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Sample records for 12s rrna gene

  1. Mutations in the mitochondrial 12S rRNA gene in elderly Chinese people.

    PubMed

    Zhu, Yuhua; Zhao, Jiandong; Feng, Bo; Su, Yu; Kang, Dongyang; Yuan, Huijun; Zhai, Suoqiang; Dai, Pu

    2015-01-01

    Our data indicate that the mitochondrial 12S rRNA gene, and particularly the A827G mutation, may be associated with susceptibility to age-related hearing loss. Hearing loss associated with aging is common among elderly persons. In all genetic backgrounds, mitochondrial DNA (mtDNA) mutations may be one of the most important factors contributing to aging and age-related hearing loss. The mitochondrial 12S rRNA is a hot spot for deafness-associated mutations in Chinese populations. The purpose of the present study was to elucidate the relationship of 12S rRNA gene polymorphisms and age-related hearing loss. The 12S rRNA gene polymorphisms were detected by direct sequencing. Statistical analyses were performed to assess the associations between age-related hearing loss and 12S rRNA gene variants. We report here a systematic mutational screening of the mitochondrial 12S rRNA gene in 662 elderly subjects from the general population with various hearing threshold levels (211 controls and 451 age-related hearing loss subjects). Mutational screening of the mitochondrial 12S rRNA gene identified 55 nucleotide changes, including 4 mutations localized at highly conserved sites and 51 known variants. Of the known deafness-associated mutations in the mitochondrial 12S rRNA gene, the incidence of the A1555G mutation was 0.15%, A827G was 4.38%, T1095C was 0.45%, and T1005C was 3.78%. The incidence of the other known variants was 0.15-99.85%. We found statistically significant differences in the proportions of subjects with the A827G mutation among the various age-related hearing loss groups and normal controls.

  2. 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.

  3. Identification of goat cashmere and sheep wool by PCR-RFLP analysis of mitochondrial 12S rRNA gene.

    PubMed

    Geng, Rong-Qing; Yuan, Chao; Chen, Yu-Lin

    2012-12-01

    The efficacy of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial 12S rRNA gene in identification of goat cashmere and sheep wool samples was evaluated. The specific fragments of the mitochondrial 12S rRNA gene, which were about 440 bp, were obtained using the PCR. Restriction enzyme digestion of the PCR products with endonucleases BspT I and Hinf I revealed species-specific RFLP patterns. Application of this technique on mixed samples could identify goat cashmere and sheep wool from each other within the proportion of 8:1. The technique, however, could detect only one species when the proportion of mixture was more than 9:1. The PCR-RFLP technique was demonstrated to possess potential value in precise identification of goat cashmere and sheep wool.

  4. [Polymorphism of the 12S rRNA gene and phylogeography of the Central Asian tortoises Agrionemys horsfieldii gray, 1844].

    PubMed

    Vasil'ev, V A; Bondarenko, D A; Peregortsev, E A; Voronov, A S; Ryskov, A P; Semenova, S K

    2008-06-01

    Based on intraspecific polymorphism of 12S rRNA gene, genetic variation of isolated populations of the Central Asian tortoise, Agrionemys horsfieldii, was for the first time investigated on a large part of the species distribution range, encompassing Uzbekistan, southern Kazakhstan, and northern and eastern Iran. In 59 tortoises, four haplotypes were discovered, including two (AH1 and AH2), described earlier. Haplotype AH1 was detected in 52 tortoises, inhabiting southern Kazakhstan and Uzbekistan. Haplotype AH2 was found in four tortoises from the border territory between Uzbekistan, Tajikistan, and Afghanistan. Two novel haplotypes, AH3 and AH4, were detected in the three tortoises from Iran. Based on nucleotide substitutions in the 12S rDNA sequence, the possible divergence time between the tortoises from different parts of the range was estimated. Possible pathways of the formation of modern intraspecific groups of A. horsfieldii are discussed.

  5. [Nuclear gene involves in phenotype of non-syndromic deafness associated with mitochondrial 12S rRNA mutation].

    PubMed

    Zhao, Su Ying; Zhang, Hai Jun; Xu, Chun Hong; Shan, Xiang Nian

    2006-02-01

    The human mitochondrial 12S rRNA gene mutation at position 1555 associated with non-syndromic deafness and aminoglycoside-induced deafness. Family of Huaiyin in Jiangsu is one of the biggest non-syndromic deafness family in the world. In this family, deafness is maternally inherited. After establishing immortal lymphoblastoid cell lines of the family by EB virus, we analysed 17 lymphoblastoid cell lines derived, respectively, from symptomatic, asymptomatic and controll members of the family. Compared with control members, symptomatic and asymptomatic members both exhibited significant decreases in the rate of growth as well as in the rates of mitochondrial protein synthesis. But the extent of decreases is different and the severity of mitochondrial defect is related with its clinical phenotype. These results supported that the nuclear factor involves in the phenotypic manifestation of the non-syndromic deafness associated with the A1555G mutation.

  6. Mutational analysis of the mitochondrial 12S rRNA and tRNA{sup Ser(UCN)} genes in Tunisian patients with nonsyndromic hearing loss

    SciTech Connect

    Mkaouar-Rebai, Emna . E-mail: emna_mkaouar@mail2world.com; Tlili, Abdelaziz; Masmoudi, Saber; Louhichi, Nacim; Charfeddine, Ilhem; Amor, Mohamed Ben; Lahmar, Imed; Driss, Nabil; Drira, Mohamed; Ayadi, Hammadi; Fakhfakh, Faiza

    2006-02-24

    We explored the mitochondrial 12S rRNA and the tRNA{sup Ser(UCN)} genes in 100 Tunisian families affected with NSHL and in 100 control individuals. We identified the mitochondrial A1555G mutation in one out of these 100 families and not in the 100 control individuals. Members of this family harbouring the A1555G mutation showed phenotypic heterogeneity which could be explained by an eventual nuclear-mitochondrial interaction. So, we have screened three nuclear genes: GJB2, GJB3, and GJB6 but we have not found correlation between the phenotypic heterogeneity and variants detected in these genes. We explored also the entire mitochondrial 12S rRNA and the tRNA{sup Ser(UCN)} genes. We detected five novel polymorphisms: T742C, T794A, A813G, C868T, and C954T, and 12 known polymorphisms in the mitochondrial 12S rRNA gene. None of the 100 families or the 100 controls were found to carry mutations in the tRNA{sup Ser(UCN)} gene. We report here First mutational screening of the mitochondrial 12S rRNA and the tRNA{sup Ser(UCN)} genes in the Tunisian population which describes the second family harbouring the A1555G mutation in Africa and reveals novel polymorphisms in the mitochondrial 12S rRNA gene.

  7. New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

    SciTech Connect

    Mkaouar-Rebai, Emna Tlili, Abdelaziz; Masmoudi, Saber; Charfeddine, Ilhem; Fakhfakh, Faiza

    2008-05-09

    The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene.

  8. Further involvement of the mitochondrial 12S rRNA gene in aminoglycoside-induced deafness: A novel type of heteroplasmy

    SciTech Connect

    Bacino, C.; Prezant, T.R.; Bu, X.

    1994-09-01

    Aminoglycoside-induced deafness has been linked recently to a predisposing mutation in the 3{prime} end of the small ribosomal RNA (rRNA) gene of human mitochondria (1555 A{yields}G) that makes the mitochondrial rRNA structurally more similar to its bacterial counterpart. This mutation was found in Chinese families in which the susceptibility to develop ototoxic deafness was inherited through the maternal lineage. However, the 1555 A{yields}G mutation was rarely found in sporadic patients in China, where aminoglycosides are commonly used. To further characterize the mutations predisposing to aminoglycoside ototoxicity, we analyzed the 12S rRNA gene in 35 sporadic patients without the 1555 mutation. Using single stranded conformational polymorphism (SSCP) analysis, heteroduplex (HD) analysis, sequencing, and allele-specific oligonucleotide hybridization, we found that 3 of 35 sporadic patients had unique sequence changes in the 12S rRNA gene. Two of these changes were homoplasmic. One of the patients displayed a novel type of heteroplasmy, which we term multiplasmy, with one base deletion at nt 961 and different populations of mitochondrial DNA with varying numbers of inserted cytosines at that site.

  9. Genetic polymorphism of 12S rRNA gene among Dermacentor reticulatus Fabricius ticks in the Chernobyl Nuclear Power Plant Exclusion Zone.

    PubMed

    Movila, Alexandru; Morozov, Alexandr; Sitnicova, Natalia

    2013-02-01

    The molecular genetic variability of the 12S rRNA gene, on the basis of partial primary sequence and in silico-predicted secondary structures among Dermacentor reticulatus (Fabricius 1794) ticks, was studied in the Chernobyl Nuclear Power Plant (ChNPP) Exclusion Zone. In total, 20, 20, and 25 ethanol-preserved specimens, previously collected at 3 sites with 0.76, 1.91, and 4.5 millisievert (mSv)/hr ionizing radiation background, were examined. The primary sequence analysis generated 4 haplotypes defined by 3 polymorphic sites. The most common haplotype 1 was found in all 3 locations, representing 86.2% of all sampled individuals. Haplotype 4 (10.8%) was detected at the 1.91 and 4.50 mSv/hr sites. The unique haplotypes 2 (1.5%) and 3 (1.5%) were detected only at the 1.91 and 4.50 mSv/hr sites, respectively. The haplotype diversity, nucleotide diversity, and pairwise nucleotide differences for 2 tick populations at the 1.90 and 4.50 mSv/hr sites were 0.279, 0.00085, and 0.289, and 0.397, 0.00122, 0.413, respectively. No polymorphism was detected in ticks collected at the 0.76 mSv/hr site. The primary sequences of 12S rRNA were folded into the secondary structures and the free energy of haplotypes was calculated. The free energy at 37 C (ΔG) of the nonmutant haplotype 1 and the mutant haplotypes 2, 3, and 4 were -45.79, -44.17, -39.56, and -45.79 kcal/mol, respectively. Considering the correlation between the structural profile similarity of 12S rRNA and point mutations, haplotypes 1 and 4 have similar secondary structure profiles and have received a 0.999219 similarity score in the cluster tree. The unique haplotypes 2 and 3 have differences in the secondary structure in comparison with haplotypes 1 and 4; the similarity scores were 0.914747 and 0.169431, respectively. Further studies using more genetic markers are warranted to ascertain the genetic variability and population genetic structure within D. reticulatus tick populations in the ChNPP Exclusion Zone and to

  10. Phylogenetic reconstruction of the wolf spiders (Araneae: Lycosidae) using sequences from the 12S rRNA, 28S rRNA, and NADH1 genes: implications for classification, biogeography, and the evolution of web building behavior.

    PubMed

    Murphy, Nicholas P; Framenau, Volker W; Donnellan, Stephen C; Harvey, Mark S; Park, Yung-Chul; Austin, Andrew D

    2006-03-01

    Current knowledge of the evolutionary relationships amongst the wolf spiders (Araneae: Lycosidae) is based on assessment of morphological similarity or phylogenetic analysis of a small number of taxa. In order to enhance the current understanding of lycosid relationships, phylogenies of 70 lycosid species were reconstructed by parsimony and Bayesian methods using three molecular markers; the mitochondrial genes 12S rRNA, NADH1, and the nuclear gene 28S rRNA. The resultant trees from the mitochondrial markers were used to assess the current taxonomic status of the Lycosidae and to assess the evolutionary history of sheet-web construction in the group. The results suggest that a number of genera are not monophyletic, including Lycosa, Arctosa, Alopecosa, and Artoria. At the subfamilial level, the status of Pardosinae needs to be re-assessed, and the position of a number of genera within their respective subfamilies is in doubt (e.g., Hippasa and Arctosa in Lycosinae and Xerolycosa, Aulonia and Hygrolycosa in Venoniinae). In addition, a major clade of strictly Australasian taxa may require the creation of a new subfamily. The analysis of sheet-web building in Lycosidae revealed that the interpretation of this trait as an ancestral state relies on two factors: (1) an asymmetrical model favoring the loss of sheet-webs and (2) that the suspended silken tube of Pirata is directly descended from sheet-web building. Paralogous copies of the nuclear 28S rRNA gene were sequenced, confounding the interpretation of the phylogenetic analysis and suggesting that a cautionary approach should be taken to the further use of this gene for lycosid phylogenetic analysis.

  11. Correspondence regarding Ballana et al., "Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment".

    PubMed

    Abreu-Silva, R S; Batissoco, A C; Lezirovitz, K; Romanos, J; Rincon, D; Auricchio, M T B M; Otto, P A; Mingroni-Netto, R C

    2006-05-12

    Ballana et al. [E. Ballana, E. Morales, R. Rabionet, B. Montserrat, M. Ventayol, O. Bravo, P. Gasparini, X. Estivill, Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment, Biochem. Biophys. Res. Commun. 341 (2006) 950-957] detected a T1291C mutation segregating in a Cuban pedigree with hearing impairment. They interpreted it as probably pathogenic, based on family history, RNA conformation prediction and its absence in a control group of 95 Spanish subjects. We screened a sample of 203 deaf subjects and 300 hearing controls (110 "European-Brazilians" and 190 "African-Brazilians") for the mitochondrial mutations A1555G and T1291C. Five deaf subjects had the T1291C substitution, three isolated cases and two familial cases. In the latter, deafness was paternally inherited or segregated with the A1555G mutation. This doesn't support the hypothesis of T1291C mutation being pathogenic. Two "African-Brazilian" controls also had the T1291C substitution. Six of the seven T1291C-carriers (five deaf and two controls) had mitochondrial DNA of African origin, belonging to macrohaplogroup L1/L2. Therefore, these data point to T1291C substitution as most probably an African non-pathogenic polymorphism.

  12. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    SciTech Connect

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin . E-mail: min-xin.guan@chmcc.org

    2006-04-21

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.

  13. Maternally Inherited Aminoglycoside-Induced and Nonsyndromic Deafness Is Associated with the Novel C1494T Mutation in the Mitochondrial 12S rRNA Gene in a Large Chinese Family

    PubMed Central

    Zhao, Hui; Li, Ronghua; Wang, Qiuju; Yan, Qingfeng; Deng, Jian-Hong; Han, Dongyi; Bai, Yidong; Young, Wie-Yen; Guan, Min-Xin

    2004-01-01

    We report here the characterization of a large Chinese family with maternally transmitted aminoglycoside-induced and nonsyndromic deafness. In the absence of aminoglycosides, some matrilineal relatives in this family exhibited late-onset/progressive deafness, with a wide range of severity and age at onset. Notably, the average age at onset of deafness has changed from 55 years (generation II) to 10 years (generation IV). Clinical data reveal that the administration of aminoglycosides can induce or worsen deafness in matrilineal relatives. The age at the time of drug administration appears to be correlated with the severity of hearing loss experienced by affected individuals. Sequence analysis of mitochondrial DNA in this pedigree identified a homoplasmic C-to-T transition at position 1494 (C1494T) in the 12S rRNA gene. The C1494T mutation is expected to form a novel U1494-1555A base pair, which is in the same position as the C1494-1555G pair created by the deafness-associated A1555G mutation, at the highly conserved A site of 12S rRNA. Exposure to a high concentration of paromomycin or neomycin caused a variable but significant average increase in doubling time in lymphoblastoid cell lines derived from four symptomatic and two asymptomatic individuals in this family carrying the C1494T mutation when compared to four control cell lines. Furthermore, a significant decrease in the rate of total oxygen consumption was observed in the mutant cell lines. Thus, our data strongly support the idea that the A site of mitochondrial 12S rRNA is the primary target for aminoglycoside-induced deafness. These results also strongly suggest that the nuclear background plays a role in the aminoglycoside ototoxicity and in the development of the deafness phenotype associated with the C1494T mutation in the mitochondrial 12S rRNA gene. PMID:14681830

  14. Relationships between parasitoid wasps (Hymenoptera: Braconidae: Opiinae), fruit flies (Diptera: Tephritidae) and their host plants based on 16S rRNA, 12S rRNA, and ND1 gene sequences

    NASA Astrophysics Data System (ADS)

    Ibrahim, N. J.; Md-Zain, B. M.; Yaakop, S.

    2013-11-01

    Opiinae is among the l0 largest subfamilies under the family Braconidae. Opiines species have great potential as natural enemies against fruit fly pests. Before using them as a biological control agent, construction of the phylogenetic trees could facilitate in the molecular identification of individual species and their relationships among members of the Opiines, as well as between Opiines and their host plants. Larval specimens of tephritids were collected from four crop species at five localities throughout the Peninsular Malaysia. A total of 44 specimens of opiines had successfully emerged from the hosts, fruit fly larvae. The DNA sequences of 12S and 16S rRNA were obtained for the braconids while the mitochondrial ND1 sequences were obtained for the tephritids species through polymerase chain reaction. Maximum Parsimony and Bayesian trees were constructed by using PAUP 4.0b10 and MrBayes 3.1.2 to identify the relationships among the taxa. This study illustrates the phylogenetic relationships among parasitoid opiines collected and reared from parasitized fruit flies. The phylogenetic trees constructed based on the mitochondrial 12S and 16S rRNA sequences exhibited similar topology and sequence divergence. The opiines were divided into several clades and subclades according to the genus and species. Each clade also was supported by the similar host plants with high support values. However, their pests were not specific, except for Bactrocera cucurbitae. This study has reconfirmed the associations between Opiinae, tephritids, and host plants based on molecular data.

  15. Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene.

    PubMed

    Fajardo, V; González, I; López-Calleja, I; Martín, I; Rojas, M; Hernández, P E; García, T; Martín, Rosario

    2007-06-01

    Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.

  16. [Investigation into the relationship between mitochondrial 12 S rRNA gene, tRNA gene and cytochrome oxidase Ⅱ gene variations and the risk of noise-induced hearing loss].

    PubMed

    Jiao, J; Gu, G Z; Chen, G S; Li, Y H; Zhang, H L; Yang, Q Y; Xu, X R; Zhou, W H; Wu, H; He, L H; Zheng, Y X; Yu, S F

    2017-01-06

    Objective: To explore the relationship between mitochondrial 12 S rRNA gene variation, tRNA gene variation and cytochrome oxidase Ⅱ gene point mutations and the risk of noise-induced hearing loss (NIHL). Methods: A nested case-control study was performed that followed a cohort of 7 445 noise-exposed workers in a steel factory in Henan province, China, from January 1, 2006 to December 31, 2015. Subjects whose average hearing threshold was more than 40 dB(A) in high frequency were defined as the case group, and subjects whose average hearing threshold was less than 35 dB(A) in high frequency and less than 25 dB (A) in speech frequency were defined as the control group. Subjects was recruited into the case group (n=286) and the control group (n=286) according to gender, age, job category and time of exposure to noise, and a 1∶1 case-control study was carried out. We genotyped eight single nucleotide polymorphisms in the mitochondrial 12 S rRNA gene, the mitochondrial tRNA gene and the mitochondrial cytochrome oxidase Ⅱ gene using SNPscan high-throughput genotyping technology from the recruited subjects. The relationship between polymorphic sites and NIHL, adjusted for covariates, was analyzed using conditional logistic regression analysis, as were the subgroup data. Results: The average age of the recruited subjects was (40.3±8.1) years and the length of service exposure to noise was (18.6±8.9) years. The range of noise exposed levels and cumulative noise exposure (CNE) was 80.1- 93.4 dB (A) and 86.8- 107.9 dB (A) · year, respectively. For workers exposed to noise at a CNE level<98 dB (A) · year, smokers showed an increased risk of NIHL of 1.88 (1.16-3.05) compared with non-smokers; for workers exposed to noise at a CNE level ≥98 dB(A) · year, smokers showed an increased risk of NIHL of 2.53 (1.49- 4.30) compared with non-smokers. For workers exposed to noise at a CNE level<98 dB (A) · year, the results of univariate analysis and multifactor analysis

  17. 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.

  18. 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

  19. 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

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

    2006-10-01

    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

  20. 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…

  1. 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…

  2. The A1555G mutation in the 12S rRNA gene of human mtDNA: recurrent origins and founder events in families affected by sensorineural deafness.

    PubMed

    Torroni, A; Cruciani, F; Rengo, C; Sellitto, D; López-Bigas, N; Rabionet, R; Govea, N; López De Munain, A; Sarduy, M; Romero, L; Villamar, M; del Castillo, I; Moreno, F; Estivill, X; Scozzari, R

    1999-11-01

    The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to >/=30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments.

  3. The A1555G Mutation in the 12S rRNA Gene of Human mtDNA: Recurrent Origins and Founder Events in Families Affected by Sensorineural Deafness

    PubMed Central

    Torroni, Antonio; Cruciani, Fulvio; Rengo, Chiara; Sellitto, Daniele; López-Bigas, Núria; Rabionet, Raquel; Govea, Nancy; López de Munain, Adolfo; Sarduy, Maritza; Romero, Lourdes; Villamar, Manuela; del Castillo, Ignacio; Moreno, Felipe; Estivill, Xavier; Scozzari, Rosaria

    1999-01-01

    Summary The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to ⩾30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments. PMID:10521300

  4. Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

    SciTech Connect

    Xing Guangqian; Chen Zhibin; Wei Qinjun; Tian Huiqin; Li Xiaolu; Zhou Aidong; Bu Xingkuan; Cao Xin . E-mail: caoxin@njmu.edu.cn

    2006-08-11

    We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA{sup Ser(UCN)} genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families.

  5. Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss

    PubMed Central

    Lu, Jianxin; Li, Zhiyuan; Zhu, Yi; Yang, Aifen; Li, Ronghua; Zheng, Jing; Cai, Qin; Peng, Guanghua; Zheng, Wuwei; Tang, Xiaowen; Chen, Bobei; Chen, Jianfu; Liao, Zhisu; Yang, Li; Li, Yongyan; You, Junyan; Ding, Yu; Yu, Hong; Wang, Jindan; Sun, Dongmei; Zhao, Jianyue; Xue, Ling; Wang, Jieying; Guan, Min-Xin

    2010-01-01

    In this report, we investigated the frequency and spectrum of mitochondrial 12S rRNA variants in a large cohort of 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mutational analysis of 12S rRNA gene in these subjects identified 68 (54 known and 14 novel) variants. The frequencies of known 1555A>G and 1494C>T mutations were 3.96% and 0.18%, respectively, in this cohort with nonsyndromic and aminoglycoside-induced hearing loss. Prevalence of other putative deafness-associated mutation at positions 1095 and 961 were 0.61% and 1.7% in this cohort, respectively. Furthermore, the 745A>G, 792C>T, 801A>G, 839A>G, 856A>G, 1027A>G, 1192C>T, 1192C>A, 1310C>T, 1331A>G, 1374A>G and 1452T>C variants conferred increased sensitivity to ototoxic drugs or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants appeared to be polymorphisms. Moreover, 65 Chinese subjects carrying the 1555A>G mutation exhibited bilateral and sensorineural hearing loss. A wide range of severity, age-of-onset and audiometric configuration was observed among these subjects. In particular, the sloping and flat shaped patterns were the common audiograms in individuals carrying the 1555A>G mutation. The phenotypic variability in subjects carrying these 12S rRNA mutations indicated the involvement of nuclear modifier genes, mitochondrial haplotypes, epigenetic and environmental factors in the phenotypic manifestation of these mutations. Therefore, our data demonstrated that mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity. PMID:20100600

  6. 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.

  7. Chicken rRNA Gene Cluster Structure.

    PubMed

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

    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.

  8. 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

  9. Large differences in substitutional pattern and evolutionary rate of 12S ribosomal RNA genes.

    PubMed

    Simon, C; Nigro, L; Sullivan, J; Holsinger, K; Martin, A; Grapputo, A; Franke, A; McIntosh, C

    1996-09-01

    We demonstrate using Drosophila, periodical cicadas, and hominid primates, that the molecular clock based on animal mitochondrial small-subunit (12S) rRNA genes ticks at significantly different relative rates depending on which taxa and which region of the gene are examined. Drosophila, which are commonly used as model taxa, are evolving in a highly peculiar manner with the majority of sites in the 3' half of the 12S gene apparently invariant. The analogous 3' half of the mitochondrial large-subunit rRNA gene (16S) appears to be similarly constrained. It is surprising that these regions that are already highly constrained in all animals should be even more constrained in Drosophila, especially when the Drosophila mitochondrial genome as a whole does not display a similar rate slowdown. This extreme 12S rate slowdown is not apparent in periodical cicadas or hominid primates and appears to be related to strong structural and functional constraints rather than a depressed mutation rate. Finally, the slow average rate of evolution in the third domain of Drosophila does not imply that the few variable sites lack multiple hits.

  10. 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

  11. Molecular phylogeny of mitochondrial cytochrome b and 12S rRNA sequences in the Felidae: ocelot and domestic cat lineages.

    PubMed

    Masuda, R; Lopez, J V; Slattery, J P; Yuhki, N; O'Brien, S J

    1996-12-01

    Molecular phylogeny of the cat family Felidae is derived using two mitochondrial genes, cytochrome b and 12S rRNA. Phylogenetic methods of weighted maximum parsimony and minimum evolution estimated by neighbor-joining are employed to reconstruct topologies among 20 extant felid species. Sequence analyses of 363 bp of cytochrome b and 376 bp of the 12S rRNA genes yielded average pair-wise similarity values between felids ranging from 94 to 99% and from 85 to 99%, respectively. Phylogenetic reconstruction supports more recent, intralineage associations but fails to completely resolve interlineage relationships. Both genes produce a monophyletic group of Felis species but vary in the placement of the pallas cat. The ocelot lineage represents an early divergence within the Felidae, with strong associations between ocelot and margay, Geoffroy's cat and kodkod, and pampas cat and tigrina. Implications of the relative recency of felid evolution, presence of ancestral polymorphisms, and influence of outgroups in placement of the topological root are discussed.

  12. Interordinal mammalian relationships: evidence for paenungulate monophyly is provided by complete mitochondrial 12S rRNA sequences.

    PubMed

    Lavergne, A; Douzery, E; Stichler, T; Catzeflis, F M; Springer, M S

    1996-10-01

    The complete mitochondrial 12S rRNA sequences of 5 placental mammals belonging to the 3 orders Sirenia, Proboscidea, and Hyracoidea are reported together with phylogenetic analyses (distance and parsimony) of a total of 51 mammalian orthologues. This 12S rRNA database now includes the 2 extant proboscideans (the African and Asiatic elephants Loxodonta africana and Elephas maximus), 2 of the 3 extant sirenian genera (the sea cow Dugong dugon and the West Indian manatee Trichechus manatus), and 2 of the 3 extant hyracoid genera (the rock and tree hyraxes Procavia capensis and Dendrohyrax dorsalis). The monophyly of the 3 orders Sirenia, Proboscidea, and Hyracoidea is supported by all kinds of analysis. There are 23 and 3 diagnostic subsitutions shared by the 2 proboscideans and the 2 hyracoids, respectively, but none by the 2 sirenians. The 2 proboscideans exhibit the fastest rates of 12S rRNA evolution among the 11 placental orders studied. Based on various taxonomic sampling methods among eutherian orders and marsupial outgroups, the most strongly supported clade in our comparisons clusters together the 3 orders Sirenia, Proboscidea, and Hyracoidea in the superorder Paenungulata. Within paenungulates, the grouping of sirenians and proboscideans within the mirorder Tethytheria is observed. This branching pattern is supported by all analyses by high bootstrap percentages (BPs) and decay indices. When only one species is selected per order or suborder, the taxonomic sampling leads to a relative variation in bootstrap support of 53% for Tethytheria (BPs ranging from 44 to 93%) and 7% for Paernungulata (92-99%). When each order or suborder is represented by two species, this relative variation decreased to 10% for Tethytheria (78-87%) and 3% for Paenungulata (96-99%). Two nearly exclusive synapomorphies for paenungulates are identified in the form of one transitional compensatory change, but none were detected for tethytherians. Such a robust and reliable resolution of

  13. High-resolution melting of 12S rRNA and cytochrome b DNA sequences for discrimination of species within distinct European animal families.

    PubMed

    Naue, Jana; Hansmann, Tobias; Schmidt, Ulrike

    2014-01-01

    The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM) of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible "modular" manner depending on the question to be solved.

  14. Mitochondrial tRNAArg T10454C variant may not influence the clinical expression of deafness associated 12S rRNA A1555G mutation.

    PubMed

    Luo, Zhiyi

    2016-01-01

    In this study, we examined the "pathogenic" role of the T10454C mutation in mitochondrial tRNA(Arg) gene in deafness expression as increasing reports provided an active role of this mutation in clinical manifestation of deafness associated 12S rRNA A1555G mutation. For this purpose, we reanalyzed the complete mitochondrial DNA (mtDNA) sequence data containing the T10454C mutation. Moreover, we analyzed the reported "polymorphisms" of mtDNA in the proband using the phylogentic approach. To our surprise, other mutations which occurred at protein-coding genes played more important roles in resulting mitochondrial dysfunctions by using the bioinformatic tool. In addition, evolutionary conservation analysis of the T10454C mutation indicated that this mutation was not conserved between different species. To our knowledge, this is the first report that the T10454C variant may not modulate the phenotypic expression of the deafness associated A1555G mutation.

  15. Mitochondrial m.1584A 12S m62A rRNA methylation in families with m.1555A>G associated hearing loss

    PubMed Central

    O'Sullivan, Mary; Rutland, Paul; Lucas, Deirdre; Ashton, Emma; Hendricks, Sebastian; Rahman, Shamima; Bitner-Glindzicz, Maria

    2015-01-01

    The mitochondrial DNA mutation m.1555A>G predisposes to hearing loss following aminoglycoside antibiotic exposure in an idiosyncratic dose-independent manner. However, it may also cause maternally inherited hearing loss in the absence of aminoglycoside exposure or any other clinical features (non-syndromic hearing loss). Although m.1555A>G was identified as a cause of deafness more than twenty years ago, the pathogenic mechanism of this mutation of ribosomal RNA remains controversial. Different mechanistic concepts have been proposed. Most recently, evidence from cell lines and animal models suggested that patients with m.1555A>G may have more 12S rRNA N6, N6–dimethyladenosine (m62A) methylation than controls, so-called ‘hypermethylation’. This has been implicated as a pathogenic mechanism of mitochondrial dysfunction but has yet to be validated in patients. 12S m62A rRNA methylation, by the mitochondrial transcription factor 1 (TFB1M) enzyme, occurs at two successive nucleotides (m.1584A and m.1583A) in close proximity to m.1555A>G. We examined m62A methylation in 14 patients with m.1555A>G, and controls, and found all detectable 12S rRNA transcripts to be methylated in both groups. Moreover, different RNA samples derived from the same patient (lymphocyte, fibroblast and lymphoblast) revealed that only transformed cells contained some unmethylated 12S rRNA transcripts, with all detectable 12S rRNA transcripts derived from primary samples m62A-methylated. Our data indicate that TFB1M 12S m62A rRNA hypermethylation is unlikely to be a pathogenic mechanism and may be an artefact of previous experimental models studied. We propose that RNA methylation studies in experimental models should be validated in primary clinical samples to ensure that they are applicable to the human situation. PMID:25305075

  16. The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential

    PubMed Central

    Smith, Paul M.; Elson, Joanna L.; Greaves, Laura C.; Wortmann, Saskia B.; Rodenburg, Richard J.T.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

    2014-01-01

    Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool. PMID:24092330

  17. Mitochondrial haplotypes may modulate the phenotypic manifestation of the deafness-associated 12S rRNA 1555A>G mutation.

    PubMed

    Lu, Jianxin; Qian, Yaping; Li, Zhiyuan; Yang, Aifen; Zhu, Yi; Li, Ronghua; Yang, Li; Tang, Xiaowen; Chen, Bobei; Ding, Yu; Li, Yongyan; You, Junyan; Zheng, Jing; Tao, Zhihua; Zhao, Fuxin; Wang, Jindan; Sun, Dongmei; Zhao, Jianyue; Meng, Yanzi; Guan, Min-Xin

    2010-01-01

    Mitochondrial 12S rRNA 1555A>G mutation is one of the important causes of aminoglycoside-induced and nonsyndromic deafness. Our previous investigations showed that the A1555G mutation was a primary factor underlying the development of deafness but was insufficient to produce deafness phenotype. However, it has been proposed that mitochondrial haplotypes modulate the phenotypic manifestation of the 1555A>G mutation. Here, we performed systematic and extended mutational screening of 12S rRNA gene in a cohort of 1742 hearing-impaired Han Chinese pediatric subjects from Zhejiang Province, China. Among these, 69 subjects with aminoglycoside-induced and nonsyndromic deafness harbored the homoplasmic 1555A>G mutation. These translated to a frequency of approximately 3.96% for the 1555A>G mutation in this hearing-impaired population. Clinical and genetic characterizations of 69 Chinese families carrying the 1555A>G mutation exhibited a wide range of penetrance and expressivity of hearing impairment. The average penetrances of deafness were 29.5% and 17.6%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Furthermore, the average age-of-onset for deafness without aminoglycoside exposure ranged from 5 and 30years old, with the average of 14.5years. Their mitochondrial genomes exhibited distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups A, B, C, D, F, G, M, N, R and Y, respectively. These indicated that the 1555A>G mutation occurred through recurrent origins and founder events. The haplogroup D accounted for 40.6% of the patient's mtDNA samples but only 25.8% of the Chinese control mtDNA samples. Strikingly, these Chinese families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss. In addition, the mitochondrial haplogroup specific variants: 15927G>A of haplogroup B5b, 12338T>C of haplogroup F2, 7444G>A of haplogroup B4, 5802T>C, 10454T>C, 12224C>T and 11696G>A of D4

  18. Variable rRNA gene copies in extreme halobacteria

    SciTech Connect

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

    1988-08-25

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

  19. Prevalence of the A1555G (12S rRNA) and tRNASer(UCN) mitochondrial mutations in hearing-impaired Brazilian patients.

    PubMed

    Abreu-Silva, R S; Lezirovitz, K; Braga, M C C; Spinelli, M; Pirana, S; Della-Rosa, V A; Otto, P A; Mingroni-Netto, R C

    2006-02-01

    Mitochondrial mutations are responsible for at least 1% of the cases of hereditary deafness, but the contribution of each mutation has not yet been defined in African-derived or native American genetic backgrounds. A total of 203 unselected hearing-impaired patients were screened for the presence of the mitochondrial mutation A1555G in the 12S rRNA gene and mutations in the tRNASer(UCN) gene in order to assess their frequency in the ethnically admixed Brazilian population. We found four individuals with A1555G mutation (2%), which is a frequency similar to those reported for European-derived populations in unselected samples. On the other hand, complete sequencing of the tRNASer(UCN) did not reveal reported pathogenic substitutions, namely A7445G, 7472insC, T7510C, or T7511C. Instead, other rare substitutions were found such as T1291C, A7569G, and G7444A. To evaluate the significance of these findings, 110 "European-Brazilians" and 190 "African-Brazilians" unrelated hearing controls were screened. The T1291C, A7569G and G7444A substitutions were each found in about 1% (2/190) of individuals of African ancestry, suggesting that they are probably polymorphic. Our results indicate that screening for the A1555G mutation is recommended among all Brazilian deaf patients, while testing for mutations in the tRNASer(UCN) gene should be considered only when other frequent deafness-causing mutations have been excluded or in the presence of a maternal transmission pattern.

  20. 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

  1. Molecular phylogenetic inference of the woolly mammoth Mammuthus primigenius, based on complete sequences of mitochondrial cytochrome b and 12S ribosomal RNA genes.

    PubMed

    Noro, M; Masuda, R; Dubrovo, I A; Yoshida, M C; Kato, M

    1998-03-01

    Complete sequences of cytochrome b (1,137 bases) and 12S ribosomal RNA (961 bases) genes in mitochondrial DNA were successfully determined from the woolly mammoth (Mammuthus primigenius), African elephant (Loxodonta africana), and Asian elephant (Elephas maximus). From these sequence data, phylogenetic relationships among three genera were examined. Molecular phylogenetic trees reconstructed by the neighbor-joining and the maximum parsimony methods provided an identical topology both for cytochrome b and 12S rRNA genes. These results support the "Mammuthus-Loxodonta" clade, which is contrary to some previous morphological reports that Mammuthus is more closely related to Elephas than to Loxodonta.

  2. Reconstructing 16S rRNA genes in metagenomic data.

    PubMed

    Yuan, Cheng; Lei, Jikai; Cole, James; Sun, Yanni

    2015-06-15

    Metagenomic data, which contains sequenced DNA reads of uncultured microbial species from environmental samples, provide a unique opportunity to thoroughly analyze microbial species that have never been identified before. Reconstructing 16S ribosomal RNA, a phylogenetic marker gene, is usually required to analyze the composition of the metagenomic data. However, massive volume of dataset, high sequence similarity between related species, skewed microbial abundance and lack of reference genes make 16S rRNA reconstruction difficult. Generic de novo assembly tools are not optimized for assembling 16S rRNA genes. In this work, we introduce a targeted rRNA assembly tool, REAGO (REconstruct 16S ribosomal RNA Genes from metagenOmic data). It addresses the above challenges by combining secondary structure-aware homology search, zproperties of rRNA genes and de novo assembly. Our experimental results show that our tool can correctly recover more rRNA genes than several popular generic metagenomic assembly tools and specially designed rRNA construction tools. The source code of REAGO is freely available at https://github.com/chengyuan/reago. © The Author 2015. Published by Oxford University Press.

  3. Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA{sup Ser(UCN)} G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

    SciTech Connect

    Yuan Huijun; Chen Jing; Liu Xin; Cheng Jing; Wang Xinjian; Yang Li; Yang Shuzhi; Cao Juyang; Kang Dongyang; Dai Pu; Zha, Suoqiang; Han Dongyi Young Wieyen Guan Minxin

    2007-10-12

    Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA{sup Ser(UCN)} G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA{sup Ser(UCN)} G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.

  4. Extremely low penetrance of deafness associated with the mitochondrial 12S rRNA mutation in 16 Chinese families: Implication for early detection and prevention of deafness

    SciTech Connect

    Dai Pu; Liu Xin; Han Dongyi . E-mail: hdy301@263.net; Qian Yaping; Huang Deliang; Yuan Huijun; Li Weiming; Yu Fei; Zhang Ruining; Lin Hongyan; He Yong; Yu Youjun; Sun Quanzhu; Qin Huaiyi; Li Ronghua; Zhang Xin; Kang Dongyang; Cao Juyang; Young Wieyen . E-mail: ywy301@163.net; Guan Minxin |. E-mail: min-xin.guan@cchmc.org

    2006-02-03

    Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of 16 Chinese pedigrees (a total of 246 matrilineal relatives) with aminoglycoside-induced impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: being bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss, ranging from 4% to 18%, with an average of 8%. In particular, nineteen of 246 matrilineal relatives in these pedigrees had aminoglycoside-induced hearing loss. Mutational analysis of the mtDNA in these pedigrees showed the presence of homoplasmic 12S rRNA A1555G mutation, which has been associated with hearing impairment in many families worldwide. The extremely low penetrance of hearing loss in these Chinese families carrying the A1555G mutation strongly supports the notion that the A1555G mutation itself is not sufficient to produce the clinical phenotype. Children carrying the A1555G mutation are susceptible to the exposure of aminoglycosides, thereby inducing or worsening hearing impairment, as in the case of these Chinese families. Using those genetic and molecular approaches, we are able to diagnose whether children carry the ototoxic mtDNA mutation. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside therapy, and eventually to decrease the incidence of deafness.

  5. Regulation of Arabidopsis thaliana 5S rRNA Genes.

    PubMed

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

    2007-05-01

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

  6. Mitochondrial COX2 G7598A mutation may have a modifying role in the phenotypic manifestation of aminoglycoside antibiotic-induced deafness associated with 12S rRNA A1555G mutation in a Han Chinese pedigree.

    PubMed

    Chen, Tianbin; Liu, Qicai; Jiang, Ling; Liu, Can; Ou, Qishui

    2013-02-01

    Recent studies suggest that certain mitochondrial haplogroup markers and some specific variants in mitochondrial haplogroup may also influence the phenotypic expression of particular mitochondrial disorders. In this report, the clinical, genetic, and molecular characterization were identified in a Chinese pedigree with the aminoglycoside antibiotic (AmAn)-induced deafness and nonsyndromic hearing loss (NSHL). The pathogenic gene responsible for this hereditary NSHL pedigree was determined by Microarray chip, which possessed the nine NSHL hot-spot mutations, including GJB2 (35delG, 176dell6bp, 235de1C, and 299delAT), GJB3 (538C>T), SLC26A4 (IVS7-2A>G and 2168A>G), and mitochondrial DNA (mtDNA) 12S rRNA (C1494T and A1555G). Only the homoplasmic A1555G mutation was detected, which was confirmed by direct sequencing. Also, real-time amplification refractory mutation system quantitative polymerase chain reaction methodology was performed to calculate the A1555G mutation load. The proband's complete mtDNA genome were amplified and direct sequencing was performed to determine the mitochondrial haplogroup and private mutations. The proband's mitochondrial haplogroup belonges to M7b1 and a private mutation MTCOX2 G7598A (p.Ala 5 Thr) is found. Phylogenetic analysis of COX2 polypeptide sequences demonstrates that the alanine residue is relatively conserved, but owing to the missense mutation (p.Ala 5 Thr), its side chain hydrophobicity will be changed, and what is more, as it is adjacent to a glutamine residue, which is highly conserved and hydrophilic, in an evolutionary stable domain; G7598A (p.Ala 5 Thr) may alter the protein secondary structure and physiological function of COX2 and, thus, aggravate the mitochondrial dysfunction conferred by the A1555G mutation. Furthermore, the G7598A mutation is absent in 100 unrelated healthy controls; therefore, G7598A (p.Ala 5 Thr) in the mitochondrial haplogoup M7b1 may have a modifying role, enhancing its penetrance and severity

  7. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    USDA-ARS?s Scientific Manuscript database

    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...

  8. The coexistence of mitochondrial ND6 T14484C and 12S rRNA A1555G mutations in a Chinese family with Leber's hereditary optic neuropathy and hearing loss

    SciTech Connect

    Wei Qiping; Zhou Xiangtian; Yang Li; Sun Yanhong; Zhou Jian; Li Guang; Jiang, Robert; Lu Fan; Qu Jia . E-mail: jqu@wzmc.net; Guan Minxin . E-mail: min-xin.guan@cchmc.org

    2007-06-15

    We report here the clinical, genetic and molecular characterization of one three-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON) and hearing loss. Four of 14 matrilineal relatives exhibited the moderate central vision loss at the average age of 12.5 years. Of these, one subject exhibited both LHON and mild hearing impairment. Sequence analysis of the complete mitochondrial genomes in the pedigree showed the presence of homoplasmic LHON-associated ND6 T14484C mutation, deafness-associated 12S rRNA A1555 mutation and 47 other variants belonging to Eastern Asian haplogroup H2. None of other mitochondrial variants was evolutionarily conserved and functional significance. Therefore, the coexistence of the A1555G mutation and T14484C mutations in this Chinese family indicate that the A1555G mutation may play a synergistic role in the phenotypic manifestation of LHON associated ND6 T14484C mutation. However, the incomplete penetrance of vision and hearing loss suggests the involvement of nuclear modifier genes and environmental factors in the phenotypic expression of these mtDNA mutations.

  9. Biochemical Evidence for a Nuclear Modifier Allele (A10S) in TRMU (Methylaminomethyl-2-thiouridylate-methyltransferase) Related to Mitochondrial tRNA Modification in the Phenotypic Manifestation of Deafness-associated 12S rRNA Mutation.

    PubMed

    Meng, Feilong; Cang, Xiaohui; Peng, Yanyan; Li, Ronghua; Zhang, Zhengyue; Li, Fushan; Fan, Qingqing; Guan, Anna S; Fischel-Ghosian, Nathan; Zhao, Xiaoli; Guan, Min-Xin

    2017-02-17

    Nuclear modifier gene(s) was proposed to modulate the phenotypic expression of mitochondrial DNA mutation(s). Our previous investigations revealed that a nuclear modifier allele (A10S) in TRMU (methylaminomethyl-2-thiouridylate-methyltransferase) related to tRNA modification interacts with 12S rRNA 1555A→G mutation to cause deafness. The A10S mutation resided at a highly conserved residue of the N-terminal sequence. It was hypothesized that the A10S mutation altered the structure and function of TRMU, thereby causing mitochondrial dysfunction. Using molecular dynamics simulations, we showed that the A10S mutation introduced the Ser(10) dynamic electrostatic interaction with the Lys(106) residue of helix 4 within the catalytic domain of TRMU. The Western blotting analysis displayed the reduced levels of TRMU in mutant cells carrying the A10S mutation. The thermal shift assay revealed the Tm value of mutant TRMU protein, lower than that of the wild-type counterpart. The A10S mutation caused marked decreases in 2-thiouridine modification of U34 of tRNA(Lys), tRNA(Glu) and tRNA(Gln) However, the A10S mutation mildly increased the aminoacylated efficiency of tRNAs. The altered 2-thiouridine modification worsened the impairment of mitochondrial translation associated with the m.1555A→G mutation. The defective translation resulted in the reduced activities of mitochondrial respiration chains. The respiratory deficiency caused the reduction of mitochondrial ATP production and elevated the production of reactive oxidative species. As a result, mutated TRMU worsened mitochondrial dysfunctions associated with m.1555A→G mutation, exceeding the threshold for expressing a deafness phenotype. Our findings provided new insights into the pathophysiology of maternally inherited deafness that was manifested by interaction between mtDNA mutation and nuclear modifier gene. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Fragmentary 5S rRNA gene in the human mitochondrial genome

    SciTech Connect

    Nierlich, D.P.

    1982-02-01

    The human mitochondrial genoma contains a 23-nucleodtide sequence that is homologous to a part of the 5S rRNA's of bacteria. This homology, the structure of the likely transcript, and the location of the sequence relative to the mitochondrial rRNA genes suggest that the sequence represents a fragmentary 5S rRNA gene.

  11. The mitochondrial 12S gene is a suitable marker of populations of Sarcoptes scabiei from wombats, dogs and humans in Australia.

    PubMed

    Skerratt, L F; Campbell, N J H; Murrell, A; Walton, S; Kemp, D; Barker, S C

    2002-04-01

    We sequenced part of the mitochondrial 12S ribosomal RNA gene of 23 specimens of Sarcoptes scabiei from eight wombats, one dog and three humans. Twelve of the 326 nucleotide positions varied among these mites and there were nine haplotypes (sequences) that differed by 1-8 nucleotides. Phylogenetic analyses indicated that these mites were from two lineages: (1) mites from wombats from Victoria, Australia, and mites from the humans and dog from the Northern Territory, Australia (haplotypes 1-4, 9); and (2) mites from the humans and dog from the Northern Territory (haplotypes 5-8). Mites from the three different hosts (wombats, a dog and humans) had not diverged phylogenetically; rather, these mites had similar 12S sequences. Thus, we conclude that these mites from wombats, humans and a dog are closely related, and that they diverged from a common ancestor relatively recently. This conclusion is consistent with the argument that people and/or their dogs introduced to Australia the S. scabiei mites that infect wombats in Australia . So, S. scabiei, which has been blamed for the extinction of populations of wombats in Australia, may be a parasitic mite that was introduced to Australia with people and/or their dogs. These data show that the mitochondrial 12S rRNA gene may be a suitable population marker of S. scabiei from wombats, dogs and humans in Australia.

  12. Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species.

    PubMed

    Mechergui, Arij; Achour, Wafa; Hassen, Assia Ben

    2014-05-01

    In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains. © 2013 APMIS. Published by John Wiley & Sons Ltd.

  13. Teenage-onset non-syndromic deafness associated with a mutation and a polymorphism in the mitochondrial 12S ribsomal RNA gene in a large Zairese pedigree

    SciTech Connect

    Matthijs, G.; Claes, S.; Cassiman, J.J.

    1994-09-01

    Non-syndromic deafness has been described as both an autosomal dominant and a recessive trait. Recently, Prezant et al. have identified a 1555 A to G substitution in the mitcohondrial 12S rRNA gene associated with deafness, either in an early-onset form with a postulated recessive nuclear defect for phenotypic expression, or after treatment with aminoglycosides. We have analyzed samples from a large pedigree originating from the village KAI SINGINI in Bas-Zaire. Patients have a maternally inherited, sudden-onset and bilateral sensineuronal deafness before the age of 20. To the best of our knowledge, no aminoglycosides have been given to these individuals. Sequencing of the mitochondrial 12S rRNA gene revealed the presence of a homoplasmic 1555 A-G mutation in 8 patients tested. This mutation is invariably associated with a newly described T-C transition at 1420 in the same gene. The 1420 mutation was also found in 1 of 30 unrelated black individuals. It is phylogenetically less conserved than its neighboring bases and than the 1555 mutation. It has been speculated that the 1555 mutation results in greater susceptibility to the effects of aminoglycosides on translational fidelity in the mitochondrial ribosome. It remains to be investigated whether the substitution at 1420 contributes to the ribosomal malfunction and the disease phenotype. Our results add to previous evidence for the 1555 mutation as a pathogenic mutation in non-syndromic deafness. We are currently collecting further family data and samples. The analysis of the entire pedigree will shed light on the possible contribution of nuclear defects in the cochlear-specific damage by impairment of mitochondrial translation.

  14. Transformation of tetrahymena thermophila with hypermethylated rRNA genes

    SciTech Connect

    Karrer, K.M.; Yao, M.C.

    1988-04-01

    The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N/sup 6/-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3'', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

  15. Dicistronic tRNA-5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes.

    PubMed

    Acker, Joël; Ozanne, Christophe; Kachouri-Lafond, Rym; Gaillardin, Claude; Neuvéglise, Cécile; Marck, Christian

    2008-10-01

    In eukaryotes, genes transcribed by RNA polymerase III (Pol III) carry their own internal promoters and as such, are transcribed as individual units. Indeed, a very few cases of dicistronic Pol III genes are yet known. In contrast to other hemiascomycetes, 5S rRNA genes of Yarrowia lipolytica are not embedded into the tandemly repeated rDNA units, but appear scattered throughout the genome. We report here an unprecedented genomic organization: 48 over the 108 copies of the 5S rRNA genes are located 3' of tRNA genes. We show that these peculiar tRNA-5S rRNA dicistronic genes are expressed in vitro and in vivo as Pol III transcriptional fusions without the need of the 5S rRNA gene-specific factor TFIIIA, the deletion of which displays a viable phenotype. We also report the existence of a novel putative non-coding Pol III RNA of unknown function about 70 nucleotide-long (RUF70), the 13 genes of which are devoid of internal Pol III promoters and located 3' of the 13 copies of the tDNA-Trp (CCA). All genes embedded in the various dicistronic genes, fused 5S rRNA genes, RUF70 genes and their leader tRNA genes appear to be efficiently transcribed and their products correctly processed in vivo.

  16. Dicistronic tRNA–5S rRNA genes in Yarrowia lipolytica: an alternative TFIIIA-independent way for expression of 5S rRNA genes

    PubMed Central

    Acker, Joël; Ozanne, Christophe; Kachouri-Lafond, Rym; Gaillardin, Claude; Neuvéglise, Cécile; Marck, Christian

    2008-01-01

    In eukaryotes, genes transcribed by RNA polymerase III (Pol III) carry their own internal promoters and as such, are transcribed as individual units. Indeed, a very few cases of dicistronic Pol III genes are yet known. In contrast to other hemiascomycetes, 5S rRNA genes of Yarrowia lipolytica are not embedded into the tandemly repeated rDNA units, but appear scattered throughout the genome. We report here an unprecedented genomic organization: 48 over the 108 copies of the 5S rRNA genes are located 3′ of tRNA genes. We show that these peculiar tRNA–5S rRNA dicistronic genes are expressed in vitro and in vivo as Pol III transcriptional fusions without the need of the 5S rRNA gene-specific factor TFIIIA, the deletion of which displays a viable phenotype. We also report the existence of a novel putative non-coding Pol III RNA of unknown function about 70 nucleotide-long (RUF70), the 13 genes of which are devoid of internal Pol III promoters and located 3′ of the 13 copies of the tDNA-Trp (CCA). All genes embedded in the various dicistronic genes, fused 5S rRNA genes, RUF70 genes and their leader tRNA genes appear to be efficiently transcribed and their products correctly processed in vivo. PMID:18790808

  17. Multiple independent insertions of 5S rRNA genes in the spliced-leader gene family of trypanosome species.

    PubMed

    Beauparlant, Marc A; Drouin, Guy

    2014-02-01

    Analyses of the 5S rRNA genes found in the spliced-leader (SL) gene repeat units of numerous trypanosome species suggest that such linkages were not inherited from a common ancestor, but were the result of independent 5S rRNA gene insertions. In trypanosomes, 5S rRNA genes are found either in the tandemly repeated units coding for SL genes or in independent tandemly repeated units. Given that trypanosome species where 5S rRNA genes are within the tandemly repeated units coding for SL genes are phylogenetically related, one might hypothesize that this arrangement is the result of an ancestral insertion of 5S rRNA genes into the tandemly repeated SL gene family of trypanosomes. Here, we use the types of 5S rRNA genes found associated with SL genes, the flanking regions of the inserted 5S rRNA genes and the position of these insertions to show that most of the 5S rRNA genes found within SL gene repeat units of trypanosome species were not acquired from a common ancestor but are the results of independent insertions. These multiple 5S rRNA genes insertion events in trypanosomes are likely the result of frequent founder events in different hosts and/or geographical locations in species having short generation times.

  18. 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

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

    PubMed

    Woolnough, Jessica L; Atwood, Blake L; 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.

  20. Diversity of 23S rRNA genes within individual prokaryotic genomes.

    PubMed

    Pei, Anna; Nossa, Carlos W; Chokshi, Pooja; Blaser, Martin J; Yang, Liying; Rosmarin, David M; Pei, Zhiheng

    2009-01-01

    The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons. Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%-4.04%). Significant (1.17%-4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes. These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.

  1. 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.

  2. The 12S rRNA A1555G mutation in the mitochondrial haplogroup D5a is responsible for maternally inherited hypertension and hearing loss in two Chinese pedigrees.

    PubMed

    Chen, Hong; Zheng, Jing; Xue, Ling; Meng, Yanzi; Wang, Yan; Zheng, Bingjiao; Fang, Fang; Shi, Suxue; Qiu, Qiaomeng; Jiang, Pingping; Lu, Zhongqiu; Mo, Jun Qin; Lu, Jianxin; Guan, Min-Xin

    2012-06-01

    We reported here clinical, genetic evaluations and molecular analysis of mitochondrial DNA (mtDNA) in two Han Chinese families carrying the known mitochondrial 12S rRNA A1555G mutation. In contrast with the previous data that hearing loss as a sole phenotype was present in the maternal lineage of other families carrying the A1555G mutation, matrilineal relatives among these two Chinese families exhibited both hearing loss and hypertension. Of 21 matrilineal relatives, 9 subjects exhibited both hearing loss and hypertension, 2 individuals suffered from only hypertension and 1 member had only hearing loss. The average age at onset of hypertension in the affected matrilineal relatives of these families was 60 and 46 years, respectively, whereas those of hearing loss in these two families were 33 and 55 years, respectively. Molecular analysis of their mtDNA identified distinct sets of variants belonging to the Eastern Asian haplogroup D5a. In contrast, the A1555G mutation occurred among other mtDNA haplogroups D, B, R, F, G, Y, M and N, respectively. Our data further support that the A1555G mutation is necessary but by itself insufficient to produce the clinical phenotype. The other modifiers are responsible for the phenotypic variability of matrilineal relatives within and among these families carrying the A1555G mutation. Our investigation provides the first evidence that the 12S rRNA A1555G mutation leads to both of hearing loss and hypertension. Thus, our findings may provide the new insights into the understanding of pathophysiology and valuable information for management and treatment of maternally inherited hearing loss and hypertension.

  3. 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.

  4. 5S rRNA gene arrangements in protists: a case of nonadaptive evolution.

    PubMed

    Drouin, Guy; Tsang, Corey

    2012-06-01

    Given their high copy number and high level of expression, one might expect that both the sequence and organization of eukaryotic ribosomal RNA genes would be conserved during evolution. Although the organization of 18S, 5.8S and 28S ribosomal RNA genes is indeed relatively well conserved, that of 5S rRNA genes is much more variable. Here, we review the different types of 5S rRNA gene arrangements which have been observed in protists. This includes linkages to the other ribosomal RNA genes as well as linkages to ubiquitin, splice-leader, snRNA and tRNA genes. Mapping these linkages to independently derived phylogenies shows that these diverse linkages have repeatedly been gained and lost during evolution. This argues against such linkages being the primitive condition not only in protists but also in other eukaryote species. Because the only characteristic the diverse genes with which 5S rRNA genes are found linked with is that they are tandemly repeated, these arrangements are unlikely to provide any selective advantage. Rather, the observed high variability in 5S rRNA genes arrangements is likely the result of the fact that 5S rRNA genes contain internal promoters, that these genes are often transposed by diverse recombination mechanisms and that these new gene arrangements are rapidly homogenized by unequal crossingovers and/or by gene conversions events in species with short generation times and frequent founder events.

  5. Over-representation of the G12S polymorphism of the SDHD gene in patients with MEN2A syndrome

    PubMed Central

    Lendvai, Nikoletta; Tóth, Miklos; Valkusz, Zsuzsanna; Bekő, Gabriella; Szücs, Nikolette; Csajbók, Éva; Igaz, Péter; Kriszt, Balázs; Kovács, Balázs; Rácz, Károly; Patócs, Attila

    2012-01-01

    OBJECTIVE: To evaluate whether germline variants of the succinate dehydrogenase genes might be phenotypic modifiers in patients with multiple endocrine neoplasia type 2. Mutations of genes encoding subunits of the succinate dehydrogenase are associated with hereditary paraganglioma/pheochromocytoma syndrome. Pheochromocytoma is one of the main manifestations of multiple endocrine neoplasia type 2 caused by germline mutation of the rearranged during transfection proto-oncogene. METHODS: Polymorphisms of the succinate dehydrogenase genes were analyzed in 77 rearranged during transfection mutation carriers, 47 patients with sporadic medullary thyroid cancer, 48 patients with sporadic Pheo, and 100 healthy individuals. Exons 10–16 of the rearranged during transfection proto-oncogene were analyzed by direct DNA sequencing, and all exons of the von Hippel-Lindau, succinate dehydrogenase B, and succinate dehydrogenase subunit D genes were tested by direct DNA sequencing and multiple ligation probe analysis. The G12S polymorphism of the succinate dehydrogenase subunit D gene was determined by restriction fragment length polymorphism. RESULTS: Of the 77 rearranged during transfection mutation carriers, 55 from 16 families had multiple endocrine neoplasia type 2A, three from three families had multiple endocrine neoplasia type 2B, and 19 from two families had familial medullary thyroid carcinoma. Eight of 55 (14.5%) patients with multiple endocrine neoplasia type 2A had this variant whereas it was absent in multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, sporadic medullary thyroid carcinoma, and sporadic pheochromocytoma groups, and its prevalence in controls was 1% (p<0.002 multiple endocrine neoplasia type 2A versus controls). No associations between G12S and age of manifestation, incidence of pheochromocytoma or hyperparathyroidism, or level of serum calcitonin were observed. CONCLUSION: The high prevalence of the G12S variant in patients

  6. 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

  7. Phylogenetic relatedness determined between antibiotic resistance and 16S rRNA genes in actinobacteria.

    PubMed

    Sagova-Mareckova, Marketa; Ulanova, Dana; Sanderova, Petra; Omelka, Marek; Kamenik, Zdenek; Olsovska, Jana; Kopecky, Jan

    2015-04-01

    Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite

  8. 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

  9. Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity.

    PubMed

    Sun, Dong-Lei; Jiang, Xuan; Wu, Qinglong L; Zhou, Ning-Yi

    2013-10-01

    Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the "gold standard" in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions.

  10. Visual analysis of the yeast 5S rRNA gene transcriptome: regulation and role of La protein.

    PubMed

    French, Sarah L; Osheim, Yvonne N; Schneider, David A; Sikes, Martha L; Fernandez, Cesar F; Copela, Laura A; Misra, Vikram A; Nomura, Masayasu; Wolin, Sandra L; Beyer, Ann L

    2008-07-01

    5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of approximately 1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.

  11. 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

  12. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-10-01

    We examined intragenomic variation of paralogous 5S rRNA genes to evaluate the concept of ribosomal constraints. In a dataset containing 1161 genomes from 779 unique species, 96 species exhibited > 3% diversity. Twenty-seven species with > 10% diversity contained a total of 421 mismatches between all pairs of the most dissimilar copies of 5S rRNA genes. The large majority (401 of 421) of the diversified positions were conserved at the secondary structure level. The high diversity was associated with partial rRNA operon, split operon, or spacer length-related divergence. In total, these findings indicated that there are tight ribosomal constraints on paralogous 5S rRNA genes in a genome despite of the high degree of diversity at the primary structure level. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes▿ †

    PubMed Central

    Pei, Anna Y.; Oberdorf, William E.; Nossa, Carlos W.; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A.; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M.; Sotero, Steven; DeSantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

    2010-01-01

    Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases. PMID:20418441

  16. Diversity of 16S rRNA genes within individual prokaryotic genomes.

    PubMed

    Pei, Anna Y; Oberdorf, William E; Nossa, Carlos W; Agarwal, Ankush; Chokshi, Pooja; Gerz, Erika A; Jin, Zhida; Lee, Peng; Yang, Liying; Poles, Michael; Brown, Stuart M; Sotero, Steven; Desantis, Todd; Brodie, Eoin; Nelson, Karen; Pei, Zhiheng

    2010-06-01

    Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 +/- 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% +/- 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2 degrees structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in "Candidatus Protochlamydia amoebophila." Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.

  17. Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

    SciTech Connect

    Wang Qiuju; Li Qingzhong; Han Dongyi . E-mail: hdy301@263.net; Zhao Yali; Zhao Lidong; Qian Yaping; Yuan Hu; Li Ronghua; Zhai Suoqiang; Young Wieyen . E-mail: ywy301@263.net; Guan Minxin . E-mail: min-xin.guan@chmcc.org

    2006-02-10

    We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family.

  18. Histone methyltransferases regulating rRNA gene dose and dosage control in Arabidopsis

    PubMed Central

    Pontvianne, Frédéric; Blevins, Todd; Chandrasekhara, Chinmayi; Feng, Wei; Stroud, Hume; Jacobsen, Steven E.; Michaels, Scott D.; Pikaard, Craig S.

    2012-01-01

    Eukaryotes have hundreds of nearly identical 45S ribosomal RNA (rRNA) genes, each encoding the 18S, 5.8S, and 25S catalytic rRNAs. Because cellular demands for ribosomes and protein synthesis vary during development, the number of active rRNA genes is subject to dosage control. In genetic hybrids, one manifestation of dosage control is nucleolar dominance, an epigenetic phenomenon in which the rRNA genes of one progenitor are repressed. For instance, in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, the A. thaliana-derived rRNA genes are selectively silenced. An analogous phenomenon occurs in nonhybrid A. thaliana, in which specific classes of rRNA gene variants are inactivated. An RNA-mediated knockdown screen identified SUVR4 {SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR)3-9]-RELATED 4} as a histone H3 Lys 9 (H3K9) methyltransferase required for nucleolar dominance in A. suecica. H3K9 methyltransferases are also required for variant-specific silencing in A. thaliana, but SUVH5 [SU(VAR)3-9 HOMOLOG 5] and SUVH6, rather than SUVR4, are the key activities in this genomic context. Mutations disrupting the H3K27 methyltransferases ATXR5 or ATXR6 affect which rRNA gene variants are expressed or silenced, and in atxr5 atxr6 double mutants, dominance relationships among variants are reversed relative to wild type. Interestingly, these changes in gene expression are accompanied by changes in the relative abundance of the rRNA gene variants at the DNA level, including overreplication of the normally silenced class and decreased abundance of the normally dominant class. Collectively, our results indicate that histone methylation can affect both the doses of different variants and their differential silencing through the choice mechanisms that achieve dosage control. PMID:22549957

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

    PubMed

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

    2016-01-01

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

  20. 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

  1. 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.

  2. 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.

  3. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis

    PubMed Central

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S.

    2016-01-01

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. PMID:26744421

  4. Chromosome-specific NOR inactivation explains selective rRNA gene silencing and dosage control in Arabidopsis.

    PubMed

    Chandrasekhara, Chinmayi; Mohannath, Gireesha; Blevins, Todd; Pontvianne, Frederic; Pikaard, Craig S

    2016-01-15

    In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2. © 2016 Chandrasekhara et al.; Published by Cold Spring Harbor Laboratory Press.

  5. 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

  6. 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

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

    PubMed

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

    2017-04-03

    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. Copyright © 2017 Rabanal et al.

  8. 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.

  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. Taxonomic Resolutions Based on 18S rRNA Genes: A Case Study of Subclass Copepoda

    PubMed Central

    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. PMID:26107258

  11. Rare Events of Intragenus and Intraspecies Horizontal Transfer of the 16S rRNA Gene

    PubMed Central

    Tian, Ren-Mao; Cai, Lin; Zhang, Wei-Peng; Cao, Hui-Luo; Qian, Pei-Yuan

    2015-01-01

    Horizontal gene transfer (HGT) of operational genes has been widely reported in prokaryotic organisms. However, informational genes such as those involved in transcription and translation processes are very difficult to be horizontally transferred, as described by Woese’s complexity hypothesis. Here, we analyzed all of the completed prokaryotic genome sequences (2,143 genomes) in the NCBI (National Center for Biotechnology Information) database, scanned for genomes with high intragenomic heterogeneity of 16S rRNA gene copies, and explored potential HGT events of ribosomal RNA genes based on the phylogeny, genomic organization, and secondary structures of the ribosomal RNA genes. Our results revealed 28 genomes with relatively high intragenomic heterogeneity of multiple 16S rRNA gene copies (lowest pairwise identity <98.0%), and further analysis revealed HGT events and potential donors of the heterogeneous copies (such as HGT from Chlamydia suis to Chlamydia trachomatis) and mutation events of some heterogeneous copies (such as Streptococcus suis JS14). Interestingly, HGT of the 16S rRNA gene only occurred at intragenus or intraspecies levels, which is quite different from the HGT of operational genes. Our results improve our understanding regarding the exchange of informational genes. PMID:26220935

  12. 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

  13. High or low correlation between co-occuring gene clusters and 16S rRNA gene phylogeny.

    PubMed

    Rudi, Knut; Sekelja, Monika

    2013-02-01

    Ribosomal RNA (rRNA) genes are universal for all living organisms. Yet, the correspondence between genome composition and rRNA phylogeny remains poorly known. The aim of this study was to use the information from genome sequence databases to address the correlation between rRNA gene phylogeny and total gene composition in bacteria. This was done by analysing 327 genomes with TIGRFAM functional gene annotations. Our approach consisted of two steps. First, we searched for discriminatory clusters of co-occurring genes. Using a multivariate statistical approach, we identified 11 such clusters which contain genes that were co-occurring only in a subset of genomes and contributed to explain the gene content differences between genome subsets. Second, we mapped the discovered clusters to 16S rRNA-based phylogeny and calculated the correlation between co-occuring genes and phylogeny. Six of the 11 clusters exhibited significant correlation with 16S rRNA gene phylogeny. The most distinct phylogenetic finding was a high correlation between iron-sulfur oxidoreductases in combination with carbon nitrogen ligases and Chlorobium. The other correlations identified covered relatively large phylogroups: Actinobacteria were positively associated with kinases, while Gammaproteobacteria were positively associated with methylases and acyltransferases. The suggested functional differences between higher phylogroups, however, need experimental verification.

  14. The distribution, diversity, and importance of 16S rRNA gene introns in the order Thermoproteales.

    PubMed

    Jay, Zackary J; Inskeep, William P

    2015-07-09

    Intron sequences are common in 16S rRNA genes of specific thermophilic lineages of Archaea, specifically the Thermoproteales (phylum Crenarchaeota). Environmental sequencing (16S rRNA gene and metagenome) from geothermal habitats in Yellowstone National Park (YNP) has expanded the available datasets for investigating 16S rRNA gene introns. The objectives of this study were to characterize and curate archaeal 16S rRNA gene introns from high-temperature habitats, evaluate the conservation and distribution of archaeal 16S rRNA introns in geothermal systems, and determine which "universal" archaeal 16S rRNA gene primers are impacted by the presence of intron sequences. Several new introns were identified and their insertion loci were constrained to thirteen locations across the 16S rRNA gene. Many of these introns encode homing endonucleases, although some introns were short or partial sequences. Pyrobaculum, Thermoproteus, and Caldivirga 16S rRNA genes contained the most abundant and diverse intron sequences. Phylogenetic analysis of introns revealed that sequences within the same locus are distributed biogeographically. The most diverse set of introns were observed in a high-temperature, circumneutral (pH 6) sulfur sediment environment, which also contained the greatest diversity of different Thermoproteales phylotypes. The widespread presence of introns in the Thermoproteales indicates a high probability of misalignments using different "universal" 16S rRNA primers employed in environmental microbial community analysis.

  15. Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

    PubMed Central

    Whitney, Anne M.; Mayer, Leonard W.; Morey, Roger; Steigerwalt, Arnold; Boras, Ariana; Weyant, Robin S.; Popovic, Tanja

    2002-01-01

    In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax. PMID:12396926

  16. [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.

  17. Nucleolin Is Required for DNA Methylation State and the Expression of rRNA Gene Variants in Arabidopsis thaliana

    PubMed Central

    Pontvianne, Frédéric; Abou-Ellail, Mohamed; Douet, Julien; Comella, Pascale; Matia, Isabel; Chandrasekhara, Chinmayi; DeBures, Anne; Blevins, Todd; Cooke, Richard; Medina, Francisco J.; Tourmente, Sylvette; Pikaard, Craig S.; Sáez-Vásquez, Julio

    2010-01-01

    In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis. PMID:21124873

  18. Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

    PubMed Central

    Barney, Michael; Volgyi, Antonia; Navarro, Alfonso; Ryder, David

    2001-01-01

    A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation. PMID:11157216

  19. 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.

  20. Unsuitability of quantitative Bacteroidales 16S rRNA gene assays for discerning fecal contamination of drinking water.

    PubMed

    van der Wielen, Paul W J J; Medema, Gertjan

    2010-07-01

    Bacteroidales species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for Bacteroidales 16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.

  1. Novel essential gene Involved in 16S rRNA processing in Escherichia coli.

    PubMed

    Kurata, Tatsuaki; Nakanishi, Shinobu; Hashimoto, Masayuki; Taoka, Masato; Yamazaki, Yukiko; Isobe, Toshiaki; Kato, Jun-ichi

    2015-02-27

    Biogenesis of ribosomes is a complex process mediated by many factors. While its transcription proceeds, ribosomal RNA (rRNA) folds itself into a characteristic three-dimensional structure through interaction with ribosomal proteins, during which its ends are processed. Here, we show that the essential protein YqgF, a RuvC family protein with an RNase-H-like motif, is involved in the processing of pre-16S rRNA during ribosome maturation. Indeed, pre-16S rRNA accumulated in cells of a temperature-sensitive yqgF mutant (yqgF(ts)) cultured at a non-permissive temperature. In addition, purified YqgF was shown to process the 5' end of pre-16S rRNA within 70S ribosomes in vitro. Mass spectrometry analysis of the total proteins in the yqgF(ts) mutant cells showed that the expression of genes containing multiple Shine-Dalgarno-like sequences was observed to be lower than in wild type. These results are interpreted to indicate that YqgF is involved in a novel enzymic activity necessary for the processing of pre-16S rRNA, thereby affecting elongation of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis.

    PubMed

    Yang, Bo; Wang, Yong; Qian, Pei-Yuan

    2016-03-22

    Prokaryotic 16S ribosomal RNA (rRNA) sequences are widely used in environmental microbiology and molecular evolution as reliable markers for the taxonomic classification and phylogenetic analysis of microbes. Restricted by current sequencing techniques, the massive sequencing of 16S rRNA gene amplicons encompassing the full length of genes is not yet feasible. Thus, the selection of the most efficient hypervariable regions for phylogenetic analysis and taxonomic classification is still debated. In the present study, several bioinformatics tools were integrated to build an in silico pipeline to evaluate the phylogenetic sensitivity of the hypervariable regions compared with the corresponding full-length sequences. The correlation of seven sub-regions was inferred from the geodesic distance, a parameter that is applied to quantitatively compare the topology of different phylogenetic trees constructed using the sequences from different sub-regions. The relationship between different sub-regions based on the geodesic distance indicated that V4-V6 were the most reliable regions for representing the full-length 16S rRNA sequences in the phylogenetic analysis of most bacterial phyla, while V2 and V8 were the least reliable regions. Our results suggest that V4-V6 might be optimal sub-regions for the design of universal primers with superior phylogenetic resolution for bacterial phyla. A potential relationship between function and the evolution of 16S rRNA is also discussed.

  4. Growth increase of Arabidopsis by forced expression of rice 45S rRNA gene.

    PubMed

    Makabe, So; Motohashi, Reiko; Nakamura, Ikuo

    2017-02-01

    Forced expression of rice 45S rRNA gene conferred ca. 2-fold increase of above-ground growth in transgenic Arabidopsis . This growth increase was probably brought by cell proliferation, not by cell enlargement. Recent increase in carbon dioxide emissions is causing global climate change. The use of plant biomass as alternative energy source is one way to reduce these emissions. Therefore, reinforcement of plant biomass production is an urgent key issue to overcome both depletion of fossil energies and emission of carbon dioxide. Here, we created transgenic Arabidopsis with a 2-fold increase in above-ground growth by forced expression of the rice 45S rRNA gene using the maize ubiquitin promoter. Although the size of guard cells and ploidy of leaf-cells were similar between transgenic and control plants, numbers of stomata and pavement cells were much increased in the transgenic leaf. This data suggested that cell number, not cell expansion, was responsible for the growth increase, which might be brought by the forced expression of exogenous and full-length 45S rRNA gene. The expression level of rice 45S rRNA transcripts was very low, possibly triggering unknown machinery to enhance cell proliferation. Although microarray analysis showed enhanced expression of ethylene-responsive transcription factors, these factors might respond to ethylene induced by abiotic/biotic stresses or genomic incompatibility, which might be involved in the expression of species-specific internal transcribed spacer (ITS) sequences within rice 45S rRNA transcripts. Further analysis of the mechanism underlying the growth increase will contribute to understanding the regulation of the cell proliferation and the mechanism of hybrid vigor.

  5. 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

  6. Fragmentation of 23S rRNA in Strains of Proteus and Providencia Results from Intervening Sequences in the rrn (rRNA) Genes

    PubMed Central

    Miller, Wayne L.; Pabbaraju, Kanti; Sanderson, Kenneth E.

    2000-01-01

    Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred. PMID:10648538

  7. 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

  8. Diversity and evolution of 5S rRNA gene family organization in Pythium.

    PubMed

    Bedard, James E J; Schurko, Andrew M; de Cock, Arthur W A M; Klassen, Glen R

    2006-01-01

    The 5S rRNA gene family organization among 87 species and varieties of Pythium was investigated to assess evolutionary stability of the two patterns detected and to determine which pattern is likely the ancestral state in the genus. Species with filamentous sporangia (Groups A-C according to the ITS phylogenetic tree for Pythium) had 5S genes linked to the rDNA repeat that were predominantly coded for on the DNA strand opposite to the one with the other rRNA genes ('inverted' orientation). A small group of species with contiguous sporangia (Group D) is related to Groups A-C but had unlinked 5S genes. The main group of species with spherical zoosporangia (Groups E-J) generally had unlinked 5S genes in tandem arrays. The six species in Group K, although they also have spherical sporangia, had linked genes on the same strand as the other rRNA genes 'non-inverted' and most of them had pairs of tandem 5S genes. The evolutionary stability of 5S sequence organization was compared with the stability of morphological characters as interpreted from a phylogeny based on ITS sequence analysis. Features of 5S sequence organization were found to be just as consistent within groups as were the morphological characters. To determine the ancestral type of 5S family organization, a survey of Phytophthora strains was conducted to supply an outgroup reference. The most parsimonious interpretation of the data in this survey yielded the tentative conclusion that the linked condition of the 5S sequences was ancestral.

  9. 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.

  10. Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing.

    PubMed

    Wang, Tao; Kong, Fanrong; Chen, Sharon; Xiao, Meng; Sorrell, Tania; Wang, Xiaoyan; Wang, Shuo; Sintchenko, Vitali

    2011-01-01

    The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

  11. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    PubMed

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. [Phylogenetic comparison between Spirulina and Arthrospira based on 16S rRNA and rpoC1 gene].

    PubMed

    Wu, Yuemei; Wang, Suying; Dong, Shirui

    2016-02-04

    Based on 16S rRNA and rpoC1 gene sequences, the phylogenetic relationship between Spirulina and Arthrospira were studied and compared. We amplified, sequenced and analyzed 16S rRNA and rpoC1 of 84 strains. Then the phylogenetic trees were constructed and compared. The conserved sites percentage, average G+C content and sequence identity of rpoC1 were 49.7%, 47.7%, 76%-100% respectively, significantly lower than 79.4%, 55.6% and 91%-100% of 16S rRNA, and the heterogeneity degree was higher. The trees generated with two different genes showed similar topologies and thus inferred consistent phylogenetic relationships. Eighty-four experimental strains were divided into 3 groups belonging to 2 genera: F-35 1, F-904-2, F-1070 and TJBC14 were Spirulina and the rest were Arthrospira. Although morphospecies and geographical species could not be distinguished based on 16S rRNA and rpoC1 gene sequences, the bootstrap value of rpoC1 (100%) was higher than that of 16S rRNA (99%). Moreover, clustering effect of rpoC1 for Spirulina and Arthrospirai was better than 16S rRNA. Spirulina and Arthrospira were different genera, rpoC1 gene has more advantage to distinguish the strains in the same genus than that of 16S rRNA gene.

  18. 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 Central

    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. PMID:24277737

  19. 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.

  20. Assembly of Regulatory Factors on rRNA and Ribosomal Protein Genes in Saccharomyces cerevisiae▿ †

    PubMed Central

    Kasahara, Koji; Ohtsuki, Kazushige; Ki, Sewon; Aoyama, Kayo; Takahashi, Hiroyuki; Kobayashi, Takehiko; Shirahige, Katsuhiko; Kokubo, Tetsuro

    2007-01-01

    HMO1 is a high-mobility group B protein that plays a role in transcription of genes encoding rRNA and ribosomal proteins (RPGs) in Saccharomyces cerevisiae. This study uses genome-wide chromatin immunoprecipitation to study the roles of HMO1, FHL1, and RAP1 in transcription of these genes as well as other RNA polymerase II-transcribed genes in yeast. The results show that HMO1 associates with the 35S rRNA gene in an RNA polymerase I-dependent manner and that RPG promoters (138 in total) can be classified into several distinct groups based on HMO1 abundance at the promoter and the HMO1 dependence of FHL1 and/or RAP1 binding to the promoter. FHL1, a key regulator of RPGs, binds to most of the HMO1-enriched and transcriptionally HMO1-dependent RPG promoters in an HMO1-dependent manner, whereas it binds to HMO1-limited RPG promoters in an HMO1-independent manner, irrespective of whether they are transcribed in an HMO1-dependent manner. Reporter gene assays indicate that these functional properties are determined by the promoter sequence. PMID:17646381

  1. Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

    PubMed

    Wagner, Josef; Coupland, Paul; Browne, Hilary P; Lawley, Trevor D; Francis, Suzanna C; Parkhill, Julian

    2016-11-14

    Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The Pac

  2. 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.

  3. Effect of DNA methylation on 18S rRNA gene sequences during culture of Taxus chinensis cells.

    PubMed

    Xiang, Fu; Li, Liqing; Yin, Rui; Jin, Wenwen; Yu, Longjiang

    2009-01-01

    * Author for correspondence and reprint requests Z. Naturforsch. 64c, 418-420 (2009); received December 15, 2008 Cell suspension culture has rapidly become an alternative source of taxol, an anticancer compound. To investigate the role of DNA methylation in the cultural course of Taxus chinensis cells, analyses of 18S rRNA gene sequences of cultured T chinensis cells and related species were conducted. The phylogenetic analysis of 18S rRNA gene sequences indicated that HG-1 (the cultured T chinensis cells), like T mairei (the natural variety of T chinensis), should be a new variety of T chinensis, and cell culture can change the 18S rRNA gene sequence at the level of species despite 18S rRNA is the most conserved gene. The analyses of the CpG and TpG+CpA relative abundance and GC content of the 18S rRNA gene sequences made clear that DNA methylation contributed to changes of the 18S rRNA gene sequence of HG-1 at the level of species, which can make HG-1 to become a new variety of 7 chinensis.

  4. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases.

    PubMed

    Clarridge, Jill E

    2004-10-01

    The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

  5. 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

  6. The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science.

    PubMed

    Ames, Nancy J; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R

    As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

  7. 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.

  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. Hypomethylation and hypermethylation of the tandem repetitive 5S rRNA genes in Arabidopsis.

    PubMed

    Vaillant, Isabelle; Tutois, Sylvie; Jasencakova, Zuzana; Douet, Julien; Schubert, Ingo; Tourmente, Sylvette

    2008-04-01

    5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.

  10. Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis.

    PubMed

    Douet, J; Tourmente, S

    2007-07-01

    5S ribosomal DNA is a highly conserved tandemly repeated multigenic family. As suggested for a long time, we have shown that only a fraction of the 5S rRNA genes are expressed in Arabidopsis thaliana. In Xenopus laevis, there is a developmental control of the expression of the 5S rRNA genes with only one of the two 5S rDNA families expressed during oogenesis. For both Arabidopsis and Xenopus, the strongest transcription of 5S rRNA, respectively in the seed and during oogenesis is correlated with heterogeneity in the transcribed 5S rRNAs. Epigenetic mechanisms such as modification of the chromatin structure are involved in the transcriptional regulation of the 5S rRNA genes in both organisms. In Arabidopsis, two silencing pathways, methylation-dependent (RNAi) and methylation-independent (MOM pathway), are involved in the silencing of a 5S rDNA fraction.

  11. Genotyping of commensal Neisseria spp strains by pulsed-field gel electrophoresis and 16S rRNA gene sequencing.

    PubMed

    Mechergui, Arij; Achour, Wafa; Ben Hassen, Assia

    2017-04-04

    We investigated the diversity of the primary sequences of the 16S rRNA genes among 46 commensal Neisseria strains and evaluated the use of this approach as a molecular typing tool in comparison with PFGE analysis. Identification to the genus was done using conventional methods and API NH (bio-Mérieux(®) ). Identification to species level was based on 16S rRNA gene sequencing. PFGE analysis was done using SpeI. Fourteen, two, three and fourteen 16S rRNA sequence types were found among twenty Neisseria flavescens, two Neisseria sicca, five Neisseria macacae and nineteen Neisseria mucosa clinical isolates. Forty-three different PFGE patterns were found among the tested strains. We demonstrated a high diversity among 16S rRNA genes which was reflected by PFGE analysis. © 2017 Wiley Periodicals, Inc.

  12. 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.

  13. 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.

  14. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome.

    PubMed

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; Desantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-09-07

    To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut

  15. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    PubMed Central

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; DeSantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their

  16. 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.

  17. 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

  18. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

    PubMed Central

    Langille, Morgan G. I.; Zaneveld, Jesse; Caporaso, J. Gregory; McDonald, Daniel; Knights, Dan; Reyes, Joshua A.; Clemente, Jose C.; Burkepile, Deron E.; Vega Thurber, Rebecca L.; Knight, Rob; Beiko, Robert G.; Huttenhower, Curtis

    2013-01-01

    Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community’s functional capabilities. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this ‘predictive metagenomic’ approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available. PMID:23975157

  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. 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.

  1. 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.

  2. Identification of Swertia mussotii and its adulterant Swertia species by 5S rRNA gene spacer.

    PubMed

    Yu, Man-Tang; Wong, Ka-Lok; Zong, Yu-Ying; Shaw, Pang-Chui; Che, Chun-Tao

    2008-03-01

    This research focused on analyzing the differences of 5S rRNA gene spacer sequences on Swertia mussotii and its commonly used adulterants, including S. franchetiana, S. wolfangiana and S. chirayita. DNA was extracted from the collected Swertia samples. 5S rRNA intergenic spacers were amplified by PCR, sequenced and analyzed. 5S rRNA gene spacer sequences were different between S. mussotii and its other three adulterants. Sequence divergence among species ranged from 30.6% to 65.0%. 5S rRNA spacers may be used as molecular authentication markers to differentiate S. mussotii and other commonly used Swertia adulterants. This result provides reliable and simple reference for the authentication of Swertia genus species.

  3. Conserved Curvature of RNA Polymerase I Core Promoter Beyond rRNA Genes: The Case of the Tritryps

    PubMed Central

    Smircich, Pablo; Duhagon, María Ana; Garat, Beatriz

    2015-01-01

    In trypanosomatids, the RNA polymerase I (RNAPI)-dependent promoters controlling the ribosomal RNA (rRNA) genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no conformational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such conserved curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes. PMID:26718450

  4. Molecular Evolution of Mycoplasma capricolum subsp. capripneumoniae Strains, Based on Polymorphisms in the 16S rRNA Genes

    PubMed Central

    Pettersson, Bertil; Bölske, Göran; Thiaucourt, François; Uhlén, Mathias; Johansson, Karl-Erik

    1998-01-01

    Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP. PMID:9573185

  5. rRNA Gene Expression of Abundant and Rare Activated-Sludge Microorganisms and Growth Rate Induced Micropollutant Removal.

    PubMed

    Vuono, David C; Regnery, Julia; Li, Dong; Jones, Zackary L; Holloway, Ryan W; Drewes, Jörg E

    2016-06-21

    The role of abundant and rare taxa in modulating the performance of wastewater-treatment systems is a critical component of making better predictions for enhanced functions such as micropollutant biotransformation. In this study, we compared 16S rRNA genes (rDNA) and rRNA gene expression of taxa in an activated-sludge-treatment plant (sequencing batch membrane bioreactor) at two solids retention times (SRTs): 20 and 5 days. These two SRTs were used to influence the rates of micropollutant biotransformation and nutrient removal. Our results show that rare taxa (<1%) have disproportionally high ratios of rRNA to rDNA, an indication of higher protein synthesis, compared to abundant taxa (≥1%) and suggests that rare taxa likely play an unrecognized role in bioreactor performance. There were also significant differences in community-wide rRNA expression signatures at 20-day SRT: anaerobic-oxic-anoxic periods were the primary driver of rRNA similarity. These results indicate differential expression of rRNA at high SRTs, which may further explain why high SRTs promote higher rates of micropollutant biotransformation. An analysis of micropollutant-associated degradation genes via metagenomics and direct measurements of a suite of micropollutants and nutrients further corroborates the loss of enhanced functions at 5-day SRT operation. This work advances our knowledge of the underlying ecosystem properties and dynamics of abundant and rare organisms associated with enhanced functions in engineered systems.

  6. Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

    PubMed Central

    Stancheva, I; Lucchini, R; Koller, T; Sogo, J M

    1997-01-01

    By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity. PMID:9108154

  7. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    PubMed

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

  8. Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium.

    PubMed

    Parker, Matthew A; Lafay, Benedicte; Burdon, Jeremy J; van Berkum, Peter

    2002-08-01

    Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.

  9. Characterization of the 18S rRNA Gene for Designing Universal Eukaryote Specific Primers

    PubMed Central

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M.; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies. PMID:24516555

  10. Characterization of the 18S rRNA gene for designing universal eukaryote specific primers.

    PubMed

    Hadziavdic, Kenan; Lekang, Katrine; Lanzen, Anders; Jonassen, Inge; Thompson, Eric M; Troedsson, Christofer

    2014-01-01

    High throughput sequencing technology has great promise for biodiversity studies. However, an underlying assumption is that the primers used in these studies are universal for the prokaryotic or eukaryotic groups of interest. Full primer universality is difficult or impossible to achieve and studies using different primer sets make biodiversity comparisons problematic. The aim of this study was to design and optimize universal eukaryotic primers that could be used as a standard in future biodiversity studies. Using the alignment of all eukaryotic sequences from the publicly available SILVA database, we generated a full characterization of variable versus conserved regions in the 18S rRNA gene. All variable regions within this gene were analyzed and our results suggested that the V2, V4 and V9 regions were best suited for biodiversity assessments. Previously published universal eukaryotic primers as well as a number of self-designed primers were mapped to the alignment. Primer selection will depend on sequencing technology used, and this study focused on the 454 pyrosequencing GS FLX Titanium platform. The results generated a primer pair yielding theoretical matches to 80% of the eukaryotic and 0% of the prokaryotic sequences in the SILVA database. An empirical test of marine sediments using the AmpliconNoise pipeline for analysis of the high throughput sequencing data yielded amplification of sequences for 71% of all eukaryotic phyla with no isolation of prokaryotic sequences. To our knowledge this is the first characterization of the complete 18S rRNA gene using all eukaryotes present in the SILVA database, providing a robust test for universal eukaryotic primers. Since both in silico and empirical tests using high throughput sequencing retained high inclusion of eukaryotic phyla and exclusion of prokaryotes, we conclude that these primers are well suited for assessing eukaryote diversity, and can be used as a standard in biodiversity studies.

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

    USDA-ARS?s Scientific Manuscript database

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

  12. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    USDA-ARS?s Scientific Manuscript database

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  13. Cryptic anuran biodiversity in Bangladesh revealed by mitochondrial 16S rRNA gene sequences.

    PubMed

    Hasan, Mahmudul; Islam, Mohammed Mafizul; Khan, Mukhlesur Rahman; Alam, Mohammad Shafiqul; Kurabayashi, Atsushi; Igawa, Takeshi; Kuramoto, Mitsuru; Sumida, Masayuki

    2012-03-01

    To survey the diversity of anuran species in Bangladesh, we compared mitochondrial 16S rRNA gene sequences (approximately 1.4 kbp) from 107 Bangladesh frog specimens. The results of genetic divergence and phylogenetic analyses incorporating data from related species revealed the occurrence of at least eight cryptic species. Hoplobatrachus tigerinus from two districts diverged considerably, indicating the involvement of a cryptic species. Two Fejervarya sp. (large and medium types) and Hylarana cf. taipehensis formed lineages distinct from related species and are probably new species. Microhyla cf. ornata differed from M. ornata with respect to type locality area and involved two distinct species. In addition, we found that Hylarana sp. and Microhyla sp. did not match congeners examined to date in either morphology or 16S rRNA sequence. The occurrence of M. fissipes was tentatively suggested. Consequently, at least, 19 species were found from Bangladesh in this study. These findings revealed a rich anuran biodiversity in Bangladesh, which is unexpected considering the rather simple topographic features of the country.

  14. 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.

  15. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses

    PubMed Central

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Background Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. Methods The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Results Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Conclusion Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3–100%. However, the

  16. Intra-Genomic Heterogeneity in 16S rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses.

    PubMed

    Chen, Jiazhen; Miao, Xinyu; Xu, Meng; He, Junlin; Xie, Yi; Wu, Xingwen; Chen, Gang; Yu, Liying; Zhang, Wenhong

    2015-01-01

    Members of the genera Prevotella, Veillonella and Fusobacterium are the predominant culturable obligate anaerobic bacteria isolated from periodontal abscesses. When determining the cumulative number of clinical anaerobic isolates from periodontal abscesses, ambiguous or overlapping signals were frequently encountered in 16S rRNA gene sequencing chromatograms, resulting in ambiguous identifications. With the exception of the genus Veillonella, the high intra-chromosomal heterogeneity of rrs genes has not been reported. The 16S rRNA genes of 138 clinical, strictly anaerobic isolates and one reference strain were directly sequenced, and the chromatograms were carefully examined. Gene cloning was performed for 22 typical isolates with doublet sequencing signals for the 16S rRNA genes, and four copies of the rrs-ITS genes of 9 Prevotella intermedia isolates were separately amplified by PCR, sequenced and compared. Five conserved housekeeping genes, hsp60, recA, dnaJ, gyrB1 and rpoB from 89 clinical isolates of Prevotella were also amplified by PCR and sequenced for identification and phylogenetic analysis along with 18 Prevotella reference strains. Heterogeneity of 16S rRNA genes was apparent in clinical, strictly anaerobic oral bacteria, particularly in the genera Prevotella and Veillonella. One hundred out of 138 anaerobic strains (72%) had intragenomic nucleotide polymorphisms (SNPs) in multiple locations, and 13 strains (9.4%) had intragenomic insertions or deletions in the 16S rRNA gene. In the genera Prevotella and Veillonella, 75% (67/89) and 100% (19/19) of the strains had SNPs in the 16S rRNA gene, respectively. Gene cloning and separate amplifications of four copies of the rrs-ITS genes confirmed that 2 to 4 heterogeneous 16S rRNA copies existed. Sequence alignment of five housekeeping genes revealed that intra-species nucleotide similarities were very high in the genera Prevotella, ranging from 94.3-100%. However, the inter-species similarities were

  17. 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

  18. International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    PubMed

    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

    2015-03-01

    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.

  19. Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas

    PubMed Central

    Bodilis, Josselin; Nsigue-Meilo, Sandrine; Besaury, Ludovic; Quillet, Laurent

    2012-01-01

    Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies. PMID:22545126

  20. 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.

  1. 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.

  2. Emergence of Macrolide-Resistant Mycoplasma pneumoniae with a 23S rRNA Gene Mutation

    PubMed Central

    Morozumi, Miyuki; Hasegawa, Keiko; Kobayashi, Reiko; Inoue, Nagako; Iwata, Satoshi; Kuroki, Haruo; Kawamura, Naohisa; Nakayama, Eiichi; Tajima, Takeshi; Shimizu, Kouichi; Ubukata, Kimiko

    2005-01-01

    A total of 195 Mycoplasma pneumoniae strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183 M. pneumoniae isolates were susceptible to all agents and had excellent MIC90s in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin, 0.0625 μg/ml for sitafloxacin, 0.5 μg/ml for minocycline, and 1 μg/ml for levofloxacin. Notably, 12 ML-resistant M. pneumoniae strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of ≥1 μg/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in Escherichia coli, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as described previously (A. Cousin-Allery, A. Charron, B. D. Barbeyrac, G. Fremy, J. S. Jensen, H. Renaudin, and C. Bebear, Epidemiol. Infect. 124:103-111, 2000). These findings suggest that excessive usage of MLs acts as a trigger to select mutations on the corresponding 23S rRNA gene with the resultant occurrence of ML-resistant M. pneumoniae. Monitoring ML susceptibilities for M. pneumoniae is necessary in the future. PMID:15917525

  3. Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

    PubMed Central

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  4. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing.

    PubMed

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating.

  5. 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.

  6. Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.

    PubMed Central

    Borrell, N; Acinas, S G; Figueras, M J; Martínez-Murcia, A J

    1997-01-01

    Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities. Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas. The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin. Most results from the two approaches were in agreement, but some discrepancies were discerned. Advantages over previous phenotypic and genetic methods are discussed. PMID:9196171

  7. 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

  8. 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

  9. Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences.

    PubMed

    Del Casale, Antonio; Flanagan, Paul V; Larkin, Michael J; Allen, Christopher C R; Kulakov, Leonid A

    2011-04-01

    Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences.

  10. Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia.

    PubMed

    Al Sheikh, Yazeed A; Marie, Mohammed Ali M; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M

    2014-01-01

    Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were blaTEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced b-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae.

  11. Prevalence of 16S rRNA methylase genes among β-lactamase-producing Enterobacteriaceae clinical isolates in Saudi Arabia.

    PubMed

    Al Sheikh, Yazeed A; Marie, Mohammed Ali M; John, James; Krishnappa, Lakshmana Gowda; Dabwab, Khaled Homoud M

    2014-01-01

    Background Co production of 16S rRNA methylases gene and β-Lactamase gene among Enterobacteriaceae isolates conferring resistance to both therapeutic options has serious implications for clinicians worldwide. Methods To study co existence of 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, and npmA) and β-Lactamase (blaTEM-1, blaSHV-12, blaCTX-M-14) genes, we screened all phenotypic positive β-Lactamase producing enterobacteriaceae by polymerase chain reaction (PCR) targeting above genes. A total of 330 enterobacteriaceae strains were collected during study period out of that 218 isolates were identified phenotypically as β-Lactamase producers, which include 50 (22.9%) Escherichia coli; 92 (42.2%) Klebsiella pneumoniae, 44 (20.2%), Citrobactor freundii and 32 (14.7%) Enterobacter spp. Results Among this 218, only 188 isolates harbored the resistant gene for β-Lactamase production. Major β-Lactamase producing isolates were blaTEM-1 type. 122 (56 %) isolates were found to produce any one of the 16S rRNA methylase genes. A total of 116 isolates co produced β-Lactamase and at least one 16S rRNA methylases gene Co production of armA gene was found in 26 isolates with rmtB and in 4 isolates with rmtC. The rmtA and rmtD genes were not detected in any of the tested isolates. Six isolates were positive for a 16S rRNA methylase gene alone. Conclusion β-Lactamase producing isolates appears to coexist with 16S rRNA methylase predominantly armA and rmtB genes in the same isolate. We conclude the major β-Lactamase and 16S rRNA methylases co-producer was K. pneumoniae followed by E. coli. We suggest further work on evaluating other β-lactamases types and novel antibiotic resistance mechanisms among Enterobacteriaceae.

  12. Two different 16S rRNA genes in a mycobacterial strain.

    PubMed Central

    Ninet, B; Monod, M; Emler, S; Pawlowski, J; Metral, C; Rohner, P; Auckenthaler, R; Hirschel, B

    1996-01-01

    Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately. PMID:8880515

  13. 16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

    PubMed

    Watts, George S; Youens-Clark, Ken; Slepian, Marvin J; Wolk, Donna M; Oshiro, Marc M; Metzger, Gregory S; Dhingra, Dalia; Cranmer, Lee D; Hurwitz, Bonnie L

    2017-09-20

    Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0. 1% of the mixture. Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0.1% of a mixture, suggests it has sufficient sensitivity for samples in which important bacteria may be rare. We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. 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.

  15. Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription

    PubMed Central

    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

    2017-01-01

    Abstract 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. PMID:27924000

  16. Genetic variation in 12S and 16S mitochondrial rDNA genes of four geographically isolated populations of Gulf Coast ticks (Acari: Ixodidae).

    PubMed

    Ketchum, H R; Teel, P D; Coates, C J; Strey, O F; Longnecker, M T

    2009-05-01

    Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei's haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42-59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.

  17. 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.

  18. Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

    PubMed Central

    Jakubczak, J L; Burke, W D; Eickbush, T H

    1991-01-01

    A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer. Images PMID:1849649

  19. Phylogenetic placement of the spider genus Nephila (Araneae: Araneoidea) inferred from rRNA and MaSp1 gene sequences.

    PubMed

    Pan, Hong-Chun; Zhou, Kai-Ya; Song, Da-Xiang; Qiu, Yang

    2004-03-01

    The family status of the genus Nephila, which belongs to Tetragnathidae currently but Araneidae formerly, was reexamined based on molecular phylogenetic analyses. In the present study, 12S and 18S rRNA gene fragments of eight species of spiders were amplified and sequenced. In addition, 3'-end partial cDNA of major ampullate spidroin-1 (MaSp1) gene of Argiope amoena was cloned and sequenced, and the 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and the predicted amino acid sequence of C-terminal non-repetitive region of MaSp1 were aligned with some previously known sequences. The resulting phylogeny showed that Araneidae and Tetragnathidae are not a sister group in the superfamily Araneoidea, and the genus Nephila is closer to the genera of the family Araneidae rather than to those of Tetragnathidae. We suggest that the genus Nephila should be transferred back to Araneidae. Or the subfamily Nephilinae might be elevated to family level after it was redefined and redelimited. Furthermore, the study showed that 3'-end non-repetitive region's cDNA sequence of MaSp1 gene and C-terminal non-repetitive region's amino acid sequence of MaSp1 are useful molecular markers for phylogenetic analysis of spiders.

  20. Accurate, Rapid Taxonomic Classification of Fungal Large-Subunit rRNA Genes

    PubMed Central

    Liu, Kuan-Liang; Porras-Alfaro, Andrea; Eichorst, Stephanie A.

    2012-01-01

    Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp). PMID:22194300

  1. Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes.

    PubMed

    Liu, Kuan-Liang; Porras-Alfaro, Andrea; Kuske, Cheryl R; Eichorst, Stephanie A; Xie, Gary

    2012-03-01

    Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project.

  2. Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere.

    PubMed

    Kimura, Hiroyuki; Ishibashi, Jun-Ichiro; Masuda, Harue; Kato, Kenji; Hanada, Satoshi

    2007-04-01

    International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

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

    SciTech Connect

    Butler, D.K.; Yasuda, L.E.; Yao, Meng-Chao

    1995-12-01

    This report discusses the formation of rRNA gene palindrome in Tetrahymena thermophila and the involvement of intramolecular recombination. This, along with the authors` previous study, is the first to define a molecular pathway of palindrome formation. 48 refs., 6 figs.

  4. A group IIC-type intron interrupts the rRNA methylase gene of Geobacillus stearothermophilus strain 10.

    PubMed

    Moretz, Samuel E; Lampson, Bert C

    2010-10-01

    Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from Geobacillus stearothermophilus were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.

  5. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    USDA-ARS?s Scientific Manuscript database

    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. Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

    PubMed Central

    Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

    2012-01-01

    Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

  9. 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...

  10. Evolutionary and Diagnostic Implications of Intragenomic Heterogeneity in the 16S rRNA Gene in Aeromonas Strains

    PubMed Central

    Morandi, Alessia; Zhaxybayeva, Olga; Gogarten, J. Peter; Graf, Joerg

    2005-01-01

    Sequencing 16S rRNA genes (SSU) cloned from Aeromonas strains revealed that strains contained up to six copies differing by ≤1.5%. The SSU copies from Aeromonas veronii LMG13695 clustered with sequences from four Aeromonas species. These results demonstrate intragenomic heterogeneity of SSU and suggest caution when using SSU to identify aeromonads. PMID:16159790

  11. 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...

  12. Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus

    PubMed Central

    2011-01-01

    Background Streptococcus is an economically important genus as a number of species belonging to this genus are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene sequence similarity. The variability observed among the members of these groups is low and it is difficult to distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that can be used for preliminary identification and can supplement the existing methods for identification of clinically-relevant isolates of the genus Streptococcus. Methods 16S rRNA gene sequences belonging to the isolates of S. dysgalactiae, S. equi, S. pyogenes, S. agalactiae, S. bovis, S. gallolyticus, S. mutans, S. sobrinus, S. mitis, S. pneumoniae, S. thermophilus and S. anginosus were analyzed with the purpose to define genetic variability within each species to generate a phylogenetic framework, to identify species-specific signatures and in-silico restriction enzyme analysis. Results The framework based analysis was used to segregate Streptococcus spp. previously identified upto genus level. This segregation was validated using species-specific signatures and in-silico restriction enzyme analysis. 43 uncharacterized Streptococcus spp. could be identified using this approach. Conclusions The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus Streptococcus. PMID:21702978

  13. Campylobacter jejuni, an uncommon cause of splenic abscess diagnosed by 16S rRNA gene sequencing.

    PubMed

    Seng, Piseth; Quenard, Fanny; Menard, Amélie; Heyries, Laurent; Stein, Andreas

    2014-12-01

    Splenic abscess is a rare disease that primarily occurs in patients with splenic trauma, endocarditis, sickle cell anemia, or other diseases that compromise the immune system. This report describes a culture-negative splenic abscess in an immunocompetent patient caused by Campylobacter jejuni, as determined by 16S rRNA gene sequencing.

  14. R4, a non-LTR retrotransposon specific to the large subunit rRNA genes of nematodes.

    PubMed Central

    Burke, W D; Müller, F; Eickbush, T H

    1995-01-01

    A 4.7 kb sequence-specific insertion in the 26S ribosomal RNA gene of Ascaris lumbricoides, named R4, is shown to be a non-long terminal repeat (non-LTR) retrotransposable element. The R4 element inserts at a site in the large subunit rRNA gene which is midway between two other sequence-specific non-LTR retrotransposable elements, R1 and R2, found in most insect species. Based on the structure of its open reading frame and the sequence of its reverse transcriptase domain, R4 elements do not appear to be a family of R1 or R2 elements that have changed their insertion site. R4 is most similar in structure and in sequence to the element Dong, which is not specialized for insertion into rRNA units. Thus R4 represents a separate non-LTR retrotransposable element that has become specialized for insertion in the rRNA genes of its host. Using oligonucleotide primers directed to a conserved region of the reverse transcriptase encoding domain, insertions in the R4 site were also amplified from Parascaris equorum and Haemonchus contortus. Why several non-LTR retrotransposable elements have become specialized for insertion into a short (87 bp) region of the large subunit rRNA gene is discussed. PMID:8524653

  15. 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; Mainardi, Jean-Luc

    2007-09-01

    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.

  16. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. 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.

  19. 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

  20. Selective expression of two types of 28S rRNA paralogous genes in the chaetognath Spadella cephaloptera.

    PubMed

    Barthélémy, R-M; Casanova, J-P; Grino, M; Faure, E

    2007-09-13

    Significant intra-individual variation in the sequences of the ribosomal RNA (rRNA) genes is highly unusual in animal genomes; however, two classes of both 18S and 28S rRNA gene sequences have been detected in chaetognaths, a small phylum of marine invertebrates. One species, Spadella cephaloptera Busch, 1851, is well-suited to the methods of in situ analysis of gene expression, since it is totally transparent. To test our hypothesis of a possible functional division of the two classes of genes, we carried out in situ hybridization. Our results indicated that 28S class II genes are expressed intensively in the oocytes of chaetognaths. In contrast, hybridization using an heterologous probe of 28S class I genes revealed only a single and relatively weak signal in a distinct area of intestinal cells. Our results suggest that the S. cephaloptera genome contains at least three different types of rRNA 28S genes; however, those which are expressed during housekeeping conditions could not be detected in our experiments.

  1. Differential rRNA genes expression in bread wheat and its inheritance.

    PubMed

    Carvalho, Ana; Polanco, Carlos; Guedes-Pinto, Henrique; Lima-Brito, José

    2013-09-01

    The expression of the ribosomal RNA (rRNA) genes from rye, located within the nucleolus organizer regions (NORs), is repressed by cytosine methylation in wheat x rye hybrids and in triticale, as consequence of nucleolar dominance. Our previous study revealed that bread wheat cultivars with a maximum number of four Ag-NORs presented high level of rDNA cytosine methylation when compared to others with a maximum of six Ag-NORs. In order to evaluate the inheritance of the Ag-NORs number and NOR methylation patterns, we produced F1 hybrids between bread wheat cultivars with four Ag-NORs and bread wheat cultivars with six Ag-NORs (in the direct and reciprocal senses). The F2 progenies of these F1 hybrids were also evaluated for the NOR number and methylation patterns. Parent bread wheat cultivars with a maximum of four Ag-NORs after treated with 5-azacytidine evidenced a maximum of six Ag-NORs per metaphase cell and a maximum of six nucleoli per interphase nucleus, confirming that the expression of the rRNA genes in bread wheat is related to cytosine methylation. Most of the F1 hybrids showed a maximum number of four or six Ag-NORs, similarly to that of the female parent suggesting a non-mendelian inheritance, while other hybrids presented four or six Ag-NORs in both senses of the cross. The F1 NOR methylation patterns showed some fragments common to their parents but also novel fragments suggesting genomic and/or chromosome rearrangements after hybridization. Despite the different NOR patterns among the parents, an invariable NOR pattern was found among the F1 plants suggesting a tendency to stability, which was also transmitted to the F2. The F2 progenies showed plants with a maximum of four, five and/or six Ag-NORs. The ratio of plants with four, five and/or six Ag-NORs per F2 progeny was variable and did not follow any specific mendelian proportion. These results allowed us to suggest that the inheritance of the number of Ag-NORs by the F1 and F2 plants did not

  2. 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.

  3. Taxonomic classification of bacterial 16S rRNA genes using short sequencing reads: evaluation of effective study designs.

    PubMed

    Mizrahi-Man, Orna; Davenport, Emily R; Gilad, Yoav

    2013-01-01

    Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.

  4. 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.

  5. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    PubMed

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera.

    PubMed

    Kyselková, Martina; Kopecký, Jan; Felföldi, Tamás; Cermák, Ladislav; Omelka, Marek; Grundmann, Geneviève L; Moënne-Loccoz, Yvan; Ságová-Marecková, Markéta

    2008-10-01

    Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.

  7. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  8. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  9. Development of a Multiplex PCR Method for Detection of the Genes Encoding 16S rRNA, Coagulase, Methicillin Resistance and Enterotoxins in Staphylococcus aureus

    USDA-ARS?s Scientific Manuscript database

    A multiplex PCR method was developed for simultaneous detection of the genes encoding methicillin resistance (mecA), staphylococcal enterotoxins A, B and C (sea, seb and sec), coagulase (coa) and 16S rRNA. The primers for amplification of the 16S rRNA gene were specific for Staphylococcus spp., and ...

  10. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  11. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  12. Two Cases of Mycoplasma pneumoniae Pneumonia with A2063G Mutation in the 23S rRNA Gene in Siblings

    PubMed Central

    Hong, Joo Hee; Chun, Jin Kyong; Oh, Ki Jin; Kim, Juwon; Yoon, Kap Jun

    2013-01-01

    We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene. PMID:23301225

  13. rRNA gene restriction patterns and biotypes of Shigella sonnei.

    PubMed Central

    Nastasi, A.; Pignato, S.; Mammina, C.; Giammanco, G.

    1993-01-01

    Shigella sonnei is a major agent of diarrhoeal disease in developed as well as in developing countries. Several phenotypic methods to define strain differences have been applied to this species of Shigella including, more recently, analysis of extrachromosomal and chromosomal DNA. In this study, 432 endemic and epidemic strains isolated between 1975 and 1991 in Italy, France and Switzerland were submitted to rRNA gene restriction pattern analysis, after digestion of whole-cell DNA by Hinc II, and to concomitant biotyping. Thirteen ribotypes, H1 to H13, and five biotypes, a, d, e, f, g, were detected. Ninety-five percent of the sporadic strains were assigned to ribotypes H1 to H4, which could be subtyped, except for H4, in different biotypes. Strains from each of seven different outbreaks had indistinguishable ribotype-biotype patterns. In contrast, 65 strains, isolated in Sicily in 1980 over an extended period of apparently epidemic increase of isolations and which had previously been considered to be a single bacterial clone on the basis of resistance pattern and phage type, were found to belong to two different and scarcely related ribotypes. Ribotyping and biochemical subtyping appear to be a useful epidemiological tool in studies on the circulation and distribution of strains of S. sonnei. Images Fig. 1 PMID:7679353

  14. Phylogeny of the malarial genus Plasmodium, derived from rRNA gene sequences.

    PubMed Central

    Escalante, A A; Ayala, F J

    1994-01-01

    Malaria is among mankind's worst scourges, affecting many millions of people, particularly in the tropics. Human malaria is caused by several species of Plasmodium, a parasitic protozoan. We analyze the small subunit rRNA gene sequences of 11 Plasmodium species, including three parasitic to humans, to infer their evolutionary relationships. Plasmodium falciparum, the most virulent of the human species, is closely related to Plasmodium reichenowi, which is parasitic to chimpanzee. The estimated time of divergence of these two Plasmodium species is consistent with the time of divergence (6-10 million years ago) between the human and chimpanzee lineages. The falciparum-reichenowi clade is only remotely related to two other human parasites, Plasmodium malariae and Plasmodium vivax, which are also only remotely related to each other. Thus, the parasitic associations of the Plasmodium species with their human hosts are phylogenetically independent. The remote phylogenetic relationship between the two bird parasites, Plasmodium gallinaceum and Plasmodium lophurae, and any of the human parasites provides no support for the hypothesis that infection by Plasmodium falciparum is a recent acquisition of humans, possibly coincident with the onset of agriculture. PMID:7972067

  15. Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome.

    PubMed

    Santos, Susana S; Nunes, Inês; Nielsen, Tue K; Jacquiod, Samuel; Hansen, Lars H; Winding, Anne

    2017-07-01

    Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. 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.

  17. Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

    PubMed Central

    Xiao, Lihua; Escalante, Lillian; Yang, Chunfu; Sulaiman, Irshad; Escalante, Anannias A.; Montali, Richard J.; Fayer, Ronald; Lal, Altaf A.

    1999-01-01

    Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed. PMID:10103253

  18. Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.

    PubMed Central

    Haltiner, M M; Smale, S T; Tjian, R

    1986-01-01

    A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription. Images PMID:3785147

  19. Mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance.

    PubMed Central

    Kim, Kyung Suk; Kang, Jung Oak; Eun, Chang Soo; Han, Dong Soo; Choi, Tae Yeal

    2002-01-01

    Among 12 clarithromycin-resistant Helicobacter pylori strains isolated in Guri, Korea, 8 showed an adenine to guanine mutation at position 2143 (formerly A2144G or E. coli 2059) in the 23S rRNA gene by the PCR-restriction fragment length polymorphism (RFLP) method. The remaining 4 strains, digested by neither BsaI nor BbsI, showed a thymine to cytosine mutation at position 2182 (T2182C) by direct sequencing of the PCR products. The T2182C mutants showed a tendency of higher levels of minimum inhibitory concentration to clarithromycin than the A2143G mutants. In conclusion, either the A2143G or the T2182C mutation was present in 100% of clarithromycin-resistant H. pylori isolates examined. The PCR-RFLP technique with restriction enzymes BbsI and BsaI was a rapid and relatively simple method to detect the clarithromycin resistance. But undigested isolates were quite frequent among our isolates (33.3%), the PCR-RFLP method with restriction enzymes BbsI and BsaI should not be used alone, and development of other rapid detection method for clarithromycin resistance is mandatory. PMID:12378008

  20. Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element.

    PubMed Central

    Hasan, G; Turner, M J; Cordingley, J S

    1982-01-01

    An ordered restriction map of the ribosomal RNA genes of Trypanosoma brucei brucei is presented. Bgl II fragments of T.b.brucei genomic DNA were cloned into pAT 153, and the clones containing rDNA identified. Restriction maps were established and the sense strands identified. One clone was shown by heteroduplex mapping to contain a 1.1 kb inserted sequence which was demonstrated to be widely distributed throughout the genomes of members of the subgenus Trypanozoon. However, in two other subgenera of Trypanosoma, Nannomonas and Schizotrypanum, the sequence is far less abundant. Analysis of the genomic DNA from two serodemes of T.b.brucei showed that the sequence was present in the rRNA of only one of them, implying that the sequence is a mobile element and that its appearance in rDNA is a comparitively recent occurrence. Images PMID:6294613

  1. 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells.

    PubMed

    Kuchipudi, Suresh V; Tellabati, Meenu; Nelli, Rahul K; White, Gavin A; Perez, Belinda Baquero; Sebastian, Sujith; Slomka, Marek J; Brookes, Sharon M; Brown, Ian H; Dunham, Stephen P; Chang, Kin-Chow

    2012-10-08

    One requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes. The relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells. Based on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA

  2. EzEditor: a versatile sequence alignment editor for both rRNA- and protein-coding genes.

    PubMed

    Jeon, Yoon-Seong; Lee, Kihyun; Park, Sang-Cheol; Kim, Bong-Soo; Cho, Yong-Joon; Ha, Sung-Min; Chun, Jongsik

    2014-02-01

    EzEditor is a Java-based molecular sequence editor allowing manipulation of both DNA and protein sequence alignments for phylogenetic analysis. It has multiple features optimized to connect initial computer-generated multiple alignment and subsequent phylogenetic analysis by providing manual editing with reference to biological information specific to the genes under consideration. It provides various functionalities for editing rRNA alignments using secondary structure information. In addition, it supports simultaneous editing of both DNA sequences and their translated protein sequences for protein-coding genes. EzEditor is, to our knowledge, the first sequence editing software designed for both rRNA- and protein-coding genes with the visualization of biologically relevant information and should be useful in molecular phylogenetic studies. EzEditor is based on Java, can be run on all major computer operating systems and is freely available from http://sw.ezbiocloud.net/ezeditor/.

  3. Diagnosis of Pierce's disease using biomarkers specific to Xylella fastidiosa rRNA and Vitis vinifera gene expression.

    PubMed

    Choi, H-K; Goes da Silva, F; Lim, H-J; Iandolino, A; Seo, Y-S; Lee, S-W; Cook, D R

    2010-10-01

    Pierce's disease (PD), caused by Xylella fastidiosa, represents one of the most damaging diseases of cultivated grape. Management of PD in the vineyard often relies on the removal of infected individuals, which otherwise serve as a source of inoculum for nearby healthy vines. Effective implementation of such control measures requires early diagnosis, which is complicated by the fact that infected vines often harbor high titers of the pathogen in advance of visual symptom development. Here, we report a biomarker system that simultaneously monitors Xylella-induced plant transcripts as well as Xylella ribosomal (r)RNA. Plant biomarker genes were derived from a combination of in silico analysis of grape expressed sequence tags and validation by means of reverse-transcriptase polymerase chain reaction (RT-PCR). Four genes upregulated upon PD infection were individually multiplexed with an X. fastidiosa marker rRNA and scored using either real-time RT-PCR or gel-based conventional RT-PCR techniques. The system was sufficiently sensitive to detect both host gene transcript and pathogen rRNA in asymptomatic infected plants. Moreover, these plant biomarker genes were not induced by water deficit, which is a component of PD development. Such biomarker genes could have utility for disease control by aiding early detection and as a screening tool in breeding programs.

  4. 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

  5. Phylogenetic relationships of Indian caecilians (Amphibia: Gymnophiona) inferred from mitochondrial rRNA gene sequences.

    PubMed

    Wilkinson, Mark; A Sheps, Jonathan; Oommen, Oommen V; Cohen, Bernard L

    2002-06-01

    India has a diverse caecilian fauna, including representatives of three of the six currently recognized families, the Caeciliidae, Ichthyophiidae, the endemic Uraeotyphlidae, but previous molecular phylogenetic studies of caecilians have not included sequences for any Indian caecilians. Partial 12S and 16S mitochondrial gene sequences were obtained for a single representative of each of the caecilian families found in India and aligned against previously reported sequences for 13 caecilian species. The resulting alignment (16 taxa, 1200 sites, of which 288 cannot be aligned unambiguously) was analyzed using parsimony, maximum-likelihood, and distance methods. As judged by bootstrap proportions, decay indices, and leaf stabilities, well-supported relationships of the Indian caecilians are recovered from the alignment. The data (1) corroborate the hypothesis, based on morphology, that the Uraeotyphlidae and Ichthyophiidae are sister taxa, (2) recover a monophyletic Ichthyophiidae, including Indian and South East Asian representatives, and (3) place the Indian caeciliid Gegeneophis ramaswamii as the sister group of the caeciliid caecilians of the Seychelles. Rough estimates of divergence times suggest an origin of the Uraeotyphlidae and Ichthyophiidae while India was isolated from Laurasia and Africa and are most consistent with an Indian origin of these families and subsequent dispersal of ichthyophiids into South East Asia.

  6. Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

    PubMed Central

    Leon, W; Fouts, D L; Manning, J

    1978-01-01

    Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb. Images PMID:634795

  7. Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean.

    PubMed

    Shi, Xiao Li; Lepère, Cécile; Scanlan, David J; Vaulot, Daniel

    2011-04-28

    The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

  8. Species-specificity of rRNA gene transcription in plants manifested as a switch in RNA polymerase specificity.

    PubMed Central

    Doelling, J H; Pikaard, C S

    1996-01-01

    Rapid evolution of ribosomal RNA (rRNA) gene promoters often prevents their recognition in a foreign species. Unlike animal systems, we show that foreign plant rRNA gene promoters are recognized in an alien species, but tend to program transcription by a different polymerase. In plants, RNA polymerase I transcripts initiate at a TATATA element (+1 is underlined) important for promoter strength and start-site selection. However, transcripts initiate from +32 following transfection of a tomato promoter into Arabidopsis. The rRNA gene promoter of a more closely related species, Brassica oleracea, programs both +1 and +29 transcription. A point mutation at +2 improving the identity between the Brassica and Arabidopsis promoters increases +1 transcription, indicating a role for the initiator element in species-specificity. Brassica +29 transcripts can be translated to express a luciferase reporter gene, implicating RNA polymerase II. TATA mutations that disrupt TATA-binding protein (TBP) interactions inhibit +29 transcription and luciferase expression. Co-expressed TBP proteins bearing compensatory mutations restore +29 transcription and luciferase activity, suggesting a direct TBP-TATA interaction. Importantly, +1 transcription is unaffected by the TATA mutations, suggesting that in the context of pol I recognition, the TATA-containing initiator element serves a function other than TBP binding. PMID:8972859

  9. Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene phylochip and mapping of signal intensities.

    PubMed

    Metfies, Katja; Medlin, Linda K

    2008-05-01

    DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the

  10. Molecular characterization of the full-length 23S and 5S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis.

    PubMed

    Tazumi, Akihiro; Saito, Satoru; Sekizuka, Tsuyoshi; Murayama, Ohoshi; Takamiya, Shinzaburo; Moore, John E; Millar, B Cherie; Matsuda, Motoo

    2007-08-01

    An approximately 4.2 kbp region encoding 23S and 5S rRNA genes was identified when recombinant plasmid DNAs from two genomic DNA libraries and an inverse PCR product of Taylorella asinigenitalis UK-1 isolate were analyzed. Full-length genes of 23S rRNA (3,225 bp) and 5S rRNA (117 bp) of T. asinigenitalis are described. The present sequence analysis identified a non-coding hypothetically intrinsic transcription terminator region downstream of the 5S rRNA gene. The sequence, however, downstream of the 5S rRNA gene did not show any distal tRNA genes. Surprisingly, an intervening sequence (IVS) of 270 bp in length, including two specific tandem repeat units of 80 bp and one partial unit of 48 bp with unknown functions was identified in the first quarter of the 23S rRNA gene sequence. A second IVS of 70 bp in length was also identified in the central region of the 23S rRNA gene. In addition, by using PCR and sequencing procedures, two T. asinigenitalis isolates, UK-1 and UK-2, carried multiple IVSs in the first quarter and central regions. Moreover, the 23S rRNA fragmentation occurred in the UK-1 isolate. A phylogenetic analysis was first carried out based on the 23S rRNA sequence data from T. asinigenitalis UK-1 and 13 other beta-Proteobacteria. This is the first report of IVSs in the 23S rRNA gene from the beta-Proteobacteria.

  11. 16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns

    PubMed Central

    Rundell, Ethan A.; Banta, Lois M.; Ward, Doyle V.; Watts, Corey D.; Birren, Bruce; Esteban, David J.

    2014-01-01

    A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities. PMID:25101630

  12. Analysis of rRNA gene methylation in Arabidopsis thaliana by CHEF-Conventional 2D gel electrophoresis

    PubMed Central

    Mohannath, Gireesha; Pikaard, Craig S.

    2017-01-01

    Summary Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb to 9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes and sub-chromosomal DNA fragments, etc. Here we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~ 4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes. PMID:27576719

  13. [Phylogeny of protostome moulting animals (Ecdysozoa) inferred from 18 and 28S rRNA gene sequences].

    PubMed

    Petrov, N B; Vladychenskaia, N S

    2005-01-01

    Reliability of reconstruction of phylogenetic relationships within a group of protostome moulting animals was evaluated by means of comparison of 18 and 28S rRNA gene sequences sets both taken separately and combined. Reliability of reconstructions was evaluated by values of the bootstrap support of major phylogenetic tree nodes and by degree of congruence of phylogenetic trees inferred by various methods. By both criteria, phylogenetic trees reconstructed from the combined 18 and 28S rRNA gene sequences were better than those inferred from 18 and 28S sequences taken separately. Results obtained are consistent with phylogenetic hypothesis separating protostome animals into two major clades, moulting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, Crustacea + Hexapoda) and unmoulting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, Sipuncula). Clade Cephalorhyncha does not include nematomorphs (Nematomorpha). Conclusion was taken that it is necessary to use combined 18 and 28S data in phylogenetic studies.

  14. Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.

    PubMed

    Mohannath, Gireesha; Pikaard, Craig S

    2016-01-01

    Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb-9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

  15. Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

    PubMed Central

    Laguerre, Gisèle; Allard, Marie-Reine; Revoy, Françoise; Amarger, Noelle

    1994-01-01

    Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. Images PMID:16349165

  16. 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

  17. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB

    PubMed Central

    DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G. L.

    2006-01-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses 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. PMID:16820507

  18. A comprehensive evaluation of the sl1p pipeline for 16S rRNA gene sequencing analysis.

    PubMed

    Whelan, Fiona J; Surette, Michael G

    2017-08-14

    Advances in next-generation sequencing technologies have allowed for detailed, molecular-based studies of microbial communities such as the human gut, soil, and ocean waters. Sequencing of the 16S rRNA gene, specific to prokaryotes, using universal PCR primers has become a common approach to studying the composition of these microbiota. However, the bioinformatic processing of the resulting millions of DNA sequences can be challenging, and a standardized protocol would aid in reproducible analyses. The short-read library 16S rRNA gene sequencing pipeline (sl1p, pronounced "slip") was designed with the purpose of mitigating this lack of reproducibility by combining pre-existing tools into a computational pipeline. This pipeline automates the processing of raw 16S rRNA gene sequencing data to create human-readable tables, graphs, and figures to make the collected data more readily accessible. Data generated from mock communities were compared using eight OTU clustering algorithms, two taxon assignment approaches, and three 16S rRNA gene reference databases. While all of these algorithms and options are available to sl1p users, through testing with human-associated mock communities, AbundantOTU+, the RDP Classifier, and the Greengenes 2011 reference database were chosen as sl1p's defaults based on their ability to best represent the known input communities. sl1p promotes reproducible research by providing a comprehensive log file, and reduces the computational knowledge needed by the user to process next-generation sequencing data. sl1p is freely available at https://bitbucket.org/fwhelan/sl1p .

  19. Assessing hog lagoon waste contamination in the Cape Fear Watershed using Bacteroidetes 16S rRNA gene pyrosequencing.

    PubMed

    Arfken, Ann M; Song, Bongkeun; Mallin, Michael A

    2015-09-01

    Hog lagoons can be major sources of waste and nutrient contamination to watersheds adjacent to pig farms. Fecal source tracking methods targeting Bacteroidetes 16S rRNA genes in pig fecal matter may underestimate or fail to detect hog lagoon contamination in riverine environments. In order to detect hog lagoon wastewater contamination in the Cape Fear Watershed, where a large number of hog farms are present, we conducted pyrosequencing analyses of Bacteroidetes 16S rRNA genes in hog lagoon waste and identified new hog lagoon-specific marker sequences. Additional pyrosequencing analyses of Bacteroidetes 16S rRNA genes were conducted with surface water samples collected at 4 sites during 5 months in the Cape Fear Watershed. Using an operational taxonomic unit (OTU) identity cutoff value of 97 %, these newly identified hog lagoon markers were found in 3 of the river samples, while only 1 sample contained the pig fecal marker. In the sample containing the pig fecal marker, there was a relatively high percentage (14.1 %) of the hog lagoon markers and a low pig fecal marker relative abundance of 0.4 % in the Bacteroidetes 16S rRNA gene sequences. This suggests that hog lagoon contamination must be somewhat significant in order for pig fecal markers to be detected, and low levels of hog lagoon contamination cannot be detected targeting only pig-specific fecal markers. Thus, new hog lagoon markers have a better detection capacity for lagoon waste contamination, and in conjunction with a pig fecal marker, provide a more comprehensive and accurate detection of hog lagoon waste contamination in susceptible watersheds.

  20. Identification of culturable stream water bacteria from urban, agricultural, and forested watersheds using 16S rRNA gene sequencing

    Treesearch

    Kenneth T. Belt; Christina Hohn; Aiah Gbakima; James A. Higgins

    2007-01-01

    Bacteria present in water samples taken on a weekly basis, from June 2004 through June 2005, from three streams, were cultured on Coliscan® Easygel® agar plates. Colonies representative of a variety of colors and morphologies were subjected to amplification and sequencing of a 1000-1100 nt portion of the 16S rRNA gene. A total of 528 colonies were...

  1. 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

  2. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras.

    PubMed

    Ashelford, Kevin E; Chuzhanova, Nadia A; Fry, John C; Jones, Antonia J; Weightman, Andrew J

    2006-09-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

  3. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  4. New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain Chimeras†

    PubMed Central

    Ashelford, Kevin E.; Chuzhanova, Nadia A.; Fry, John C.; Jones, Antonia J.; Weightman, Andrew J.

    2006-01-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/. PMID:16957188

  5. 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.

  6. Testing evolutionary models to explain the process of nucleotide substitution in gut bacterial 16S rRNA gene sequences.

    PubMed

    Garcia-Mazcorro, Jose F

    2013-09-01

    The 16S rRNA gene has been widely used as a marker of gut bacterial diversity and phylogeny, yet we do not know the model of evolution that best explains the differences in its nucleotide composition within and among taxa. Over 46 000 good-quality near-full-length 16S rRNA gene sequences from five bacterial phyla were obtained from the ribosomal database project (RDP) by study and, when possible, by within-study characteristics (e.g. anatomical region). Using alignments (RDPX and MUSCLE) of unique sequences, the FINDMODEL tool available at http://www.hiv.lanl.gov/ was utilized to find the model of character evolution (28 models were available) that best describes the input sequence data, based on the Akaike information criterion. The results showed variable levels of agreement (from 33% to 100%) in the chosen models between the RDP-based and the MUSCLE-based alignments among the taxa. Moreover, subgroups of sequences (using either alignment method) from the same study were often explained by different models. Nonetheless, the different representatives of the gut microbiota were explained by different proportions of the available models. This is the first report using evolutionary models to explain the process of nucleotide substitution in gut bacterial 16S rRNA gene sequences. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  7. Clinical Fusobacterium mortiferum Isolates Cluster with Undifferentiated Clostridium rectum Species Based on 16S rRNA Gene Phylogenetic Analysis.

    PubMed

    Lee, Yangsoon; Eun, Chang Soo; Han, Dong Soo

    2016-05-01

    The most commonly encountered clinical Fusobacterium species are F. nucleatum and F. necrophorum; other Fusobacteria, such as F. mortiferum and F. varium, have occasionally been isolated from human specimens. Clostridium rectum is a gram-positive species characterized as a straight bacillus with oval sub-terminal spores. The close 16S rRNA gene sequence relationship of C. rectum with the genus Fusobacterium is unexpected given their very different phenotypic characteristics. Between 2014 and 2015, a total of 19 Fusobacterium isolates were recovered from the colonic tissue of 10 patients at a university hospital. All isolates were identified based on 16S rRNA gene sequencing. The phylogenetic relationship among these isolates was estimated using the neighbor-joining method and the Molecular Evolutionary Genetic Analysis (MEGA) version 6. Based on phylogenetic analysis, the F. mortiferum isolates clustered into two groups - F. mortiferum DSM 19809 (group I) and F. mortiferum ATCC 25557 (group II) - even though they are of the same species. Furthermore, the F. mortiferum DSM 19809 (group I) showed a close phylogenetic relationship with C. rectum, even though C. rectum is classified as a gram-positive spore-producing bacillus. C. rectum is clearly unrelated to the genus Clostridium as it shows highest 16S rRNA gene sequence similarity with species from the genus Fusobacterium Therefore, additional methods such as Gram staining and other biochemical methods should be performed for Fusobacterium identification. © 2016 by the Association of Clinical Scientists, Inc.

  8. Concurrent speciation in the eastern woodland salamanders (Genus Plethodon):DNA sequences of the complete albumin nuclear and partialmitochondrial 12s genes

    USGS Publications Warehouse

    Highton, Richard; Hastings, Amy Picard; Palmer, Catherine; Watts, Richard; Hass, Carla A.; Culver, Melanie; Arnold, Stevan

    2012-01-01

    Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.

  9. Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes.

    PubMed

    Highton, Richard; Hastings, Amy Picard; Palmer, Catherine; Watts, Richard; Hass, Carla A; Culver, Melanie; Arnold, Stevan J

    2012-05-01

    Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time. Copyright © 2012. Published by Elsevier Inc.

  10. Diversity and depth-specific distribution of SAR11 cluster rRNA genes from marine planktonic bacteria

    SciTech Connect

    Field, K.G.; Gordon, D.; Wright, T.

    1997-01-01

    Small-subunit (SSU) ribosomal DNA (rDNA) gene clusters are phylogenetically related sets of SSU rRNA genes, commonly encountered in genes amplified from natural populations. Genetic variability in gene clusters could result form artifacts (polymerase error or PCR chimera formation), microevolution (variation among rrn copies within strains), or macroevolution (genetic divergence correlated with long-term evolutionary divergence). To better understand gene clusters, this study assessed genetic diversity and distribution of a single environmental SSU rDNA gene cluster, the SAR11 cluster. SAR11 cluster genes, from an uncultured group of the {alpha} subclass of the class Proteobacteria, have been recovered from coastal and midoceanic waters of the North Atlantic and Pacific. We cloned and bidirectionally sequenced 23 new SAR11 cluster 16S rRNA genes, from 80 and 250 m im the Sargasso Sea and from surface coastal waters of the Atlantic and Pacific, and analyzed them with previously published sequences. Two SAR11 genes were obviously PCR chimeras, but the biological (nonchimeric) origins of most subgroups within the cluster were confirmed by independent recovery from separate gene libraries. Using group-specific oligonucleotide probes, we analyzed depth profiles of nucleic acids, targeting both amplified rDNAs and bulk RNAs. Two subgroups within the SAR11 cluster showed different highly depth-specific distributions. We conclude that some of the genetic diversity within the SAR11 gene cluster represents macroevolutionary divergence correlated with niche specialization. Furthermore, we demonstrate the utility for marine microbial ecology of oligonucleotide probes based on gene sequences amplified from natural populations and show that a detailed knowledge of sequence variability may be needed to effectively design these probes. 48 refs., 7 figs., 3 tabs.

  11. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis.

    PubMed

    Virtanen, Seppo; Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal microbiota studies employing different

  12. Comparative analysis of vaginal microbiota sampling using 16S rRNA gene analysis

    PubMed Central

    Kalliala, Ilkka; Nieminen, Pekka; Salonen, Anne

    2017-01-01

    Background Molecular methods such as next-generation sequencing are actively being employed to characterize the vaginal microbiota in health and disease. Previous studies have focused on characterizing the biological variation in the microbiota, and less is known about how factors related to sampling contribute to the results. Our aim was to investigate the impact of a sampling device and anatomical sampling site on the quantitative and qualitative outcomes relevant for vaginal microbiota research. We sampled 10 Finnish women representing diverse clinical characteristics with flocked swabs, the Evalyn® self-sampling device, sterile plastic spatulas and a cervical brush that were used to collect samples from fornix, vaginal wall and cervix. Samples were compared on DNA and protein yield, bacterial load, and microbiota diversity and species composition based on Illumina MiSeq sequencing of the 16S rRNA gene. We quantified the relative contributions of sampling variables versus intrinsic variables in the overall microbiota variation, and evaluated the microbiota profiles using several commonly employed metrics such as alpha and beta diversity as well as abundance of major bacterial genera and species. Results The total DNA yield was strongly dependent on the sampling device and to a lesser extent on the anatomical site of sampling. The sampling strategy did not affect the protein yield or the bacterial load. All tested sampling methods produced highly comparable microbiota profiles based on MiSeq sequencing. The sampling method explained only 2% (p-value = 0.89) of the overall microbiota variation, markedly surpassed by intrinsic factors such as clinical status (microscopy for bacterial vaginosis 53%, p = 0.0001), bleeding (19%, p = 0.0001), and the variation between subjects (11%, p-value 0.0001). Conclusions The results indicate that different sampling strategies yield comparable vaginal microbiota composition and diversity. Hence, past and future vaginal

  13. Increased expression of LD1 genes transcribed by RNA polymerase I in Leishmania donovani as a result of duplication into the rRNA gene locus

    SciTech Connect

    Lodes, M.J.; Merlin, G.; DeVos, T.

    1995-12-01

    This report investigates the duplication of two LD1 genes into the rRNA locus and the resultant transcription by RNA polymerase I, which has a faster transcription rate than that of RNA polymerase II. This was conducted using a 2.2-Mb chromosome in Leishmania donovani. 55 refs., 6 figs.

  14. Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR.

    PubMed

    Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; Schrenzel, Jacques

    2016-04-23

    Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators. For in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3-4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures. Removal of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.

  15. Complete ecological isolation and cryptic diversity in Polynucleobacter bacteria not resolved by 16S rRNA gene sequences

    PubMed Central

    Hahn, Martin W; Jezberová, Jitka; Koll, Ulrike; Saueressig-Beck, Tanja; Schmidt, Johanna

    2016-01-01

    Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences. PMID:26943621

  16. Nearly complete rRNA genes from 371 Animalia: updated structure-based alignment and detailed phylogenetic analysis.

    PubMed

    Mallatt, Jon; Craig, Catherine Waggoner; Yoder, Matthew J

    2012-09-01

    This study presents a manually constructed alignment of nearly complete rRNA genes from most animal clades (371 taxa from ~33 of the ~36 metazoan phyla), expanded from the 197 sequences in a previous study. This thorough, taxon-rich alignment, available at http://www.wsu.edu/~jmallatt/research/rRNAalignment.html and in the Dryad Repository (doi: http://dx.doi.org/10.5061/dryad.1v62kr3q), is based rigidly on the secondary structure of the SSU and LSU rRNA molecules, and is annotated in detail, including labeling of the erroneous sequences (contaminants). The alignment can be used for future studies of the molecular evolution of rRNA. Here, we use it to explore if the larger number of sequences produces an improved phylogenetic tree of animal relationships. Disappointingly, the resolution did not improve, neither when the standard maximum-likelihood method was used, nor with more sophisticated methods that partitioned the rRNA into paired and unpaired sites (stem, loop, bulge, junction), or accounted for the evolution of the paired sites. For example, no doublet model of paired-site substitutions (16-state, 16A and 16B, 7A-F, or 6A-C models) corrected the placement of any rogue taxa or increased resolution. The following findings are from the simplest, standard, ML analysis. The 371-taxon tree only imperfectly supported the bilaterian clades of Lophotrochozoa and Ecdysozoa, and this problem remained after 17 taxa with unstably positioned sequences were omitted from the analysis. The problem seems to stem from base-compositional heterogeneity across taxa and from an overrepresentation of highly divergent sequences among the newly added taxa (e.g., sequences from Cephalopoda, Rotifera, Acoela, and Myxozoa). The rogue taxa continue to concentrate in two locations in the rRNA tree: near the base of Arthropoda and of Bilateria. The approximately uncertain (AU) test refuted the monophyly of Mollusca and of Chordata, probably due to long-branch attraction of the highly

  17. Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa.

    PubMed

    Bhoora, Raksha; Franssen, Linda; Oosthuizen, Marinda C; Guthrie, Alan J; Zweygarth, Erich; Penzhorn, Barend L; Jongejan, Frans; Collins, Nicola E

    2009-02-05

    A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximately 1600bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.

  18. Effects of Growth Conditions on Expression of Mycobacterial murA and tyrS Genes and Contributions of Their Transcripts to Precursor rRNA Synthesis

    PubMed Central

    Gonzalez-y-Merchand, J. A.; Colston, M. J.; Cox, R. A.

    1999-01-01

    All mycobacteria studied to date have an rRNA operon, designated rrnA, located downstream from a single copy of the murA gene, which encodes an enzyme (EC 2.5.1.7) important for peptidoglycan synthesis. The rrnA operon has a promoter, P1(A), located within the coding region of murA, near the 3′ end. Samples of RNA were isolated from Mycobacterium tuberculosis at different stages of the growth cycle and from Mycobacterium smegmatis grown under different conditions. RNase protection assays were used to investigate transcripts of both murA and rrnA. Transcription of murA was found to continue into the 16S rRNA gene, as if murA and rrnA form a hybrid (protein coding-rRNA coding) operon. During the growth of M. tuberculosis, the hybrid operon contributed approximately 2% to total pre-rRNA. Analysis of M. smegmatis RNA revealed that the level of murA RNA depended on the growth rate and that the patterns of expression during the growth cycle were different for murA and rrnA. M. smegmatis has a second rRNA operon, rrnB, located downstream from a single copy of the tyrS gene, encoding tyrosyl-tRNA synthetase. Transcription of tyrS was found to continue into the 16S rRNA gene rrnB. The hybrid tyrS-rrnB operon contributed 0.2 to 0.6% to rrnB transcripts. The pattern of tyrS expression during the growth cycle matched the pattern of rrnB expression, reflecting the essential role of TyrS and rRNA in protein biosynthesis. PMID:10419962

  19. Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group

    PubMed Central

    Hakovirta, Janetta R.; Prezioso, Samantha; Hodge, David; Pillai, Segaran P.

    2016-01-01

    Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group, i.e., Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis. These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific to B. anthracis was detected in some B. cereus strains. However, a different SNP, at position 1139, was identified as being specific to B. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories. PMID:27582514

  20. Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group.

    PubMed

    Hakovirta, Janetta R; Prezioso, Samantha; Hodge, David; Pillai, Segaran P; Weigel, Linda M

    2016-11-01

    Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group, i.e., Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific to B. anthracis was detected in some B. cereus strains. However, a different SNP, at position 1139, was identified as being specific to B. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  1. A Bayesian taxonomic classification method for 16S rRNA gene sequences with improved species-level accuracy.

    PubMed

    Gao, Xiang; Lin, Huaiying; Revanna, Kashi; Dong, Qunfeng

    2017-05-10

    Species-level classification for 16S rRNA gene sequences remains a serious challenge for microbiome researchers, because existing taxonomic classification tools for 16S rRNA gene sequences either do not provide species-level classification, or their classification results are unreliable. The unreliable results are due to the limitations in the existing methods which either lack solid probabilistic-based criteria to evaluate the confidence of their taxonomic assignments, or use nucleotide k-mer frequency as the proxy for sequence similarity measurement. We have developed a method that shows significantly improved species-level classification results over existing methods. Our method calculates true sequence similarity between query sequences and database hits using pairwise sequence alignment. Taxonomic classifications are assigned from the species to the phylum levels based on the lowest common ancestors of multiple database hits for each query sequence, and further classification reliabilities are evaluated by bootstrap confidence scores. The novelty of our method is that the contribution of each database hit to the taxonomic assignment of the query sequence is weighted by a Bayesian posterior probability based upon the degree of sequence similarity of the database hit to the query sequence. Our method does not need any training datasets specific for different taxonomic groups. Instead only a reference database is required for aligning to the query sequences, making our method easily applicable for different regions of the 16S rRNA gene or other phylogenetic marker genes. Reliable species-level classification for 16S rRNA or other phylogenetic marker genes is critical for microbiome research. Our software shows significantly higher classification accuracy than the existing tools and we provide probabilistic-based confidence scores to evaluate the reliability of our taxonomic classification assignments based on multiple database matches to query sequences. Despite

  2. Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences.

    PubMed

    Kuhnert, P; Capaul, S E; Nicolet, J; Frey, J

    1996-10-01

    The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

  3. Mutations in 23S rRNA gene associated with decreased susceptibility to tiamulin and valnemulin in Mycoplasma gallisepticum.

    PubMed

    Li, Bei-Bei; Shen, Jian-Zhong; Cao, Xing-Yuan; Wang, Yang; Dai, Lei; Huang, Si-Yang; Wu, Cong-Ming

    2010-07-01

    Mycoplasma gallisepticum is a major etiological agent of chronic respiratory disease (CRD) in chickens and sinusitis in turkeys. The pleuromutilin antibiotics tiamulin and valnemulin are currently used in the treatment of M. gallisepticum infection. We studied the in vitro development of pleuromutilin resistance in M. gallisepticum and investigated the molecular mechanisms involved in this process. Pleuromutilin-resistant mutants were selected by serial passages of M. gallisepticum strains PG31 and S6 in broth medium containing subinhibitory concentrations of tiamulin or valnemulin. A portion of the gene encoding 23S rRNA gene (domain V) and the gene encoding ribosome protein L3 were amplified and sequenced. No mutation could be detected in ribosome protein L3. Mutations were found at nucleotide positions 2058, 2059, 2061, 2447 and 2503 of 23S rRNA gene (Escherichia coli numbering). Although a single mutation could cause elevation of tiamulin and valnemulin MICs, combinations of two or three mutations were necessary to produce high-level resistance. All the mutants were cross-resistant to lincomycin, chloramphenicol and florfenicol. Mutants with the A2058G or the A2059G mutation exhibited cross-resistance to macrolide antibiotics erythromycin, tilmicosin and tylosin.

  4. Morphology and small subunit (SSU) rRNA gene sequence of the new brackish water ciliate Neobakuella flava n. g., n. sp. (Ciliophora, Spirotricha, Bakuellidae) and SSU rRNA gene sequences of six additional hypotrichs from Korea.

    PubMed

    Li, Liqiong; Khan, Sadia Nawroz; Ji, Daode; Shin, Mann Kyoon; Berger, Helmut

    2011-01-01

    The morphology and the small subunit (SSU) rRNA gene sequence of the hypotrich Neobakuella flava n. g., n. sp. from the estuary of the Taehwagang River (Ulsan, South Korea) were investigated. The three frontal cirri, the composition of the midventral complex of cirral pairs and rows, and the simple dorsal kinety pattern of three bipolar kineties assign it to the urostyloid taxon Bakuellidae. The increased number of buccal and parabuccal cirri, the presence of transverse cirri, and more than one left marginal row, as well as the lack of caudal cirri separate Neobakuella n. g. from the other bakuellids. Neobakuella flava n. sp. has many 0.3 μm sized green and/or yellow usually dark-green cortical granules and some sparsely distributed, 2 × 1 μm sized grass green with yellowish shimmer granules. The gene sequence data indicate a close relationship with Diaxonella and a distinct separation from the bakuellid Metaurostylopsis and parabirojimid Parabirojimia. The SSU rRNA gene sequences of four further urostyloids (i.e. Diaxonella pseudorubra, Anteholosticha monilata, Metaurostylopsis struederkypkeae, Pseudourostyla cristata) and two stylonychines (i.e. Sterkiella cavicola, Sterkiella histriomuscorum) from Korea were analyzed. Anteholosticha monilata, type of the genus, is clearly separated from the Holosticha clade, supporting the morphological separation from Holosticha. Sterkiella cavicola, type of Sterkiella, clusters within the stylonychines and is obviously closely related with S. histriomuscorum.

  5. Accurate identification of fastidious Gram-negative rods: integration of both conventional phenotypic methods and 16S rRNA gene analysis

    PubMed Central

    2013-01-01

    Background Accurate identification of fastidious Gram-negative rods (GNR) by conventional phenotypic characteristics is a challenge for diagnostic microbiology. The aim of this study was to evaluate the use of molecular methods, e.g., 16S rRNA gene sequence analysis for identification of fastidious GNR in the clinical microbiology laboratory. Results A total of 158 clinical isolates covering 20 genera and 50 species isolated from 1993 to 2010 were analyzed by comparing biochemical and 16S rRNA gene sequence analysis based identification. 16S rRNA gene homology analysis identified 148/158 (94%) of the isolates to species level, 9/158 (5%) to genus and 1/158 (1%) to family level. Compared to 16S rRNA gene sequencing as reference method, phenotypic identification correctly identified 64/158 (40%) isolates to species level, mainly Aggregatibacter aphrophilus, Cardiobacterium hominis, Eikenella corrodens, Pasteurella multocida, and 21/158 (13%) isolates correctly to genus level, notably Capnocytophaga sp.; 73/158 (47%) of the isolates were not identified or misidentified. Conclusions We herein propose an efficient strategy for accurate identification of fastidious GNR in the clinical microbiology laboratory by integrating both conventional phenotypic methods and 16S rRNA gene sequence analysis. We conclude that 16S rRNA gene sequencing is an effective means for identification of fastidious GNR, which are not readily identified by conventional phenotypic methods. PMID:23855986

  6. Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1.

    PubMed

    Fairley, Jennifer A; Mitchell, Louise E; Berg, Tracy; Kenneth, Niall S; von Schubert, Conrad; Silljé, Herman H W; Medema, René H; Nigg, Erich A; White, Robert J

    2012-02-24

    Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  8. Phylogeny of intestinal ciliates, including Charonina ventriculi, and comparison of microscopy and 18S rRNA gene pyrosequencing for rumen ciliate community structure analysis.

    PubMed

    Kittelmann, Sandra; Devente, Savannah R; Kirk, Michelle R; Seedorf, Henning; Dehority, Burk A; Janssen, Peter H

    2015-04-01

    The development of high-throughput methods, such as the construction of 18S rRNA gene clone or pyrosequencing libraries, has allowed evaluation of ciliate community composition in hundreds of samples from the rumen and other intestinal habitats. However, several genera of mammalian intestinal ciliates have been described based only on morphological features and, to date, have not been identified using molecular methods. Here, we isolated single cells of one of the smallest but widely distributed intestinal ciliates, Charonina ventriculi, and sequenced its 18S rRNA gene. We verified the sequence in a full-cycle rRNA approach using fluorescence in situ hybridization and thereby assigned an 18S rRNA gene sequence to this species previously known only by its morphology. Based on its full-length 18S rRNA gene sequence, Charonina ventriculi was positioned within the phylogeny of intestinal ciliates in the subclass Trichostomatia. The taxonomic framework derived from this phylogeny was used for taxonomic assignment of trichostome ciliate 18S rRNA gene sequence data stemming from high-throughput amplicon pyrosequencing of rumen-derived DNA samples. The 18S rRNA gene-based ciliate community structure was compared to that obtained from microscopic counts using the same samples. Both methods allowed identification of dominant members of the ciliate communities and classification of the rumen ciliate community into one of the types first described by Eadie in 1962. Notably, each method is associated with advantages and disadvantages. Microscopy is a highly accurate method for evaluation of total numbers or relative abundances of different ciliate genera in a sample, while 18S rRNA gene pyrosequencing represents a valuable alternative for comparison of ciliate community structure in a large number of samples from different animals or treatment groups. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Using DGGE and 16S rRNA Gene Sequence Analysis to Evaluate Changes in Oral Bacterial Composition

    PubMed Central

    CHEN, Zhou; TRIVEDI, Harsh M.; CHHUN, Nok; BARNES, Virginia M.; SAXENA, Deepak; XU, Tao; LI, Yihong

    2015-01-01

    Objective To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Methods Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles of PCR-amplified and 16S rRNA gene segments sequence analysis. Results Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified six phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. Conclusion The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/0.243% sodium fluoride may promote a healthier composition within the oral bacterial community. PMID:22319750

  10. Bacteria and Bacterial rRNA Genes Associated with the Development of Colitis in IL-10–/– Mice

    PubMed Central

    Presley, Laura L.; Bent, Elizabeth; Wei, Bo; Braun, Jonathan; Schiller, Neal L.; Straus, Daniel S.; Borneman, James

    2013-01-01

    Background Microorganisms appear to play important yet ill-defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population-based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10–/– mice. Methods Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. Results Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture-independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). Conclusions The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease-associated immunity in murine immune colitis and human Crohn's disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host–microbe interactions underlying IBD. PMID:18381614

  11. Microbial Dark Matter: Unusual intervening sequences in 16S rRNA genes of candidate phyla from the deep subsurface

    SciTech Connect

    Jarett, Jessica; Stepanauskas, Ramunas; Kieft, Thomas; Onstott, Tullis; Woyke, Tanja

    2014-03-17

    The Microbial Dark Matter project has sequenced genomes from over 200 single cells from candidate phyla, greatly expanding our knowledge of the ecology, inferred metabolism, and evolution of these widely distributed, yet poorly understood lineages. The second phase of this project aims to sequence an additional 800 single cells from known as well as potentially novel candidate phyla derived from a variety of environments. In order to identify whole genome amplified single cells, screening based on phylogenetic placement of 16S rRNA gene sequences is being conducted. Briefly, derived 16S rRNA gene sequences are aligned to a custom version of the Greengenes reference database and added to a reference tree in ARB using parsimony. In multiple samples from deep subsurface habitats but not from other habitats, a large number of sequences proved difficult to align and therefore to place in the tree. Based on comparisons to reference sequences and structural alignments using SSU-ALIGN, many of these ?difficult? sequences appear to originate from candidate phyla, and contain intervening sequences (IVSs) within the 16S rRNA genes. These IVSs are short (39 - 79 nt) and do not appear to be self-splicing or to contain open reading frames. IVSs were found in the loop regions of stem-loop structures in several different taxonomic groups. Phylogenetic placement of sequences is strongly affected by IVSs; two out of three groups investigated were classified as different phyla after their removal. Based on data from samples screened in this project, IVSs appear to be more common in microbes occurring in deep subsurface habitats, although the reasons for this remain elusive.

  12. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

    PubMed

    Schloss, Patrick D; Jenior, Matthew L; Koumpouras, Charles C; Westcott, Sarah L; Highlander, Sarah K

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

  13. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    PubMed

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  14. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

    PubMed Central

    Jenior, Matthew L.; Koumpouras, Charles C.; Westcott, Sarah L.; Highlander, Sarah K.

    2016-01-01

    Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting. PMID:27069806

  15. 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.

  16. Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes

    SciTech Connect

    Gihring, Thomas; Green, Stefan; Schadt, Christopher Warren

    2011-01-01

    Technologies for massively parallel sequencing are revolutionizing microbial ecology and are vastly increasing the scale of ribosomal RNA (rRNA) gene studies. Although pyrosequencing has increased the breadth and depth of possible rRNA gene sampling, one drawback is that the number of reads obtained per sample is difficult to control. Pyrosequencing libraries typically vary widely in the number of sequences per sample, even within individual studies, and there is a need to revisit the behaviour of richness estimators and diversity indices with variable gene sequence library sizes. Multiple reports and review papers have demonstrated the bias in non-parametric richness estimators (e.g. Chao1 and ACE) and diversity indices when using clone libraries. However, we found that biased community comparisons are accumulating in the literature. Here we demonstrate the effects of sample size on Chao1, ACE, CatchAll, Shannon, Chao-Shen and Simpson's estimations specifically using pyrosequencing libraries. The need to equalize the number of reads being compared across libraries is reiterated, and investigators are directed towards available tools for making unbiased diversity comparisons.

  17. Formation of nucleolar polymorphisms in trisomic chickens and subsequent microevolution of rRNA gene clusters in diploids.

    PubMed

    Delany, M E; Muscarella, D E; Bloom, S E

    1991-01-01

    Variations in nucleolar size are common in animals and man, yet the basis and significance of this variation are not well understood. In this report, we describe the generation de novo of individuals that express nucleolar size variations (polymorphisms) and the underlying basis for this phenotype in a vertebrate animal system (Gallus domesticus). Individuals that express nucleolar size polymorphisms were produced from mating chickens trisomic for the nucleolar organizer (NO) chromosome; 10%-18% of progeny demonstrated nucleolar polymorphisms. These progeny were incorporated into a diploid genetic line in which the polymorphic trait was observed to segregate in Mendelian fashion. An even more dramatic nucleolar size polymorphism (one macro- plus one micronucleolus) evolved in one diploid family over the course of only two generations. These individuals were used to ascertain that the polymorphic-nucleoli phenotype was expressed in tissues derived from the three primary embryonic cell layers in embryos and neonates. Image analysis was conducted on cells of these birds to quantitate the size differences between macro- and micronucleoli (5 mu2 versus 1 mu2, respectively). Finally, these birds were studied with the technique of in situ hybridization, which showed that gene number differences between homologous NO chromosomes (i.e., heterozygosity for rRNA gene copy number), underlies the polymorphic-nucleoli phenotype. Thus, the chicken emerges as an experimental system through which heterozygosity for the rRNA gene copy number can be induced, easily identified, transmitted, and expressed in all somatic tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair.

    PubMed

    Meier, Andreas; Thoma, Fritz

    2005-03-01

    UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

  19. Application of SmartGene IDNS software to partial 16S rRNA gene sequences for a diverse group of bacteria in a clinical laboratory.

    PubMed

    Simmon, Keith E; Croft, Ann C; Petti, Cathy A

    2006-12-01

    Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories.

  20. Application of SmartGene IDNS Software to Partial 16S rRNA Gene Sequences for a Diverse Group of Bacteria in a Clinical Laboratory▿

    PubMed Central

    Simmon, Keith E.; Croft, Ann C.; Petti, Cathy A.

    2006-01-01

    Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories. PMID:17050811

  1. Molecular phylogenetics of the spider family Micropholcommatidae (Arachnida: Araneae) using nuclear rRNA genes (18S and 28S).

    PubMed

    Rix, Michael G; Harvey, Mark S; Roberts, J Dale

    2008-03-01

    The spider family Micropholcommatidae is an enigmatic taxon of uncertain limits and uncertain affinities. Various phylogenetic hypotheses have been proposed for the family, but these hypotheses have never been tested with a robust phylogenetic analysis. The existence of similar Australasian and New World taxa, the possibility of morphological convergence associated with extreme 'smallness', and the apparent paucity of synapomorphic morphological characters, have all clouded generic relationships in this group. We used fragments from two nuclear ribosomal RNA genes (18S and 28S) to test the monophyly and phylogenetic position of the Micropholcommatidae. The analyses incorporated 50 ingroup spider species, including 23 micropholcommatid species and representatives from 14 other spider families. Ribosomal RNA secondary structures were inferred for the V3-V5 region of the 18S rRNA gene, and Domain II of the 28S rRNA gene of Hickmania troglodytes [Higgins, E.T., Petterd, W.F., 1883. Description of a new cave-inhabiting spider, together with notes on mammalian remains from a recently discovered cave in the Chudleigh district. Pap. Proc. R. Soc. Tasman. 1882, 191-192]. These secondary structures were used to guide multiple sequence alignments, and determine the position and nature of indels in different taxa. Secondary structure information was also incorporated into a structurally partitioned rRNA analysis in MrBayes Version 3.1.2, using a doublet model of nucleotide substitution. This structurally partitioned rRNA analysis provided a less resolved but more conservative and informative estimate of phylogeny than an otherwise identical, unpartitioned rDNA analysis. With the exception of the Chilean species Teutoniella cekalovici [Platnick, N.I., Forster, R.R., 1986. On Teutoniella, an American genus of the spider family Micropholcommatidae (Araneae, Palpimanoidea). Am. Mus. Novit. 2854, 1-9], the family Micropholcommatidae was found to be monophyletic with three

  2. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences

    PubMed Central

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-01-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species. PMID:25313278

  3. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons

    PubMed Central

    Fischer, Sandra; Kumar, Neeraj

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea. PMID:28097056

  4. Rhea: a transparent and modular R pipeline for microbial profiling based on 16S rRNA gene amplicons.

    PubMed

    Lagkouvardos, Ilias; Fischer, Sandra; Kumar, Neeraj; Clavel, Thomas

    2017-01-01

    The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps, alpha- and beta-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.

  5. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    NASA Astrophysics Data System (ADS)

    Ma, Haiyan; Jiang, Guoliang; Wu, Zhiqiang; Wang, Xin

    2009-06-01

    Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

  6. Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.

    PubMed

    Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

    2014-10-01

    Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach.

  7. Burden of emerging anaerobes in the MALDI-TOF and 16S rRNA gene sequencing era.

    PubMed

    La Scola, Bernard; Fournier, Pierre-Edouard; Raoult, Didier

    2011-06-01

    The isolation of anaerobes from patients has declined in recent years, whereas their detection by molecular techniques has increased. In the present work, we analyzed the application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing to routine identification of anaerobes in clinical microbiology laboratory. We identified 544 isolates of 79 species by routine culture from deep samples in our hospital. MALDI-TOF MS allowed identification of 332 isolates (61%). The remaining 212 (39%) were identified by 16S rRNA gene sequencing, allowing identification of 202 at the species level. The most common anaerobes were Propionibacterium spp. (12%), Finegoldia magna (4%), Fusobacterium spp. (6%) and Bacteroides spp. (6%). However, among the 79 identified species, seven were new species or genera, including two Prevotella conceptionensis, a species previously detected by our team by amplification and sequencing, five Anaerococcus sp. and one Prevotella sp. Beyond the identification of these new species, we also identified several uncommon or previously not described associations between species and specific pathologic conditions. MALDI-TOF MS-based identification, which will become more effective with future spectra database improvement, will be likely responsible of a burden of emerging anaerobes in clinical microbiology. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing.

    PubMed

    Han, Mi-Soon; Kim, Hyunsoo; Lee, Yangsoon; Kim, Myungsook; Ku, Nam Su; Choi, Jun Yong; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Yunsop

    2017-01-01

    Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica, 18 (20.9%) were Elizabethkingia miricola, and 51 (59.3%) were Elizabethkingia anophelis Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.

  9. Identification and phylogeny of Arabian snakes: Comparison of venom chromatographic profiles versus 16S rRNA gene sequences.

    PubMed

    Al Asmari, Abdulrahman; Manthiri, Rajamohammed Abbas; Khan, Haseeb Ahmad

    2014-11-01

    Identification of snake species is important for various reasons including the emergency treatment of snake bite victims. We present a simple method for identification of six snake species using the gel filtration chromatographic profiles of their venoms. The venoms of Echis coloratus, Echis pyramidum, Cerastes gasperettii, Bitis arietans, Naja arabica, and Walterinnesia aegyptia were milked, lyophilized, diluted and centrifuged to separate the mucus from the venom. The clear supernatants were filtered and chromatographed on fast protein liquid chromatography (FPLC). We obtained the 16S rRNA gene sequences of the above species and performed phylogenetic analysis using the neighbor-joining method. The chromatograms of venoms from different snake species showed peculiar patterns based on the number and location of peaks. The dendrograms generated from similarity matrix based on the presence/absence of particular chromatographic peaks clearly differentiated Elapids from Viperids. Molecular cladistics using 16S rRNA gene sequences resulted in jumping clades while separating the members of these two families. These findings suggest that chromatographic profiles of snake venoms may provide a simple and reproducible chemical fingerprinting method for quick identification of snake species. However, the validation of this methodology requires further studies on large number of specimens from within and across species.

  10. Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments.

    PubMed

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.

  11. Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

    PubMed Central

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

  12. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  13. Comparative Analysis of the Methanogen Diversity in Horse and Pony by Using mcrA Gene and Archaeal 16S rRNA Gene Clone Libraries

    PubMed Central

    Lwin, Khin-Ohnmar

    2014-01-01

    Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing the α-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. The mcrA clone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P < 0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both the mcrA and the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds of Equus caballus. PMID:24678264

  14. Recognition of Potentially Novel Human Disease-Associated Pathogens by Implementation of Systematic 16S rRNA Gene Sequencing in the Diagnostic Laboratory▿ †

    PubMed Central

    Keller, Peter M.; Rampini, Silvana K.; Büchler, Andrea C.; Eich, Gerhard; Wanner, Roger M.; Speck, Roberto F.; Böttger, Erik C.; Bloemberg, Guido V.

    2010-01-01

    Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease. PMID:20631113

  15. Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21

    PubMed Central

    Gregory, Steven T.; Cate, Jamie H. D.; Dahlberg, Albert E.

    2001-01-01

    Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus. PMID:11418580

  16. [Composition of the oil-slime microbial community determined by analysis of the 16S rRNA gene].

    PubMed

    Grigor'eva, T V; Laĭkov, A V; Rizvanov, A A; Il'inskaia, O N; Naumova, R P

    2013-01-01

    Analysis of the 16S rRNA genes of the cultured microorganisms of industrial oil-slime revealed predominance (-85-90%) of the Gammaproteobacteria in the community of aerobic heterotrophs and specific oil-slime degraders. Relation of the isolated strains with members of the genera Pseudomonas, Stenotrophomonas, and Enterobacter was established. Analysis of the same gene in the total DNA from the oil-slime revealed greater microbial diversity (-20 operative taxonomic units determined by T-RFLP) than in the cultured part of the community, which included -12 different colony types. Three major restriction fragments were found, with their total area -50%. These results demonstrated the low morphological and phylogenetic diversity of the oil-slime bacterial community.

  17. 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. Copyright 1999 Academic Press.

  18. 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

  19. [Bacteria closely related to Phyllobacterium trifolii according to their 16S rRNA gene are discovered in the nodules of Hungarian sainfoin].

    PubMed

    Baĭmiev, Al Kh; Baĭmiev, An Kh; Gubaĭdullin, I I; Kulikova, O L; Chemeris, A V

    2007-05-01

    The population genetic diversity and phylogeny of the bacteria entering the symbiosis with sainfoin that grows on the Chesnokovskaya Mountain, Ufa region, Republic of Bashkortostan, have been studied. RAPD analysis of DNA polymorphism of the microbial strains grown from the nodules of 20 plants using several random primers detected a high degree of genetic homogeneity in their population as compared with the populations of rhizobia of other leguminous plants growing at the same site. Sequencing of 16S rRNA genes of the three most different samples have demonstrated that these genes were identical and display 99.9% homology with the sequence of Phyllobacterium trifolii 16S rRNA gene.

  20. Analysis of 525 Samples To Determine the Usefulness of PCR Amplification and Sequencing of the 16S rRNA Gene for Diagnosis of Bone and Joint Infections

    PubMed Central

    Fenollar, Florence; Roux, Véronique; Stein, Andréas; Drancourt, Michel; Raoult, Didier

    2006-01-01

    The 16S rRNA gene PCR in the diagnosis of bone and joint infections has not been systematically tested. Five hundred twenty-five bone and joint samples collected from 525 patients were cultured and submitted to 16S rRNA gene PCR detection of bacteria in parallel. The amplicons with mixed sequences were also cloned. When discordant results were observed, culture and PCR were performed once again. Bacteria were detected in 139 of 525 samples. Culture and 16S rRNA gene PCR yielded identical documentation in 475 samples. Discrepancies were linked to 13 false-positive culture results, 5 false-positive PCR results, 9 false-negative PCR results, 16 false-negative culture results, and 7 mixed infections. Cloning and sequencing of 16S rRNA gene amplicons in 6 of 8 patients with mixed infections identified 2 to 8 bacteria per sample. Rarely described human pathogens such as Alcaligenes faecalis, Comamonas terrigena, and 21 anaerobes were characterized. We also detected, by 16S rRNA gene PCR, four previously identified bacteria never reported in human infection, Alkanindiges illinoisensis, dehydroabietic acid-degrading bacterium DhA-73, unidentified Hailaer soda lake bacterium, and uncultured bacterium clone HuCa4. Seven organisms representing new potential species were also detected. PCR followed by cloning and sequencing may help to identify new pathogens involved in mixed bone infection. PMID:16517890

  1. Clinical and Microbiological Aspects of Linezolid Resistance Mediated by the cfr Gene Encoding a 23S rRNA Methyltransferase▿

    PubMed Central

    Arias, Cesar A.; Vallejo, Martha; Reyes, Jinnethe; Panesso, Diana; Moreno, Jaime; Castañeda, Elizabeth; Villegas, Maria V.; Murray, Barbara E.; Quinn, John P.

    2008-01-01

    The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method. PMID:18174304

  2. First description of heterogeneity in 18S rRNA genes in the haploid genome of Cryptosporidium andersoni Kawatabi type.

    PubMed

    Ikarashi, Makoto; Fukuda, Yasuhiro; Honma, Hajime; Kasai, Kenji; Kaneta, Yoshiyasu; Nakai, Yutaka

    2013-09-01

    The Apicomplexan Cryptosporidium andersoni, is a species of gastric Cryptosporidium, is frequently detected in older calves and adult cattle. Genotyping analyses based on 18S ribosomal RNA gene sequences have been performed on a novel C. andersoni genotype, namely the Kawatabi type, and the oocysts were classified into two distinct groups genotypically: Type A (the sequence in GenBank) and Type B (with a thymine nucleotide insertion not in Type A). This study analyzed 3775 cattle at a slaughterhouse and 310 cattle at a farm using microscopy and found 175 Cryptosporidium-positive animals: 171 from the slaughterhouse and four from the farm, and all infecting parasites were determined to be C. andersoni from 18S rRNA gene sequences determined from fecal DNA. In genotyping analyses with single isolated oocysts, about a half of analyzed ones were clearly classified into well known two genotypes (Type A and B). In addition to these two known genotypes, we have detected some oocysts showing mixed signals of Types A and B in the electropherogram from the automated sequencer (the Type C genotype). To determine the genotypic composition of sporozoites carried by the Type C oocysts, we analyzed their 18S rRNA gene sequences using a single sporozoite isolation procedure. Some sporozoites were classified as either Type A or Type B. However, more than half of the analyzed isolated sporozoites showed a mixed signal identical to that of Type C oocysts, and both the Type A and B signals were surely detectable from such sporozoites after a cloning procedure. In conclusion, C. andersoni carries two different genotypes heterogeneously in its haploid genome. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. 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.

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

    PubMed

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

    1996-09-01

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

  5. Differential gene expression in Neurospora crassa cell types: heterogeneity and multiple copies of rRNA genes. Annual progress report, July 1981-June 1982

    SciTech Connect

    Dutta, S.K.

    1982-01-01

    The significant results obtained were as follows: (I) Multiple copies of isolated rRNA genes from N. crassa were tested for heterogeneity by rRNA: rDNA reassociation kinetics. More than 90% of rDNA copies were identical. The possible heterogeneity of a small fraction of rDNAs could not be attributed to inclusion of any tDNA sequences. (II) Two approaches to study gross differences between rRNA genes from N. crassa cell types-conidia, germinated conidia, and mycelia were undertaken. No difference was seen in either the restriction patterns nor the autoradiographs. Either gross differences between rDNAS of N. crassa cell types were not present or they were not detected by these two approaches. (III) Using similar DNA restriction analysis procedures, differences between closely related heterothallic and homothallic species of Neurospora were detected. (IV) Successful sequencing of 317 bases of the N. crassa slime mutant pMF2 clone which includes the 5.8S rDNA and it's flanking internal spacer regions was achieved. (ERB)

  6. Pyrosequencing of mcrA and Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

    PubMed Central

    Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng

    2014-01-01

    Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield. PMID:25381241

  7. Correlation of Dehalococcoides 16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater ▿ †

    PubMed Central

    van der Zaan, Bas; Hannes, Fredericke; Hoekstra, Nanne; Rijnaarts, Huub; de Vos, Willem M.; Smidt, Hauke; Gerritse, Jan

    2010-01-01

    Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 106 gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp. PMID:20008170

  8. Correlation of Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenase genes with geochemical conditions in chloroethene-contaminated groundwater.

    PubMed

    van der Zaan, Bas; Hannes, Fredericke; Hoekstra, Nanne; Rijnaarts, Huub; de Vos, Willem M; Smidt, Hauke; Gerritse, Jan

    2010-02-01

    Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 10(6) gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp.

  9. Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae)

    PubMed Central

    Dobešová, E; Malinská, H; Matyášek, R; Leitch, A R; Soltis, D E; Soltis, P S; Kovařík, A

    2015-01-01

    To study the relationship between uniparental rDNA (encoding 18S, 5.8S and 26S ribosomal RNA) silencing (nucleolar dominance) and rRNA gene dosage, we studied a recently emerged (within the last 80 years) allotetraploid Tragopogon mirus (2n=24), formed from the diploid progenitors T. dubius (2n=12, D-genome donor) and T. porrifolius (2n=12, P-genome donor). Here, we used molecular, cytogenetic and genomic approaches to analyse rRNA gene activity in two sibling T. mirus plants (33A and 33B) with widely different rRNA gene dosages. Plant 33B had ~400 rRNA genes at the D-genome locus, which is typical for T. mirus, accounting for ~25% of total rDNA. We observed characteristic expression dominance of T. dubius-origin genes in all organs. Its sister plant 33A harboured a homozygous macrodeletion that reduced the number of T. dubius-origin genes to about 70 copies (~4% of total rDNA). It showed biparental rDNA expression in root, flower and callus, but not in leaf where D-genome rDNA dominance was maintained. There was upregulation of minor rDNA variants in some tissues. The RNA polymerase I promoters of reactivated T. porrifolius-origin rRNA genes showed reduced DNA methylation, mainly at symmetrical CG and CHG nucleotide motifs. We hypothesise that active, decondensed rDNA units are most likely to be deleted via recombination. The silenced homeologs could be used as a ‘first reserve' to ameliorate mutational damage and contribute to evolutionary success of polyploids. Deletion and reactivation cycles may lead to bidirectional homogenisation of rRNA arrays in the long term. PMID:25537492

  10. Community analysis of chronic wound bacteria using 16S rRNA gene-based pyrosequencing: impact of diabetes and antibiotics on chronic wound microbiota.

    PubMed

    Price, Lance B; Liu, Cindy M; Melendez, Johan H; Frankel, Yelena M; Engelthaler, David; Aziz, Maliha; Bowers, Jolene; Rattray, Rogan; Ravel, Jacques; Kingsley, Chris; Keim, Paul S; Lazarus, Gerald S; Zenilman, Jonathan M

    2009-07-31

    Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.

  11. Technical considerations in the use of 18s rRNA in gene expression studies

    EPA Science Inventory

    Gene expression analysis is now commonly used in ecotoxicological studies to indicate exposure of an organism to xenobiotics. For example, the vitellogenin gene is used to diagnose exposure of fish to environmental estrogens. Reverse transcription polymerase chain reaction (RT-PC...

  12. Technical considerations in the use of 18s rRNA in gene expression studies

    EPA Science Inventory

    Gene expression analysis is now commonly used in ecotoxicological studies to indicate exposure of an organism to xenobiotics. For example, the vitellogenin gene is used to diagnose exposure of fish to environmental estrogens. Reverse transcription polymerase chain reaction (RT-PC...

  13. [Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

    PubMed

    Nazina, T N; Shumkova, E S; Sokolova, D Sh; Babich, T L; Zhurina, M V; Xue, Yan-Fen; Osipov, G A; Poltaraus, A B; Tourova, T P

    2015-01-01

    The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

  14. Phylogenetic relationships between Vorticella convallaria and other species inferred from small subunit rRNA gene sequences.

    PubMed

    Itabashi, Takeshi; Mikami, Kazuyuki; Fang, Jie; Asai, Hiroshi

    2002-08-01

    Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

  15. Next-Generation Sequencing of the Bacterial 16S rRNA Gene for Forensic Soil Comparison: A Feasibility Study.

    PubMed

    Jesmok, Ellen M; Hopkins, James M; Foran, David R

    2016-05-01

    Soil has the potential to be valuable forensic evidence linking a person or item to a crime scene; however, there is no established soil individualization technique. In this study, the utility of soil bacterial profiling via next-generation sequencing of the 16S rRNA gene was examined for associating soils with their place of origin. Soil samples were collected from ten diverse and nine similar habitats over time, and within three habitats at various horizontal and vertical distances. Bacterial profiles were analyzed using four methods: abundance charts and nonmetric multidimensional scaling provided simplification and visualization of the massive datasets, potentially aiding in expert testimony, while analysis of similarities and k-nearest neighbor offered objective statistical comparisons. The vast majority of soil bacterial profiles (95.4%) were classified to their location of origin, highlighting the potential of bacterial profiling via next-generation sequencing for the forensic analysis of soil samples.

  16. Rapid Identification of Clinically Relevant Nocardia Species to Genus Level by 16S rRNA Gene PCR

    PubMed Central

    Laurent, Frederic J.; Provost, Frederique; Boiron, Patrick

    1999-01-01

    Two regions of the gene coding for 16S rRNA in Nocardia species were selected as genus-specific primer sequences for a PCR assay. The PCR protocol was tested with 60 strains of clinically relevant Nocardia isolates and type strains. It gave positive results for all strains tested. Conversely, the PCR assay was negative for all tested species belonging to the most closely related genera, including Dietzia, Gordona, Mycobacterium, Rhodococcus, Streptomyces, and Tsukamurella. Besides, unlike the latter group of isolates, all Nocardia strains exhibited one MlnI recognition site but no SacI restriction site. This assay offers a specific and rapid alternative to chemotaxonomic methods for the identification of Nocardia spp. isolated from pathogenic samples. PMID:9854071

  17. Identification of Atypical Rhodococcus-Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing▿

    PubMed Central

    Pilares, Lilian; Agüero, Jesús; Vázquez-Boland, José A.; Martínez-Martínez, Luis; Navas, Jesús

    2010-01-01

    Rhodococcus equi and Dietzia spp. are closely related actinomycetes that show similar phenotypic properties. In humans, R. equi is an opportunistic pathogen associated with severe immunodeficiency. Dietzia spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as R. equi by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true R. equi strains, whereas the other eight were identified as members of the genus Dietzia, including Dietzia maris (four isolates), Dietzia natronolimnaea (two isolates), and Dietzia timorensis and Dietzia sp. (one isolate each). The eight Dietzia isolates were highly sensitive to 12 antimicrobial compounds. PMID:20220156

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

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

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

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

    PubMed Central

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

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

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

    PubMed

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

    2004-03-01

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

  1. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia.

    PubMed

    Xia, Li-Ping; Bian, Long-Yan; Xu, Min; Liu, Ying; Tang, Ai-Ling; Ye, Wen-Qin

    2015-01-01

    Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.

  2. Bacterial rRNA genes associated with soil suppressiveness against the plant-parasitic nematode Heterodera schachtii.

    PubMed

    Yin, Bei; Valinsky, Lea; Gao, Xuebiao; Becker, J Ole; Borneman, James

    2003-03-01

    The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, alpha-Proteobacteria, beta-Proteobacteria, and gamma-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured alpha-proteobacterial clones was consistently associated with the

  3. Bacterial rRNA Genes Associated with Soil Suppressiveness against the Plant-Parasitic Nematode Heterodera schachtii

    PubMed Central

    Yin, Bei; Valinsky, Lea; Gao, Xuebiao; Becker, J. Ole; Borneman, James

    2003-01-01

    The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured α-proteobacterial clones was consistently associated with the highly

  4. Distribution of 5-methylcytosine residues in 5S rRNA genes in Arabidopsis thaliana and Secale cereale.

    PubMed

    Fulnecek, J; Matyásek, R; Kovarík, A

    2002-12-01

    Bisulfite genomic sequencing was used to localise 5-methylcytosine residues (mC) in 5S rRNA genes of Arabidopsis thaliana and Secale cereale. The maps of mC distribution were compared with the previously published map of the corresponding region in Nicotiana tabacum. In all three species, the level of methylation of 5S rRNA genes was generally higher than the average for the entire genome. The ratio of 5S rDNA methylation to average overall methylation was 44%/30-33% for N. tabacum, 27%/4-6% for A. thaliana and 24%/20-22% for S. cereale. With the exception of one clone from S. cereale, no methylation-free 5S rDNA was detected. The level of methylation at different sequence motifs in 5S rDNA was calculated for N. tabacum/A. thaliana/ S. cereale, and this analysis yielded the following values (expressed as a percentage of total C): mCG 90%/78%/85%, mCWG 89%/41%/53%, mCmCG 72%/32%/16%, mCCG 4%/2%/0%, mCHH 15%/6%/1%, where W=A or T, and H=A or C or T. Non-symmetrical methylation was almost negligible in the large genome of S. cereale but relatively frequent in N. tabacum and A. thaliana, suggesting that the strict correlation between genome size and cytosine methylation might be violated for this type of methylation. Among non-symmetrical motifs the mCWA triplets were significantly over-represented in Arabidopsis, while in tobacco this preference was not as pronounced. The differences in methylation levels in different sequence contexts might be of phylogenetic significance, but further species in related and different taxa need to be studied before firm conclusions can be drawn.

  5. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGES

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; ...

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  6. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  7. Identification of polybacterial communities in patients with postoperative, posttraumatic, and endogenous endophthalmitis through 16S rRNA gene libraries.

    PubMed

    Jayasudha, Rajagopalaboopathi; Narendran, Venkatapathy; Manikandan, Palanisamy; Prabagaran, Solai Ramatchandirane

    2014-05-01

    Endophthalmitis is a potential vision-threatening complication following surgical procedures (postoperative endophthalmitis [POE]), trauma (posttraumatic endophthalmitis [PTE]), and bacteremic seeding of the eye from a distant infection site (endogenous endophthalmitis [EE]). Several studies have revealed the polybacterial characteristics of endophthalmitis, which make the administration of antibiotics to treat the disease challenging. However, until now, the polybacterial communities of POE, PTE, and EE have not been precisely studied. Hence, the present study was designed to identify the bacterial community of endophthalmitis through 16S rRNA gene libraries. Of the 40 intraocular samples tested, 30 libraries were constructed with bacterial nested-PCR-positive samples. The obtained recombinant clones were screened through amplified rRNA gene restriction analysis (ARDRA) to identify unique clones. The multiple types of ARDRA patterns (P=0.345) and diverse bacterial sequences (P=0.277) within the libraries revealed the polybacterial nature of infection in POE, PTE, and EE. Moreover, to the best of our knowledge, this is the first report on polybacterial infection in EE. Gram-positive bacteria, including Bacillus spp. (n=19), Streptococcus spp. (n=18), Staphylococcus spp. (n=6), Exiguobacterium spp. (n=3), Gemella spp. (n=2), Enterococcus spp. (n=2), a Lysinibacillus sp. (n=1), a Clostridium sp. (n=1), and a Nocardia sp. (n=1), and Gram-negative bacteria, including Serratia spp. (n=18), Pseudomonas spp. (n=10), Enterobacter spp. (n=8), Acinetobacter spp. (n=3), Pantoea spp. (n=3), a Haemophilus sp. (n=1), and a Massilia sp. (n=1), were identified. Interestingly, among them, 10 bacterial species were not previously reported to be associated with endophthalmitis or other ocular infections. Besides, the presence of 4 unidentifiable clones suggests the possibility of novel organisms that might cause eye infections. Therefore, it is recommended that, in addition to the

  8. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  9. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.

    PubMed

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L; Lynch, Susan V

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  10. B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

    PubMed

    Ruiz-Estévez, Mercedes; Badisco, Liesbeth; Broeck, Jozef Vanden; Perfectti, Francisco; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M

    2014-12-01

    The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

  11. Analysis of microbiota associated with peri-implantitis using 16S rRNA gene clone library

    PubMed Central

    Koyanagi, Tatsuro; Sakamoto, Mitsuo; Takeuchi, Yasuo; Ohkuma, Moriya; Izumi, Yuichi

    2010-01-01

    Background Peri-implantitis (PI) is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified. Objective The purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences. Design Three subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique. Results A total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46%) were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions. Conclusions The biofilm in PI showed a more complex microbiota when compared to periodontitis and periodontally healthy teeth

  12. 16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

    PubMed Central

    Suchodolski, Jan S.; Dowd, Scot E.; Wilke, Vicky; Steiner, Jörg M.; Jergens, Albert E.

    2012-01-01

    Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation. PMID:22720094

  13. Composition of fecal microbiota of laboratory mice derived from Japanese commercial breeders using 16S rRNA gene clone libraries

    PubMed Central

    NOZU, Ryoko; UENO, Masami; HAYASHIMOTO, Nobuhito

    2016-01-01

    The fecal microbiota of six mice derived from three Japanese commercial breeders was analyzed by using 16S rRNA gene clone libraries to construct a database for analyzing the gut microbiota of laboratory mice. The 566 clones were obtained from the clone libraries generated from the fecal DNA samples derived from BALB/c, C57BL/6N, DBA/2 and ICR mice. Among these 566 clones, there were 446 unique 16S rRNA gene sequences. When grouped at the 98% similarity level, the 446 unique sequences consisted of 103 Clostridiales, 43 Bacteroidales, 5 Lactobacillus and 3 Erysipelotricaceae, as well as sequences from 11 other phyla. PMID:26902692

  14. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  15. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns.

    PubMed

    Atherly, Todd; Ziemer, Cherie J

    2014-04-01

    One-hundred-and-three isolates of Bacteroides ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. Phylogenetic relationships of basal hexapods reconstructed from nearly complete 18S and 28S rRNA gene sequences.

    PubMed

    Gao, Yan; Bu, Yun; Luan, Yun-Xia

    2008-11-01

    This study combined nearly complete 28S and 18S rRNA gene sequences (>4100 nt long) to investigate the phylogenetic relationships of basal hexapods (Protura, Collembola, and Diplura). It sequenced more 28S genes, to expand on a previous study from this lab that used 18S plus only a tiny part of the 28S gene. Sixteen species of basal hexapods, five insects, six crustaceans, two myriapods, and two chelicerates were included in the analyses. Trees were constructed with maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded consistent results: (1) Hexapoda was monophyletic and nested in a paraphyletic Crustacea, and Hexapoda was divided into Entognatha [Collembola+Nonoculata (Protura plus Diplura)] and Insecta (=Ectognatha), but the Nonoculata clade must be accepted with caution because of its strong nonstationarity of nucleotide composition. (2) Within Diplura, the monophyly of Campodeoidea and of Japygoidea were supported respectively, and all methods united Projapygoidea with Japygoidea. (3) Within Protura, Sinentomidae was the sister group to Acerentomata. (4) Within Collembola, the modern taxonomical hierarchy of Collembola (Poduromorpha, Entomobryomorpha, Symphypleona and Neelipleona) was confirmed.

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

    PubMed

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

    2015-01-01

    Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

  18. 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.

  19. Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences.

    PubMed

    Yan, Ying; Xu, Yuan; Yi, Zhenzhen; Warren, Alan

    2013-09-01

    Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

  20. [Detection of bacterial signal of 16S rRNA gene in prostate tissues obtained by perineal approach from patient with chronic pelvic pain syndrome].

    PubMed

    Zhu, Qing-Feng; Xie, Hui; Weng, Zhi-Liang; Yang, Yi-Rong; Chen, Bi-Cheng

    2009-03-31

    To explore the role of bacteria in etiology of chronic pelvic pain syndrome (CPPS), i.e., chronic prostatitis and the correlation between presence of bacterial signal of 16S ribosomal RNA (16S rRNA) gene and the response to antibiotics. Samples of prostatic and subcutaneous tissues were obtained by biopsy via perineal approach from 112 CPPS patients, aged 20 - 48. Polymerase chain reaction was conducted to detect the 16S rRNA gene of bacteria. The patients were treated with gatifloxacin 0.4 g once a day for 4 weeks and then 4 weeks later the effects of treatment were assessed by the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). PCR was completed in 94 of the 112 patients, and 18 were excluded because their subcutaneous biopsies were positive for 16S rRNA, showing the possible contamination of their prostatic tissues. The total positive rate of bacterial 16S rRNA gene was 63.8% (60/94). The positive rate of bacterial 16S rRNA gene in the patients with IIIa CPPS and IIIb CPPS were 68.3% and 60.3% respectively. The total gatifloxacin effective rate of positive bacterial signal group after the was 55.0%, significantly higher than that of the negative bacterial signal group (14.7%, P < 0.001). The gatifloxacin effective rate of the 16S rRNA positive IIIa CPPS patients was 75%, significantly higher than that of the 6S rRNA negative IIIa CPPS patients (23.1%, P < 0.001), and the gatifloxacin effective rate of the 16S rRNA positive IIIb CPPS patients was 37.5%, significantly higher than that of the 6S rRNA negative IIIb CPPS patients (9.5%, P < 0.05). Bacterial infection is related to CPPS in part of the patients. Bacterial signal detection helps predict the effect of antimicrobial therapy.

  1. Prevalence of 16S rRNA methylase, modifying enzyme, and extended-spectrum beta-lactamase genes among Acinetobacter baumannii isolates.

    PubMed

    Liu, Zhenru; Ling, Baodong; Zhou, Liming

    2015-08-01

    Multidrug-resistant Acinetobacter baumannii has become a worldwide problem, and methylation of 16S rRNA has recently emerged as a new mechanism of resistance to aminoglycosides, which is mediated by a newly recognized group of 16S rRNA methylases. 16S rRNA methylase confers a high-level resistance to all 4,6-substituted deoxystreptamine aminoglycosides that are currently used in clinical practice. Some of the A. baumannii isolates have been found to coproduce extended-spectrum beta-lactamases (ESBLs), contributing to their multidrug resistance. The aim of this study was to detect the determinants of the 16S rRNA methylase genes armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA, the modifying enzyme genes aac(6')-Ib, ant(3″)-Ia, aph(3')-I, and the extended-spectrum beta-lactamase genes bla(TEM), bla(SHV), and bla(CTX-M-3) among A. baumannii isolates in northeastern Sichuan, China. Minimum inhibitory concentrations (MICs) of 21 different antimicrobial agents against the A. baumannii isolates were determined. The clinical isolates showed a high level of resistance (MIC≧256 μg/ml) to aminoglycosides, which ranged from 50·1 to 83·8%. The resistances to meropenem and imipenem, two of the beta-lactam antibiotics and the most active antibiotics against A. baumannii, were 9·1 and 8·2%, respectively. Among 60 amikacin-resistant isolates, only the 16S rRNA methylase gene armA was found to be prevalent (66·7%), but the other 16S rRNA methylase genes rmtA, rmtB, rmtC, rmtD, rmtE, and npmA were not detected. The prevalences of the modifying enzyme genes aac (6')-Ib, ant (3″)-Ia, and aph (3')-I were 51·7, 81·7, and 58·3%, respectively, which are different from a previous study in which the occurrences of these genes were 3, 64, and 72%, respectively. Among the 40 isolates that were armA-positive, the prevalences of bla(TEM), bla(SHV), and bla(CTX-M-3) genes were detected for the first time in China, and their occurrences were 45, 65, and 52·5%, respectively. In all, A

  2. Nearly Complete 28S rRNA Gene Sequences Confirm New Hypotheses of Sponge Evolution

    PubMed Central

    Thacker, Robert W.; Hill, April L.; Hill, Malcolm S.; Redmond, Niamh E.; Collins, Allen G.; Morrow, Christine C.; Spicer, Lori; Carmack, Cheryl A.; Zappe, Megan E.; Pohlmann, Deborah; Hall, Chelsea; Diaz, Maria C.; Bangalore, Purushotham V.

    2013-01-01

    The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges. PMID:23748742

  3. Nearly complete 28S rRNA gene sequences confirm new hypotheses of sponge evolution.

    PubMed

    Thacker, Robert W; Hill, April L; Hill, Malcolm S; Redmond, Niamh E; Collins, Allen G; Morrow, Christine C; Spicer, Lori; Carmack, Cheryl A; Zappe, Megan E; Pohlmann, Deborah; Hall, Chelsea; Diaz, Maria C; Bangalore, Purushotham V

    2013-09-01

    The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges.

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

    PubMed

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

    2008-10-01

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

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

    PubMed

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

    2017-05-01

    Spontaneous alcoholic fermentation of grape must is a complex process, carried out by indigenous yeast populations arising from the vineyard or the winery environment and therefore representing an autochthonous microbial terroir of the production area. Microbial diversity at species and biotype level is extremely important in order to develop the composite and typical flavour profile of DOCG (Appellation of Controlled and Guaranteed Origin) wines. In this study, we monitored fungal populations involved in spontaneous fermentations of Aglianico and Greco di Tufo grape must by high-throughput sequencing (HTS) of 18S rRNA gene amplicons. We firstly proposed an alternative/addition to ITS as target gene in HTS studies and highlighted consistency between the culture-dependent and -independent approaches. A complex mycobiota was found at the beginning of the fermentation, mainly characterized by non-Saccharomyces yeasts and several moulds, with differences between the two types of grapes. Moreover, Interdelta patterns revealed a succession of several Saccharomyces cerevisiae biotypes and a high genetic diversity within this species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene.

    PubMed

    Kobayashi, S; Suzuki, J; Takeuchi, T

    2009-06-01

    We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli.

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

    PubMed

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

    2007-01-01

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

  8. Toxocara canis and Toxocara vitulorum: molecular characterization, discrimination, and phylogenetic analysis based on mitochondrial (ATP synthase subunit 6 and 12S) and nuclear ribosomal (ITS-2 and 28S) genes.

    PubMed

    Wickramasinghe, Susiji; Yatawara, Lalani; Rajapakse, R P V J; Agatsuma, Takeshi

    2009-06-01

    Toxocara canis and Toxocara vitulorum are two important parasites of dogs and buffaloes with public health concern. The objectives of the present study are to identify molecular markers to discriminate these closely related parasites and to determine their phylogenetic position and genetic diversity within the genus Toxocara. Thus, two mitochondrial genes (complete ATPase 6 and partial small subunit ribosomal RNA (12S rDNA)), two nuclear ribosomal genes (second internal transcribed spacer region (ITS-2)), and part of the large subunit 28S region were analyzed. Nucleotide sequence (597 bp) and predicted amino acid sequences of the complete ATPase 6 gene (199 amino acids) of both species (T. canis and T. vitulorum) are similar in size with the Toxocara cati and Toxocara malaysiensis. There was 88% nucleotide similarity between T. canis and T. vitulorum and many transversions present in the 12S gene. Analyses of the ITS-2 and 28S regions revealed that the 28S region was more conserved (95% nucleotide similarity between T. canis and T. vitulorum) than the ITS-2 region (85%). This study has provided useful molecular markers for the molecular epidemiological investigation of Toxocara species. Further, phylogenetic analyses of the ITS-2 and 28S genes have indicated that the members of the genus Toxocara form a distinct group with reference to their definitive hosts.

  9. Expression and Function of Different Guanine-Plus-Cytosine Content 16S rRNA Genes in Haloarcula hispanica at Different Temperatures

    PubMed Central

    Sato, Yu; Fujiwara, Taketomo; Kimura, Hiroyuki

    2017-01-01

    The halophilic archaeon Haloarcula hispanica harbors three ribosomal RNA (rRNA) operons (rrnA, rrnB, and rrnC) that contain the 16S rRNA genes rrsA, rrsB, and rrsC, respectively. Although rrsB and rrsC (rrsBC) have almost identical sequences, the rrsA and rrsBC sequences differ by 5.4%, and they differ by 2.5% with respect to guanine-plus-cytosine content (PGC). The strong correlation between the typical growth temperatures of archaea and PGC of their 16S rRNA genes suggests that H. hispanica may harbor different 16S rRNA genes having different PGC to maintain rapid growth in a wide range of temperatures. We therefore performed reverse transcription-coupled quantitative PCR to assess expression levels of rrsA (PGC, 58.9%) and rrsBC (PGC, 56.4–56.5%) at various temperatures. The expression ratio of rrsA to rrsBC increased with culture temperature. Mutants with complete deletions of one or two of the three rRNA operons were constructed and their growth rates at different temperatures compared to that of the wild-type. The growth characteristics of the rRNA operon single-mutant strains were indistinguishable from the wild-type. The rRNA operon double-mutant strains maintained the same temperature range as wild-type but displayed reduced growth rates. In particular, the double-mutant strains grew much slower than wild-type at low temperature related to minimum growth temperature of the wild-type. On the other hand, at physiologically high temperatures the wild-type and the double-mutant strain which harbors only rrnA with high-PGC rrsA grew significantly faster than the double-mutant strain which harbors only rrnC with low-PGC rrsC. These findings suggest the importance of 16S rRNAs transcribed from rrsA with high-PGC in maintaining rapid growth of this halophilic archaeon at raised growth temperatures. PMID:28400752

  10. Expression and Function of Different Guanine-Plus-Cytosine Content 16S rRNA Genes in Haloarcula hispanica at Different Temperatures.

    PubMed

    Sato, Yu; Fujiwara, Taketomo; Kimura, Hiroyuki

    2017-01-01

    The halophilic archaeon Haloarcula hispanica harbors three ribosomal RNA (rRNA) operons (rrnA, rrnB, and rrnC) that contain the 16S rRNA genes rrsA, rrsB, and rrsC, respectively. Although rrsB and rrsC (rrsBC) have almost identical sequences, the rrsA and rrsBC sequences differ by 5.4%, and they differ by 2.5% with respect to guanine-plus-cytosine content (PGC). The strong correlation between the typical growth temperatures of archaea and PGC of their 16S rRNA genes suggests that H. hispanica may harbor different 16S rRNA genes having different PGC to maintain rapid growth in a wide range of temperatures. We therefore performed reverse transcription-coupled quantitative PCR to assess expression levels of rrsA (PGC, 58.9%) and rrsBC (PGC, 56.4-56.5%) at various temperatures. The expression ratio of rrsA to rrsBC increased with culture temperature. Mutants with complete deletions of one or two of the three rRNA operons were constructed and their growth rates at different temperatures compared to that of the wild-type. The growth characteristics of the rRNA operon single-mutant strains were indistinguishable from the wild-type. The rRNA operon double-mutant strains maintained the same temperature range as wild-type but displayed reduced growth rates. In particular, the double-mutant strains grew much slower than wild-type at low temperature related to minimum growth temperature of the wild-type. On the other hand, at physiologically high temperatures the wild-type and the double-mutant strain which harbors only rrnA with high-PGCrrsA grew significantly faster than the double-mutant strain which harbors only rrnC with low-PGCrrsC. These findings suggest the importance of 16S rRNAs transcribed from rrsA with high-PGC in maintaining rapid growth of this halophilic archaeon at raised growth temperatures.

  11. Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

    PubMed Central

    Peters, Sabine; Koschinsky, Stefanie; Schwieger, Frank; Tebbe, Christoph C.

    2000-01-01

    A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process. PMID:10698754

  12. Constitutive and Inducible Expression of the rRNA Methylase Gene erm(B) in Campylobacter

    PubMed Central

    Deng, Fengru; Shen, Jianzhong; Zhang, Maojun; Wu, Congming

    2015-01-01

    Macrolides are the antimicrobials of choice for treating human campylobacteriosis. The recent emergence of erm(B) in Campylobacter bacteria threatens the utility of this class of antibiotics. Here we report the constitutive and inducible expression of erm(B) in Campylobacter isolates derived from diarrheal patients and food-producing animals. Constitutive expression of erm(B) was associated with insertion and deletion in the regulatory region of the gene, providing the first documentation of the differential expression of erm(B) in Campylobacter bacteria. PMID:26259800

  13. Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood.

    PubMed

    Moriyama, Kosei; Ando, Chie; Tashiro, Kosuke; Kuhara, Satoru; Okamura, Seiichi; Nakano, Shuji; Takagi, Yasumitsu; Miki, Takeyoshi; Nakashima, Yoshiyuki; Hirakawa, Hideki

    2008-07-01

    Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.

  14. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  15. 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 ...

  16. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  17. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  18. True microbiota involved in chronic lung infection of cystic fibrosis patients found by culturing and 16S rRNA gene analysis.

    PubMed

    Rudkjøbing, Vibeke B; Thomsen, Trine R; Alhede, Morten; Kragh, Kasper N; Nielsen, Per H; Johansen, Ulla R; Givskov, Michael; Høiby, Niels; Bjarnsholt, Thomas

    2011-12-01

    Patients suffering from cystic fibrosis (CF) develop chronic lung infection. In this study, we investigated the microorganisms present in transplanted CF lungs (n = 5) by standard culturing and 16S rRNA gene analysis. A correspondence between culturing and the molecular methods was observed. In conclusion, standard culturing seems reliable for the identification of the dominating pathogens.

  19. Analysis of partial sequences of genes coding for 16S rRNA of actinomycetes isolated from Casuarina equisetifolia nodules in Mexico.

    PubMed Central

    Niner, B M; Brandt, J P; Villegas, M; Marshall, C R; Hirsch, A M; Valdés, M

    1996-01-01

    Filamentous bacteria isolated from surface-sterilized nodules of Casuarina equisetifolia trees in México were capable of reducing acetylene, a diagnostic test for nitrogenase, but were unable to nodulate their host. Analysis of partial 16S rRNA gene sequences suggests that the Mexican isolates are not Frankia strains but members of a novel clade. PMID:8702297

  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. 18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae)

    USDA-ARS?s Scientific Manuscript database

    In the southeastern United States, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA gene (SSU rRNA). Howe...

  2. 16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting

    PubMed Central

    Navarro-Noya, Yendi; Hernández-Rodríguez, César; Zenteno, Juan C.; Buentello-Volante, Beatriz; Cancino-Díaz, Mario E.; Jan-Roblero, Janet; Cancino-Díaz, Juan C.

    2012-01-01

    Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases. PMID:24031830

  3. Avoidance and Potential Remedy Solutions of Chimeras in Reconstructing the Phylogeny of Aphids Using the 16S rRNA Gene of Buchnera: A Case in Lachninae (Hemiptera).

    PubMed

    Chen, Rui; Wang, Zhe; Chen, Jing; Qiao, Ge-Xia

    2015-08-25

    It is known that PCR amplification of highly homologous genes from complex DNA mixtures can generate a significant proportion of chimeric sequences. The 16S rRNA gene is not only widely used in estimating the species diversity of endosymbionts in aphids but also used to explore the co-diversification of aphids and their endosymbionts. Thus, chimeric sequences may lead to the discovery of non-existent endosymbiont species and mislead Buchnera-based phylogenetic analysis that lead to false conclusions. In this study, a high probability (6.49%) of chimeric sequence occurrence was found in the amplified 16S rRNA gene sequences of endosymbionts from aphid species in the subfamily Lachninae. These chimeras are hybrid products of multiple parent sequences from the dominant species of endosymbionts in each corresponding host. It is difficult to identify the chimeric sequences of a new or unidentified species due to the high variability of their main parent, Buchnera aphidicola, and because the chimeric sequences can confuse the phylogenetic analysis of 16S rRNA gene sequences. These chimeras present a challenge to Buchnera-based phylogenetic research in aphids. Thus, our study strongly suggests that using appropriate methods to detect chimeric 16S rRNA sequences may avoid some false conclusions in endosymbiont-based aphid research.

  4. A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq.

    PubMed

    Burke, Catherine M; Darling, Aaron E

    2016-01-01

    The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

  5. The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

    PubMed

    Xie, Wenbing; Ling, Te; Zhou, Yonggang; Feng, Weijun; Zhu, Qiaoyun; Stunnenberg, Henk G; Grummt, Ingrid; Tao, Wei

    2012-05-22

    rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

  6. [Archaeal diversity in permafrost deposits of Bunger Hills Oasis and King George Island (Antarctica) according to the 16S rRNA gene sequencing].

    PubMed

    Karaevskaia, E S; Demchenko, L S; Demidov, N É; Rivkina, E M; Bulat, S A; Gilichinskiĭ, D A

    2014-01-01

    Archaeal communities of permafrost deposits of King George Island and Bunger Hills Oasis (Antarctica) differing in the content of biogenic methane were analyzed using clone libraries of two 16S rRNA gene regions. Phylotypes belonging to methanogenic archaea were identified in all horizons.

  7. A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

    PubMed Central

    Darling, Aaron E.

    2016-01-01

    Background The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. Results We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. Conclusions This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution. PMID:27688981

  8. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  9. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  10. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  11. 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

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

    PubMed Central

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

    1996-01-01

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

  13. Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes.

    PubMed Central

    Escalante, A A; Ayala, F J

    1995-01-01

    We have explored the evolutionary history of the Apicomplexa and two related protistan phyla, Dinozoa and Ciliophora, by comparing the nucleotide sequences of small subunit ribosomal RNA genes. We conclude that the Plasmodium lineage, to which the malarial parasites belong, diverged from other apicomplexan lineages (piroplasmids and coccidians) several hundred million years ago, perhaps even before the Cambrian. The Plasmodium radiation, which gave rise to several species parasitic to humans, occurred approximately 129 million years ago; Plasmodium parasitism of humans has independently arisen several times. The origin of apicomplexans (Plasmodium), dinoflagellates, and ciliates may be > 1 billion years old, perhaps older than the three multicellular kingdoms of animals, plants, and fungi. Digenetic parasitism independently evolved several times in the Apicomplexa. PMID:7597031

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

    PubMed

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

    2015-06-01

    Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89-95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73-5.04 μm) × 3.94 μm (3.50-4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-01

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

  17. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

  18. Combined Analyses of the ITS Loci and the Corresponding 16S rRNA Genes Reveal High Micro- and Macrodiversity of SAR11 Populations in the Red Sea

    PubMed Central

    Ngugi, David Kamanda; Stingl, Ulrich

    2012-01-01

    Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium. PMID:23185592

  19. New Primers Targeting Full-Length Ciliate 18S rRNA Genes and Evaluation of Dietary Effect on Rumen Ciliate Diversity in Dairy Cows.

    PubMed

    Zhang, Jun; Zhao, Shengguo; Zhang, Yangdong; Sun, Peng; Bu, Dengpan; Wang, Jiaqi

    2015-12-01

    Analysis of the full-length 18S rRNA gene sequences of rumen ciliates is more reliable for taxonomical classification and diversity assessment than the analysis of partial hypervariable regions only. The objective of this study was to develop new oligonucleotide primers targeting the full-length 18S rRNA genes of rumen ciliates, and to evaluate the effect of different sources of dietary fiber (corn stover or a mixture of alfalfa hay and corn silage) and protein (mixed rapeseed, cottonseed, and/or soybean meals) on rumen ciliate diversity in dairy cows. Primers were designed based on a total of 137 previously reported ciliate 18S rRNA gene sequences. The 3'-terminal sequences of the newly designed primers, P.1747r_2, P.324f, and P.1651r, demonstrated >99% base coverage. Primer pair D (P.324f and P.1747r_2) was selected for the cloning and sequencing of ciliate 18S rRNA genes because it produced a 1423-bp amplicon, and did not amply the sequences of other eukaryotic species, such as yeast. The optimal species-level cutoff value for distinguishing between the operational taxonomic units of different ciliate species was 0.015. The phylogenetic analysis of full-length ciliate 18S rRNA gene sequences showed that distinct ciliate profiles were induced by the different sources of dietary fiber and protein. Dasytricha and Entodinium were the predominant genera in the ruminal fluid of dairy cattle, and Dasytricha was significantly more abundant in cows fed with corn stover than in cows fed with alfalfa hay and corn silage.

  20. Combined analyses of the ITS loci and the corresponding 16S rRNA genes reveal high micro- and macrodiversity of SAR11 populations in the Red Sea.

    PubMed

    Ngugi, David Kamanda; Stingl, Ulrich

    2012-01-01

    Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24 °C throughout the year, and a remarkable uniform temperature (~22 °C) and salinity (~41 psu) from the mixed layer (~200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea's water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.

  1. First report on the bacterial diversity in the distal gut of dholes (Cuon alpinus) by using 16S rRNA gene sequences analysis.

    PubMed

    Chen, Lei; Zhang, Honghai; Liu, Guangshuai; Sha, Weilai

    2016-05-01

    The aim of this study was to investigate the bacterial community in the distal gut of dholes (Cuon alpinus) based on the analysis of bacterial 16S rRNA gene sequences. Fecal samples were collected from five healthy unrelated dholes captured from Qilian Mountain in Gansu province of China. The diversity of the fecal bacteria community was investigated by constructing a polymerase chain reaction (PCR)-amplified 16S rRNA gene clone library. Bacterial 16S rRNA gene was amplified by using universal bacterial primers 27F and 1492R. A total of 275 chimera-free near full length 16S rRNA gene sequences were collected, and 78 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified according to the 97 % sequence similarity. Forty-two OTUs (53.8 %) showed less than 98 % sequence similarity to 16S rRNA gene sequences reported previously. Phylogenetic analysis demonstrated that dhole bacterial community comprised five different phyla, with the majority of sequences being classified within the phylum Bacteroidetes (64.7 %), followed by Firmicutes (29.8 %), Fusobacteria (4.7 %),Proteobacteria (0.4 %), and Actinobacteria (0.4 %). The only order Bacteroidales in phylum Bacteroidetes was the most abundant bacterial group in the intestinal bacterial community of dholes. Firmicutes and Bacteroidetes were the two most diverse bacterial phyla with 46.2 and 44.9 % of OTUs contained, respectively. Bacteroidales and Clostridiales were the two most diverse bacterial orders that contained 44.9 and 39.7 % of OTUs, respectively.

  2. 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.

  3. Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

    PubMed

    Fatahi-Bafghi, Mehdi; Heidarieh, Parvin; Rasouli-Nasab, Masoumeh; Habibnia, Shadi; Hashemi-Shahraki, Abdorazagh; Eshraghi, Seyyed Saeed

    2016-10-01

    Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

  4. 16S rRNA Gene Mutations Associated with Decreased Susceptibility to Tetracycline in Mycoplasma bovis

    PubMed Central

    Amram, E.; Mikula, I.; Schnee, C.; Ayling, R. D.; Nicholas, R. A. J.; Rosales, R. S.; Harrus, S.

    2014-01-01

    Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 μg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 μg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P < 0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline. PMID:25403668

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

    PubMed

    Berbee, M L; Taylor, J W

    1992-01-01

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

  6. Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp.

    PubMed

    Kageyama, Yasushi; Takaki, Yoshihiro; Shimamura, Shigeru; Nishi, Shinro; Nogi, Yuichi; Uchimura, Kohsuke; Kobayashi, Tohru; Hitomi, Jun; Ozaki, Katsuya; Kawai, Shuji; Ito, Susumu; Horikoshi, Koki

    2007-07-01

    Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.

  7. 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

  8. Ribosomal gene polymorphism in small genomes: analysis of different 16S rRNA sequences expressed in the honeybee parasite Nosema ceranae (Microsporidia).

    PubMed

    Sagastume, Soledad; Martín-Hernández, Raquel; Higes, Mariano; Henriques-Gil, Nuno

    2014-01-01

    To date, few organisms have been shown to possess variable ribosomal RNA, otherwise considered a classic example of uniformity by concerted evolution. The polymorphism for the 16S rRNA in Nosema ceranae analysed here is striking as Microsporidia are intracellular parasites which have suffered a strong reduction in their genomes and cellular organization. Moreover, N. ceranae infects the honeybee Apis mellifera, and has been associated with the colony-loss phenomenon during the last decade. The variants of 16S rRNA include single nucleotide substitutions, one base insertion-deletion, plus a tetranucleotide indel. We show that different gene variants are expressed. The polymorphic sites tend to be located in particular regions of the rRNA molecule, and the comparison to the Escherichia coli 16S rRNA secondary structure indicates that most variations probably do not preclude ribosomal activity. The fact that the polymorphisms in such a minimal organism as N. ceranae are maintained in samples collected worldwide suggest that the existence of differently expressed rRNA may play an adaptive role in the microsporidian. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  9. Origin and inheritance of group I introns in 26S rRNA genes of Gaeumannomyces graminis.

    PubMed

    Tan, M K

    1997-06-01

    Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes.

  10. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

    PubMed

    Vierheilig, J; Savio, D; Ley, R E; Mach, R L; Farnleitner, A H; Reischer, G H

    2015-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems.

  11. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

    PubMed Central

    Vierheilig, J.; Savio, D.; Ley, R. E.; Mach, R. L.; Farnleitner, A. H.

    2016-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multicompartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  12. Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

    PubMed Central

    Pajarillo, Edward Alain B.; Chae, Jong Pyo; Balolong, Marilen P.; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

    2015-01-01

    This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

  13. Clarithromycin resistance and 23S rRNA gene point mutations of Helicobacter pylori infection in children.

    PubMed

    Çağan-Appak, Yeliz; Gazi, Hörü; Ayhan, Semin; Cengiz-Özyurt, Beyhan; Kurutepe, Semra; Kasırga, Erhun

    2016-01-01

    This study aims to evaluate Helicobacter pylori with clarithromycin resistant genotypes in Manisa region, Turkey. Two hundred patients, who received diagnosis of Helicobacter pylori infection histopathologically, were included. The sex, age and endoscopy indications of the patients were recorded. Polymerase chain reaction method was applied to determine the clarithromycin resistance rate and resistance genotypes at the histologic sections prepared from gastric biopsies that had been embedded in paraffin after fixation by formalin. Helicobacter pylori resistance to clarithromycin was found in 19/200 (9.5%) patients. 10/19 (52.6%) of these clarithromycin-resistant patients had A2143G mutation and 9/19 (47.4%) had A2142G mutation. A2142C mutation on 23S rRNA gene was not detected for any of the patients. Clarithromycin can be used as a first step treatment in the eradication of Helicobacter pylori for the children in our region; if the treatment fails for some patients, clarithromycin resistance, especially A2143G and A2142G mutations should be considered.

  14. Identification of culturable stream water bacteria from urban, agricultural, and forested watersheds using 16S rRNA gene sequencing.

    PubMed

    Belt, Kenneth T; Hohn, Christina; Gbakima, Aiah; Higgins, James A

    2007-09-01

    Bacteria present in water samples taken on a weekly basis, from June 2004 through June 2005, from three streams, were cultured on Coliscan Easygel agar plates. Colonies representative of a variety of colors and morphologies were subjected to amplification and sequencing of a 1000-1100 nt portion of the 16S rRNA gene. A total of 528 colonies were sequenced; these categorized into 26 genera and 78 species. Of 175 dark blue/purple colonies presumed to be E. coli, sequence analysis indicated that 45 (25%) were actually other genera. For the urban stream Gwynns Falls Gwynns Run, E. coli was the most common genus/species encountered, followed by Klebsiella and Aeromonas. For Pond Branch, a stream located in a forested watershed, it was Serratia, followed by Yersinia and Aeromonas. For McDonogh (MCDN), a stream associated with Zea mays (corn) row crop agriculture, E. coli was the most frequently isolated genus/species, followed by Aeromonas and Enterobacter. ERIC-PCR genotyping of isolates from the most prevalent genera/species, indicated a high degree of diversity within-stream for E. coli and K. pneumoniae. Conversely, genotyping of Y. enterocolitica isolates indicated that some were shared between different streams.

  15. A molecular phylogeny of the marine red algae (Rhodophyta) based on the nuclear small-subunit rRNA gene.

    PubMed Central

    Ragan, M A; Bird, C J; Rice, E L; Gutell, R R; Murphy, C A; Singh, R K

    1994-01-01

    A phylogeny of marine Rhodophyta has been inferred by a number of methods from nucleotide sequences of nuclear genes encoding small subunit rRNA from 39 species in 15 orders. Sequence divergences are relatively large, especially among bangiophytes and even among congeners in this group. Subclass Bangiophycidae appears polyphyletic, encompassing at least three lineages, with Porphyridiales distributed between two of these. Subclass Florideophycidae is monophyletic, with Hildenbrandiales, Corallinales, Ahnfeltiales, and a close association of Nemaliales, Acrochaetiales, and Palmariales forming the four deepest branches. Cermiales may represent a convergence of vegetative and reproductive morphologies, as family Ceramiaceae is at best weakly related to the rest of the order, and one of its members appears to be allied to Gelidiales. Except for Gigartinales, for which more data are required, the other florideophyte orders appear distinct and taxonomically justified. A good correlation was observed with taxonomy based on pit-plug ultrastructure. Tests under maximum-likelihood and parsimony of alternative phylogenies based on structure and chemistry refuted suggestions that Acrochaetiales is the most primitive florideophyte order and that Gelidiales and Hildenbrandiales are sister groups. PMID:8041780

  16. Phylogenetic position of Loricifera inferred from nearly complete 18S and 28S rRNA gene sequences.

    PubMed

    Yamasaki, Hiroshi; Fujimoto, Shinta; Miyazaki, Katsumi

    2015-01-01

    Loricifera is an enigmatic metazoan phylum; its morphology appeared to place it with Priapulida and Kinorhyncha in the group Scalidophora which, along with Nematoida (Nematoda and Nematomorpha), comprised the group Cycloneuralia. Scarce molecular data have suggested an alternative phylogenetic hypothesis, that the phylum Loricifera is a sister taxon to Nematomorpha, although the actual phylogenetic position of the phylum remains unclear. Ecdysozoan phylogeny was reconstructed through maximum-likelihood (ML) and Bayesian inference (BI) analyses of nuclear 18S and 28S rRNA gene sequences from 60 species representing all eight ecdysozoan phyla, and including a newly collected loriciferan species. Ecdysozoa comprised two clades with high support values in both the ML and BI trees. One consisted of Priapulida and Kinorhyncha, and the other of Loricifera, Nematoida, and Panarthropoda (Tardigrada, Onychophora, and Arthropoda). The relationships between Loricifera, Nematoida, and Panarthropoda were not well resolved. Loricifera appears to be closely related to Nematoida and Panarthropoda, rather than grouping with Priapulida and Kinorhyncha, as had been suggested by previous studies. Thus, both Scalidophora and Cycloneuralia are a polyphyletic or paraphyletic groups. In addition, Loricifera and Nematomorpha did not emerge as sister groups.

  17. Deciphering bacterial community changes in zucker diabetic fatty rats based on 16S rRNA gene sequences analysis.

    PubMed

    Gu, Chunyan; Yang, Ye; Xiang, Hong; Li, Shu; Liang, Lina; Sui, Hua; Zhan, Libin; Lu, Xiaoguang

    2016-08-02

    The aim of the present pilot study was deciphering bacterial community changes in Zucker diabetic fatty rats (ZDF rats), a model of type 2 diabetes. Recent studies unmasked that the status of gastrointestinal tract microbiota has a marked impact on nutrition-related syndromes such as obesity and type-2 diabetes (T2D). In this study, samples taken from the gastrointestinal tracts (GI tracts) of ZDF and their lean littermates (ZL rats) were subjected to 16S rRNA gene sequence-based analysis to examine the characteristic bacterial communities, including those located in the stomach, duodenum, jejunum, ileum, cecum and feces. Results revealed that the Firmicutes/Bacteroidetes ratio was increased and greater numbers of Lactobacillus were detected along GI tracts in ZDF rats compared to ZL rats. In conclusion, this work is the first study to systematically characterize bacterial communities along ZDF rat GI tract and provides substantial evidence supporting a prospective strategy to alter the GI microbial communities improving obesity and T2D.

  18. Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys

    PubMed Central

    Thijs, Sofie; Op De Beeck, Michiel; Beckers, Bram; Truyens, Sascha; Stevens, Vincent; Van Hamme, Jonathan D.; Weyens, Nele; Vangronsveld, Jaco

    2017-01-01

    Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies. PMID:28400755

  19. [Localization of nucleolar DNA and transcription sites of rRNA genes in situ in wheat cells].

    PubMed

    He, Jie; Tao, Wei; Hao, Shui

    2008-02-01

    By using the conventional electron microscopic technique and DNA specific cytochemical staining method (NAMA-Ur), we directly observed the arrangement and location of intranucleolar DNA in situ in wheat (Triticum aestivum L.) cells. The results showed that nucleolar DNA was found in Fibrillar Centers (FC), Dense Fibrillar Component (DFC) and the transitional region between FC and DFC. Moreover, the nucleolar DNA was distributed along the periphery of FC and by surrounding FC. We employed RNP preference staining (Bernhard staining) method to visualize the distribution and position of RNP in situ in nucleoli of wheat cells. The results directly showed that RNP mainly located in the transitional region between FC and DFC, in DFC and in Granular Component (GC). Moreover, RNP was irregularly distributed around FC. By employing anti-RNA/DNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites was not only in the transitional region between DFC and FC but also in DFC of nucleoli in wheat cells.

  20. High-throughput 16S rRNA gene sequencing reveals alterations of mouse intestinal microbiota after radiotherapy.

    PubMed

    Kim, Young Suk; Kim, Jinu; Park, Soo-Je

    2015-06-01

    The mammalian gastrointestinal tract harbors a highly complex microbial community that comprises hundreds of different types of bacterial cells. The gastrointestinal microbiota plays an important role in the function of the host intestine. Most cancer patients undergoing pelvic irradiation experience side effects such as diarrhea; however, little is currently known about the effects of irradiation on the microorganisms colonizing the mucosal surfaces of the gastrointestinal tract. The aim of this study was to investigate the effects of gamma irradiation on the compositions of the large and small intestinal microbiotas. The gut microbiotas in control mice and mice receiving irradiation treatment were characterized by high-throughput sequencing of the bacterial 16S rRNA gene. Irradiation treatment induced significant alterations in the bacterial compositions of the large and small intestines at the genus level. Unexpectedly, irradiation treatment increased the number of operational taxonomic units in the small intestine but not the large intestine. In particular, irradiation treatment increased the level of the genera Alistipes in the large intestine and increased the level of the genus Corynebacterium in the small intestine. By contrast, compared with that in the corresponding control group, the level of the genera Prevotella was lower in the irradiated large intestine, and the level of the genera Alistipes was lower in the irradiated small intestine. Overall, the data presented here reveal the potential microbiological effects of pelvic irradiation on the gastrointestinal tracts of cancer patients.

  1. Deciphering bacterial community changes in zucker diabetic fatty rats based on 16S rRNA gene sequences analysis

    PubMed Central

    Xiang, Hong; Li, Shu; Liang, Lina; Sui, Hua; Zhan, Libin; Lu, Xiaoguang

    2016-01-01

    The aim of the present pilot study was deciphering bacterial community changes in Zucker diabetic fatty rats (ZDF rats), a model of type 2 diabetes. Recent studies unmasked that the status of gastrointestinal tract microbiota has a marked impact on nutrition-related syndromes such as obesity and type-2 diabetes (T2D). In this study, samples taken from the gastrointestinal tracts (GI tracts) of ZDF and their lean littermates (ZL rats) were subjected to 16S rRNA gene sequence-based analysis to examine the characteristic bacterial communities, including those located in the stomach, duodenum, jejunum, ileum, cecum and feces. Results revealed that the Firmicutes/Bacteroidetes ratio was increased and greater numbers of Lactobacillus were detected along GI tracts in ZDF rats compared to ZL rats. In conclusion, this work is the first study to systematically characterize bacterial communities along ZDF rat GI tract and provides substantial evidence supporting a prospective strategy to alter the GI microbial communities improving obesity and T2D. PMID:27418144

  2. The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis.

    PubMed

    Song, Wei; Li, Lingzhi; Huang, Hongliang; Jiang, Keji; Zhang, Fengying; Chen, Xuezhong; Zhao, Ming; Ma, Lingbo

    2016-01-01

    Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics.

  3. The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis

    PubMed Central

    Song, Wei; Li, Lingzhi; Huang, Hongliang; Jiang, Keji; Chen, Xuezhong

    2016-01-01

    Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics. PMID:27957494

  4. Identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp.

    PubMed

    Fernández-No, I C; Böhme, K; Caamaño-Antelo, S; Barros-Velázquez, J; Calo-Mata, P

    2015-04-01

    The main goal of this work was the identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp. that may be useful for typing purposes. These species include, among others, Bacillus cereus, an important pathogenic species involved in food poisoning, and Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus, which are causative agents of food spoilage described as responsible for foodborne disease outbreaks. With this purpose in mind, 52 Bacillus strains isolated from culture collections and fresh and processed food were considered. SNP type "Y" at sites 212 and 476 appeared in the majority of B. licheniformis studied strains. SNP type "R" at site 278 was detected in many strains of the B. subtilis/Bacillus amyloliquefaciens group, while polymorphism "Y" at site 173 was characteristic of the majority of strains of B. cereus/Bacillus thuringiensis group. The analysis of SNPs provided more intra-specific information than phylogenetic analysis in the cases of B. cereus and B. subtilis. Moreover, this study describes novel SNPs that should be considered when designing 16S rRNA-based primers and probes for multiplex-PCR, Real-Time PCR and microarray systems for foodborne Bacillus spp.

  5. Identification of sulfate-reducing bacteria in methylmercury-contaminated mine tailings by analysis of SSU rRNA genes.

    PubMed

    Winch, Susan; Mills, Heath J; Kostka, Joel E; Fortin, Danielle; Lean, David R S

    2009-04-01

    Sulfate-reducing bacteria (SRB) are often used in bioremediation of acid mine drainage because microbial sulfate reduction increases pH and produces sulfide that binds with metals. Mercury methylation has also been linked with sulfate reduction. Previous geochemical analysis indicated the occurrence of sulfate reduction in mine tailings, but no molecular characterization of the mine tailings-associated microbial community has determined which SRB are present. This study characterizes the bacterial communities of two geochemically contrasting, high-methylmercury mine tailing environments, with emphasis on SRB, by analyzing small subunit (SSU) rRNA genes present in the tailings sediments and in enrichment cultures inoculated with tailings. Novel Deltaproteobacteria and Firmicutes-related sequences were detected in both the pH-neutral gold mine tailings and the acidic high-sulfide base-metal tailings. At the subphylum level, the SRB communities differed between sites, suggesting that the community structure was dependent on local geochemistry. Clones obtained from the gold tailings and enrichment cultures were more similar to previously cultured isolates whereas clones from acidic tailings were more closely related to uncultured lineages identified from other acidic sediments worldwide. This study provides new insights into the novelty and diversity of bacteria colonizing mine tailings, and identifies specific organisms that warrant further investigation with regard to their roles in mercury methylation and sulfur cycling in these environments.

  6. Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454-pyrosequencing.

    PubMed

    Lopes, Ana R; Manaia, Célia M; Nunes, Olga C

    2014-03-01

    Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data.

  7. Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes

    PubMed Central

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-01-01

    Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future. PMID:26404329

  8. Physical Localization and DNA Methylation of 45S rRNA Gene Loci in Jatropha curcas L.

    PubMed Central

    Gong, Zhiyun; Xue, Chao; Zhang, Mingliang; Guo, Rui; Zhou, Yong; Shi, Guoxin

    2013-01-01

    In eukaryotes, 45S rRNA genes are arranged in tandem arrays of repeat units, and not all copies are transcribed during mitosis. DNA methylation is considered to be an epigenetic marker for rDNA activation. Here, we established a clear and accurate karyogram for Jatropha curcas L. The chromosomal formula was found to be 2n = 2x = 22 = 12m+10sm. We found that the 45S rDNA loci were located at the termini of chromosomes 7 and 9 in J. curcas. The distribution of 45S rDNA has no significant difference in J. curcas from different sources. Based on the hybridization signal patterns, there were two forms of rDNA - dispersed and condensed. The dispersed type of signals appeared during interphase and prophase, while the condensed types appeared during different stages of mitosis. DNA methylation analysis showed that when 45S rDNA stronger signals were dispersed and connected to the nucleolus, DNA methylation levels were lower at interphase and prophase. However, when the 45S rDNA loci were condensed, especially during metaphase, they showed different forms of DNA methylation. PMID:24386362

  9. RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits.

    PubMed Central

    Fabian, G R; Hopper, A K

    1987-01-01

    The Saccharomyces cerevisiae mutant ts351 had been shown to affect processing of 27S pre-rRNA to mature 25S and 5.8S rRNAs (C. Andrew, A. K. Hopper, and B. D. Hall, Mol. Gen. Genet. 144:29-37, 1976). We showed that this strain contains two mutations leading to temperature-sensitive lethality. The rRNA-processing defect, however, is a result of only one of the two mutations. We designated the lesion responsible for the rRNA-processing defect rrp1 and showed that it is located on the right arm of chromosome IV either allelic to or tightly linked to mak21. This rrp1 lesion also results in hypersensitivity to aminoglycoside antibiotics and a reduced 25S/18S rRNA ratio at semipermissive temperatures. We cloned the RRP1 gene and provide evidence that it encodes a moderately abundant mRNA which is in lower abundance and larger than most mRNAs encoding ribosomal proteins. Images PMID:3549696

  10. 16S rRNA gene-targeted TTGE in determining diversity of gut microbiota during acute diarrhoea and convalescence.

    PubMed

    Monira, Shirajum; Shabnam, Syeda Antara; Alam, Nur Haque; Endtz, Hubert Ph; Cravioto, Alejandro; Alam, Munirul

    2012-09-01

    The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea [patients vs healthy siblings: 11±0.9 vs 21.8±1.1 (mean ± standard error), p<0.01]. The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings (15±0.9, p<0.01). These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery.

  11. 16S rRNA Gene-targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

    PubMed Central

    Monira, Shirajum; Shabnam, Syeda Antara; Alam, Nur Haque; Endtz, Hubert Ph.; Cravioto, Alejandro

    2012-01-01

    The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea [patients vs healthy siblings: 11±0.9 vs 21.8±1.1 (mean±standard error), p<0.01]. The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings (15±0.9, p<0.01). These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery. PMID:23082626

  12. Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan.

    PubMed

    Khan, Muhammad Kasib; He, Lan; Hussain, Altaf; Azam, Sabita; Zhang, Wen-Jie; Wang, Li-Xia; Zhang, Qing-Li; Hu, Min; Zhou, Yan-Qin; Zhao, Junlong

    2013-01-01

    A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

    PubMed Central

    Price, Lance B.; Liu, Cindy M.; Melendez, Johan H.; Frankel, Yelena M.; Engelthaler, David; Aziz, Maliha; Bowers, Jolene; Rattray, Rogan; Ravel, Jacques; Kingsley, Chris; Keim, Paul S.; Lazarus, Gerald S.; Zenilman, Jonathan M.

    2009-01-01

    Background Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others. PMID:19649281

  14. Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

    PubMed

    Yuan, Youhua; Wen, Yan; You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.

  15. Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes

    PubMed Central

    Baker, Brett J.; Hugenholtz, Philip; Dawson, Scott C.; Banfield, Jillian F.

    2003-01-01

    During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name “Candidatus Captivus acidiprotistae.” To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat. PMID:12957940

  16. Characterization of Mycobacterium leprae Genotypes in China—Identification of a New Polymorphism C251T in the 16S rRNA Gene

    PubMed Central

    You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China. PMID:26196543

  17. Design and application of an internal amplification control to improve Dehalococcoides mccartyi 16S rRNA gene enumeration by qPCR.

    PubMed

    Hatt, Janet K; Ritalahti, Kirsti M; Ogles, Dora M; Lebrón, Carmen A; Löffler, Frank E

    2013-10-01

    Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of chlorinated ethenes to nontoxic ethene in contaminated aquifers. Enumeration of Dhc biomarker genes using quantitative real-time PCR (qPCR) in groundwater is a key component of site assessment and bioremediation monitoring. Unfortunately, standardized qPCR procedures that recognize impaired measurements due to PCR inhibition, low template DNA concentrations, or analytical error are not available, thus limiting confidence in qPCR data. To improve contemporary approaches for enumerating Dhc in environmental samples, multiplex qPCR assays were designed to quantify the Dhc 16S rRNA gene and one of two different internal amplification controls (IACs): a modified Dhc 16S rRNA gene fragment (Dhc*) and the firefly luciferase gene luc. The Dhc* IAC exhibited competitive inhibition in qPCR with the Dhc 16S rRNA gene template when the ratio of either target was 100-fold greater than the other target. A multiplex qPCR assay with the luc IAC avoided competitive inhibition and accurately quantified Dhc abundances ranging from ∼10 to 10(7) 16S rRNA gene copies per reaction. The addition of ∼10(6) E. coli luc IAC to simulated groundwater amended with the Dhc-containing consortium KB-1 yielded reproducible luc counts after DNA extraction and multiplex qPCR enumeration. The application of the luc IAC assay improved Dhc biomarker gene quantification from simulated groundwater samples and is a valuable approach for "ground truthing" qPCR data obtained in different laboratories, thus reducing ambiguity associated with qPCR enumeration and reproducibility.

  18. Morphometric and molecular data on two mitochondrial genes of a newly discovered chimaeran fish ( Hydrolagus melanophasma, Chondrichthyes)

    NASA Astrophysics Data System (ADS)

    De La Cruz-Agüero, José; García-Rodríguez, Francisco Javier; Cota-Gómez, Víctor Manuel; Melo-Barrera, Felipe Neri; González-Armas, Rogelio

    2012-06-01

    Fresh and preserved (type material) specimens of the black ghost chimaera Hydrolagus melanophasma were compared for morphometric characteristics. A molecular comparison was also performed on two mitochondrial gene sequences (12S rRNA and 16S rRNA gene sequences). While significant differences in measurements were found, the differences were not attributable to sexual dimorphism or the quality of the specimens, but to the sample size and the type of statistical tests. The result of the genetic characterization showed that 12S rRNA and 16S rRNA genes represented robust molecular markers that characterized the species.

  19. Phylogenetic analysis based on 18S rRNA gene and matK gene sequences of Panax vietnamensis and five related species.

    PubMed

    Komatsu, K; Zhu, S; Fushimi, H; Qui, T K; Cai, S; Kadota, S

    2001-07-01

    Panax vietnamensis was discovered recently in Vietnam. Its bamboo-like rhizomes, called Vietnamese Ginseng, have attracted considerable attention because of their specific pharmacological activities. In order to define the taxonomic position of this new species and include it in the molecular authentication of Ginseng drugs, the 18S ribosomal RNA gene and matK gene sequences of P. vietnamensis were determined and compared with those of its related taxa, P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus, besides previously reported P. ginseng, P. japonicus and P. quinquefolius. The 18S rRNA gene sequences were found to be 1809 bps in length. The sequence of P. vietnamensis was identical to that of P. quinquefolius, and presented one base substitution from those of both P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus. The matK gene sequences of 6 taxa were found to be 1509 bps in length. The sequence of P. vietnamensis differed from those of P. japonicus var. major, P. pseudo-ginseng subsp. himalaicus, P. ginseng, P. japonicus and P. quinquefolius at 4, 5, 9, 9 and 10 nucleotide positions, respectively. The phylogenetic tree reconstructed by the combined 18S rRNA-matK gene analysis using the maximum parsimony method showed that P. vietnamensis was sympatric with other Panax species and had a close relationship with P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus.

  20. Mapping of the human gene for a melanocyte protein Pmel 17 (D12S53E) to chromosome 12q13-q14

    SciTech Connect

    Kubota, Ryo; Wang, Yimin; Minoshima, Shinsei

    1995-03-20

    We have isolated several new cDNA clones that are differentially expressed in retina from a human retina cDNA library using a new combined method of subtractive hybridization and differential hybridization. Among these cDNAs, we found that cDNA clone BA7 was identical to the previously reported Pmel 17 cDNA in its nucleotide sequence. Pmel 17 mRNA is preferentially expressed in melanocytes, and the protein expressed in Escherichia coli cross-reacts with anti-tyrosinase antibodies. The mouse Pmel 17 gene was mapped to the pter-q21 region of human chromosome 12. In this paper, we present evidence precisely locating the human Pmel 17 gene to chromosome 12q13-q14. 7 refs., 1 fig.

  1. Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study.

    PubMed

    Minard, Guillaume; Tran, Florence-Hélène; Dubost, Audrey; Tran-Van, Van; Mavingui, Patrick; Moro, Claire Valiente

    2014-01-01

    The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 × 10(0.01) and 5.47 × 10(0.1) for wAlbA and between 2.03 × 10(0.1) and 1.4 × 10(1) for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area.

  2. Phylogenetic relationships among Linguatula serrata isolates from Iran based on 18S rRNA and mitochondrial cox1 gene sequences.

    PubMed

    Ghorashi, Seyed Ali; Tavassoli, Mousa; Peters, Andrew; Shamsi, Shokoofeh; Hajipour, Naser

    2016-01-01

    The phylogenetic relationships among seven Linguatula serrata (L. serrata) isolates collected from cattle, goats, sheep, dogs and camels in different geographical locations of Iran were investigated using partial 18S ribosomal RNA (rRNA) and partial mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequences. The nucleotide sequences were analysed in order to determine the phylogenetic relationships between the isolates. Higher sequence diversity and intraspecies variation was observed in the cox1 gene compared to 18S rRNA sequences. Phylogenetic analysis of the cox1 gene placed all L. serrata isolates in a sister clade to L. arctica. The Mantel regression analysis revealed no association between genetic variations and host species or geographical location, perhaps due to the small sample size. However, genetic variations between L. serrata isolates in Iran and those isolated in other parts of the world may exist and could reveal possible evolutionary relationships.

  3. Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and phylogenetic comparison with rRNA genes.

    PubMed

    Pilhofer, Martin; Rappl, Kristina; Eckl, Christina; Bauer, Andreas Peter; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Petroni, Giulio

    2008-05-01

    In the past, studies on the relationships of the bacterial phyla Planctomycetes, Chlamydiae, Lentisphaerae, and Verrucomicrobia using different phylogenetic markers have been controversial. Investigations based on 16S rRNA sequence analyses suggested a relationship of the four phyla, showing the branching order Planctomycetes, Chlamydiae, Verrucomicrobia/Lentisphaerae. Phylogenetic analyses of 23S rRNA genes in this study also support a monophyletic grouping and their branching order--this grouping is significant for understanding cell division, since the major bacterial cell division protein FtsZ is absent from members of two of the phyla Chlamydiae and Planctomycetes. In Verrucomicrobia, knowledge about cell division is mainly restricted to the recent report of ftsZ in the closely related genera Prosthecobacter and Verrucomicrobium. In this study, genes of the conserved division and cell wall (dcw) cluster (ddl, ftsQ, ftsA, and ftsZ) were characterized in all verrucomicrobial subdivisions (1 to 4) with cultivable representatives (1 to 4). Sequence analyses and transcriptional analyses in Verrucomicrobia and genome data analyses in Lentisphaerae suggested that cell division is based on FtsZ in all verrucomicrobial subdivisions and possibly also in the sister phylum Lentisphaerae. Comprehensive sequence analyses of available genome data for representatives of Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes strongly indicate that their last common ancestor possessed a conserved, ancestral type of dcw gene cluster and an FtsZ-based cell division mechanism. This implies that Planctomycetes and Chlamydiae may have shifted independently to a non-FtsZ-based cell division mechanism after their separate branchings from their last common ancestor with Verrucomicrobia.

  4. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

    PubMed

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping

    2016-07-01

    The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae.

  5. Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes.

    PubMed

    French, Sarah L; Sikes, Martha L; Hontz, Robert D; Osheim, Yvonne N; Lambert, Tashima E; El Hage, Aziz; Smith, Mitchell M; Tollervey, David; Smith, Jeffrey S; Beyer, Ann L

    2011-02-01

    To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.

  6. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    NASA Astrophysics Data System (ADS)

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg-1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  7. Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. Contrasting evolutionary patterns of 28S and ITS rRNA genes reveal high intragenomic variation in Cephalenchus (Nematoda): Implications for species delimitation.

    PubMed

    Pereira, Tiago José; Baldwin, James Gordon

    2016-05-01

    Concerted evolution is often assumed to be the evolutionary force driving multi-family genes, including those from ribosomal DNA (rDNA) repeat, to complete homogenization within a species, although cases of non-concerted evolution have been also documented. In this study, sequence variation of 28S and ITS ribosomal RNA (rRNA) genes in the genus Cephalenchus is assessed at three different levels, intragenomic, intraspecific, and interspecific. The findings suggest that not all Cephalenchus species undergo concerted evolution. High levels of intraspecific polymorphism, mostly due to intragenomic variation, are found in Cephalenchus sp1 (BRA-01). Secondary structure analyses of both rRNA genes and across different species show a similar substitution pattern, including mostly compensatory (CBC) and semi-compensatory (SBC) base changes, thus suggesting the functionality of these rRNA copies despite the variation found in some species. This view is also supported by low sequence variation in the 5.8S gene in relation to the flanking ITS-1 and ITS-2 as well as by the existence of conserved motifs in the former gene. It is suggested that potential cross-fertilization in some Cephalenchus species, based on inspection of female reproductive system, might contribute to both intragenomic and intraspecific polymorphism of their rRNA genes. These results reinforce the potential implications of intragenomic and intraspecific genetic diversity on species delimitation, especially in biodiversity studies based solely on metagenetic approaches. Knowledge of sequence variation will be crucial for accurate species diversity estimation using molecular methods.

  10. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites

    PubMed Central

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-01-01

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg−1 were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 103 to 2.28 × 106 and 4.17 × 102 to 1.99 × 105, respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites. PMID:26184609

  11. Diversity and distribution of 16S rRNA and phenol monooxygenase genes in the rhizosphere and endophytic bacteria isolated from PAH-contaminated sites.

    PubMed

    Peng, Anping; Liu, Juan; Ling, Wanting; Chen, Zeyou; Gao, Yanzheng

    2015-07-17

    This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81 mg·kg(-1) were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83 × 10(3) to 2.28 × 10(6) and 4.17 × 10(2) to 1.99 × 10(5), respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.

  12. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    PubMed

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  13. Sequence Diversity of the oprI Gene, Coding for Major Outer Membrane Lipoprotein I, among rRNA Group I Pseudomonads

    PubMed Central

    De Vos, Daniel; Bouton, Christiane; Sarniguet, Alain; De Vos, Paul; Vauterin, Marc; Cornelis, Pierre

    1998-01-01

    The sequence of oprI, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on Pseudomonas aeruginosa and Pseudomonas fluorescens. Within the P. aeruginosa species, oprI sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The oprI sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with P. aeruginosa oprI). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the HaeIII, PvuII, and SphI sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of P. fluorescens isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the oprI gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads. PMID:9851998

  14. Comparison of PCR-based restriction length polymorphism analysis of urease genes with rRNA gene profiling for monitoring Helicobacter pylori infections in patients on triple therapy.

    PubMed Central

    Owen, R J; Bickley, J; Hurtado, A; Fraser, A; Pounder, R E

    1994-01-01

    Multiple isolates of Helicobacter pylori from antral biopsies of nine patients were examined by DNA fingerprinting. Analysis of rRNA gene patterns and HaeIII restriction fragment length polymorphism of PCR-amplified urease genes were compared and used to study colonization before and after failed triple therapy. H. pylori isolates from a single biopsy shared the same HaeIII DNA fingerprint regardless of the isolation method (plate or broth). DNA pattern types of paired strains of H. pylori were distinct between patients and were not grossly affected by treatment except for one patient with an altered strain type. H. pylori infections were generally associated with several subpopulations of strains, evident from the subtypic variation before and after treatment, detectable by both DNA fingerprinting methods. The urease gene patterns also provided evidence that some cultures of H. pylori probably contained a mixture of genomic subtypes. The study suggests that triple therapy has the effect either of inducing minor genomic variations or of changing the proportions of different subtypes of H. pylori. It was concluded that urease gene profiling provides a simple yet reliable method of establishing whether treatment failures are attributable to incomplete eradication of H. pylori. Images PMID:7914204

  15. Composition of the bacterial population of refrigerated beef, identified with direct 16S rRNA gene analysis and pure culture technique.

    PubMed

    Olofsson, T C; Ahrné, S; Molin, G

    2007-09-30

    The composition of the dominating population of freshly cut beef, and beef stored at 4 degrees C for 8 d, was studied by direct analysis of the 16S rRNA gene (PCR amplification, cloning and sequencing) and compared with pure culture technique where the isolates picked from the viable plate count were identified by sequencing of the 16S rRNA gene. The composition of the bacterial population was recorded at two different time points, at the start when the viable plate count of the meat was 4 x 10(2) colony forming unit (cfu) per cm(2) and when it was 5 x 10(7) cfu per cm(2). Direct gene analysis by PCR amplification generated 30 clones, and 79 isolates were picked from the plate count, and identified by 16S rRNA gene sequencing. At the low initial bacterial load of the beef, the two sampling strategies showed variations in the composition of species. Direct 16S rRNA gene analysis revealed a domination of Bacillus-like sequences while no such sequences were found in isolates from the viable plate count. Instead the population of the plate count was dominated by Chryseobacterium spp. In contrast, the two sampling strategies matched on the multiplying beef population, where both methods indicated Pseudomonas spp. as the dominating group (99% of the population-sequences), irrespectively of sampling strategy. Pseudomonas panacis/Pseudomons brennerii was the dominating taxon (99% similarity to type strain), but sequences with highest similarity to Pseudomonas lundensis (99%), Pseudomonas beteli (99%) and Pseudomonas koreensis (100%) were also found.

  16. A sequence dimorphism in a conserved domain of human 28S rRNA. Uneven distribution of variant genes among individuals. Differential expression in HeLa cells.

    PubMed Central

    Qu, L H; Nicoloso, M; Bachellerie, J P

    1991-01-01

    In humans, cellular 28S rRNA displays a sequence dimorphism within an evolutionarily conserved motif, with the presence, at position +60, of either a A (like the metazoan consensus) or a G. The relative abundance of the two forms of variant genes in the genome exhibit large differences among individuals. The two variant forms are generally represented in cellular 28S rRNA in proportion of their relative abundance in the genome, at least for leucocytes. However, in some cases, one form of variant may be markedly underexpressed as compared to the other. Thus, in HeLa cells, A-form genes contribute to only 1% of the cellular content in mature 28S rRNA although amounting to 15% of the ribosomal genes. The differential expression seems to result from different transcriptional activities rather than from differences in pre-rRNA processing efficiency or in stabilities of mature rRNAs. G-form ribosomal genes were not detected in other mammals, including chimpanzee, which suggests that the fixation of this variant type is a rather recent event in primate evolution. Images PMID:2020541

  17. The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies

    PubMed Central

    Heylen, Kim; Sessitsch, Angela; De Vos, Paul

    2013-01-01

    Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment. PMID:23977026

  18. Two Theileria cervi SSU RRNA gene sequence types found in isolates from white-tailed deer and elk in North America.

    PubMed

    Chae, J S; Waghela, S D; Craig, T M; Kocan, A A; Wagner, G G; Holman, P J

    1999-07-01

    Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.

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

    PubMed

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

    2016-01-01

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

  20. Detection of genetic homogeneity of Panax notoginseng cultivars by sequencing nuclear 18S rRNA and plastid matK genes.

    PubMed

    Zhang, Ying; Zhang, Jin-Chao; Huang, Ming-Hui; Yang, Meng-Su; Cao, Hui

    2006-07-01

    The nuclear 18S rRNA and chloroplast MATK genes of 18 samples of Panax notoginseng and its processed material Sanqi (Radix Notoginseng) were analyzed. The two genes, regardless of cultivar origin, were found to be identical to genotype R1 and M1, respectively, of the published sequences (GenBank accession no. D85171 and AB027526). This phenomenon implies that the species is highly conserved, which is probably caused by the use of the same strain in cultivation and the lack of active mutation in these two genes.

  1. 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.