tobramycin derivatives show increased rRNA A-site binding and higher potency against resistant bacteria.

    PubMed

    Fair, Richard J; McCoy, Lisa S; Hensler, Mary E; Aguilar, Bernice; Nizet, Victor; Tor, Yitzhak

    2014-09-01

    Semisynthetic derivatives of the clinically useful aminoglycosides tobramycin and amikacin were prepared by selectively modifying their 6'' positions with a variety of hydrogen bond donors and acceptors. Their binding to the rRNA A-site was probed using an in vitro FRET-based assay, and their antibacterial activities against several resistant strains (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, MRSA) were quantified by determining minimum inhibitory concentrations (MICs). The most potent derivatives were evaluated for their eukaryotic cytotoxicity. Most analogues displayed higher affinity for the bacterial A-site than the parent compounds. Although most tobramycin analogues exhibited no improvement in antibacterial activity, several amikacin analogues showed potent and broad-spectrum antibacterial activity against resistant bacteria. Derivatives tested for eukaryotic cytotoxicity exhibited minimal toxicity, similar to the parent compounds.

  9. Molecular Methods for Identification of Acinetobacter Species by Partial Sequencing of the rpoB and 16S rRNA Genes

    PubMed Central

    Khosravi, Azar Dokht; Shahraki, Abdolrazagh Hashemi; Heidarieh, Parvin; Sheikhi, Nasrin

    2015-01-01

    Background Acinetobacter spp. is a diverse group of Gram-negative bacteria which are ubiquitous in soil and water, and an important cause of nosocomial infections. The purpose of this study was to identify a collection of Acinetobacter spp. clinical isolates accurately and to investigate their antibiotic susceptibility patterns. Materials and Methods A total of 197 non-duplicate clinical isolates of Acinetobacter spp. isolates identified using conventional biochemical tests. The molecular technique of PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes was applied for species identification. Antimicrobial susceptibility test was performed with a disk diffusion assay. Results Based on 16S rRNA and rpoB genes analysis separately, most of clinical isolates can be identified with high bootstrap values. However, the identity of the isolate 555T was uncertain due to high similarity of A. grimontii and A. junii. Identification by concatenation of 16S rRNA and rpoB confirmed the identity of clinical isolates of Acenitobacer to species level confidently. Accordingly, the isolate 555T assigned as A. grimontii due to 100% similarity to A. grimontii. Moreover, this isolate showed 98.64% to A. junii. Besides, the identity of the isolates 218T and 364T was confirmed as Genomic species 3 and A. calcoaceticus respectively. So, the majority of Acinetobacter spp. isolates, were identified as: A. baumannii (131 isolates, 66%), A. calcoaceticus (9 isolates, 4.5%), and A. genomosp 16 (8 isolates, 4%). The rest of identified species showed the lower frequencies. In susceptibility test, 105 isolates (53%), presented high antibiotic resistance of 90% to ceftriaxone, piperacillin, piperacillin tazobactam, amikacin, and 81% to ciprofloxacin. Conclusion Sequence analysis of the 16S rRNA and rpoB spacer simultaneously was able to do identification of Acinetobacter spp. to species level. A.baumannii was identified as the most prevalent species with high antibiotic resistance. Other

  10. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  11. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes.

    PubMed

    Petrova, Olga E; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

    2017-01-24

    Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.

  12. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    PubMed Central

    Petrova, Olga E.; Garcia-Alcalde, Fernando; Zampaloni, Claudia; Sauer, Karin

    2017-01-01

    Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested. PMID:28117413

  13. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  14. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  15. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  16. Fitness cost due to mutations in the 16S rRNA associated with spectinomycin resistance in Chlamydia psittaci 6BC.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2005-11-01

    The fitness cost of a resistance determinant is the primary parameter that determines its frequency in vivo. As a model for analysis of the impact of drug resistance mutations on the intracellular life cycle of Chlamydia spp., we studied the growth of four genetically defined spectinomycin-resistant (Spc(r)) clonal variants of Chlamydia psittaci 6BC isolated in the plaque assay. The development of each variant was monitored over 46 h postinfection in the absence of drug, either in pure culture or in 1:1 competition with the parent strain. Spc(r) mutations in the 16S rRNA gene at positions 1191 and 1193 were associated with a marked impairment of C.psittaci biological fitness, and the bacteria were severely out-competed by the wild-type parent. In contrast, mutations at position 1192 had minor effects on the bacterial life cycle, allowing the resistant isolates to compete more efficiently with the wild-type strain. Thus, mutations with a wide range of fitness costs can be selected in the plaque assay, providing a new strategy for prediction and monitoring of the emergence of antibiotic resistance in chlamydiae. So far, drug resistance has not been a serious threat for the treatment of chlamydial infections. Tetracycline is an effective antichlamydial drug that targets 16S rRNA. Attempts to isolate spontaneous tetracycline-resistant mutants of C. psittaci 6BC revealed a frequency <3 x 10(-9). We suggest that the rarity of genotypic antibiotic resistance among chlamydial clinical isolates reflects the deleterious effects of such mutations on the fitness of these obligate intracellular bacteria in the host.

  17. Phylogenetic Sequence Variations in Bacterial rRNA Affect Species-Specific Susceptibility to Drugs Targeting Protein Synthesis▿‡

    PubMed Central

    Akshay, Subramanian; Bertea, Mihai; Hobbie, Sven N.; Oettinghaus, Björn; Shcherbakov, Dimitri; Böttger, Erik C.; Akbergenov, Rashid

    2011-01-01

    Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket. PMID:21730122

  18. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  19. Survival assays using Caenorhabditis elegans

    PubMed Central

    Park, Hae-Eun H.; Jung, Yoonji; Lee, Seung-Jae V.

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

  20. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  1. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  2. 16S rRNA beacons for bacterial monitoring during human space missions.

    PubMed

    Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

    2007-04-01

    Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

  3. Phylogeny of Metschnikowia species estimated from partial rRNA sequences.

    PubMed

    Mendonça-Hagler, L C; Hagler, A N; Kurtzman, C P

    1993-04-01

    Phylogenetic relationships of species assigned to the genus Metschnikowia were estimated from the extents of divergence among partial sequences of rRNA. The data suggest that the aquatic species (Metschnikowia australis, Metschnikowia bicuspidata, Metschnikowia krissii, and Metschnikowia zobellii) and the terrestrial species (Metschnikowia hawaiiensis, Metschnikowia lunata, Metschnikowia pulcherrima, and Metschnikowia reukaufii) form two groups within the genus. M. lunata and M. hawaiiensis are well separated from other members of the genus, and M. hawaiiensis may be sufficiently divergent that it could be placed in a new genus. Species of the genus Metschnikowia are unique compared with other ascomycetous yeasts because they have a deletion in the large-subunit rRNA sequence that includes nucleotides 434 to 483.

  4. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology

    PubMed Central

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James

    2012-01-01

    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  5. A renaissance for the pioneering 16S rRNA gene

    SciTech Connect

    Tringe, Susannah; Hugenholtz, Philip

    2008-09-07

    Culture-independent molecular surveys using the 16S rRNA gene have become a mainstay for characterizing microbial community structure over the last quarter century. More recently this approach has been overshadowed by metagenomics, which provides a global overview of a community's functional potential rather than just an inventory of its inhabitants. However, the pioneering 16S rRNA gene is making a comeback in its own right thanks to a number of methodological advancements including higher resolution (more sequences), analysis of multiple related samples (e.g. spatial and temporal series) and improved metadata and use of metadata. The standard conclusion that microbial ecosystems are remarkably complex and diverse is now being replaced by detailed insights into microbial ecology and evolution based only on this one historically important marker gene.

  6. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment

    PubMed Central

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  7. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    PubMed

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  8. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    SciTech Connect

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  9. DNA sequencing analysis of ITS and 28S rRNA of Poria cocos.

    PubMed

    Atsumi, Toshiyuki; Kakiuchi, Nobuko; Mikage, Masayuki

    2007-08-01

    We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype.

  10. Application of 12S rRNA gene for the identification of animal-derived drugs.

    PubMed

    Luo, Jiaoyang; Yan, Dan; Zhang, Da; Han, Yumei; Dong, Xiaoping; Yang, Yong; Deng, Kejun; Xiao, Xiaohe

    2011-01-01

    PURPOSE. Animal-derived drugs are the major source of biological products and traditional medicine, but they are often difficult to identify, causing confusion in the clinical application. Among these medicinal animals, a number of animal species are endangered, leading to the destruction of biodiversity. The identification of animal-derived drugs and their alternatives would be a first step toward biodiversity conservation and safe medication. Until now, no effective method for identifying animal-derived drugs has been demonstrated; DNA-based species identification presents a brand-new technique. METHODS. We designed primers to amplify a 523-bp fragment of 12S rRNA and generated sequences for 13 individuals within six medicinal animal species. We examined the efficiency of species recognition based on this sequence, and we also tested the taxonomic affiliations against the GenBank database. RESULTS. All the tested drugs were identified successfully, and a visible gap was found between the inter-specific and intra-specific variation. We further demonstrated the importance of data exploration in DNA-based species identification practice by examining the sequence characteristics of relative genera in GenBank. This region of the 12S rRNA gene had a 100% success rate of species recognition within the six medicinal animal species. CONCLUSIONS. We propose that the 12S rRNA locus might be universal for identifying animal-derived drugs and their adulterants. The development of 12S rRNA for indentifying animal-derived drugs that share a common gene target would contribute significantly to the clinical application of animal-derived drugs and the conservation of medicinal animal species. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  11. Phenotypic characterisation and 16S rRNA sequence analysis of veterinary isolates of Streptococcus pluranimalium.

    PubMed

    Twomey, D F; Carson, T; Foster, G; Koylass, M S; Whatmore, A M

    2012-05-01

    Forty-two isolates of Streptococcus pluranimalium were identified from cattle (n=38), sheep (n=2), an alpaca (n=1) and a pheasant (n=1) in the United Kingdom. The isolates were confirmed as S. pluranimalium by 16S rRNA sequence analysis but could not be differentiated reliably from Streptococcus acidominimus by phenotypic characterisation using commercial kits routinely used in veterinary laboratories. The alanyl-phenylalanyl-proline arylamidase reaction could be used to differentiate S. pluranimalium (positive) from Aerococcus urinae (negative).

  12. GJB2 and mitochondrial 12S rRNA susceptibility mutations in sudden deafness.

    PubMed

    Chen, Kaitian; Sun, Liang; Zong, Ling; Wu, Xuan; Zhan, Yuan; Dong, Chang; Cao, Hui; Tang, Haocheng; Jiang, Hongyan

    2016-06-01

    Genetic susceptibility may play an important role in the pathogenesis of sudden deafness. However, the specific genes involved are largely unknown. We sought to explore the frequency of GJB2 and mitochondrial 12S rRNA susceptibility mutations in patients with sudden deafness. Between September 2011 and May 2012, 62 consecutive patients with sudden deafness were seen. In 50 of these, no etiological factors for sudden deafness were found. We detected GJB2 and mitochondrial 12S rRNA variants by direct sequencing in these 50 patients and in 53-aged matched controls with normal hearing. In addition, we undertook functional analyses of the mitochondrial mutations which we detected, applying structural and phylogenetic analysis. GJB2 sequencing identified six mutations, including three pathogenic mutations (c.235delC, c.299-300delAT, c.109G>A) and three polymorphisms, in the study participants, giving an allele frequency of 15.0 %. A homozygous c.109G>A mutation was detected in two participants. A total of 16 variants in mitochondrial 12S rRNA gene were identified in the participants. No significant differences were found in GJB2 heterozygosity or in mitochondrial 12S rRNA variants between patients with sudden deafness and in controls. Our results suggest that the homozygous GJB2 c.109G>A mutation may be a cause of sudden deafness involving both ears. This finding should increase awareness of the likely role of genetic factors in the etiology of sudden deafness in general.

  13. A multiplex real-time polymerase chain reaction assay to diagnose Epiphyas postvittana (Lepidoptera: Tortricidae).

    PubMed

    Barr, N B; Ledezma, L A; Farris, R E; Epstein, M E; Gilligan, T M

    2011-10-01

    A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.

  14. Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

    PubMed Central

    Berthier, Yvette; Verdier, Valérie; Guesdon, Jean-Luc; Chevrier, Danièle; Denis, Jean-Baptiste; Decoux, Guy; Lemattre, Monique

    1993-01-01

    Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity. Images PMID:16348894

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

    PubMed

    Nakada, Takashi; Misawa, Kazuharu; Nozaki, Hisayoshi

    2008-07-01

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

  16. rRNA Binding Sites and the Molecular Mechanism of Action of the Tetracyclines

    PubMed Central

    2016-01-01

    The tetracycline antibiotics are known to be effective in the treatment of both infectious and noninfectious disease conditions. The 16S rRNA binding mechanism currently held for the antibacterial action of the tetracyclines does not explain their activity against viruses, protozoa that lack mitochondria, and noninfectious conditions. Also, the mechanism by which the tetracyclines selectively inhibit microbial protein synthesis against host eukaryotic protein synthesis despite conservation of ribosome structure and functions is still questionable. Many studies have investigated the binding of the tetracyclines to the 16S rRNA using the small ribosomal subunit of different bacterial species, but there seems to be no agreement between various reports on the exact binding site on the 16S rRNA. The wide range of activity of the tetracyclines against a broad spectrum of bacterial pathogens, viruses, protozoa, and helminths, as well as noninfectious conditions, indicates a more generalized effect on RNA. In the light of recent evidence that the tetracyclines bind to various synthetic double-stranded RNAs (dsRNAs) of random base sequences, suggesting that the double-stranded structures may play a more important role in the binding of the tetracyclines to RNA than the specific base pairs, as earlier speculated, it is imperative to consider possible alternative binding modes or sites that could help explain the mechanisms of action of the tetracyclines against various pathogens and disease conditions. PMID:27246781

  17. Two Distinct Mechanisms Cause Heterogeneity of 16S rRNA

    PubMed Central

    Ueda, Kumiko; Seki, Tatsuji; Kudo, Takuji; Yoshida, Toshiomi; Kataoka, Masakazu

    1999-01-01

    To investigate the frequency of heterogeneity among the multiple 16S rRNA genes within a single microorganism, we determined directly the 120-bp nucleotide sequences containing the hypervariable α region of the 16S rRNA gene from 475 Streptomyces strains. Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains. The heterogeneous loci were detected only in the stem region designated helix 10. All of the substitutions conserved the relevant secondary structure. The 33 strains were divided into two groups: one group, including 22 strains, had less than two heterogeneous bases; the other group, including 11 strains, had five or more heterogeneous bases. The two groups were different in their combinations of heterogeneous bases. The former mainly contained transitional substitutions, and the latter was mainly composed of transversional substitutions, suggesting that at least two mechanisms, possibly misincorporation during DNA replication and horizontal gene transfer, cause rRNA heterogeneity. PMID:9864315

  18. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

    PubMed

    Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

    2015-11-13

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

  19. Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

    PubMed Central

    Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

    2015-01-01

    To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

  20. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays.

    PubMed

    Pitkänen, Tarja; Ryu, Hodon; Elk, Michael; Hokajärvi, Anna-Maria; Siponen, Sallamaari; Vepsäläinen, Asko; Räsänen, Pia; Santo Domingo, Jorge W

    2013-01-01

    In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

  1. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

    PubMed

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen

    2016-10-07

    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

  2. A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.

    PubMed

    Boonyayatra, S; Fox, L K; Besser, T E; Sawant, A; Gay, J M; Raviv, Z

    2012-01-01

    The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

  3. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

    PubMed Central

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru

    2016-01-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes. PMID:27245411

  4. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis.

    PubMed

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2016-08-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

  5. Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

    PubMed Central

    Lioy, Virginia S.; Goussard, Sylvie; Guerineau, Vincent; Yoon, Eun-Jeong; Courvalin, Patrice; Galimand, Marc; Grillot-Courvalin, Catherine

    2014-01-01

    In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution—ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness. PMID:24398977

  6. Comparison of 16S rRNA Gene PCR and BACTEC 9240 for Detection of Neonatal Bacteremia

    PubMed Central

    Jordan, J. A.; Durso, M. B.

    2000-01-01

    Ten percent of infants born in the United States are admitted to neonatal intensive care units (NICU) annually. Approximately one-half of these admissions are from term infants (>34 weeks of gestation) at risk for systemic infection. Most of the term infants are not infected but rather have symptoms consistent with other medical conditions that mimic sepsis. The current standard of care for evaluating bacterial sepsis in the newborn is performing blood culturing and providing antibiotic therapy while awaiting the 48-h preliminary result of culture. Implementing a more rapid means of ruling out sepsis in term newborns could result in shorter NICU stays and less antibiotic usage. The purpose of this feasibility study was to compare the utility of PCR to that of conventional culture. To this end, a total of 548 paired blood samples collected from infants admitted to the NICU for suspected sepsis were analyzed for bacterial growth using the BACTEC 9240 instrument and for the bacterial 16S rRNA gene using a PCR assay which included a 5-h preamplification culturing step. The positivity rates by culture and PCR were 25 (4.6%) and 27 (4.9%) positive specimens out of a total of 548 specimens, respectively. The comparison revealed sensitivity, specificity, and positive and negative predictive values of 96.0, 99.4, 88.9, and 99.8%, respectively, for PCR. In summary, this PCR-based approach, requiring as little as 9 h of turnaround time and blood volumes as small as 200 μl, correlated well with conventional blood culture results obtained for neonates suspected of having bacterial sepsis. PMID:10878046

  7. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  8. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  9. Organization, Structure, and Variability of the rRNA Operon of the Whipple's Disease Bacterium (Tropheryma whippelii)

    PubMed Central

    Maiwald, Matthias; von Herbay, Axel; Lepp, Paul W.; Relman, David A.

    2000-01-01

    Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete, Tropheryma whippelii. We determined a nearly complete rRNA operon sequence of T. whippelii from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features. PMID:10809715

  10. High power system for ECRH at 140Ghz, 2MW, 0.5s on FTU tokamak

    SciTech Connect

    Sozzi, C.; Bozzi, R.; Bruschi, A.; Cirant, S.; Gandini, F.; Granucci, G.; Mellera, V.; Muzzini, V.; Nardone, A.; Simonetto, A.; Spinicchia, N.; Berardi, B.; Ciccone, G.; DiGiovenale, S.; Iannone, F.; Lupini, S.; Mantovani, S.; Pesci, E.

    1999-09-20

    The 140GHz, 2MW, 0.5s ECRH system on FTU tokamak integrates closed waveguide transmission lines ({approx_equal}30 m) with quasi optical systems at both ends for efficient coupling from the 4 gyrotrons to the 4 waveguides and from these to the plasma through a single access port. Poloidal and toroidal control of the beam's launching angles and polarization is performed without movable components close to the plasma. Most of the components of each generation and transmission system were designed to operate at a power level higher than 0.5 MW, and a possible up-grade to a full 1 MW, 0.5 s capability is discussed.

  11. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  12. A Tale of Two Lines: Searching for the 5s - 5p Resonance Lines in Pm-like Ion Spectra

    SciTech Connect

    Trabert, E; Vilkas, M J; Ishikawa, Y

    2008-10-24

    Highly charged ions in the promethium sequence have been suggested to show spectral features resembling the alkali sequence ions. Guided by calculations, the 5s-5p resonance lines have been sought in a variety of experiments. In the light of the most extensive calculations of Pm-like ions yet, applying relativistic multi-reference Moeller-Plesset second-order perturbation theory, the experimental evidence is reviewed and the line identification problem assessed.

  13. Karyotypic features including organizations of the 5S, 45S rDNA loci and telomeres of Scadoxus multiflorus (Amaryllidaceae)

    PubMed Central

    Monkheang, Pansa; Chaveerach, Arunrat; Sudmoon, Runglawan; Tanee, Tawatchai

    2016-01-01

    Abstract Scadoxus multiflorus Martyn, 1795 is an ornamental plant with brilliantly colored flowers. Even though its chromosomes are rather large, there is no karyotype description reported so far. Therefore, conventional and molecular cytogenetic studies including fluorescence in situ hybridization (FISH) with 45S and 5S rDNA, and human telomere sequence (TTAGGG)n probes (Arabidopsis-type telomere probes yielded negative results) were carried out. The chromosome number is as reported previously, 2n = 18. The nine chromosome pairs include two large submetacentric, five large acrocentric, one medium acrocentric, two small metacentric and eight small submetacentric chromosomes. Hybridization sites of the 45S rDNA signals were on the short arm ends of chromosomes #1, #3 and #8, while 5S rDNA signals appeared on the long arm of chromosome 3, in one homologue as a double signal. The telomere signals were restricted to all chromosome ends. Three chromosome pairs could be newly identified, chromosome pair 3 by 5S rDNA and chromosomes #1, #3 and #8 by 45S rDNA loci. In addition to new information about rDNA locations we show that the ends of Scadoxus multiflorus chromosomes harbor human instead of Arabidopsis-type telomere sequences. Overall, the Scadoxus multiflorus karyotype presents chromosomal heteromorphy concerning size, shape and 45S and 5S rDNA positioning. As Scadoxus Rafinesque, 1838 and related species are poorly studied on chromosomal level the here presented data is important for better understanding of evolution in Amaryllidaceae. PMID:28123684

  14. Low-temperature crystal structures of stibnite implying orbital overlap of Sb 5s2 inert pair electrons

    NASA Astrophysics Data System (ADS)

    Kyono, A.; Kimata, M.; Matsuhisa, M.; Miyashita, Y.; Okamoto, K.

    The crystal structure of stibnite [Sb2S3, Pnma, a=11.314(2), b=3.837(2), c=11.234(3) Å, V= 487.7(3) Å3 at 293 K] was refined in situ at 230, 173, and 128 K. It is a major characteristic of the structure that the Sb-S secondary bonds enclosing Sb 5s2 inert lone-pair electrons at 293 K are significantly shorter than the corresponding sum of the Sb and S van der Waals radii. Concerning the temperature dependence, although both the polyhedral volume and the cation eccentricity of the two SbS7 polyhedra exhibit continuous contractions with decreasing temperature, the sphericity values remain constant, indicating isotropic shrinkage. Consequently, the geometries of Sb 5s2 inert lone-pair electrons and ligand atoms remain unchanged at low temperatures. This is because the crystal structure of stibnite at low temperature induces contraction with attractive interactions, which is called the orbital overlap between Sb 5s2 inert lone-pair electrons and ligand orbitals to maintain the coordination environment. In this case, Sb 5s2 lone-pair electrons are not inert, but active. Such orbital overlaps of inert lone-electron pairs can provide a reasonable explanation for shorter secondary bonds and lower band gap energy of the binary compounds containing heavy elements such as Sb, Te, Pb, and Bi, which are key factors in tracing the origins of color, luster, and semiconductivity of their minerals or compounds.

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

    PubMed Central

    1976-01-01

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

  16. Minimally Invasive Transforaminal Lumbar Interbody Fusion at L5-S1 through a Unilateral Approach: Technical Feasibility and Outcomes

    PubMed Central

    Choi, Won-Suh; Kim, Jin-Sung; Ryu, Kyeong-Sik; Hur, Jung-Woo; Seong, Ji-Hoon

    2016-01-01

    Background. Minimally invasive spinal transforaminal lumbar interbody fusion (MIS-TLIF) at L5-S1 is technically more demanding than it is at other levels because of the anatomical and biomechanical traits. Objective. To determine the clinical and radiological outcomes of MIS-TLIF for treatment of single-level spinal stenosis low-grade isthmic or degenerative spondylolisthesis at L5-S1. Methods. Radiological data and electronic medical records of patients who underwent MIS-TLIF between May 2012 and December 2014 were reviewed. Fusion rate, cage position, disc height (DH), disc angle (DA), disc slope angle, segmental lordotic angle (SLA), lumbar lordotic angle (LLA), and pelvic parameters were assessed. For functional assessment, the visual analogue scale (VAS), Oswestry disability index (ODI), and patient satisfaction rate (PSR) were utilized. Results. A total of 21 levels in 21 patients were studied. DH, DA, SLA, and LLA had increased from their preoperative measures at the final follow-up. Fusion rate was 86.7% (18/21) at 12 months' follow-up. The most common cage position was anteromedial (15/21). The mean VAS scores for back and leg pain mean ODI scores improved significantly at the final follow-up. PSR was 88%. Cage subsidence was observed in 33.3% (7/21). Conclusions. The clinical and radiologic outcomes after MIS-TLIF at L5-S1 in patients with spinal stenosis or spondylolisthesis are generally favorable. PMID:27433472

  17. Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes.

    PubMed

    Mori, Hiroshi; Maruyama, Fumito; Kato, Hiromi; Toyoda, Atsushi; Dozono, Ayumi; Ohtsubo, Yoshiyuki; Nagata, Yuji; Fujiyama, Asao; Tsuda, Masataka; Kurokawa, Ken

    2014-01-01

    The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.

  18. Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin.

    PubMed

    Witek, Marta A; Conn, Graeme L

    2014-09-01

    The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m(1)A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m(1)A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m(1)A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical enzyme is a functional 16S rRNA (m(1)A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family.

  19. Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

    PubMed Central

    Phister, Trevor G.; Mills, David A.

    2003-01-01

    Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis. PMID:14660395

  20. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences.

    PubMed

    Yarza, Pablo; Yilmaz, Pelin; Pruesse, Elmar; Glöckner, Frank Oliver; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Whitman, William B; Euzéby, Jean; Amann, Rudolf; Rosselló-Móra, Ramon

    2014-09-01

    Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.

  1. How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

    16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.

  2. RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.

    PubMed

    Lindahl, Lasse; Bommankanti, Ananth; Li, Xing; Hayden, Lauren; Jones, Adrienne; Khan, Miriam; Oni, Tolulope; Zengel, Janice M

    2009-07-01

    RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

  3. Abiotrophia defectiva infection of a total hip arthroplasty diagnosed by 16S rRNA gene sequencing.

    PubMed

    Rozemeijer, Wouter; Jiya, Timothy U; Rijnsburger, Martine; Heddema, Edou; Savelkoul, Paul; Ang, Wim

    2011-05-01

    We describe a case of a total hip arthroplasty infection caused by Abiotrophia defectiva, identified by 16S rRNA gene sequencing. Removal of the prosthesis followed by antibiotic treatment resulted in a good clinical outcome. 16S rRNA gene sequencing can be a useful tool in diagnosing infection with this fastidious microorganism that can easily be misidentified using phenotypic identification methods.

  4. Two linear regression models predicting cumulative dynamic L5/S1 joint moment during a range of lifting tasks based on static postures.

    PubMed

    Xu, Xu; Chang, Chien-Chi; Lu, Ming-Lun

    2012-01-01

    Previous studies have indicated that cumulative L5/S1 joint load is a potential risk factor for low back pain. The assessment of cumulative L5/S1 joint load during a field study is challenging due to the difficulty of continuously monitoring the dynamic joint load. This study proposes two regression models predicting cumulative dynamic L5/S1 joint moment based on the static L5/S1 joint moment of a lifting task at lift-off and set-down and the lift duration. Twelve men performed lifting tasks at varying lifting ranges and asymmetric angles in a laboratory environment. The cumulative L5/S1 joint moment was calculated from continuous dynamic L5/S1 moments as the reference for comparison. The static L5/S1 joint moments at lift-off and set-down were measured for the two regression models. The prediction error of the cumulative L5/S1 joint moment was 21 ± 14 Nm × s (12% of the measured cumulative L5/S1 joint moment) and 14 ± 9 Nm × s (8%) for the first and the second models, respectively. Practitioner Summary: The proposed regression models may provide a practical approach for predicting the cumulative dynamic L5/S1 joint loading of a lifting task for field studies since it requires only the lifting duration and the static moments at the lift-off and/or set-down instants of the lift.

  5. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed Central

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-01-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994). PMID:7651413

  6. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-09-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

  7. Bacillus nanhaiisediminis sp. nov., an alkalitolerant member of Bacillus rRNA group 6.

    PubMed

    Zhang, Jianli; Wang, Jiewei; Song, Fei; Fang, Caiyuan; Xin, Yuhua; Zhang, Yabo

    2011-05-01

    A Gram-stain-positive, rod-shaped bacterium, designated strain NH3(T), was isolated from a sediment sample from the South China Sea and was subjected to a polyphasic taxonomic study. The isolate grew optimally at 37 °C and pH 9. Strain NH3(T) had cell-wall peptidoglycan based on meso-diaminopimelic acid and MK-7 as the predominant menaquinone. The cellular fatty acid profile included significant amounts of iso-C(15 : 0) and iso-C(14 : 0). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of strain NH3(T) was 40.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NH3(T) was a member of rRNA group 6 of the genus Bacillus, which includes alkalitolerant, alkaliphilic and halotolerant species. The closest phylogenetic relatives were Bacillus akibai 1139(T) (96.82 % 16S rRNA gene sequence similarity), B. pseudofirmus DSM 8715(T) (96.76 %), B. okhensis Kh10-101(T) (96.76 %) and B. alkalidiazotrophicus MS 6(T) (96.47 %). Strain NH3(T) could be distinguished from these phylogenetically close neighbours based on a number of phenotypic properties. On the basis of phenotypic and chemotaxonomic characteristics and phylogenetic data, we conclude that strain NH3(T) ( = CGMCC 1.10116(T)  = JCM 16507(T)) merits classification as the type strain of a novel species, for which the name Bacillus nanhaiisediminis sp. nov. is proposed.

  8. Analysis, Optimization and Verification of Illumina-Generated 16S rRNA Gene Amplicon Surveys

    PubMed Central

    Nelson, Michael C.; Morrison, Hilary G.; Benjamino, Jacquelynn; Grim, Sharon L.; Graf, Joerg

    2014-01-01

    The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity. PMID:24722003

  9. Multi-site-specific 16S rRNA Methyltransferase RsmF from Thermus thermophilus

    SciTech Connect

    Demirci, H.; Larsen, L; Hansen, T; Rasmussen, A; Cadambi, A; Gregory, S; Kirpekar, F; Jogl, G

    2010-01-01

    Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m{sup 5}C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m{sup 5}C967. In contrast to E. coli RsmF, which introduces a single m{sup 5}C1407 modification, T. thermophilus RsmF modifies three positions, generating m{sup 5}C1400 and m{sup 5}C1404 in addition to m{sup 5}C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 {angstrom} resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.

  10. The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2012-03-26

    Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides (1531{sup AUCACCUCC}1539) near the 3{prime} end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530{sup GAUCA}1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530{sup GA}1531 to 1530{sup AG}1531 double mutation severely affects the viability of bacteria. However, whether Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.

  11. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  12. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies.

    PubMed

    Rössler, D; Ludwig, W; Schleifer, K H; Lin, C; McGill, T J; Wisotzkey, J D; Jurtshuk, P; Fox, G E

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  13. Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

    NASA Technical Reports Server (NTRS)

    Rossler, D.; Ludwig, W.; Schleifer, K. H.; Lin, C.; McGill, T. J.; Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1991-01-01

    Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

  14. Campylobacter species identification based on polymorphism of DNA encoding rRNA.

    PubMed Central

    Moureau, P; Derclaye, I; Gregoire, D; Janssen, M; Cornelis, G R

    1989-01-01

    Total DNA from five Campylobacter species was digested with a mixture of XhoI and BglII restriction endonucleases and analyzed by Southern hybridization by using a probe complementary to the DNA coding for the 16S rRNA. Each of the Campylobacter species, including C. jejuni, C. coli, C. laridis, C. fetus, and C. upsaliensis, displayed a characteristic pattern. Although some bands may be common to different species, the simplicity of the hybridization pattern enabled us to discriminate among the different species of Campylobacter. Images PMID:2570080

  15. Greengenes, a Chimera-checked 16S rRNA gene database and workbenchcompatible with ARB

    SciTech Connect

    DeSantis, Todd Z.; Hugenholtz, Philip; Larsen, Neils; Rojas,Mark; Brodie, Eoin L.; Keller, Keith; Huber, Thomas; Dalevi, Daniel; Hu,Ping; Andersen, Gary L.

    2006-04-10

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that in congruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3 percent of environmental sequences and 0.2 percent of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  16. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  17. A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products.

    PubMed

    Nakano, Shigeru; Matsumura, Atsushi; Yamada, Toshihiro

    2004-03-01

    A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei. For all other bacterial strains and eukaryote tested, results were negative. Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0). To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L. fructivorans was inoculated into several acidic food as an indicator. Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.

  18. Comparison of Gen-probe transcription-mediated amplification, Abbott PCR, and Roche PCR assays for detection of wild-type and mutant plasmid strains of Chlamydia trachomatis in Sweden.

    PubMed

    Møller, Jens Kjølseth; Pedersen, Lisbeth Nørum; Persson, Kenneth

    2008-12-01

    The clinical performance of two nucleic acid amplification assays targeting the cryptic plasmid and two assays targeting rRNA molecules in Chlamydia trachomatis was examined. First-catch urine samples from Malmoe, Sweden, were tested for C. trachomatis with the Abbott real-time PCR assay m2000 and an in-house PCR for the new variant strain of C. trachomatis with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark, and further examined with the Roche COBAS Amplicor CT (RCA) PCR, the Gen-Probe Aptima Combo 2 assay (AC2) targeting the C. trachomatis 23S rRNA, and the Aptima C. trachomatis assay (ACT) targeting the 16S rRNA molecule. A positive prevalence of 9% (163/1,808 urine samples examined) was detected according to the combined reference standard. The clinical sensitivity and specificity of the four assays were as follows: for ACT, 100% (163/163) and 99.9% (1,643/1,645), respectively; for AC2, 100% (163/163) and 99.6% (1,640/1,645); for m2000, 68.7% (112/163) and 99.9% (1,644/1,645); for RCA, 63.8% (104/163) and 99.9% (1,643/1,645). The two Gen-Probe assays detected all mutant strains characterized by the in-house PCR as having the deletion in the cryptic plasmid, whereas the Roche and the Abbott PCRs targeting the plasmid were both unable to detect the plasmid mutant. The difference in clinical sensitivity between the plasmid PCR assays m2000 and RCA, on the one hand, and the rRNA assays AC2 and ACT, on the other, could be attributed almost exclusively to the presence of the plasmid mutant in about one-quarter of the Chlamydia-positive samples examined.

  19. Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

    PubMed

    Radax, C; Gruber, C; Stan-Lotter, H

    2001-08-01

    Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

  20. Functional Specialization of Domains Tandemly Duplicated Witin 16S rRNA Methyltransferase RsmC

    SciTech Connect

    Sunita,S.; Purta, E.; Durawa, M.; Tkaczuk, K.; Swaathi, J.; Bujnicki, J.; Sivaraman, J.

    2007-01-01

    RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Angstroms resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

  1. Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis.

    PubMed

    Drygin, Denis; Siddiqui-Jain, Adam; O'Brien, Sean; Schwaebe, Michael; Lin, Amy; Bliesath, Josh; Ho, Caroline B; Proffitt, Chris; Trent, Katy; Whitten, Jeffrey P; Lim, John K C; Von Hoff, Daniel; Anderes, Kenna; Rice, William G

    2009-10-01

    Hallmark deregulated signaling in cancer cells drives excessive ribosome biogenesis within the nucleolus, which elicits unbridled cell growth and proliferation. The rate-limiting step of ribosome biogenesis is synthesis of rRNA (building blocks of ribosomes) by RNA Polymerase I (Pol I). Numerous kinase pathways and products of proto-oncogenes can up-regulate Pol I, whereas tumor suppressor proteins can inhibit rRNA synthesis. In tumorigenesis, activating mutations in certain cancer-associated kinases and loss-of-function mutations in tumor suppressors lead to deregulated signaling that stimulates Pol I transcription with resultant increases in ribosome biogenesis, protein synthesis, cell growth, and proliferation. Certain anticancer therapeutics, such as cisplatin and 5-fluorouracil, reportedly exert, at least partially, their activity through disruption of ribosome biogenesis, yet many prime targets for anticancer drugs within the ribosome synthetic machinery of the nucleolus remain largely unexploited. Herein, we describe CX-3543, a small molecule nucleolus-targeting agent that selectively disrupts nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting Pol I transcription and inducing apoptosis in cancer cells. CX-3543 is the first G-quadruplex interactive agent to enter human clinical trials, and it is currently under evaluation against carcinoid/neuroendocrine tumors in a phase II clinical trial.

  2. DbpA: a DEAD box protein specifically activated by 23s rRNA.

    PubMed Central

    Fuller-Pace, F V; Nicol, S M; Reid, A D; Lane, D P

    1993-01-01

    The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA. Images PMID:8253085

  3. The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing

    PubMed Central

    Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

    2016-01-01

    ABSTRACT Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

  4. Greengenes: 16S rRNA Database and Workbench Compatible with ARB

    DOE Data Explorer

    DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.; Huber, T.; Dalevi, D. Hu, P. Andersen, G. L.

    Greengenes was developed, as the abstract of an AEM reprint states, to "addresse limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria....Greengenes is also a functional workbench to assist in analysis of user-generated 16S rRNA gene sequences. Batches of sequencing reads can be uploaded for quality-based trimming and creation of multiple-sequence alignments (9). Three types of non-MSA similarity searches are also available, seed extension by BLAST (1), similarity based on shared 7-mers by a tool called Simrank, and a direct degenerative pattern match for probe/primer evaluation. Results are displayed using user-preferred taxonomic nomenclature and can be saved between sessions. [Taken from DeSantis, T. Z., P. Hugenholtz, N. Larsen, M. Rojas, E. L. Brodie, K. Keller, T. Huber, D. Dalevi, P. Hu, and G. L. Andersen. 2006. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol 72:5069-72, pages 1 and 3] (Specialized Interface)

  5. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    PubMed

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  6. Control of rRNA Synthesis in Escherichia coli: a Systems Biology Approach†

    PubMed Central

    Dennis, Patrick P.; Ehrenberg, Mans; Bremer, Hans

    2004-01-01

    The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (Vmax/Km) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation. PMID:15590778

  7. Phylogenetic relationships of conifers inferred from partial 28S rRNA gene sequences.

    PubMed

    Stefanoviac, S; Jager, M; Deutsch, J; Broutin, J; Masselot, M

    1998-05-01

    The conifers, which traditionally comprise seven families, are the largest and most diverse group of living gymnosperms. Efforts to systematize this diversity without a cladistic phylogenetic framework have often resulted in the segregation of certain genera and/or families from the conifers. In order to understand better the relationships between the families, we performed cladistic analyses using a new data set obtained from 28S rRNA gene sequences. These analyses strongly support the monophyly of conifers including Taxaceae. Within the conifers, the Pinaceae are the first to diverge, being the sister group of the rest of conifers. A recently discovered Australian genus Wollemia is confirmed to be a natural member of the Araucariaceae. The Taxaceae are nested within the conifer clade, being the most closely related to the Cephalotaxaceae. The Taxodiaceae and Cupressaceae together form a monophyletic group. Sciadopitys should be considered as constituting a separate family. These relationships are consistent with previous cladistic analyses of morphological and molecular (18S rRNA, rbcL) data. Furthermore, the well-supported clade linking the Araucariaceae and Podocarpaceae, which has not been previously reported, suggests that the common ancestor of these families, both having the greatest diversity in the Southern Hemisphere, inhabited Gondwanaland.

  8. Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

    PubMed Central

    Teixidò, J; Altamura, S; Londei, P; Amils, R

    1989-01-01

    The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures. Images PMID:2493632

  9. Li(V0.5Ti0.5)S2 as a 1 V lithium intercalation electrode

    NASA Astrophysics Data System (ADS)

    Clark, Steve J.; Wang, Da; Armstrong, A. Robert; Bruce, Peter G.

    2016-03-01

    Graphite, the dominant anode in rechargeable lithium batteries, operates at ~0.1 V versus Li+/Li and can result in lithium plating on the graphite surface, raising safety concerns. Titanates, for example, Li4Ti5O12, intercalate lithium at~1.6 V versus Li+/Li, avoiding problematic lithium plating at the expense of reduced cell voltage. There is interest in 1 V anodes, as this voltage is sufficiently high to avoid lithium plating while not significantly reducing cell potential. The sulfides, LiVS2 and LiTiS2, have been investigated as possible 1 V intercalation electrodes but suffer from capacity fading, large 1st cycle irreversible capacity or polarization. Here we report that the 50/50 solid solution, Li1+x(V0.5Ti0.5)S2, delivers a reversible capacity to store charge of 220 mAhg-1 (at 0.9 V), 99% of theoretical, at a rate of C/2, retaining 205 mAhg-1 at C-rate (92% of theoretical). Rate capability is excellent with 200 mAhg-1 at 3C. C-rate is discharge in 1 h. Polarization is low, 100 mV at C/2. To the best of our knowledge, the properties/performances of Li(V0.5Ti0.5)S2 exceed all previous 1 V electrodes.

  10. Thickness-Dependent and Magnetic-Field-Driven Suppression of Antiferromagnetic Order in Thin V5S8 Single Crystals.

    PubMed

    Hardy, Will J; Yuan, Jiangtan; Guo, Hua; Zhou, Panpan; Lou, Jun; Natelson, Douglas

    2016-06-28

    With materials approaching the 2D limit yielding many exciting systems with intriguing physical properties and promising technological functionalities, understanding and engineering magnetic order in nanoscale, layered materials is generating keen interest. One such material is V5S8, a metal with an antiferromagnetic ground state below the Néel temperature TN ∼ 32 K and a prominent spin-flop signature in the magnetoresistance (MR) when H∥c ∼ 4.2 T. Here we study nanoscale-thickness single crystals of V5S8, focusing on temperatures close to TN and the evolution of material properties in response to systematic reduction in crystal thickness. Transport measurements just below TN reveal magnetic hysteresis that we ascribe to a metamagnetic transition, the first-order magnetic-field-driven breakdown of the ordered state. The reduction of crystal thickness to ∼10 nm coincides with systematic changes in the magnetic response: TN falls, implying that antiferromagnetism is suppressed; and while the spin-flop signature remains, the hysteresis disappears, implying that the metamagnetic transition becomes second order as the thickness approaches the 2D limit. This work demonstrates that single crystals of magnetic materials with nanometer thicknesses are promising systems for future studies of magnetism in reduced dimensionality and quantum phase transitions.

  11. 5S program to reduce change-over time on forming department (case study on CV Piranti Works temanggung)

    NASA Astrophysics Data System (ADS)

    Rosiana Dewi, Septika; Setiawan, Budi; P, Susatyo Nugroho W.

    2013-06-01

    Productivity is one aspect that determines the success of a company in the competitive world of business. There are seven main types of activities that do not have value-added in manufacturing processes such as overproduction, waiting time, transportation, excess inventory, unnecessary motion and defects. The whole activity is a waste (waste) that can cause harm to the Company. Therefore, in production activities is important to pay attention so that the objectives of production productivity can be achieved. Problems experienced by CV Piranti Works is a production target is not achieved resulting in a lost sale raises the cost of which can cause harm to the Company. From the analysis conducted major known cause of the problem is the length of time required for changeover. This is supported by the high non-value added activity in the changeover activities. Lean Manufacturing is an approach to make system more efficient by reducing waste. This study refers to the book compiled by Takashi Osada (2004) and several other references. In this research used method 5S (Seiri, Seiton, Seiso, Seiketsu, and Shitsuke) for the of forming departement. The purpose of this research is to design a work environment using the 5S method (Seiri, Seiton, Seiso, Seiketsu, and Shitsuke) and make arrangement of equipment and working tool cabinet design with TRIZ methods. From these results, is expected to eliminate or reduce of non-value added activity and improved the changeover time so as to meet production targets completion of the company.

  12. 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

  13. Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyl-dopamine induced neurotoxicity.

    PubMed

    Vauzour, David; Corona, Giulia; Spencer, Jeremy P E

    2010-09-01

    Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra. Recent investigations have shown that conjugates such as the 5-S-cysteinyl-dopamine, possess strong neurotoxicity and may contribute to the underlying progression of the disease pathology. Although the neuroprotective actions of flavonoids are well reported, that of hydroxycinnamates and other phenolic acids is less established. We show that the hydroxycinnamates caffeic acid and p-coumaric acid, the hydroxyphenethyl alcohol, tyrosol, and a Champagne wine extract rich in these components protect neurons against injury induced by 5-S-cysteinyl-dopamine in vitro. The protection induced by these polyphenols was equal to or greater than that observed for the flavonoids, (+)-catechin, (-)-epicatechin and quercetin. For example, p-coumaric acid evoked significantly more protection at 1muM (64.0+/-3.1%) than both (-)-epicatechin (46.0+/-4.1%, p<0.05) and (+)-catechin (13.1+/-3.0%, p<0.001) at the same concentration. These data indicate that hydroxycinnamates, phenolic acids and phenolic alcohol are also capable of inducing neuroprotective effects to a similar extent to that seen with flavonoids.

  14. Neisseria Species Identification Assay for the Confirmation of Neisseria gonorrhoeae-Positive Results of the COBAS Amplicor PCR▿

    PubMed Central

    Mangold, Kathy A.; Regner, MaryAnn; Tajuddin, Mohammed; Tajuddin, Aamair M.; Jennings, Lawrence; Du, Hongyan; Kaul, Karen L.

    2007-01-01

    Screening assays for Neisseria gonorrhoeae exhibit low positive predictive values, particularly in low-prevalence populations. A new real-time PCR assay that detects and identifies individual Neisseria spp. using melt curve analysis was compared to two previously published supplementary assays. NsppID, a 16S rRNA real-time PCR/melt curve assay developed to distinguish N. gonorrhoeae from other Neisseria spp., was compared to real-time PCR assays targeting genes reportedly specific for N. gonorrhoeae, the cppB gene and the porA pseudogene. A total of 408 clinical specimens (324 female endocervical swabs and 84 male urine or urogenital swab specimens) were screened using the COBAS Amplicor assay for Chlamydia trachomatis and N. gonorrhoeae (CT/NG) (Roche Diagnostics, Indianapolis, IN) followed by confirmatory testing via real-time PCR. The NsppID assay detected Neisseria spp. in 150/181 COBAS-positive specimens (82%), including six dual infections, and identified N. gonorrhoeae in 102 (56%) specimens. Sixty-nine of 181 (38%) specimens were positive for N. gonorrhoeae by porA pseudogene, and 115/181 (64%) were positive for cppB. However, cppB was also positive in 15% of COBAS-negative specimens, more than either NsppID (4%) or porA pseudogene (2%) assays. The porA pseudogene assay had the highest specificity for both genders but the lowest sensitivity, especially in female specimens. NsppID had a slightly lower specificity but greater sensitivity and overall accuracy. The least desirable confirmatory assay was cppB, due to poor specificity. The NsppID assay is an accurate confirmatory assay for N. gonorrhoeae detection. In addition, the NsppID assay can identify the non-N. gonorrhoeae species responsible for the majority of false-positive results from the COBAS Amplicor CT/NG assay. PMID:17360838

  15. Ricin Activity Assay by Direct Analysis in Real Time Mass Spectrometry Detection of Adenine Release

    DTIC Science & Technology

    2010-02-01

    direct analysis in real time mass spectrometry. The release of adenine from the inhomo- geneous substrate herring sperm DNA by ricin was determined to...chain catalyzes cleavage at adenosine 4324 (in rat RNA) of 28S rRNA to release adenine.10 This action inhibits protein synthesis, leading to cell...death. In addition to RNA, herring sperm DNA (hsDNA) is a substrate for ricin.11 We chose to employ hsDNA for this assay because it is relatively stable

  16. Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care.

    PubMed

    Kurabayashi, Hirotaka; Kaneko, Akihiro; Sekiya, Ryo; Karakida, Kazunari; Sasaki, Masashi; Nakatogawa, Noriko; Aoki, Takayuki; Ota, Yoshihide; Sakamoto, Haruo

    2011-02-01

    After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.

  17. Development and Evaluation of Three Real-Time PCR Assays for Genotyping and Source Tracking Cryptosporidium spp. in Water.

    PubMed

    Li, Na; Neumann, Norman F; Ruecker, Norma; Alderisio, Kerri A; Sturbaum, Gregory D; Villegas, Eric N; Chalmers, Rachel; Monis, Paul; Feng, Yaoyu; Xiao, Lihua

    2015-09-01

    The occurrence of Cryptosporidium oocysts in drinking source water can present a serious public health risk. To rapidly and effectively assess the source and human-infective potential of Cryptosporidium oocysts in water, sensitive detection and correct identification of oocysts to the species level (genotyping) are essential. In this study, we developed three real-time PCR genotyping assays, two targeting the small-subunit (SSU) rRNA gene (18S-LC1 and 18S-LC2 assays) and one targeting the 90-kDa heat shock protein (hsp90) gene (hsp90 assay), and evaluated the sensitivity and Cryptosporidium species detection range of these assays. Using fluorescence resonance energy transfer probes and melt curve analysis, the 18S-LC1 and hsp90 assays could differentiate common human-pathogenic species (C. parvum, C. hominis, and C. meleagridis), while the 18S-LC2 assay was able to differentiate nonpathogenic species (such as C. andersoni) from human-pathogenic ones commonly found in source water. In sensitivity evaluations, the 18S-LC2 and hsp90 genotyping assays could detect as few as 1 Cryptosporidium oocyst per sample. Thus, the 18S-LC2 and hsp90 genotyping assays might be used in environmental monitoring, whereas the 18S-LC1 genotyping assay could be useful for genotyping Cryptosporidium spp. in clinical specimens or wastewater samples.

  18. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  19. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  20. Novel Real-Time PCR Assay for Simultaneous Detection and Differentiation of Clostridium chauvoei and Clostridium septicum in Clostridial Myonecrosis▿

    PubMed Central

    Halm, Anna; Wagner, Martin; Köfer, Josef; Hein, Ingeborg

    2010-01-01

    A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 × 103 C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method. PMID:20129968

  1. Novel real-time PCR assay for simultaneous detection and differentiation of Clostridium chauvoei and Clostridium septicum in clostridial myonecrosis.

    PubMed

    Halm, Anna; Wagner, Martin; Köfer, Josef; Hein, Ingeborg

    2010-04-01

    A real-time PCR assay based on the 16S rRNA gene sequence was designed for differentiation of blackleg-causing Clostridium chauvoei and Clostridium septicum, a phylogenetically closely related bacterium responsible for malignant edema. In order to exclude false-negative results, an internal amplification control was included in the assay. A set of three probes, one specific for C. chauvoei, one specific for C. septicum, and one specific for both species, permitted unequivocal detection of C. chauvoei in tests of 32 Clostridium sp. strains and 10 non-Clostridium strains. The assay proved to be sensitive, detecting one genome of C. chauvoei or C. septicum per PCR and 1.79 x 10(3) C. chauvoei cells/g artificially contaminated muscle tissue. In tests of 11 clinical specimens, the real-time PCR assay yielded the same results as an established conventional PCR method.

  2. Development of quantitative PCR assays targeting 16S rRNA gene of Enterococcus spp. and their application to the identification of Enterococcus species in environmental samples

    EPA Science Inventory

    The detection of environmental enterococci has primarily been determined using culture-based techniques that might exclude some enterococci species as well as those that are nonculturable. To address this, the relative abundance of enterococci was examined by challenging fecal an...

  3. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  4. Development of a PCR Assay for the Detection of Spironucleus muris

    PubMed Central

    Jackson, Glenn A; Livingston, Robert S; Riley, Lela K; Livingston, Beth A; Franklin, Craig L

    2013-01-01

    Spironucleus muris is a protozoan that can colonize the intestinal tract of many rodent species. Although its effects on animal health and research are debated, S. muris is often included on exclusion lists for rodent facilities. Common diagnostic tests for S. muris are insensitive and typically are performed at postmortem examination. We sought to develop a PCR-based diagnostic test with sufficient sensitivity and specificity for use on fecal samples from live rodents. We designed and optimized a PCR assay that targeted the 16S-like rRNA gene of S. muris. The assay was highly specific, given that samples from mice contaminated with S. muris were PCR positive, whereas samples from mice contaminated with other protozoa were negative. The assay also was highly sensitive, detecting as few as 5 template copies per microliter diluent. All mice positive for S. muris on postmortem exams also were positive by fecal PCR. Moreover, S. muris was detected by PCR in mice negative by postmortem examination but from colonies known to be contaminated as well as in rats and hamsters. To assess protozoal loads in mice of differing ages, the PCR assay was adapted to a quantitative format. Fecal loads of S. muris were highest in 4-wk-old mice and declined with age. The PCR assay developed promises to be a highly specific antemortem diagnostic assay with higher sensitivity than that of existing postmortem tests. PMID:23562099

  5. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  6. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

    PubMed

    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  7. Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

    PubMed Central

    Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

    1994-01-01

    Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

  8. 16S rRNA methyltransferase KsgA contributes to oxidative stress resistance and virulence in Staphylococcus aureus.

    PubMed

    Kyuma, Tatsuhiko; Kizaki, Hayato; Ryuno, Hiroki; Sekimizu, Kazuhisa; Kaito, Chikara

    2015-12-01

    We previously reported that the rRNA methyltransferases RsmI and RsmH, which are responsible for cytidine dimethylation at position 1402 of 16S rRNA in the decoding center of the ribosome, contribute to Staphylococcus aureus virulence. Here we evaluated other 16S rRNA methyltransferases, including KsgA (RsmA), RsmB/F, RsmC, RsmD, RsmE, and RsmG. Knockout of KsgA, which methylates two adjacent adenosines at positions 1518 and 1519 of 16S rRNA in the intersubunit bridge of the ribosome, attenuated the S. aureus killing ability against silkworms. The ksgA knockout strain was sensitive to oxidative stress and had a lower survival rate in murine macrophages than the parent strain. The ksgA knockout strain exhibited decreased translational fidelity in oxidative stress conditions. Administration of N-acetyl-l-cysteine, a free-radical scavenger, restored the killing ability of the ksgA knockout strain against silkworms. These findings suggest that the methyl-modifications of 16S rRNA by KsgA contribute to maintain ribosome function under oxidative conditions and thus to S. aureus virulence.

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

    PubMed

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

    1978-03-01

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

  10. METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

    PubMed

    Bengtsson-Palme, Johan; Hartmann, Martin; Eriksson, Karl Martin; Pal, Chandan; Thorell, Kaisa; Larsson, Dan Göran Joakim; Nilsson, Rolf Henrik

    2015-11-01

    The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The METAXA software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to METAXA - METAXA2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of METAXA2 was compared to other commonly used taxonomic classifiers, showing that METAXA2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. METAXA2 is freely available from http://microbiology.se/software/metaxa2/.

  11. Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites▿

    PubMed Central

    Basu, Abhijit; Das, Priyanka; Chaudhuri, Sujan; Bevilacqua, Elena; Andrews, Joel; Barik, Sailen; Hatzoglou, Maria; Komar, Anton A.; Mazumder, Barsanjit

    2011-01-01

    Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation. PMID:21930789

  12. RiboFR-Seq: a novel approach to linking 16S rRNA amplicon profiles to metagenomes

    PubMed Central

    Zhang, Yanming; Ji, Peifeng; Wang, Jinfeng; Zhao, Fangqing

    2016-01-01

    16S rRNA amplicon analysis and shotgun metagenome sequencing are two main culture-independent strategies to explore the genetic landscape of various microbial communities. Recently, numerous studies have employed these two approaches together, but downstream data analyses were performed separately, which always generated incongruent or conflict signals on both taxonomic and functional classifications. Here we propose a novel approach, RiboFR-Seq (Ribosomal RNA gene flanking region sequencing), for capturing both ribosomal RNA variable regions and their flanking protein-coding genes simultaneously. Through extensive testing on clonal bacterial strain, salivary microbiome and bacterial epibionts of marine kelp, we demonstrated that RiboFR-Seq could detect the vast majority of bacteria not only in well-studied microbiomes but also in novel communities with limited reference genomes. Combined with classical amplicon sequencing and shotgun metagenome sequencing, RiboFR-Seq can link the annotations of 16S rRNA and metagenomic contigs to make a consensus classification. By recognizing almost all 16S rRNA copies, the RiboFR-seq approach can effectively reduce the taxonomic abundance bias resulted from 16S rRNA copy number variation. We believe that RiboFR-Seq, which provides an integrated view of 16S rRNA profiles and metagenomes, will help us better understand diverse microbial communities. PMID:26984526

  13. Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

    PubMed

    Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

    1994-10-11

    Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years.

  14. Fission properties of the 1. 5-s spontaneous fission activity produced in bombardmentof /sup 248/Cm with /sup 18/O

    SciTech Connect

    Hoffman, D.C.; Lee, D.; Ghiorso, A.; Nurmia, M.J.; Aleklett, K.; Leino, M.

    1981-08-01

    We have measured the mass and kinetic-energy distributions of fragments from the spontaneous fission of a 1.5-s activity produced in bombardments of /sup 248/Cm with 95-MeV /sup 18/O ions. Its spontaneous fission decay exhibits a very symmetric, narrow (full width at half maximum = 12 mass units) mass distribution, a very high total kinetic energy of 234 +- 2 MeV, and increasing total kinetic energy with increasingly symmetric mass division. Based on its half-life and the similarity of its fission properties to the unique fission properties so far only observed for /sup 258/Fm and /sup 259/Fm, the most likely assignment of this activity is to the known /sup 259/Fm. However, assignment to some as yet undiscovered neutron-rich heavy element isotope such as /sup 260/Md cannot be unequivocally excluded.

  15. Inhibition of viral RNA methylation in herpes simplex virus type 1-infected cells by 5' S-isobutyl-adenosine.

    PubMed Central

    Jacquemont, B; Huppert, J

    1977-01-01

    5' S-isobutyl-adenosine (SIBA), a structural analogue of S-adenosylhomocysteine, reversibly blocks the multiplication of herpes simplex type 1 virus. In the presence of SIBA, viral protein synthesis is inhibited. After removing SIBA the synthesis of proteins starts rapidly again. The new polypeptides are mainly alpha proteins (Honess and Roizman, J. Virol. 14:8-19, 1974,), normally the first to be synthesized after infection. The rapid synthesis of proteins after release of inhibition seems to be directed by mRNA formed in the presence of SIBA as indicated by experiments using actinomycin D but which was undermethylated as shown by analysis of methyl groups on RNA. SIBA inhibits the methylation of mRNA and especially that of the 5' cap. Capping of mRNA thus seems to be essential for efficient translation. The analogue affected various methylations to different extents. Images PMID:192910

  16. Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay.

    PubMed

    Duarte, Sabrina Castilho; Linhares, Guido Fontgalland Coelho; Romanowsky, Tatiana Nunes; da Silveira Neto, Osvaldo José; Borges, Ligia Miranda Ferreira

    2008-03-25

    Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.

  17. Measurements of the exclusive decays of the upsilon(5S) to meson final states and improved B(s)* mass measurement.

    PubMed

    Aquines, O; Li, Z; Lopez, A; Mendez, H; Ramirez, J; Huang, G S; Miller, D H; Pavlunin, V; Sanghi, B; Shipsey, I P J; Xin, B; Adams, G S; Anderson, M; Cummings, J P; Danko, I; Napolitano, J; He, Q; Insler, J; Muramatsu, H; Park, C S; Thorndike, E H; Coan, T E; Gao, Y S; Liu, F; Stroynowski, R; Artuso, M; Blusk, S; Butt, J; Li, J; Menaa, N; Mountain, R; Nisar, S; Randrianarivony, K; Redjimi, R; Sia, R; Skwarnicki, T; Stone, S; Wang, J C; Zhang, K; Csorna, S E; Bonvicini, G; Cinabro, D; Dubrovin, M; Lincoln, A; Asner, D M; Edwards, K W; Briere, R A; Brock, I; Chen, J; Ferguson, T; Tatishvili, G; Vogel, H; Watkins, M E; Rosner, J L; Adam, N E; Alexander, J P; Berkelman, K; Cassel, D G; Duboscq, J E; Ecklund, K M; Ehrlich, R; Fields, L; Galik, R S; Gibbons, L; Gray, R; Gray, S W; Hartill, D L; Heltsley, B K; Hertz, D; Jones, C D; Kandaswamy, J; Kreinick, D L; Kuznetsov, V E; Mahlke-Krüger, H; Meyer, T O; Onyisi, P U E; Patterson, J R; Peterson, D; Phillips, E A; Pivarski, J; Riley, D; Ryd, A; Sadoff, A J; Schwarthoff, H; Shi, X; Stroiney, S; Sun, W M; Wilksen, T; Weinberger, M; Athar, S B; Avery, P; Breva-Newell, L; Patel, R; Potlia, V; Stoeck, H; Yelton, J; Rubin, P; Cawlfield, C; Eisenstein, B I; Karliner, I; Kim, D; Lowrey, N; Naik, P; Sedlack, C; Selen, M; White, E J; Wiss, J; Shepherd, M R; Besson, D; Pedlar, T K; Cronin-Hennessy, D; Gao, K Y; Gong, D T; Hietala, J; Kubota, Y; Klein, T; Lang, B W; Poling, R; Scott, A W; Smith, A; Dobbs, S; Metreveli, Z; Seth, K K; Tomaradze, A; Zweber, P; Ernst, J; Arms, K; Severini, H; Dytman, S A; Love, W; Mehrabyan, S; Savinov, V

    2006-04-21

    Using 420 pb(-1) of data collected on the upsilon(5S) resonance with the CLEO III detector, we reconstruct B mesons in 25 exclusive decay channels to measure or set upper limits on the decay rate of upsilon(5S) into B meson final states. We measure the inclusive B cross section to be sigma(upsilon(5S) --> BB(X)) = (0.177 +/- 0.030 +/- 0.016) nb and make the first measurements of the production rates of sigma(upsilon(5S) --> B*B*) = (0.131 +/- 0.025 +/- 0.014) nb and sigma(upsilon(5S) --> BB*) = (0.043 +/- 0.016 +/- 0.006) nb, respectively. We set 90% confidence level limits of sigma(upsilon(5S) -->BB) < 0.038 nb, sigma(upsilon(5S) --> B(*)B(*)pi) < 0.055 nb and sigma(upsilon(5S) --> BBpipi) < 0.024 nb. We also extract the most precise value of the B(s)* mass to date, M(B(s)*) = (5411.7 +/- 1.6 +/- 0.6) MeV/c2.

  18. Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays.

    PubMed

    Jenkins, Marion W; Tiwari, Sangam; Lorente, Mario; Gichaba, Charles Maina; Wuertz, Stefan

    2009-11-01

    Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and

  19. Interaction of potassium cyanide with the [Ni-4Fe-5S] active site cluster of CO dehydrogenase from Carboxydothermus hydrogenoformans.

    PubMed

    Ha, Seung-Wook; Korbas, Malgorzata; Klepsch, Mirjam; Meyer-Klaucke, Wolfram; Meyer, Ortwin; Svetlitchnyi, Vitali

    2007-04-06

    The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.

  20. Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences.

    PubMed

    Takeuchi, M; Yokota, A

    1994-11-15

    16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum, were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.

  1. 18S rRNA suggests that Entoprocta are protostomes, unrelated to Ectoprocta.

    PubMed

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

    1996-05-01

    The Ento- and Ectoprocta are sometimes placed together in the Bryozoa, which have variously been regarded as proto- or deuterostomes. However, Entoprocta have also been allied to the pseudocoelomates, while Ectoprocta are often united with the Brachiopoda and Phoronida in the (super)phylum Lophophorata. Hence, the phylogenetic relationships of these taxa are still much debated. We determined complete 18S rRNA sequences of two entoprocts, an ectoproct, an inarticulate brachiopod, a phoronid, two annelids, and a platyhelminth. Phylogenetic analyses of these data show that (1) entoprocts and lophophorates have spiralian, protostomous affinities, (2) Ento- and Ectoprocta are not sister taxa, (3) phoronids and brachiopods form a monophyletic clade, and (4) neither Ectoprocta or Annelida appear to be monophyletic. Both deuterostomous and pseudocoelomate features may have arisen at least two times in evolutionary history. These results advocate a Spiralia-Radialia-based classification rather than one based on the Protostomia-Deuterostomia concept.

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

    PubMed

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

    1999-11-01

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

  3. Identification of Scopulariopsis species by partial 28S rRNA gene sequence analysis.

    PubMed

    Jagielski, Tomasz; Kosim, Kinga; Skóra, Magdalena; Macura, Anna Barbara; Bielecki, Jacek

    2013-01-01

    The genus Scopulariopsis contains over 30 species of mitosporic moulds, which although usually saprophytic may also act as opportunistic pathogens in humans. They have mainly been associated with onychomycosis, and only sporadically reported as a cause of deep tissue infections or systemic disease. Identification of Scopulariopsis species still largely relies on phenotype-based methods. There is a need for a molecular diagnostic approach, that would allow to reliably discriminate between different Scopulariopsis species. The aim of this study was to apply sequence analysis of partial 28S rRNA gene for species identification of Scopulariopsis clinical isolates. Although the method employed did reveal some genetic polymorphism among Scopulariopsis isolates tested, it was not enough for species delineation. For this to be achieved, other genetic loci, within and beyond the rDNA operon, need to be investigated.

  4. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  5. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  6. [Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus].

    PubMed

    Fernandes, Paula Rogério; Da Silva, Andréa Caetano; Gambarini, Maria Lúcia; Linhares, Guido Fontgalland C

    2008-01-01

    Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

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

    PubMed

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

    1991-09-01

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

  8. Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis.

    PubMed

    Schuppler, M; Mertens, F; Schön, G; Göbel, U B

    1995-02-01

    The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, we have established a clone library of 16S rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNP1 detected 27 clones with 16S rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 16S rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 16S rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

  9. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

  10. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons

    PubMed Central

    Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

    2015-01-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  11. The identification of rRNA maturation sites in the microsporidian Encephalitozoon cuniculi argues against the full excision of presumed ITS1 sequence.

    PubMed

    Peyretaillade, E; Peyret, P; Metenier, G; Vivares, C P; Prensier, G

    2001-01-01

    In Encephalitozoon cuniculi like in other microsporidia, the primary transcript for SSU and LSU rRNAs includes only one internal transcribed spacer (ITS1) which separates SSU rRNA from the 5.8S region associated with LSU rRNA. The extraction of total RNA from E. cuniculi-infected MRC5 cells using a hot phenol/chloroform procedure enabled us to perform primer extension and S1 nuclease protection experiments in the aim of identifying rRNA maturation sites. Our data support a simple processing (four cleavage sites) with elimination of only nine nucleotides between SSU and LSU rRNA regions. Most of the presumed ITS1 sequence characterized by strain-dependent polymorphism therefore remains linked to SSU rRNA 3' end. A new secondary structure for the sixth domain of E. cuniculi LSU rRNA is proposed following the identification of its 3' terminus.

  12. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  13. Detection of banned ruminant-derived material in industrial feedstuffs by TaqMan real-time PCR Assay.

    PubMed

    Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

    2011-08-01

    A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.

  14. rRNA (rrn) Operon-Engineered Bacillus subtilis as a Feasible Test Organism for Antibiotic Discovery

    PubMed Central

    Tanaka, Yukinori; Nanamiya, Hideaki; Yano, Koichi; Kakugawa, Koji; Kawamura, Fujio

    2013-01-01

    Bacillus subtilis contains 10 rRNA (rrn) operons. We found that rRNA operon-engineered B. subtilis strain RIK543, with only the rrnO operon, is specifically hypersensitive to RNA polymerase inhibitors such as rifamycin SV and rifampin (80-fold and 20-fold, respectively). In pilot screening experiments, we found actinomycete isolates successfully at an incidence of 1.9% (18/945) that produced antibacterials that were detectable only with RIK543 as the test organism. Strain RIK543 may be a feasible test organism for the discovery of novel RNA polymerase inhibitors. PMID:23335737

  15. Primary Structure of 28S rRNA Gene Confirms Monophyly of Free-Living Heterotrophic and Phototrophic Apicomplexans (Alveolata).

    PubMed

    Mikhailov, K V; Tikhonenkov, D V; Janouškovec, J; Diakin, A Y; Ofitserov, M V; Mylnikov, A P; Aleshin, V V

    2015-11-01

    Phylogenetic analysis of large subunit ribosomal RNA (LSU rRNA or 28S rRNA) gene sequences from free-living predatory flagellates Colpodella angusta, Voromonas pontica, and Alphamonas edax (Apicomplexa) confirms their close relationship with chromerids Chromera velia and Vitrella brassicaformis, which possess a functional photosynthetic plastid. Together these organisms form a sister group to parasitic apicomplexans (coccidians and gregarines, or sporozoans sensu lato). This result agrees with the previous conclusion on monophyly of colpodellids and chromerids (chrompodellids) based on phylogenomic data. The revealed relationships demonstrate a complex pattern of acquisition, loss, or modification of plastids and transition to parasitism during alveolate evolution.

  16. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  17. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  18. Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.

    PubMed

    Wang, Hye-Young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok; Uh, Young; Lee, Hyeyoung

    2014-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.

  19. A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group.

    PubMed

    Nikolić, Petra; Mehle, Natasa; Gruden, Kristina; Ravnikar, Maja; Dermastia, Marina

    2010-10-01

    We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S-23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of 'Ca. P. mali' or 'Ca. P. pyri'. New panel of assays were tested on the DNA samples of 'Ca. P. mali', 'Ca. P. prunorum', 'Ca. P. pyri', other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

  20. Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR.

    PubMed

    Aparecida de Oliveira, Maria; Abeid Ribeiro, Eliana Guimarães; Morato Bergamini, Alzira Maria; Pereira De Martinis, Elaine Cristina

    2010-02-01

    Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE QPCR SYBR Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

  1. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    PubMed Central

    Kaminska, Katarzyna H.; Purta, Elzbieta; Hansen, Lykke H.; Bujnicki, Janusz M.; Vester, Birte; Long, Katherine S.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity. PMID:20007606

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

    PubMed Central

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

    1996-01-01

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

  3. Subcuticular bacteria associated with two common New Zealand echinoderms: Characterization using 16S rRNA sequence analysis and fluorescence in situ hybridization.

    PubMed

    Lawrence, Scott A; O'Toole, Ronan; Taylor, Michael W; Davy, Simon K

    2010-02-01

    Many echinoderms contain subcuticular bacteria (SCB), symbionts which reside in the lumen between the host's epidermal cells and outer cuticle. This relationship is common, existing in about 60% of echinoderms studied so far, yet the function of SCB remains largely unknown. In this study, phylogenetic analysis was carried out on 16S rRNA sequences obtained from echinoderm-associated bacteria, resulting in the identification of four species of putative SCB. All four bacteria were identified from the holothurian Stichopus mollis, and two of the four were also found in the asteroid Patiriella sp. Two of these bacteria belong to the Alphaproteobacteria, and two to the Gammaproteobacteria. In addition to phylogenetic analysis, fluorescence in situ hybridization (FISH) assays were carried out on Patiriella sp., S. mollis, and the asteroid Astrostole scabra. Results showed that Patiriella sp. and S. mollis contain SCB, in agreement with the phylogenetic analysis, while SCB were not detected in A. scabra. Of the bacteria detected using FISH, more than 80% were recognized as belonging to the Alphaproteobacteria in both host species. However, in S. mollis about 20% of the detected SCB successfully hybridized with the Gammaproteobacteria-specific probe, whereas bacteria belonging to this class were never observed in Patiriella sp. This is only the second study to characterize SCB by molecular means, and is the first to identify SCB in situ using FISH.

  4. Sensitive detection and serovar differentiation of typhoidal and nontyphoidal Salmonella enterica species using 16S rRNA Gene PCR coupled with high-resolution melt analysis.

    PubMed

    Masek, Billie J; Hardick, Justin; Won, Helen; Yang, Samuel; Hsieh, Yu-Hsiang; Rothman, Richard E; Gaydos, Charlotte A

    2014-03-01

    Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that could rapidly detect and identify S. enterica species infections as a cause of sepsis is needed. Nine typhoidal and nontyphoidal S. enterica serovars were used to establish the limit of detection (LOD) of a previously published 16S rRNA gene PCR (16S PCR) in mock whole blood specimens. In addition, 16 typhoidal and nontyphoidal S. enterica serovars were used to evaluate the serovar differentiation capability of 16S PCR coupled with high-resolution melt analysis. The overall LOD of 16S PCR for the nine typhoidal and nontyphoidal S. enterica serovars analyzed was <10 colony-forming units per milliliter (CFU/mL) in mock whole blood specimens, with the lowest and highest LOD at <1 CFU/mL and 9 CFU/mL, respectively. By high-resolution melt analysis, the typhoidal and nontyphoidal S. enterica serovar groups analyzed each generated a unique grouping code, allowing for serovar-level identification. 16S PCR coupled with high-resolution melt analysis could be a useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of S. enterica bloodstream infections.

  5. Development and evaluation of two novel oligonucleotide probes based on 16S rRNA sequence for the identification of Salmonella in foods.

    PubMed

    Lin, C K; Tsen, H Y

    1995-05-01

    DNA sequence in the V3 to V6 region of the 16S rRNA gene of Salmonella enteritidis was determined. By comparison of this sequence with those of Escherichia coli and Proteus vulgaris obtained from GenBank/EMBL database, three oligonucleotides termed as 16S I, 16S II and 16S III were synthesized. Hybridization of these oligonucleotides with 325 Salmonella isolates and some non-Salmonella isolates including the Salmonella closely related species of the family of Enterobacteriaceae showed that 16S II could not be used as a Salmonella specific-probe. 16S I and 16S III hybridized with all the Salmonella isolates tested, the former also hybridizing with Citrobacter spp. and the latter hybridizing with Klebsiella pneumoniae as well as Serratia marcescens. Since enrichment of the target cells in food samples was usually required prior to the DNA hybridization assay, the interference from those non-Salmonella isolates could be prevented by enrichment by culturing in lactose-combined tetrathionate (CTET) broth followed by Gram-negative (GN) broth at 37 degrees C and/or 43 degrees C. Such a culture step could inhibit the growth of Klebsiella spp., Ser. marcescens and/or Citrobacter spp. and allowed the specific detection of Salmonella.

  6. Survival Rates and Risk Factors for Cephalad and L5-S1 Adjacent Segment Degeneration after L5 Floating Lumbar Fusion : A Minimum 2-Year Follow-Up

    PubMed Central

    Lee, Young-Seok; Park, Seung-Won

    2015-01-01

    Objective Although the L5-S1 has distinct structural features in comparison with other lumbar spine segments, not much is known about adjacent segment degeneration (ASD) at the L5-S1 segment. The aim of study was to compare the incidence and character of ASD of the cephalad and L5-S1 segments after L5 floating lumbar fusion. Methods From 2005 to 2010, 115 patients who underwent L5 floating lumber fusion were investigated. The mean follow-up period was 46.1 months. The incidence of radiological and clinical ASD of the cephalad and the L5-S1 segments was compared using survival analysis. Risk factors affecting ASD were analyzed using a log rank test and the Cox proportional hazard model. Results Radiological ASD of the L5-S1 segment had a statistically significant higher survival rate than that of the cephalad segment (p=0.001). However, clinical ASD of the L5-S1 segment was significantly lower survival rates than that of the cephalad segment (p=0.038). Risk factor analysis showed that disc degeneration of the cephalad segment and preoperative spinal stenosis of the L5-S1 segment were risk factors. Conclusion In L5 floating fusion, radiological ASD was more common in the cephalad segment and clinical ASD was more common in the L5-S1 segment. At the L5-S1 segment, the degree of spinal stenosis appears to be the most influential risk factor in ASD incidences, unlike the cephalad segment. PMID:25733991

  7. Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR.

    PubMed

    Orlandi, Palmer A; Carter, Laurenda; Brinker, Anna Marie; da Silva, Alexandre J; Chu, Dan-My; Lampel, Keith A; Monday, Steven R

    2003-08-01

    Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.

  8. Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

    PubMed Central

    Yu, Chang-Ping; Ahuja, Rajiv; Sayler, Gary; Chu, Kung-Hui

    2005-01-01

    A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats. PMID:15746346

  9. Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay.

    PubMed

    Chen, Te-Li; Lee, Yi-Tzu; Kuo, Shu-Chen; Yang, Su-Pen; Fung, Chang-Phone; Lee, Shou-Dong

    2014-09-01

    Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus-A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S-23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those 'between 1 and 3' or 'close to 13TU'. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species.

  10. Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.

    PubMed

    Urdaci, M C; Chakroun, C; Faure, D; Bernardet, J F

    1998-01-01

    A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum.

  11. Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

    PubMed

    Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

    2016-12-06

    The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development.

  12. RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS

    PubMed Central

    Bowers, Holly A.; Tomas, Carmelo; Tengs, Torstein; Kempton, Jason W.; Lewitus, Alan J.; Oldach, David W.

    2010-01-01

    Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. PMID:20411032

  13. Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes.

    PubMed

    Hof, J V; Hernandez, P; Bjerknes, C A; Kraszewska, E K; Lamm, S S

    1987-03-01

    The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.

  14. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

    PubMed Central

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-01-01

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmAII enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmAII, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmAII in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmAII activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmAII, thereby facilitating TEL binding to the ribosome. PMID:26365244

  15. Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

    PubMed

    Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2010-06-01

    Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

  16. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  17. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  18. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  19. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  20. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  1. Molecular Cytogenetics in Digenean Parasites: Linked and Unlinked Major and 5S rDNAs, B Chromosomes and Karyotype Diversification.

    PubMed

    García-Souto, Daniel; Pasantes, Juan J

    2015-01-01

    Digenetic trematodes are the largest group of internal metazoan parasites, but their chromosomes are poorly studied. Although chromosome numbers and/or karyotypes are known for about 300 of the 18,000 described species, molecular cytogenetic knowledge is mostly limited to the mapping of telomeric sequences and/or of major rDNA clusters in 9 species. In this work we mapped major and 5S rDNA clusters and telomeric sequences in chromosomes of Bucephalus minimus, B. australis, Prosorhynchoides carvajali (Bucephaloidea), Monascus filiformis (Gymnophalloidea), Parorchis acanthus (Echinostomatoidea), Cryptocotyle lingua (Opisthorchioidea), Cercaria longicaudata, Monorchis parvus (Monorchioidea), Diphterostomum brusinae, and Bacciger bacciger (Microphalloidea). Whilst single major and minor rDNA clusters were mapped to different chromosome pairs in B. minimus and P. acanthus, overlapping signals were detected on a single chromosome pair in the remaining taxa. FISH experiments using major rDNA and telomeric probes clearly demonstrated the presence of highly stretched NORs in most of the digenean taxa analyzed. B chromosomes were detected in the B. bacciger samples hosted by Ruditapes decussatus. Although the cercariae specimens obtained from Donax trunculus, Tellina tenuis, and R. decussatus were in agreement with B. bacciger, their karyotypes showed striking morphological differences in agreement with the proposed assignation of these cercariae to different species of the genus Bacciger. Results are discussed in comparison with previous data on digenean chromosomes.

  2. Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

    PubMed

    Hansen, M C; Nielsen, A K; Molin, S; Hammer, K; Kilstrup, M

    2001-08-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

  3. 16S rRNA Amplicon Sequencing for Epidemiological Surveys of Bacteria in Wildlife

    PubMed Central

    Razzauti, Maria; Bard, Emilie; Bernard, Maria; Brouat, Carine; Charbonnel, Nathalie; Dehne-Garcia, Alexandre; Loiseau, Anne; Tatard, Caroline; Tamisier, Lucie; Vayssier-Taussat, Muriel; Vignes, Helene

    2016-01-01

    ABSTRACT The human impact on natural habitats is increasing the complexity of human-wildlife interactions and leading to the emergence of infectious diseases worldwide. Highly successful synanthropic wildlife species, such as rodents, will undoubtedly play an increasingly important role in transmitting zoonotic diseases. We investigated the potential for recent developments in 16S rRNA amplicon sequencing to facilitate the multiplexing of the large numbers of samples needed to improve our understanding of the risk of zoonotic disease transmission posed by urban rodents in West Africa. In addition to listing pathogenic bacteria in wild populations, as in other high-throughput sequencing (HTS) studies, our approach can estimate essential parameters for studies of zoonotic risk, such as prevalence and patterns of coinfection within individual hosts. However, the estimation of these parameters requires cleaning of the raw data to mitigate the biases generated by HTS methods. We present here an extensive review of these biases and of their consequences, and we propose a comprehensive trimming strategy for managing these biases. We demonstrated the application of this strategy using 711 commensal rodents, including 208 Mus musculus domesticus, 189 Rattus rattus, 93 Mastomys natalensis, and 221 Mastomys erythroleucus, collected from 24 villages in Senegal. Seven major genera of pathogenic bacteria were detected in their spleens: Borrelia, Bartonella, Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia. Mycoplasma, Ehrlichia, Rickettsia, Streptobacillus, and Orientia have never before been detected in West African rodents. Bacterial prevalence ranged from 0% to 90% of individuals per site, depending on the bacterial taxon, rodent species, and site considered, and 26% of rodents displayed coinfection. The 16S rRNA amplicon sequencing strategy presented here has the advantage over other molecular surveillance tools of dealing with a large spectrum of bacterial

  4. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  5. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences.

    PubMed Central

    Robison-Cox, J F; Bateson, M M; Ward, D M

    1995-01-01

    Detection of chimeric artifacts formed when PCR is used to retrieve naturally occurring small-subunit (SSU) rRNA sequences may rely on demonstrating that different sequence domains have different phylogenetic affiliations. We evaluated the CHECK_CHIMERA method of the Ribosomal Database Project and another method which we developed, both based on determining nearest neighbors of different sequence domains, for their ability to discern artificially generated SSU rRNA chimeras from authentic Ribosomal Database Project sequences. The reliability of both methods decreases when the parental sequences which contribute to chimera formation are more than 82 to 84% similar. Detection is also complicated by the occurrence of authentic SSU rRNA sequences that behave like chimeras. We developed a naive statistical test based on CHECK_CHIMERA output and used it to evaluate previously reported SSU rRNA chimeras. Application of this test also suggests that chimeras might be formed by retrieving SSU rRNAs as cDNA. The amount of uncertainty associated with nearest-neighbor analyses indicates that such tests alone are insufficient and that better methods are needed. PMID:7538272

  6. 23S rRNA gene mutations contributing to macrolide resistance in Campylobacter jejuni and Campylobacter coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Operon specific 23S rRNA mutations affecting minimum inhibitory concentrations (MICs) of macrolides (erythromycin [ERY], azithromycin [AZM], tylosin [TYL]) and a lincosamide (clindamycin [CLI]) were examined in a collection of Campylobacter jejuni and C. coli isolates. The three copies of the Campy...

  7. Molecular Diagnosis of Kingella kingae Pericarditis by Amplification and Sequencing of the 16S rRNA Gene▿

    PubMed Central

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainardi, Jean-Luc

    2007-01-01

    Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing. PMID:17634294

  8. Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene.

    PubMed

    Matta, Matta; Wermert, Delphine; Podglajen, Isabelle; Sanchez, Olivier; Buu-Hoï, Annie; Gutmann, Laurent; Meyer, Guy; Mainard