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Sample records for rrna gene mutations

  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. 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. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. [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.

  9. 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

  10. 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.

  11. 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

  12. Mutations affecting domain V of the 23S rRNA gene in Helicobacter pylori from Cairo, Egypt.

    PubMed

    Ghaith, Doaa; Elzahry, Mohammad; Mostafa, Gehan; Mostafa, Sally; Elsherif, Rasha; Ramzy, Iman

    2016-10-01

    Clarithromycin is a main component of the recommended first-line triple therapy for Helicobacter pylori in Egypt. We aimed in our study to investigate the prevalence of clarithromycin-resistant H. pylori strains due to the point mutations at domain V of the H. pylori 23S rRNA among the Egyptian population using the polymerase chain reaction/restricted fragment length polymorphism (PCR/RFLP) assay. Gastric biopsies obtained from 100 dyspeptic patients who consecutively attended at Cairo University Hospital during the period from January to November 2013 were subjected to PCR/RFLP in order to detect the point mutations at domain V of the H. pylori 23S rRNA associated with clarithromycin resistance. The PCR amplicon of the 23S H. pylori rRNA is restricted with MboII for detection of A2142G mutation and with BsaI for A2143G mutation. The prevalence of H. pylori infection among 100 patients was 70%; clarithromycin resistance was detected in 39/70 (57.7%) of positive H. pylori isolates. Occurrence of 23S rRNA A2142G mutations resulted in two DNA fragments (418 and 350 bp) by PCR-RFLP; on the other hand, no A2143G mutations were detected. The high prevalence of clarithromycin resistance (57.7%) caused by A2142G mutations at domain V of the H. pylori 23S rRNA may mandate changing of the standard clarithromycin-containing triple therapy. The PCR/RFLP assay was a rapid and accurate method for molecular detection of H. pylori infection in addition to determination of different nucleotide mutations causing clarithromycin resistance.

  13. The study of mutation in 23S rRNA resistance gene of Helicobacter pylori to clarithromycin in patients with gastrointestinal disorders in Isfahan - Iran.

    PubMed

    Khademi, Farzad; Faghri, Jamshid; Moghim, Sharareh; Esfahani, Bahram Nasr; Fazeli, Hossein; Poursina, Farkhondeh; Adibi, Peyman; Madhi, Masoumeh; Safaei, Hajieh Ghasemian

    2014-01-01

    Helicobacter pylori antimicrobial resistance is an important factor responsible for treatment failure. The purpose of this study was evaluating the prevalence of point mutations in clarithromycin-resistant clinical isolates of H. pylori in Isfahan city of Iran. Thirty isolates of H. pylori from 130 biopsy specimens were isolated by culture and confirmed by biochemical and PCR tests. The MIC of clarithromycin antibiotic for 30 clinical isolates of H. pylori was determined by E-test method. The point mutations in the 288 bp of 23S rRNA gene of H. pylori were investigated in four clarithromycin-resistant clinical isolates by PCR followed by sequencing. Among 30 isolates of H. pylori, 4 cases were resistant to clarithromycin. One point mutation was found at position T2243C in the 23S rRNA gene in all resistance isolates. In our study, H. pylori resistance to clarithromycin associated with point mutation at position 2243 (T2243C).

  14. 23S rRNA gene mutations contributing to macrolide resistance in Campylobacter jejuni and Campylobacter coli

    USDA-ARS?s Scientific Manuscript database

    Operon specific 23S rRNA mutations affecting minimum inhibitory concentrations (MICs) of macrolides (erythromycin [ERY], azithromycin [AZM], tylosin [TYL]) and a lincosamide (clindamycin [CLI]) were examined in a collection of Campylobacter jejuni and C. coli isolates. The three copies of the Campy...

  15. The study of mutation in 23S rRNA resistance gene of Helicobacter pylori to clarithromycin in patients with gastrointestinal disorders in Isfahan — Iran

    PubMed Central

    Khademi, Farzad; Faghri, Jamshid; Moghim, Sharareh; Esfahani, Bahram Nasr; Fazeli, Hossein; Poursina, Farkhondeh; Adibi, Peyman; Madhi, Masoumeh; Safaei, Hajieh Ghasemian

    2014-01-01

    Background: Helicobacter pylori antimicrobial resistance is an important factor responsible for treatment failure. The purpose of this study was evaluating the prevalence of point mutations in clarithromycin-resistant clinical isolates of H. pylori in Isfahan city of Iran. Materials and Methods: Thirty isolates of H. pylori from 130 biopsy specimens were isolated by culture and confirmed by biochemical and PCR tests. The MIC of clarithromycin antibiotic for 30 clinical isolates of H. pylori was determined by E-test method. The point mutations in the 288 bp of 23S rRNA gene of H. pylori were investigated in four clarithromycin-resistant clinical isolates by PCR followed by sequencing. Results: Among 30 isolates of H. pylori, 4 cases were resistant to clarithromycin. One point mutation was found at position T2243C in the 23S rRNA gene in all resistance isolates. Conclusions: In our study, H. pylori resistance to clarithromycin associated with point mutation at position 2243 (T2243C). PMID:24800187

  16. Emergence of methicillin-resistant coagulase-negative staphylococci resistant to linezolid with rRNA gene C2190T and G2603T mutations.

    PubMed

    Cidral, Thiago André; Carvalho, Maria Cícera; Figueiredo, Agnes Marie Sá; de Melo, Maria Celeste Nunes

    2015-10-01

    The aim of this article were to determinate the mechanism of linezolid resistance in coagulase-negative methicillin-resistant staphylococci from hospitals in the northeast of Brazil. We identified the isolates using VITEK(®) 2 and MALDI-TOF. Susceptibility to antibiotics was measured by the disk-diffusion method and by Etest(®) . Extraction of the whole genome DNA was performed, followed by screening of all the strains for the presence of mecA and cfr genes. The domain V region of 23S rRNA gene was sequenced and then aligned with a linezolid-susceptible reference strain. Pulsed-field gel electrophoresis (PFGE) macro-restriction analysis was performed. Three linezolid-resistant Staphylococcus hominis and two linezolid-resistant Staphylococcus epidermidis strains were analyzed. The isolates showed two point mutations in the V region of the 23S rRNA gene (C2190T and G2603T). We did not detect the cfr gene in any isolate by PCR. The S. hominis showed the same pulsotype, while the S. epidermidis did not present any genetic relation to each other. In conclusion, this study revealed three S. hominis and two S. epidermidis strains with resistance to linezolid due to a double mutation (C2190T and G2603T) in the domain V of the 23S rRNA gene. For the first time, the mutation of C2190T in S. epidermidis is described. This study also revealed the clonal spread of a S. hominis pulsotype between three public hospitals in the city of Natal, Brazil. These findings highlight the importance of continued vigilance of linezolid resistance in staphylococci.

  17. 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.

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

    PubMed

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

    2016-12-01

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

  19. 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.

  20. 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

  1. A single mutation in the 15S rRNA gene confers non sense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

    PubMed Central

    Gargouri, Ali; Macadré, Catherine; Lazowska, Jaga

    2015-01-01

    We have determined the nucleotide sequence of the mim3-1 mitochondrial ribosomal suppressor, acting on ochre mitochondrial mutations and one frameshift mutation in Saccharomyces cerevisiae. The 15s rRNA suppressor gene contains a G633 to C transversion. Yeast mitochondrial G633 corresponds to G517 of the E.coli 15S rRNA, which is occupied by an invariant G in all known small rRNA sequences. Interestingly, this mutation has occurred at the same position as the known MSU1 mitochondrial suppressor which changes G633 to A. The suppressor mutation lies in a highly conserved region of the rRNA, known in E.coli as the 530-loop, interacting with the S4, S5 and S12 ribosomal proteins. We also show an interesting interaction between the mitochondrial mim3-1 and the nuclear nam3-1 suppressors, both of which have the same action spectrum on mitochondrial mutations: nam3-1 abolishes the suppressor effect when present with mim3-1 in the same haploid cell. We discuss these results in the light of the nature of Nam3, identified by 1 as the yeast mitochondrial translation release factor. A hypothetical mechanism of suppression by "ribosome shifting" is also discussed in view of the nature of mutations suppressed and not suppressed. PMID:28357310

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rRNA gene mutation in treated isolates.

    PubMed

    Brown-Elliott, Barbara A; Iakhiaeva, Elena; Griffith, David E; Woods, Gail L; Stout, Jason E; Wolfe, Cameron R; Turenne, Christine Y; Wallace, Richard J

    2013-10-01

    Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.

  10. 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…

  11. 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…

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

    PubMed Central

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

    1998-01-01

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

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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.

    PubMed

    Rosellini, D; LaFayette, P R; Barone, P; Veronesi, F; Parrott, W A

    2004-05-01

    Genes conferring resistance to kanamycin are frequently used to obtain transgenic plants as spontaneous resistance to kanamycin is not known to exist in higher plants. Nevertheless, mutations conferring kanamycin resistance have been identified in Chlamydomonas reinhardtii, raising the question as to why kanamycin-resistant mutants have not been found in higher plants. While attempting plastid transformation of alfalfa, we obtained non-transgenic but kanamycin-resistant somatic embryos following 2 months of culture in the presence of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to the decoding site of the 16S rRNA in ten independent resistant events revealed an A to C transversion at position 1357 of the 16S plastid rDNA, the same site at which an A to G conversion confers kanamycin resistance to C. reinhardtii by reducing the ability of the antibiotic to bind to its target site. All plants derived from the resistant embryos through additional cycles of somatic embryogenesis in the absence of kanamycin retained the mutant phenotype, suggesting that the mutation was homoplastomic. Resistant plants produced 85% less biomass than controls; their leaves were chlorotic during early development and over time slowly turned green. The absence of kanamycin- resistant mutants in higher plants might be explained by the requirement for a regeneration system capable of resulting in homoplastomic individuals, or it may be the result of the detrimental effect of the mutation on the phenotype.

  20. 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.

  1. Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

    PubMed

    Pernodet, J L; Boccard, F; Alegre, M T; Blondelet-Rouault, M H; Guérineau, M

    1988-01-01

    Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.

  2. 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.

  3. 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...

  4. 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.

  5. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis.

    PubMed

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2016-08-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes.

  6. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

    PubMed Central

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru

    2016-01-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes. PMID:27245411

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

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

    PubMed

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

    1985-10-01

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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

    PubMed Central

    Emperador, Sonia; Pacheu-Grau, David; Bayona-Bafaluy, M. Pilar; Garrido-Pérez, Nuria; Martín-Navarro, Antonio; López-Pérez, Manuel J.; Montoya, Julio; Ruiz-Pesini, Eduardo

    2015-01-01

    Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA. PMID:25642242

  18. 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.

  19. A ribosomal ambiguity mutation in the 530 loop of E. coli 16S rRNA.

    PubMed Central

    O'Connor, M; Göringer, H U; Dahlberg, A E

    1992-01-01

    A series of base substitution and deletion mutations were constructed in the highly conserved 530 stem and loop region of E. coli 16S rRNA involved in binding of tRNA to the ribosomal A site. Base substitution and deletion of G517 produced significant effects on cell growth rate and translational fidelity, permitting readthrough of UGA, UAG and UAA stop codons as well as stimulating +1 and -1 frameshifting in vivo. By contrast, mutations at position 534 had little or no effect on growth rate or translational fidelity. The results demonstrate the importance of G517 in maintaining translational fidelity but do not support a base pairing interaction between G517 and U534. PMID:1380697

  20. Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae.

    PubMed Central

    Lucier, T S; Heitzman, K; Liu, S K; Hu, P C

    1995-01-01

    Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for [14C]erythromycin than did ribosomes from the M129 parent strain. Sequencing of DNA amplified from the region of the 2S rRNA gene encoding domain V revealed an A-to-G transition in the central loop at position 2063 of M129-ER1 and a similar A-to-G transition at position 2064 in M129-ER2. Transitions at homologous locations in the 23S rRNA from other organisms have been shown to result in resistance to MLS antibiotics. Thus, MLS-like resistance can occur in M. pneumoniae as the result of point mutations in the 23S rRNA gene which reduce the affinity of these antibiotics for the ribosome. Since they involve only single-base changes, development of resistance to erythromycin in vivo by these mechanisms could be relatively frequent event. PMID:8593017

  1. 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

  2. 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.

  3. 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.

  4. Recurrent gene mutations in CLL.

    PubMed

    Martínez-Trillos, Alejandra; Quesada, Víctor; Villamor, Neus; Puente, Xose S; López-Otín, Carlos; Campo, Elías

    2013-01-01

    Next-generation sequencing of whole genomes and exomes in chronic lymphocytic leukemia (CLL) has provided the first comprehensive view of somatic mutations in this disease. Subsequent studies have characterized the oncogenic pathways and clinical implications of a number of these mutations. The global number of somatic mutations per case is lower than those described in solid tumors but is in agreement with previous estimates of less than one mutation per megabase in hematological neoplasms. The number and pattern of somatic mutations differ in tumors with unmutated and mutated IGHV, extending at the genomic level the clinical differences observed in these two CLL subtypes. One of the striking conclusions of these studies has been the marked genetic heterogeneity of the disease, with a relatively large number of genes recurrently mutated at low frequency and only a few genes mutated in up to 10-15 % of the patients. The mutated genes tend to cluster in different pathways that include NOTCH1 signaling, RNA splicing and processing machinery, innate inflammatory response, Wnt signaling, and DNA damage and cell cycle control, among others. These results highlight the molecular heterogeneity of CLL and may provide new biomarkers and potential therapeutic targets for the diagnosis and management of the disease.

  5. 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.

  6. 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

  7. Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage

    PubMed Central

    Tait-Kamradt, A.; Davies, T.; Cronan, M.; Jacobs, M. R.; Appelbaum, P. C.; Sutcliffe, J.

    2000-01-01

    The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (63KPWRQKGTGRAR74) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains. PMID:10898684

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

    PubMed Central

    Arkov, Alexey L.; Hedenstierna, Klas O. F.; Murgola, Emanuel J.

    2002-01-01

    Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination. PMID:12193621

  14. 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

  15. 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.

  16. Identification of a Novel G2073A Mutation in 23S rRNA in Amphenicol-Selected Mutants of Campylobacter jejuni

    PubMed Central

    Naren, Gaowa; Li, Hui; Xia, Xi; Wu, Congming; Shen, Jianzhong; Zhang, Qijing; Wang, Yang

    2014-01-01

    Objectives This study was conducted to examine the development and molecular mechanisms of amphenicol resistance in Campylobacter jejuni by using in vitro selection with chloramphenicol and florfenicol. The impact of the resistance development on growth rates was also determined using in vitro culture. Methods Chloramphenicol and florfenicol were used as selection agents to perform in vitro stepwise selection. Mutants resistant to the selective agents were obtained from the selection process. The mutant strains were compared with the parent strain for changes in MICs and growth rates. The 23S rRNA gene and the L4 and L22 ribosomal protein genes in the mutant strains and the parent strain were amplified and sequenced to identify potential resistance-associated mutations. Results C. jejuni strains that were highly resistant to chloramphenicol and florfenicol were obtained from in vitro selection. A novel G2073A mutation in all three copies of the 23S rRNA gene was identified in all the resistant mutants examined, which showed resistance to both chloramphenicol and florfenicol. In addition, all the mutants selected by chloramphenicol also exhibited the G74D modification in ribosomal protein L4, which was previously shown to confer a low-level erythromycin resistance in Campylobacter species. The mutants selected by florfenicol did not have the G74D mutation in L4. Notably, the amphenicol-resistant mutants also exhibited reduced susceptibility to erythromycin, suggesting that the selection resulted in cross resistance to macrolides. Conclusions This study identifies a novel point mutation (G2073A) in 23S rRNA in amphenicol-selected mutants of C. jejuni. Development of amphenicol resistance in Campylobacter likely incurs a fitness cost as the mutant strains showed slower growth rates in antibiotic-free media. PMID:24728007

  17. 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

  18. 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

  19. Gene mutations in Cushing's disease

    PubMed Central

    Xiong, Qi; Ge, Wei

    2016-01-01

    Cushing's disease (CD) is a severe (and potentially fatal) disease caused by adrenocorticotropic hormone (ACTH)-secreting adenomas of the pituitary gland (often termed pituitary adenomas). The majority of ACTH-secreting corticotroph tumors are sporadic and CD rarely appears as a familial disorder, thus, the genetic mechanisms underlying CD are poorly understood. Studies have reported that various mutated genes are associated with CD, such as those in menin 1, aryl hydrocarbon receptor-interacting protein and the nuclear receptor subfamily 3 group C member 1. Recently it was identified that ubiquitin-specific protease 8 mutations contribute to CD, which was significant towards elucidating the genetic mechanisms of CD. The present study reviews the associated gene mutations in CD patients. PMID:27588171

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

    PubMed Central

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

    2012-01-01

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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Fitness cost due to mutations in the 16S rRNA associated with spectinomycin resistance in Chlamydia psittaci 6BC.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2005-11-01

    The fitness cost of a resistance determinant is the primary parameter that determines its frequency in vivo. As a model for analysis of the impact of drug resistance mutations on the intracellular life cycle of Chlamydia spp., we studied the growth of four genetically defined spectinomycin-resistant (Spc(r)) clonal variants of Chlamydia psittaci 6BC isolated in the plaque assay. The development of each variant was monitored over 46 h postinfection in the absence of drug, either in pure culture or in 1:1 competition with the parent strain. Spc(r) mutations in the 16S rRNA gene at positions 1191 and 1193 were associated with a marked impairment of C.psittaci biological fitness, and the bacteria were severely out-competed by the wild-type parent. In contrast, mutations at position 1192 had minor effects on the bacterial life cycle, allowing the resistant isolates to compete more efficiently with the wild-type strain. Thus, mutations with a wide range of fitness costs can be selected in the plaque assay, providing a new strategy for prediction and monitoring of the emergence of antibiotic resistance in chlamydiae. So far, drug resistance has not been a serious threat for the treatment of chlamydial infections. Tetracycline is an effective antichlamydial drug that targets 16S rRNA. Attempts to isolate spontaneous tetracycline-resistant mutants of C. psittaci 6BC revealed a frequency <3 x 10(-9). We suggest that the rarity of genotypic antibiotic resistance among chlamydial clinical isolates reflects the deleterious effects of such mutations on the fitness of these obligate intracellular bacteria in the host.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. [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.

  9. The androgen receptor gene mutations database.

    PubMed

    Patterson, M N; Hughes, I A; Gottlieb, B; Pinsky, L

    1994-09-01

    The androgen receptor gene mutations database is a comprehensive listing of mutations published in journals and meetings proceedings. The majority of mutations are point mutations identified in patients with androgen insensitivity syndrome. Information is included regarding the phenotype, the nature and location of the mutations, as well as the effects of the mutations on the androgen binding activity of the receptor. The current version of the database contains 149 entries, of which 114 are unique mutations. The database is available from EMBL (NetServ@EMBL-Heidelberg.DE) or as a Macintosh Filemaker file (mc33001@musica.mcgill.ca).

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Gene mutations in chronic lymphocytic leukemia.

    PubMed

    Amin, Nisar A; Malek, Sami N

    2016-04-01

    The recent discovery of genes mutated in chronic lymphocytic leukemia (CLL) has stimulated new research into the role of these genes in CLL pathogenesis. CLL cases carry approximately 5-20 mutated genes per exome, a lower number than detected in many human tumors. Of the recurrently mutated genes in CLL, all are mutated in 10% or less of patients when assayed in unselected CLL cohorts at diagnosis. Mutations in TP53 are of major clinical relevance, are often associated with del17p and gain in frequency over time. TP53 mutated and associated del17p states substantially lower response rates, remission duration, and survival in CLL. Mutations in NOTCH1 and SF3B1 are recurrent, often associated with progressive CLL that is also IgVH unmutated and ZAP70-positive and are under investigation as targets for novel therapies and as factors influencing CLL outcome. There are an estimated 20-50 additional mutated genes with frequencies of 1%-5% in CLL; more work is needed to identify these and to study their significance. Finally, of the major biological aberration categories influencing CLL as a disease, gene mutations will need to be placed into context with regard to their ultimate role and importance. Such calibrated appreciation necessitates studies incorporating multiple CLL driver aberrations into biological and clinical analyses.

  3. 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

  4. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology

    PubMed Central

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James

    2012-01-01

    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  5. 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.

  6. Properties of small rRNA methyltransferase RsmD: Mutational and kinetic study

    PubMed Central

    Sergeeva, Olga V.; Prokhorova, Irina V.; Ordabaev, Yerdos; Tsvetkov, Philipp O.; Sergiev, Petr V.; Bogdanov, Alexey A.; Makarov, Alexander A.; Dontsova, Olga A.

    2012-01-01

    Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit’s involvement in translation. PMID:22535590

  7. 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.

  8. Temperature Sensitivity Caused by Mutant Release Factor 1 Is Suppressed by Mutations That Affect 16S rRNA Maturation

    PubMed Central

    Kaczanowska, Magdalena; Rydén-Aulin, Monica

    2004-01-01

    To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the rplM-rpsI operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the yrdC open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1. PMID:15126466

  9. 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.

  10. 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

  11. 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

  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. Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy.

    PubMed

    De Francesco, Vincenzo; Zullo, Angelo; Giorgio, Floriana; Saracino, Ilaria; Zaccaro, Cristina; Hassan, Cesare; Ierardi, Enzo; Di Leo, Alfredo; Fiorini, Giulia; Castelli, Valentina; Lo Re, Giovanna; Vaira, Dino

    2014-03-01

    Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8%), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88% (P<0.001; odds ratio 6.1, 95% confidence interval 2-18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

  15. Genes and mutations causing retinitis pigmentosa

    PubMed Central

    Daiger, SP; Sullivan, LS; Bowne, SJ

    2013-01-01

    Retinitis pigmentosa (RP) is a heterogeneous set of inherited retinopathies with many disease-causing genes, many known mutations, and highly varied clinical consequences. Progress in finding treatments is dependent on determining the genes and mutations causing these diseases, which includes both gene discovery and mutation screening in affected individuals and families. Despite the complexity, substantial progress has been made in finding RP genes and mutations. Depending on the type of RP, and the technology used, it is possible to detect mutations in 30–80% of cases. One of the most powerful approaches to genetic testing is high-throughput ‘deep sequencing’, that is, next-generation sequencing (NGS). NGS has identified several novel RP genes but a substantial fraction of previously unsolved cases have mutations in genes that are known causes of retinal disease but not necessarily RP. Apparent discrepancy between the molecular defect and clinical findings may warrant reevaluation of patients and families. In this review, we summarize the current approaches to gene discovery and mutation detection for RP, and indicate pitfalls and unsolved problems. Similar considerations apply to other forms of inherited retinal disease. PMID:23701314

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. [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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

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

    PubMed Central

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

    1992-01-01

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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. DHPLC screening of cystic fibrosis gene mutations.

    PubMed

    Ravnik-Glavac, Metka; Atkinson, Andrew; Glavac, Damjan; Dean, Michael

    2002-04-01

    Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Trifiro, M; Lumbroso, R; Vasiliou, D M; Pinsky, L

    1996-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).

  11. 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

  12. GJB2 gene mutations in childhood deafness.

    PubMed

    Angeli, S; Utrera, R; Dib, S; Chiossone, E; Naranjo, C; Henríquez, O; Porta, M

    2000-03-01

    The frequency of childhood deafness is estimated at 1:1,000 and at least half of these cases are genetic. Recently, mutations in the GJB2 gene have been found in a great number of familial and sporadic cases of congenital deafness in Caucasians. The most common mutation (70%) is the frameshift mutation of a single guanine in position 35 (35delG). More than 20 mutations in the GJB2 gene are associated with DFNB1, a prevalent type of autosomal recessive non-syndromic neurosensory deafness. Last year we initiated a systematic screening programme to evaluate the causes of deafness in the population of prelingually deaf children who are referred to our cochlear implant programme. All of the deaf children and their parents undergo a comprehensive medical review, directed to identify causes of acquired deafness and manifestations of syndromic hearing impairment. DNA is extracted from the blood of all of the children. The technique AS-PCR (allele-specific polymerase chain reaction) is used for the identification of the mutation 35delG. Screening for other GJB2 gene mutations is carried out by single-strand conformation polymorphisms (SSCP). Our results on the identification of DFNB1 will be presented, as well as a discussion on the implications of an aetiological diagnosis in cochlear implantation.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Mediastinal paragangliomas related to SDHx gene mutations

    PubMed Central

    Ćwikła, Jarosław; Prejbisz, Aleksander; Kwiatek, Paweł; Szperl, Małgorzata; Michalski, Wojciech; Wyrwicz, Lucjan; Kuśmierczyk, Mariusz; Januszewicz, Andrzej; Maciejczyk, Anna; Roszczynko, Marta; Pęczkowska, Mariola

    2016-01-01

    Introduction Paragangliomas (PGLs) related to hereditary syndromes are rare mediastinal tumors. Paragangliomas are caused by mutations in genes encoding subunits of succinate dehydrogenase enzyme (SDH). Aim To evaluate clinical, anatomical and functional characteristics of mediastinal paragangliomas related to SDHx gene mutations. Material and methods Retrospective analysis of 75 patients with confirmed SDHx gene mutations (24 patients with SDHB, 5 SDHC, 46 with SDHD mutations) was performed. Patients underwent evaluation using computed tomography (CT), somatostatin receptor scintigraphy (SRS) (99mTc-[HYNIC,Tyr3]-octreotide), 123I mIBG scintigraphy and urinary excretion of total methoxycatecholamines. Results Out of 75 patients, 16 (21%) patients (1 SDHB, 15 SDHD mutations) had 17 PGLs localized in the mediastinum. Fourteen PGLs were localized in the middle mediastinum (intrapericardial) and 3 PGLs in the posterior mediastinum. The median diameter of paragangliomas measured on the axial slice was 24.3 mm (interquartile range (IQR): 14.7–36.6), and the median volume was 2.78 ml (IQR: 0.87–16.16). Twelve out of 16 patients (75%) underwent SRS, and 11 of them (92.3%) had pathological uptake of the radiotracer. Eleven (68.75%) out of 16 patients underwent 123 I mIBG, with only 3 positive results. Symptoms of catecholamine excretion were observed in 3 patients with PGLs localized in the posterior mediastinum. All PGLs were benign except in 1 patient with the SDHB mutation and PGL detected in the posterior mediastinum, who had a metastatic disease. Conclusions Most mediastinal paragangliomas were related to SDHD gene mutations. They were asymptomatic, localized in the medial mediastinum, intrapericardially. PMID:27785149

  19. 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

  20. 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.

  1. 23S rRNA base pair 2057–2611 determines ketolide susceptibility and fitness cost of the macrolide resistance mutation 2058A→G

    PubMed Central

    Pfister, Peter; Corti, Natascia; Hobbie, Sven; Bruell, Christian; Zarivach, Raz; Yonath, Ada; Böttger, Erik C.

    2005-01-01

    The 23S rRNA A2058G alteration mediates macrolide, lincosamide, and streptogramin B resistance in the bacterial domain and determines the selectivity of macrolide antibiotics for eubacterial ribosomes, as opposed to eukaryotic ribosomes. However, this mutation is associated with a disparate resistance phenotype: It confers high-level resistance to ketolides in mycobacteria but only marginally affects ketolide susceptibility in streptococci. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular basis for this disparity. We show that mutational alteration of the polymorphic 2057–2611 base pair from A-U to G-C in isogenic mutants of Mycobacterium smegmatis significantly affects susceptibility to ketolides but does not influence susceptibility to other macrolide antibiotics. In addition, we provide evidence that the 2057–2611 polymorphism determines the fitness cost of the 23S rRNA A2058G resistance mutation. Supported by structural analysis, our results indicate that polymorphic nucleotides mediate the disparate phenotype of genotypically identical resistance mutations and provide an explanation for the large species differences in the epidemiology of defined drug resistance mutations. PMID:15795375

  2. 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...

  3. 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...

  4. 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.

  5. 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.

  6. PTCH gene mutations in odontogenic keratocysts.

    PubMed

    Barreto, D C; Gomez, R S; Bale, A E; Boson, W L; De Marco, L

    2000-06-01

    An odontogenic keratocyst (OKC) is a benign cystic lesion of the jaws that occurs sporadically or in association with nevoid basal cell carcinoma syndrome (NBCCS). Recently, the gene for NBCCS was cloned and shown to be the human homologue of the Drosophila segment polarity gene Patched (PTCH), a tumor suppressor gene. The PTCH gene encodes a transmembrane protein that acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues, including tooth. We investigated three cases of sporadic odontogenic keratocysts and three other cases associated with NBCCS, looking for mutations of the PTCH gene. Non-radioactive single-strand conformational polymorphism and direct sequencing of PCR products revealed a deletion of 5 base pairs (bp) in exon 3 (518delAAGCG) in one sporadic cyst as well as mutations in two cysts associated with NBCCS, a nonsense (C2760A) and a missense (G3499A) alteration. This report is the first to describe a somatic mutation of PTCH in sporadic odontogenic keratocysts as well as two novel mutations in cysts associated with NBCCS, indicating a similar pathogenesis in a subset of sporadic keratocysts.

  7. 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

  8. 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

  9. Binding Site of Macrolide Antibiotics on the Ribosome: New Resistance Mutation Identifies a Specific Interaction of Ketolides with rRNA

    PubMed Central

    Garza-Ramos, Georgina; Xiong, Liqun; Zhong, Ping; Mankin, Alexander

    2001-01-01

    Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of

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

    PubMed Central

    Olson, Nathan D.; Lund, Steven P.; Zook, Justin M.; Rojas-Cornejo, Fabiola; Beck, Brian; Foy, Carole; Huggett, Jim; Whale, Alexandra S.; Sui, Zhiwei; Baoutina, Anna; Dobeson, Michael; Partis, Lina; Morrow, Jayne B.

    2015-01-01

    This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing®, or Ion Torrent PGM®. The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies. PMID:27077030

  11. 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.

  12. The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease

    PubMed Central

    Elson, Joanna L.; Smith, Paul M.; Greaves, Laura C.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

    2015-01-01

    Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health. PMID:26349026

  13. The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease.

    PubMed

    Elson, Joanna L; Smith, Paul M; Greaves, Laura C; Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A; Taylor, Robert W; Vila-Sanjurjo, Antón

    2015-11-01

    Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  14. 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

  15. The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Trifiro, M; Lumbroso, R; Pinsky, L

    1997-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 212 to 272. We have expanded the database: (i) by adding a large amount of new data on somatic mutations in prostatic cancer tissue; (ii) by defining a new constitutional phenotype, mild androgen insensitivity (MAI); (iii) by placing additional relevant information on an internet site (http://www.mcgill.ca/androgendb/ ). The database has allowed us to examine the contribution of CpG sites to the multiplicity of reports of the same mutation in different families. The database is also available from EMBL (ftp.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker Pro or Word file (MC33@musica,mcgill.ca)

  16. The androgen receptor gene mutations database.

    PubMed Central

    Gottlieb, B; Trifiro, M; Lumbroso, R; Pinsky, L

    1997-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 212 to 272. We have expanded the database: (i) by adding a large amount of new data on somatic mutations in prostatic cancer tissue; (ii) by defining a new constitutional phenotype, mild androgen insensitivity (MAI); (iii) by placing additional relevant information on an internet site (http://www.mcgill.ca/androgendb/ ). The database has allowed us to examine the contribution of CpG sites to the multiplicity of reports of the same mutation in different families. The database is also available from EMBL (ftp.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker Pro or Word file (MC33@musica,mcgill.ca) PMID:9016528

  17. Mutations in 23S rRNA Account for Intrinsic Resistance to Macrolides in Mycoplasma hominis and Mycoplasma fermentans and for Acquired Resistance to Macrolides in M. hominis

    PubMed Central

    Pereyre, S.; Gonzalez, P.; de Barbeyrac, B.; Darnige, A.; Renaudin, H.; Charron, A.; Raherison, S.; Bébéar, C.; Bébéar, C. M.

    2002-01-01

    The mechanisms of intrinsic resistance of Mycoplasma hominis to 14- and 15-membered macrolides were investigated in comparison with those of M. pneumoniae, which is naturally susceptible to macrolides. Radiolabeled erythromycin was not accumulated by M. hominis PG21, but addition of an ABC transporter inhibitor increased the level of erythromycin uptake more than two times, suggesting the existence of an active efflux process. The affinity of [14C]erythromycin to ribosomes isolated from M. hominis was dramatically reduced relative to that to ribosomes isolated from M. pneumoniae. The nucleotide sequences of 23S rRNA of both ribosomal operons rrnA and rrnB and ribosomal proteins L4 and L22 of M. hominis were obtained. Compared to the sequence of M. pneumoniae, M. hominis harbored a G2057A transition in its 23S rRNA sequence, as did M. fermentans, another mycoplasma that is erythromycin resistant. An additional C2610U change was also found in the sequence of M. hominis. Moreover, two M. hominis clinical isolates with acquired resistance to 16-membered macrolides were examined for mutations in domain II and domain V of 23S rRNA and in ribosomal proteins L4 and L22. Compared to the sequence of reference strain PG21, one isolate harbored a A2059G transition and a C2611U transition in one of the two rrn operons, while the other one was mutated only at position 2059, also on the same operon. No mutation was found in the two ribosomal protein sequences. Overall, the present study is an exhaustive characterization of the intrinsic resistance of M. hominis to 14- and 15-membered macrolides and the first description of mycoplasma clinical isolates resistant to macrolide, lincosamide, and streptogramin antibiotics harboring a mutation at position 2611 in the 23S rRNA. PMID:12234836

  18. 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.

  19. 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.

  20. Phylogenetic analysis of the genus Microbacterium based on 16S rRNA gene sequences.

    PubMed

    Takeuchi, M; Yokota, A

    1994-11-15

    16S rRNA gene (rDNA) studies of the six species of the genus Microbacterium, M. lacticum, M. laevaniformans, M. dextranolyticum, M. imperiale, M. arborescens and M. aurum, were performed and the primary structures were compared with those of 29 representative actinobacteria and related organisms. Phylogenetic analysis indicated that six species of the genus Microbacterium and representative four species of the genus Aureobacterium appear to be phylogenetically coherent as was suggested by Rainey et al., although the peptidoglycan types of these two genera are different (peptidoglycan type B1 or B2). Thus, the phylogenetical analyses revealed that members of actinobacteria with group B-peptidoglycan do not cluster according to their peptidoglycan types, but form compact cluster different from actinobacteria or actinomycetes with group A-peptidoglycan.

  1. 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

  2. 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.

  3. From Gene Mutation to Protein Characterization

    ERIC Educational Resources Information Center

    Moffet, David A.

    2009-01-01

    A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the…

  4. From Gene Mutation to Protein Characterization

    ERIC Educational Resources Information Center

    Moffet, David A.

    2009-01-01

    A seven-week "gene to protein" laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the…

  5. Multicentric origin of hemochromatosis gene (HFE) mutations.

    PubMed Central

    Rochette, J; Pointon, J J; Fisher, C A; Perera, G; Arambepola, M; Arichchi, D S; De Silva, S; Vandwalle, J L; Monti, J P; Old, J M; Merryweather-Clarke, A T; Weatherall, D J; Robson, K J

    1999-01-01

    Genetic hemochromatosis (GH) is believed to be a disease restricted to those of European ancestry. In northwestern Europe, >80% of GH patients are homozygous for one mutation, the substitution of tyrosine for cysteine at position 282 (C282Y) in the unprocessed protein. In a proportion of GH patients, two mutations are present, C282Y and H63D. The clinical significance of this second mutation is such that it appears to predispose 1%-2% of compound heterozygotes to expression of the disease. The distribution of the two mutations differ, C282Y being limited to those of northwestern European ancestry and H63D being found at allele frequencies>5%, in Europe, in countries bordering the Mediterranean, in the Middle East, and in the Indian subcontinent. The C282Y mutation occurs on a haplotype that extends mutation has arisen during the past 2,000 years. The H63D mutation is older and does not occur on such a large extended haplotype, the haplotype in this case extending mutations on new haplotypes. In Sri Lanka we have found H63D on three new haplotypes and have found C282Y on one new haplotype, demonstrating that these mutations have arisen independently on this island. These results suggest that the HFE gene has been the subject of selection pressure. These selection pressures could be due to infectious diseases, environmental conditions, or other genetic disorders such as anemia. PMID:10090890

  6. 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.

  7. 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

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

    PubMed Central

    2014-01-01

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

  9. 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

  10. 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.

  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

    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.

  12. 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.

  13. 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

  14. 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.

  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

    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.

  16. 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

  17. INPPL1 gene mutations in opsismodysplasia

    PubMed Central

    Fradet, Anaïs; Fitzgerald, Jamie

    2016-01-01

    The INPPL1 (inositol polyphosphate phosphatase-like 1) gene encodes the inositol phosphatase, SHIP2 (for src homology 2 domain-containing inositol phosphatase 2). SHIP2 functions to dephosphorylate, and negatively regulate, the lipid second messenger phosphatidylinositol (3,4,5)P3. SHIP2 has been well studied in the area of insulin resistance and obesity but has roles in cancer and other disorders. Recently, it was reported that mutations in INPPL1 cause opsismodysplasia, a rare, autosomal recessive severe skeletal dysplasia. This review focuses on the mutations associated with opsismodysplasia and explores the role of INPPL1/ SHIP2 in skeletal development. PMID:27708270

  18. INPPL1 gene mutations in opsismodysplasia.

    PubMed

    Fradet, Anaïs; Fitzgerald, Jamie

    2017-02-01

    The INPPL1 (inositol polyphosphate phosphatase-like 1) gene encodes the inositol phosphatase, SHIP2 (for src homology 2 domain-containing inositol phosphatase 2). SHIP2 functions to dephosphorylate, and negatively regulate, the lipid second messenger phosphatidylinositol (3,4,5)P3. SHIP2 has been well studied in the area of insulin resistance and obesity but has roles in cancer and other disorders. Recently, it was reported that mutations in INPPL1 cause opsismodysplasia, a rare, autosomal recessive severe skeletal dysplasia. This review focuses on the mutations associated with opsismodysplasia and explores the role of INPPL1/ SHIP2 in skeletal development.

  19. Recurrent APC gene mutations in Polish FAP families

    PubMed Central

    Pławski, Andrzej; Podralska, Marta; Słomski, Ryszard

    2007-01-01

    The molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes. Hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the DNA repair genes. Occurrence of recurrent mutation allows improvement of molecular diagnostics. The mutation spectrum in the genes causing hereditary forms of colorectal cancers in the Polish population was previously described. In the present work an estimation of the frequency of the recurrent mutations of the APC gene was performed. Eight types of mutations occurred in 19.4% of our FAP families and these constitute 43% of all Polish diagnosed families. PMID:19725996

  20. Mutated Genes in Schizophrenia Map to Brain Networks

    MedlinePlus

    ... Matters NIH Research Matters August 12, 2013 Mutated Genes in Schizophrenia Map to Brain Networks Schizophrenia networks in the prefrontal ... Vasculitis Therapy as Effective as Standard Care Mutated Genes in Schizophrenia Map to Brain Networks Connect with Us Subscribe to ...

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. 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...

  10. 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...

  11. 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

  12. 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...

  13. 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

  14. 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...

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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...

  20. 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...

  1. 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.

  2. The Androgen Receptor Gene Mutations Database.

    PubMed

    Gottlieb, B; Lehvaslaiho, H; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M

    1998-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 272 to 309 in the past year. We have expanded the database: (i) by giving each entry an accession number; (ii) by adding information on the length of polymorphic polyglutamine (polyGln) and polyglycine (polyGly) tracts in exon 1; (iii) by adding information on large gene deletions; (iv) by providing a direct link with a completely searchable database (courtesy EMBL-European Bioinformatics Institute). The addition of the exon 1 polymorphisms is discussed in light of their possible relevance as markers for predisposition to prostate or breast cancer. The database is also available on the internet (http://www.mcgill. ca/androgendb/ ), from EMBL-European Bioinformatics Institute (ftp. ebi.ac.uk/pub/databases/androgen ), or as a Macintosh FilemakerPro or Word file (MC33@musica.mcgill.ca).

  3. The Androgen Receptor Gene Mutations Database.

    PubMed Central

    Gottlieb, B; Lehvaslaiho, H; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M

    1998-01-01

    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 272 to 309 in the past year. We have expanded the database: (i) by giving each entry an accession number; (ii) by adding information on the length of polymorphic polyglutamine (polyGln) and polyglycine (polyGly) tracts in exon 1; (iii) by adding information on large gene deletions; (iv) by providing a direct link with a completely searchable database (courtesy EMBL-European Bioinformatics Institute). The addition of the exon 1 polymorphisms is discussed in light of their possible relevance as markers for predisposition to prostate or breast cancer. The database is also available on the internet (http://www.mcgill. ca/androgendb/ ), from EMBL-European Bioinformatics Institute (ftp. ebi.ac.uk/pub/databases/androgen ), or as a Macintosh FilemakerPro or Word file (MC33@musica.mcgill.ca). PMID:9399843

  4. [Gene mutation analysis of X-linked hypophosphatemic rickets].

    PubMed

    Song, Ying; Ma, Hong-Wei; Li, Fang; Hu, Man; Ren, Shuang; Yu, Ya-Fen; Zhao, Gui-Jie

    2013-11-01

    To investigate the frequency and type of PHEX gene mutations in children with X-linked hypophosphatemic rickets (XLH), the possible presence of mutational hot spots, and the relationship between genotype and clinical phenotype. Clinical data of 10 children with XLH was retrospectively reviewed. The relationship between gene mutation type and severity of XLH was evaluated. PHEX gene mutations were detected in all 10 children with XLH, including 6 cases of missense mutation, 2 cases of splice site mutation, 1 case of frameshift mutation, and 1 case of nonsense mutation. Two new mutations, c.2048T>C and IVS14+1delAG, were found. The type of PHEX gene mutation was not associated with the degree of short stature and leg deformity (P=0.571 and 0.467), and the mutation site was also not associated with the degree of short stature and leg deformity (P=0.400 and 1.000). Missense mutation is the most common type of PHEX gene mutation in children with XLH, and c.2048T>C and IVS14+1delAG are two new PHEX gene mutations. The type and site of PHEX gene mutation are not associated with the severity of XLH.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Characterization of Acinetobacter baumannii clinical isolates carrying bla(OXA-23) carbapenemase and 16S rRNA methylase armA genes in Yemen.

    PubMed

    Bakour, Sofiane; Alsharapy, Samer Ahmed; Touati, Abdelaziz; Rolain, Jean-Marc

    2014-12-01

    The aim of this study was to investigate the molecular support of resistance to carbapenems, aminoglycosides, and fluoroquinolones in Acinetobacter baumannii clinical isolates collected from Yemen hospital. Three A. baumannii were isolated in February 2013 from three patients hospitalized at Al-Thawra University Hospital in Sana'a, Yemen. Antibiotic susceptibility testing was performed using the disk diffusion and E-test methods. Carbapenemase production was carried out by the modified Hodge test (MHT) and imipenem-ethylenediaminetetraacetic acid (EDTA) methods. Carbapenem, aminoglycoside, and fluoroquinolone resistance determinants were studied by polymerase chain reaction and sequencing. The epidemiological relatedness of the three strains was studied using multilocus sequence typing (MLST). The isolates were resistant to almost all antibiotics tested with very high imipenem, amikacin, and ciprofloxacin minimum inhibitory concentrations (>32, >256, and >32 mg/L, respectively). The microbiological tests showed that the three A. baumannii were MHT positive, besides, the activity of β-lactamases was not inhibited by EDTA. All the three isolates contained the naturally occurring bla(OXA-51)-like gene and the bla(OXA-23)-like carbapenemase-encoding gene. The 16S rRNA methylase armA gene was detected in the three isolates. In addition, screening for genes encoding the aminoglycoside-modifying enzymes (AMEs) demonstrated that one isolate contained the acetyltransferase gene aac(6')-Ib. Fluoroquinolone resistance was associated with a single mutation Ser83Leu in the quinolone resistance determining region of the gyrA gene in all isolates. The MLST showed that the sequence type (ST) obtained corresponds to ST2 for the three strains. Here we report the first identification of multidrug-resistant A. baumannii isolates harboring the bla(OXA-23)-like gene, AMEs [aac(6')-Ib], and the 16S rRNA methylase (armA) in the Yemen hospital.

  10. Hereditary sideroblastic anemia: pathophysiology and gene mutations.

    PubMed

    Harigae, Hideo; Furuyama, Kazumichi

    2010-10-01

    Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. Ring sideroblasts are erythroblasts characterized by iron accumulation in perinuclear mitochondria due to impaired iron utilization. There are two forms of sideroblastic anemia, i.e., inherited and acquired sideroblastic anemia. Inherited sideroblastic anemia is a rare and heterogeneous disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or Fe-S cluster transport, and mitochondrial metabolism. The most common inherited sideroblastic anemia is X-linked sideroblastic anemia (XLSA) caused by mutations of the erythroid-specific δ-aminolevulinate synthase gene (ALAS2), which is the first enzyme of heme biosynthesis in erythroid cells. Sideroblastic anemia due to SLC25A38 gene mutations, which is a mitochondrial transporter, is the next most common inherited sideroblastic anemia. Other forms of inherited sideroblastic anemia are very rare, and accompanied by impaired function of organs other than hematopoietic tissue, such as the nervous system, muscle, or exocrine glands due to impaired mitochondrial metabolism. Moreover, there are still significant numbers of cases with genetically undefined inherited sideroblastic anemia. Molecular analysis of these cases will contribute not only to the development of effective treatment, but also to the understanding of mitochondrial iron metabolism.

  11. 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.

  12. Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae.

    PubMed

    González, Asier; Casado, Carlos; Ariño, Joaquín; Casamayor, Antonio

    2013-01-01

    Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li(+). Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters.

  13. Ptc6 Is Required for Proper Rapamycin-Induced Down-Regulation of the Genes Coding for Ribosomal and rRNA Processing Proteins in S. cerevisiae

    PubMed Central

    González, Asier; Casado, Carlos; Ariño, Joaquín; Casamayor, Antonio

    2013-01-01

    Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li+. Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters. PMID:23704987

  14. 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.

  15. 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.

  16. 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...

  17. 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...

  18. 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 ...

  19. 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...

  20. 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...

  1. 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

  2. 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

  3. 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.

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

    PubMed

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

    2016-01-01

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

  5. 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

  6. 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

  7. [Spontaneous spectinomycin resistance mutations of the chloroplast rrn16 gene in Daucus carota callus lines].

    PubMed

    Filipenko, E A; Sidorchuk, Iu V; Deĭneko, E V

    2011-01-01

    Bioballistic transformation of carrot Daucus carota L. callus cultures with a plasmid containing the aadA (aminoglycoside 3'-adenyltransferase) gene and subsequent selection oftransformants on a selective medium containing spectinomycin (100-500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3' end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.

  8. 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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2008-03-01

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

  11. 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/.

  12. 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.

  13. Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA.

    PubMed

    Leppik, Margus; Ero, Rya; Liiv, Aivar; Kipper, Kalle; Remme, Jaanus

    2012-05-01

    Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.

  14. 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

  15. Novel recurrently mutated genes in African American colon cancers

    PubMed Central

    Guda, Kishore; Veigl, Martina L.; Varadan, Vinay; Nosrati, Arman; Ravi, Lakshmeswari; Lutterbaugh, James; Beard, Lydia; Willson, James K. V.; Sedwick, W. David; Wang, Zhenghe John; Molyneaux, Neil; Miron, Alexander; Adams, Mark D.; Elston, Robert C.; Markowitz, Sanford D.; Willis, Joseph E.

    2015-01-01

    We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors. PMID:25583493

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. [Hyperuricemia and gene mutations: a case report].

    PubMed

    Tattoli, Fabio; Falconi, Daniela; De Prisco, Ornella; Maurizio, Gherzi; Marazzi, Federico; Marengo, Marita; Serra, Ilaria; Tamagnone, Michela; Cordero di Montezemolo, Luca; Pasini, Barbara; Formica, Marco

    2017-06-01

    Hyperuricemia is frequently found in nephrology. The case presented may be useful to clarify some pathogenetic aspects. It is a patient of 18 years, hyperuricaemic. Non-consanguineous parents, hyperuricemia in the paternal line, not neuropsychiatric disorders in the family. Delay in neuromotor acquisitions, average intellectual disabilities, anxiety disorder, obsessive-compulsive personality traits. Normal renal function and renal ultrasound. Evidence of hyperuricemia in 2015. Never gouty episodes and / or lithiasis, initiated allopurinol 100 mg on alternate days, with no side effects, urea in the control range, slightly below normal uricuria. Given the complex clinical, he carried out a genetic analysis of array-CGH. He showed a deletion on the short arm of chromosome 3 (3p12.3) and a duplication of the long arm of chromosome 1 (19q13-42). The deletion 3p12.3 (paternal inheritance), involves the ROBO2 gene. Duplication 19q13.42, (maternal inheritance), includes NLRP12, DPRX, ZNF331 genes. The ROBO2 gene with its mutation, is associated with vesicoureteral reflux. The NLRP12 gene encodes proteins called "Nalps", forming a subfamily of proteins "CATERPILLAR". Many "Nalps" as well as the "Nalps 12" have an N-terminal domain (DYP) with a purin. Since uric acid is a byproduct of purine metabolism, considered the familiarity, we believe that we can hypothesize that the mutations found. In particular those concerning the NLRP-12 gene, may have a role in the presence of hyperuricemia. We believe that in patients with hyperuricemia, associated with a particular impairment of neurological picture, it is likely that there is a subtended common genetic deficiency. Copyright by Società Italiana di Nefrologia SIN, Rome, Italy.

  1. 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

  2. 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.

  3. 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

  4. [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.

  5. 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.

  6. 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

  7. 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

  8. [Screening of common deafness gene mutations in 17 000 Chinese newborns from Chengdu based on microarray analysis].

    PubMed

    Lyu, Kangmo; Xiong, Yehua; Yu, Hao; Zou, Ling; Ran, Longrong; Liu, Deshun; Yin, Qin; Xu, Yingwen; Fang, Xue; Song, Zuling; Huang, Lijia; Tan, Dayong; Zhang, Zhiwei

    2014-10-01

    To achieve early diagnosis for inheritable hearing loss and determine carrier rate of deafness causing gene mutations in order to provide information for premarital, prenatal and postnatal genetic counseling. A total of 17 000 dried heel blood spots of normal newborns in Chengdu were collected with informed consent obtained from their parents. Genomic DNA was extracted from dried blood spots using Qiagen DNA extraction kits. Microarrays with 9 common mutation loci of 4 deafness-associated genes in Chinese population were used. Nine hot mutations including GJB2 (35delG, 176del16, 235delC and 299delAT), GJB3 (538C> T), SLC26A4 (IVS 7-2A> G, 2168A> G), and mitochondrial DNA 12S rRNA (1555A> G, 1494C> T) were detected by PCR amplification and microarray hybridization. Mutations detected by microarray were verified by Sanger DNA sequencing. Of the 17 000 new-borns, 542 neonates had mutations of the 4 genes. Heterozygous mutations of GJB2, at 235delC, 299delAT, and 176del16 were identified in 254, 55, and 15 newborns, respectively. Two newborns had homozygous mutation of GJB2, 235delC. Heterozygous mutations at 538C> T of GJB3, 2168A> G and IVS 7-2A> G of SLC26A4 were found in 23, 17 and 128 newborns, respectively. For mutation analysis of mitochondrial DNA 12S rRNA, 1494C> T and 1555A> G were homogeneous mutations in 4 and 42 neonates, respectively. In addition, 6 complexity mutations were detected, which demonstrated that one newborn had heterozygous mutations at GJB2 235delC and SLC26A4, IVS7-2A> G, one had heterozygous mutation GJB2 235delC and 12S rRNA homogeneous mutation, 1555 A> G, one heterozygous mutations at GJB2, 299delAT, and GJB3, 538C> T, one at GJB2, 299delAT and 12S rRNA, 1555 A> G, two at GJB2, 299delAT, and SLC26A4, IVS7-2A> G. All mutations as above were confirmed by DNA sequencing. The total mutation carrier rate of the 4 deafness genes is 3.19% in healthy newborns at Chengdu. Mutations of GJB2 and SLAC26A4 are major ones (86.5% of total). The

  9. Mutational analysis of the human MAOA gene

    SciTech Connect

    Tivol, E.A.; Shalish, C.; Schuback, D.E.; Breakefield, X.O.; Hsu, Yun-Pung

    1996-02-16

    The monoamine oxidases (MAO-A and MAO-B) are the enzymes primarily responsible for the degradation of amine neurotransmitters, such as dopamine, norepinephrine, and serotonin. Wide variations in activity of these isozymes have been reported in control humans. The MAOA and MAOB genes are located next to each other in the p11.3-11.4 region of the human X chromosome. Our recent documentation of an MAO-A-deficiency state, apparently associated with impulsive aggressive behavior in males, has focused attention on genetic variations in the MAOA gene. In the present study, variations in the coding sequence of the MAOA gene were evaluated by RT-PCR, SSCP, and sequencing of mRNA or genomic DNA in 40 control males with >100-fold variations in MAOA activity, as measured in cultured skin fibroblasts. Remarkable conservation of the coding sequence was found, with only 5 polymorphisms observed. All but one of these were in the third codon position and thus did not alter the deduced amino acid sequence. The one amino acid alteration observed, lys{r_arrow}arg, was neutral and should not affect the structure of the protein. This study demonstrates high conservation of coding sequence in the human MAOA gene in control males, and provides primer sets which can be used to search genomic DNA for mutations in this gene in males with neuropsychiatric conditions. 47 refs., 1 fig., 2 tabs.

  10. 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

  11. 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 .

  12. 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.

  13. 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...

  14. Patterns of Somatic Mutations in Immunoglobulin Variable Genes

    PubMed Central

    Golding, G. Brian; Gearhart, Patricia J.; Glickman, Barry W.

    1987-01-01

    The mechanism responsible for somatic mutation in the variable genes of antibodies is unknown and may differ from previously described mechanisms that produce mutation in DNA. We have analyzed 421 somatic mutations from the rearranged immunoglobulin variable genes of mice to determine (1) if the nucleotide substitutions differ from those generated during meiosis and (2) if the presence of nearby direct and inverted repeated sequences could template mutations around the variable gene. The results reveal a difference in the pattern of substitutions obtained from somatic mutations vs. meiotic mutations. An increased frequency of T:A to C:G transitions and a decreased frequency of mutations involving a G in the somatic mutants compared to the meiotic mutants is indicated. This suggests that the mutational processes responsible for somatic mutation in antibody genes differs from that responsible for mutation during meiosis. An analysis of the local DNA sequences revealed many direct repeats and palindromic sequences that were capable of templating some of the known mutations. Although additional factors may be involved in targeting mutations to the variable gene, mistemplating by nearby repeats may provide a mechanism for the enhancement of somatic mutation. PMID:3557109

  15. 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

  16. Succinate dehydrogenase gene mutations in cardiac paragangliomas.

    PubMed

    Martucci, Victoria L; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M; Magoon, Bindiya T; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L; Audibert, Jennifer L; Adams, Karen T; Rosing, Douglas R; Vaidya, Anand; Dluhy, Robert G; Horvath, Keith A; Pacak, Karel

    2015-06-15

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least 1/3 of paragangliomas are related to germline mutations in 1 of 17 genes. Although these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for <0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of 15 patients with cardiac paragangliomas was performed to determine clinical presentation, genetic background, diagnostic workup, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 patients (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen patients (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; 1 additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to co-morbidities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, the investigators extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. Published by Elsevier Inc.

  17. Succinate Dehydrogenase Gene Mutations in Cardiac Paragangliomas

    PubMed Central

    Martucci, Victoria L.; Emaminia, Abbas; del Rivero, Jaydira; Lechan, Ronald M.; Magoon, Bindiya T.; Galia, Analyza; Fojo, Tito; Leung, Steve; Lorusso, Roberto; Jimenez, Camilo; Shulkin, Barry L.; Audibert, Jennifer L.; Adams, Karen T.; Rosing, Douglas R.; Vaidya, Anand; Dluhy, Robert G.; Horvath, Keith A.; Pacak, Karel

    2015-01-01

    Pheochromocytomas and paragangliomas are chromaffin cell tumors arising from neuroendocrine cells. At least one third of paragangliomas are related to germline mutations in one of 17 genes. While these tumors can occur throughout the body, cardiac paragangliomas are very rare, accounting for less than 0.3% of mediastinal tumors. The purpose of this study was to determine the clinical characteristics of patients with cardiac paragangliomas, particularly focusing on their genetic backgrounds. A retrospective chart analysis of fifteen patients with cardiac paraganglioma was performed to determine clinical presentation, genetic background, diagnostic work-up, and outcomes. The average age at diagnosis was 41.9 years. Typical symptoms of paraganglioma (e.g., hypertension, sweating, palpitations, headache) were reported at initial presentation in 13 patients (86.7%); the remaining 2, as well as 4 symptomatic patients, initially presented with cardiac-specific symptoms (e.g., chest pain, dyspnea). Genetic testing was done in 13 cases (86.7%); 10 (76.9%) were positive for mutations in succinate dehydrogenase (SDHx) subunits B, C, or D. Thirteen cases (86.7%) underwent surgery to remove the paraganglioma with no intraoperative morbidity or mortality; one additional patient underwent surgical resection but experienced intraoperative complications after removal of the tumor due to comorbities and did not survive. SDHx mutations are known to be associated with mediastinal locations and malignant behavior of paragangliomas. In this report, we extend the locations of predominantly SDHx-related paragangliomas to cardiac tumors. In conclusion, cardiac paragangliomas are frequently associated with underlying SDHx germline mutations, suggesting a need for genetic testing of all patients with this rare tumor. PMID:25896150

  18. 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/.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    PubMed

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  4. 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.

  5. Parkinson disease (PARK) genes are somatically mutated in cutaneous melanoma

    PubMed Central

    Samuels, Yardena; Azizi, Esther; Qutob, Nouar; Inzelberg, Lilah; Domany, Eytan; Schechtman, Edna; Friedman, Eitan

    2016-01-01

    Objective: To assess whether Parkinson disease (PD) genes are somatically mutated in cutaneous melanoma (CM) tissue, because CM occurs in patients with PD at higher rates than in the general population and PD is more common than expected in CM cohorts. Methods: We cross-referenced somatic mutations in metastatic CM detected by whole-exome sequencing with the 15 known PD (PARK) genes. We computed the empirical distribution of the sum of mutations in each gene (Smut) and of the number of tissue samples in which a given gene was mutated at least once (SSampl) for each of the analyzable genes, determined the 90th and 95th percentiles of the empirical distributions of these sums, and verified the location of PARK genes in these distributions. Identical analyses were applied to adenocarcinoma of lung (ADENOCA-LUNG) and squamous cell carcinoma of lung (SQUAMCA-LUNG). We also analyzed the distribution of the number of mutated PARK genes in CM samples vs the 2 lung cancers. Results: Somatic CM mutation analysis (n = 246) detected 315,914 mutations in 18,758 genes. Somatic CM mutations were found in 14 of 15 PARK genes. Forty-eight percent of CM samples carried ≥1 PARK mutation and 25% carried multiple PARK mutations. PARK8 mutations occurred above the 95th percentile of the empirical distribution for SMut and SSampl. Significantly more CM samples harbored multiple PARK gene mutations compared with SQUAMCA-LUNG (p = 0.0026) and with ADENOCA-LUNG (p < 0.0001). Conclusions: The overrepresentation of somatic PARK mutations in CM suggests shared dysregulated pathways for CM and PD. PMID:27123489

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Mutation analysis of the gene involved in adrenoleukodystrophy

    SciTech Connect

    Oost, B.A. van; Ligtenberg, M.J.L.; Kemp, S.; Bolhuis, P.A.

    1994-09-01

    A gene responsible for the X-linked genetic disorder adrenoleukodystrophy (ALD) that is characterized by demyelination of the nervous system and adrenocortical insufficiency has been identified by positional cloning. The gene encodes an ATP-binding transporter which is located in the peroxisomal membrane. Deficiency of the gene leads to accumulation of unsaturated very long chain fatty acids due to impaired peroxisomal {beta}-oxidation. A systematic analysis of the open reading frame of the ALD gene unraveled the mutations in 28 different families using reverse transcriptase-PCR followed by direct sequencing. No entire gene deletions or drastic promoter mutations have been detected. Only in one family did the mutation involved multiple exons. The remaining mutations were subtle alterations leading to missense (about 50%) or nonsense mutations, frameshifts or splice acceptor site defects. In one patient a single codon was missing. Mutations affecting a single amino acid were concentrated in the region between the third and fourth putative membrane spanning fragments and in the ATP-binding domain. This overview of mutations aids in the determination of structural and functional important regions and facilitates the screening for mutations in other ALD patients. The detection of mutations in virtually all ALD families tested indicates that the isolated gene is the only gene responsible for ALD located in Xq28.

  19. 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.

  20. 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.

  1. 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.

  2. Expression and regional assignment of Chinese hamster ESD and rRNA genes associated with translocations giving rise to chromosomes Z1 and Z6 in CHO cells.

    PubMed

    Stallings, R L; Adair, G M; Lin, J C; Siciliano, M J

    1984-01-01

    The Chinese hamster genes ADK, NP, ESD, PGM2, PEPS, PEPB, GLO, and GSR, all of which are on Chinese hamster chromosome 1, were assigned to CHO-LA chromosomes by analysis of the segregation of CHO isozymes and chromosomes from interspecific somatic cell hybrids made with CHO cells and mouse C11D cells. One allele of each of these eight loci remained linked on the normal chromosome 1 homolog. For seven loci, the other allele remained linked on chromosome Z1, but ESD was shown to have been translocated to chromosome Z6 (Chinese hamster chromosome 5q +). Ag-NOR staining of CHO chromosomes indicated that the (1;5) translocation was very likely reciprocal, since the Chinese hamster chromosome 5, which gave rise to the CHO Z6, lacks an NOR and the Z1 now has one. These data allowed regional assignment of ESD to the distal portion of Chinese hamster chromosome 1p and provided genetic evidence for the origin of CHO chromosomes Z1 and Z6 from Chinese hamster chromosomes 1 and 5. Induced electrophoretic shift mutations of ESD and positive Ag-NOR staining for the rRNA genes on the Z1 showed that the activities of the genes lying close to the translocation breakpoints were maintained.

  3. 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

  4. 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

  5. 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.

  6. Chromatin accessibility contributes to simultaneous mutations of cancer genes

    PubMed Central

    Shi, Yi; Su, Xian-Bin; He, Kun-Yan; Wu, Bing-Hao; Zhang, Bo-Yu; Han, Ze-Guang

    2016-01-01

    Somatic mutations of many cancer genes tend to co-occur (termed co-mutations) in certain patterns during tumor initiation and progression. However, the genetic and epigenetic mechanisms that contribute to the co-mutations of these cancer genes have yet to be explored. Here, we systematically investigated the association between the somatic co-mutations of cancer genes and high-order chromatin conformation. Significantly, somatic point co-mutations in protein-coding genes were closely associated with high-order spatial chromatin folding. We propose that these regions be termed Spatial Co-mutation Hotspots (SCHs) and report their occurrence in different cancer types. The conserved mutational signatures and DNA sequences flanking these point co-mutations, as well as CTCF-binding sites, are also enriched within the SCH regions. The genetic alterations that are harboured in the same SCHs tend to disrupt cancer driver genes involved in multiple signalling pathways. The present work demonstrates that high-order spatial chromatin organisation may contribute to the somatic co-mutations of certain cancer genes during tumor development. PMID:27762310

  7. Effect of Mutation to Streptomycin Resistance on Amber Suppressor Genes

    PubMed Central

    Otsuji, Nozomu; Aono, Hiroyuki

    1968-01-01

    Three classes of nonidentical streptomycin-resistant mutations were distinguished in Escherichia coli by their effect on the efficiency of suppression by an amber suppressor gene, sup E. The first class of mutation caused a strong restriction in efficiency of suppression of an amber codon in various cistrons of phage λ and in an alkaline phosphatase structural gene of E. coli. The second class caused weak restriction, and the third class caused no restriction. The restrictive effect of the streptomycin resistance mutation of the first class on the sup E gene was reduced by addition of streptomycin. This mutation had little effect on efficiencies of suppression by amber suppressor genes sup D and sup F. Analyses on the alkaline phosphatase formed in the suppressor strain indicated that mutation to restrictive streptomycin resistance causes a reduction in translation of the amber codon in the alkaline phosphatase structural gene. Images PMID:4874314

  8. [Research advances of IDH gene mutation and AML].

    PubMed

    Sun, Ming-Dong; Zheng, Yong-Qin

    2014-10-01

    The isocitrate dehydrogenase (IDH) gene mutation has been recently found, which may be involved in the occurrence of leukemia. The incidence of IDH gene mutation in the patients with adult acute myeloid leukemia (AML) is high, especially in the AML patients with normal karyotype. Different subtype and molecular biology of IDH display a different effect on the AML prognosis. This gene mutation is related with treatment response, residual, recurrence of leukemia, and it could be a sign of test and a monitoring tool of minimal residual disease (MRD). The IDH gene mutation may be an index for predicting prognosis and guiding therapy. In this article, the research progress of IDH gene mutation and its correlation with acute myeloid leukemia, especially with the clinical characteristics,are reviewed.

  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. Multiple de novo mutations in the MECP2 gene.

    PubMed

    Bunyan, David J; Robinson, David O

    2008-09-01

    Rett syndrome is an X-linked dominant disorder that usually arises following a single de novo mutation in the MECP2 gene. Point mutation testing and gene dosage analysis of a cohort of British Rett syndrome patients in our laboratory revealed four females who each had two different de novo causative mutations, presumed to be in cis because the patients showed no deviation from the classical Rett syndrome phenotype. Two of these cases had a point mutation and a small intraexonic deletion, a third had a whole exon deletion and a separate small intraexonic deletion, and a fourth case had a small intraexonic deletion and a large duplication. These findings highlight the necessity to perform both point mutation analysis and exon dosage analysis in such cases, particularly because of the possibility of undetected parental mosaicism and the implications for prenatal diagnosis in future pregnancies. These cases also suggest that the MECP2 gene may be particularly prone to multiple mutation events.

  20. 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.

  1. 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

  2. Androgen receptor gene mutation, rearrangement, polymorphism

    PubMed Central

    Eisermann, Kurtis; Wang, Dan; Jing, Yifeng; Pascal, Laura E.

    2013-01-01

    Genetic aberrations of the androgen receptor (AR) caused by mutations, rearrangements, and polymorphisms result in a mutant receptor that has varied functions compared to wild type AR. To date, over 1,000 mutations have been reported in the AR with most of these being associated with androgen insensitivity syndrome (AIS). While mutations of AR associated with prostate cancer occur less often in early stage localized disease, mutations in castration-resistant prostate cancer (CRPC) patients treated with anti-androgens occur more frequently with 10-30% of these patients having some form of mutation in the AR. Resistance to anti-androgen therapy usually results from gain-of-function mutations in the LBD such as is seen with bicalutamide and more recently with enzalutamide (MDV3100). Thus, it is crucial to investigate these new AR mutations arising from drug resistance to anti-androgens and other small molecule pharmacological agents. PMID:25045626

  3. 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

  4. High frequency of the 23S rRNA A2058G mutation of Treponema pallidum in Shanghai is associated with a current strategy for the treatment of syphilis.

    PubMed

    Lu, Haikong; Li, Kang; Gong, Weimin; Yan, Limeng; Gu, Xin; Chai, Ze; Guan, Zhifang; Zhou, Pingyu

    2015-02-01

    The preferred drugs for the treatment of syphilis, benzathine and procaine penicillin, have not been available in Shanghai for many years, and currently, the incidence of syphilis is increasing. Alternative antibiotics for patients with syphilis during the benzathine and procaine penicillin shortage include macrolides. The failure of macrolide treatment in syphilis patients has been reported in Shanghai, but the reason for this treatment failure remains unclear. We used polymerase chain reaction technology to detect a 23S rRNA A2058G mutation in Treponema pallidum in 109 specimens from syphilis patients. The use of azithromycin/erythromycin in the syphilis patients and the physicians' prescription habits were also assessed based on two questionnaires regarding the use of macrolides. A total of 104 specimens (95.4%) were positive for the A2058G mutation in both copies of the 23S rRNA gene, indicating macrolide resistance. A questionnaire provided to 122 dermatologists showed that during the penicillin shortage, they prescribed erythromycin and azithromycin for 8.24±13.95% and 3.21±6.37% of their patients, respectively, and in the case of penicillin allergy, erythromycin and azithromycin were prescribed 15.24±22.89% and 7.23±16.60% of the time, respectively. A second questionnaire provided to the syphilis patients showed that 150 (33.7%), 106 (23.8%) and 34 (7.6%) individuals had used azithromycin, erythromycin or both, respectively, although the majority did not use the drugs for syphilis treatment. Our findings suggest that macrolide resistance in Treponema pallidum is widespread in Shanghai. More than half of the syphilis patients had a history of macrolide use for other treatment purposes, which may have led to the high prevalence of macrolide resistance. Physicians in China are advised to not use azithromycin for early syphilis.

  5. The Maize Imprinted Gene Floury3 Encodes a PLATZ Protein Required for tRNA and 5S rRNA Transcription Through Interaction with RNA Polymerase III.

    PubMed

    Li, Qi; Wang, Jiechen; Ye, Jianwei; Zheng, Xixi; Xiang, Xiaoli; Li, Changsheng; Fu, Miaomiao; Wang, Qiong; Zhang, Zhi-Yong; Wu, Yongrui

    2017-09-05

    Maize (Zea mays) floury3 (fl3) is a classic semi-dominant negative mutant that exhibits severe defects in the endosperm but fl3 plants otherwise appear normal. We cloned the fl3 gene and determined that it encodes a PLATZ (plant AT-rich sequence- and zinc-binding) protein. The mutation in fl3 resulted in an Asn to His replacement in the conserved PLATZ domain, creating a dominant allele. Fl3 is specifically expressed in starchy endosperm cells and regulated by genomic imprinting, which leads to the suppressed expression of fl3 when transmitted through the male, perhaps as a consequence the semi-dominant behavior. Yeast two-hybrid screening and bimolecular luciferase complementation (BiLC) experiments revealed that FL3 interacts with the RNA polymerase III subunit 53 (RPC53) and transcription factor class C 1 (TFC1), two critical factors of the RNA polymerase III (RNAPIII) transcription complex. In the fl3 endosperm, the levels of many tRNAs and 5S rRNA that are transcribed by RNAPIII are significantly reduced, suggesting that the incorrectly folded fl3 protein may impair the function of RNAPIII. The transcriptome is dramatically altered in fl3 mutants, in which the down-regulated genes are primarily enriched in pathways related to translation, ribosome, misfolded protein responses and nutrient reservoir activity. Collectively, these changes may lead to defects in endosperm development and storage reserve filling in fl3 seeds. © 2017 American Society of Plant Biologists. All rights reserved.

  6. Screening for mutations in candidate genes for hypospadias.

    PubMed

    Nordenskjöld, A; Friedman, E; Tapper-Persson, M; Söderhäll, C; Leviav, A; Svensson, J; Anvret, M

    1999-01-01

    Hypospadias. a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5alpha-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerase chain reaction (PCR) amplification of the AR, WTI and 5alpha-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5alpha-reductase gene. In summary, mutations in the WT1, AR and 5alpha-reductase genes are not common causes of isolated hypospadias.

  7. DRUMS: a human disease related unique gene mutation search engine.

    PubMed

    Li, Zuofeng; Liu, Xingnan; Wen, Jingran; Xu, Ye; Zhao, Xin; Li, Xuan; Liu, Lei; Zhang, Xiaoyan

    2011-10-01

    With the completion of the human genome project and the development of new methods for gene variant detection, the integration of mutation data and its phenotypic consequences has become more important than ever. Among all available resources, locus-specific databases (LSDBs) curate one or more specific genes' mutation data along with high-quality phenotypes. Although some genotype-phenotype data from LSDB have been integrated into central databases little effort has been made to integrate all these data by a search engine approach. In this work, we have developed disease related unique gene mutation search engine (DRUMS), a search engine for human disease related unique gene mutation as a convenient tool for biologists or physicians to retrieve gene variant and related phenotype information. Gene variant and phenotype information were stored in a gene-centred relational database. Moreover, the relationships between mutations and diseases were indexed by the uniform resource identifier from LSDB, or another central database. By querying DRUMS, users can access the most popular mutation databases under one interface. DRUMS could be treated as a domain specific search engine. By using web crawling, indexing, and searching technologies, it provides a competitively efficient interface for searching and retrieving mutation data and their relationships to diseases. The present system is freely accessible at http://www.scbit.org/glif/new/drums/index.html.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Germline mutations of TP53 gene in breast cancer.

    PubMed

    Damineni, Surekha; Rao, Vadlamudi Raghavendra; Kumar, Satish; Ravuri, Rajasekar Reddy; Kagitha, Sailaja; Dunna, Nageswara Rao; Digumarthi, Raghunadharao; Satti, Vishnupriya

    2014-09-01

    Germline alterations of the TP53 gene encoding the p53 protein have been observed in the majority of families with the Li-Fraumeni syndrome, a rare dominantly inherited disorder with breast cancer. Genomic DNA samples of 182 breast cancer cases and 186 controls were sequenced for TP53 mutations in the exon 5-9 and intervening introns 5, 7-9. Direct sequencing was done using Applied Biosystem 3730 DNA analyzer. In the present study, we observed nine mutations in the sequenced region, of which five were novel. Hardy-Weinberg equilibrium (HWE) was done for all the mutations; C14181T, T14201G, and G13203A have shown deviation from HWE. High linkage disequilibrium (LD) was observed between C14181T (rs129547788) and T14201G (rs12951053) (r (2) = 0.98.3; D' = 1.00), whereas other observed mutations do not show strong LD with any of the other mutations. None of the intronic mutations has shown significant association with the breast cancer, two exonic mutations G13203A (rs28934578) and A14572G are significantly (P = 0.04, P = 0.007) associated with breast cancer. Germline mutations observed in DNA-binding domain of the gene showed significant association with breast cancer. This study reports five novel germline mutations in the TP53 gene out of which one mutation may confer significant risk to the breast cancer. Mutations in DNA-binding domain of TP53 gene may play role in the early onset and prognosis of breast cancer. The population-based studies of germline mutations in DNA-binding domain of TP53 gene helps in identification of individuals and families who are at risk of developing cancers.

  17. CFTR gene mutations in isolated chronic obstructive pulmonary disease

    SciTech Connect

    Pignatti, P.F.; Bombien, C.; Marigo, C.

    1994-09-01

    In order to identify a possible hereditary predisposition to the development of chronic obstructive pulmonary disease (COPD), we have looked for the presence of cystic fibrosis transmembrane regulator (CFTR) gene DNA sequence modifications in 28 unrelated patients with no signs of cystic fibrosis. The known mutations in Italian CF patients, as well as the most frequent worldwide CF mutations, were investigated. In addition, a denaturing gradient gel electrophoresis analysis of about half of the coding sequence of the gene in 56 chromosomes from the patients and in 102 chromosomes from control individuals affected by other pulmonary diseases and from normal controls was performed. Nine different CFTR gene mutations and polymorphisms were found in seven patients, a highly significant increase over controls. Two of the patients were compound heterozygotes. Two frequent CF mutations were detected: deletion F508 and R117H; two rare CF mutations: R1066C and 3667ins4; and five CF sequence variants: R75Q (which was also described as a disease-causing mutation in male sterility cases due to the absence of the vasa deferentia), G576A, 2736 A{r_arrow}G, L997F, and 3271+18C{r_arrow}T. Seven (78%) of the mutations are localized in transmembrane domains. Six (86%) of the patients with defined mutations and polymorphisms had bronchiectasis. These results indicate that CFTR gene mutations and sequence alterations may be involved in the etiopathogenesis of some cases of COPD.

  18. Somatic mutations of CASP3 gene in human cancers.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Park, Won Sang; Nam, Suk Woo; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2004-07-01

    Failure of apoptosis is one of the hallmarks of cancer. As an execution-phase caspase, caspase-3 plays a crucial role during apoptosis. To explore the possibility that the genetic alterations of CASP3, which encodes caspase-3, might be involved in the development of human tumors, we analyzed the entire coding region and all splice sites of human CASP3 gene for the detection of somatic mutations in a series of 944 human tumors, including 165 stomach carcinomas, 95 colon carcinomas, 76 breast carcinomas, 80 hepatocellular carcinomas, 181 non-small cell lung cancers, 45 acute leukemias, 28 multiple myelomas, 12 medulloblastomas, 15 Wilms' tumors, 12 renal cell carcinomas, 40 esophagus carcinomas, 33 urinary bladder carcinomas, 33 laryngeal carcinomas, and 129 non-Hodgkin lymphomas. Overall, we detected 14 somatic mutations of the CASP3 gene, including six missense and four silent mutations, two mutations in the introns, one mutation in the 5'-untranslated region, and one mutation in the 3'-untranslated region. The mutations were observed in four of 98 colon carcinomas (4.1%), four of 181 non-small cell lung cancers (2.2%), two of 129 non-Hodgkin lymphomas (1.6%), two of 165 stomach carcinomas (1.2%), one of 80 hepatocellular carcinomas (1.3%), and one of 28 multiple myelomas (3.6%). This is the first report on CASP3 gene mutations in human tumors; these data indicate that the CASP3 gene is occasionally mutated in human tumors.

  19. 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.

  20. 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

  1. Mutational specificity analysis: assay for mutations in the yeast SUP4-o gene.

    PubMed

    Kunz, Bernard A

    2014-01-01

    Mutational specificity analysis can yield valuable insights into processes that generate genetic change or maintain genetic stability. Powerful diagnostic tools for such analysis have been created by combining genetic assays for mutation with DNA sequencing. Here, steps for isolating spontaneous mutations in the yeast (Saccharomyces cerevisiae) suppressor tRNA gene SUP4-o as a prelude to sequence characterization are described (modifications of this protocol can be used to study induction of mutations by various physical or chemical agents). Mutations in SUP4-o are selected on drug-containing medium by virtue of their inactivation of suppressor activity. The small size, detailed knowledge of detectably mutable sites, and other features of the target gene facilitate subsequent analysis of these mutations.

  2. 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.

  3. Application of automatic mutation-gene pair extraction to diseases.

    PubMed

    Erdogmus, Muge; Sezerman, Osman Ugur

    2007-12-01

    To have a better understanding of the mechanisms of disease development, knowledge of mutations and the genes on which the mutations occur is of crucial importance. Information on disease-related mutations can be accessed through public databases or biomedical literature sources. However, information retrieval from such resources can be problematic because of two reasons: manually created databases are usually incomplete and not up to date, and reading through a vast amount of publicly available biomedical documents is very time-consuming. In this paper, we describe an automated system, MuGeX (Mutation Gene eXtractor), that automatically extracts mutation-gene pairs from Medline abstracts for a disease query. Our system is tested on a corpus that consists of 231 Medline abstracts. While recall for mutation detection alone is 85.9%, precision is 95.9%. For extraction of mutation-gene pairs, we focus on Alzheimer's disease. The recall for mutation-gene pair identification is estimated at 91.3%, and precision is estimated at 88.9%. With automatic extraction techniques, MuGeX overcomes the problems of information retrieval from public resources and reduces the time required to access relevant information, while preserving the accuracy of retrieved information.

  4. Association of CFTR gene mutation with bronchial asthma

    PubMed Central

    Maurya, Nutan; Awasthi, Shally; Dixit, Pratibha

    2012-01-01

    Mutation on both the copies of cystic fibrosis transmembrane conductance regulator (CFTR) gene results in cystic fibrosis (CF), which is a recessively transmitted genetic disorder. It is hypothesized that individuals heterozygous for CFTR gene mutation may develop obstructive pulmonary diseases like asthma. There is great heterogeneity in the phenotypic presentation and severity of CF lung disease. This could be due to genetic or environmental factors. Several modifier genes have been identified which may directly or indirectly interact with CFTR pathway and affect the severity of disease. This review article discusses the information related to the association of CFTR gene mutation with asthma. Association between CFTR gene mutation and asthma is still unclear. Report ranges from studies showing positive or protective association to those showing no association. Therefore, studies with sufficiently large sample size and detailed phenotype are required to define the potential contribution of CFTR in the pathogenesis of asthma. PMID:22664493

  5. 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

  6. 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

  7. [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.

  8. DKC1 gene mutation in a Taiwanese kindred with X-linked dyskeratosis congenita.

    PubMed

    Lin, Jeng-Hsien; Lee, J Yu-Yun; Tsao, Chao-Jung; Chao, Sheau-Chiou

    2002-11-01

    Dyskeratosis congenita (DKC) is a rare inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. Recent studies demonstrated mutations in the DKC1 gene encoding a protein named dyskerin, which is a component of human telomerase. In addition to the hypothesized function of pseudouridination in rRNA biosynthesis, ribosomal subunit assembly, and/or centromere/ microtubule binding, lower levels of telomerase activity in cells from patients with X-linked DKC have been observed. We report the mutation analysis of a Taiwanese family with X-linked DKC. The patient was a 19-year-old man who presented with progressive reticulate hyperpigmentation, nail dystrophy, alopecia, leukoplakia of the tongue, and pancytopenia. He died of enterocolitis and Escherichia coli sepsis at the age of 20 years. Only his mother's DNA was available for mutation analysis, which revealed a nucleotide transition of C to T (1058 C --> T), a hotspot mutation in DKC, resulting in an amino acid change from alanine to valine (A353V) in the DKC1 gene. Recent advances in the research of telomerase and its implications in the human aging process and cancer are discussed.

  9. Amelogenesis Imperfecta and Screening of Mutation in Amelogenin Gene

    PubMed Central

    Oliveira, Fernanda Veronese; Gurgel, Carla Vecchione; Kobayashi, Tatiana Yuriko; Dionísio, Thiago José; Neves, Lucimara Teixeira; Santos, Carlos Ferreira; Machado, Maria Aparecida Andrade Moreira

    2014-01-01

    The aim of this study was to report the clinical findings and the screening of mutations of amelogenin gene of a 7-year-old boy with amelogenesis imperfecta (AI). The genomic DNA was extracted from saliva of patient and his family, followed by PCR and direct DNA sequencing. The c.261C>T mutation was found in samples of mother, father, and brother, but the mutation was not found in the sequence of the patient. This mutation is a silent mutation and a single-nucleotide polymorphism (rs2106416). Thus, it is suggested that the mutation found was not related to the clinical presence of AI. Further research is necessary to examine larger number of patients and genes related to AI. PMID:25045544

  10. Novel strategies for comprehensive mutation screening of the APC gene.

    PubMed

    Wachsmannova, L; Mego, M; Stevurkova, V; Zajac, V; Ciernikova, S

    2017-03-03

    Colorectal cancer is the 4th most common cause of cancer related deaths worldwide and new possibilities in accurate diagnosis and targeted treatment are highly required. Mutations in adenomatous polyposis coli (APC) gene play a pivotal role in adenoma-carcinoma pathway of colorectal tumorigenesis. The quarter century from its´ first cloning, APC became one of the most frequently mutated, known driver genes in colorectal cancer. Intensive routine molecular testing of APC has brought the benefits for patients with family history of polyposis or colorectal cancer. Nevertheless, multiple mutational disease-causing mechanisms make the genetic testing still challenging. This minireview is focused on implementation of novel APC mutation screening diagnostic strategies for polyposis families according to the current findings. A further understanding and improved algorithms may help to increase the mutation detection rate. APC germline mutations achieve close to 100% penetrance, so more comprehensive approach followed by preventive and therapeutic strategies might reflect in decrease in burden of colorectal cancer.

  11. Cilia gene mutations cause atrioventricular septal defects by multiple mechanisms

    PubMed Central

    Burnicka-Turek, Ozanna; Steimle, Jeffrey D.; Huang, Wenhui; Felker, Lindsay; Kamp, Anna; Kweon, Junghun; Peterson, Michael; Reeves, Roger H.; Maslen, Cheryl L.; Gruber, Peter J.; Yang, Xinan H.; Shendure, Jay; Moskowitz, Ivan P.

    2016-01-01

    Atrioventricular septal defects (AVSDs) are a common severe form of congenital heart disease (CHD). In this study we identified deleterious non-synonymous mutations in two cilia genes, Dnah11 and Mks1, in independent N-ethyl-N-nitrosourea-induced mouse mutant lines with heritable recessive AVSDs by whole-exome sequencing. Cilia are required for left/right body axis determination and second heart field (SHF) Hedgehog (Hh) signaling, and we find that cilia mutations affect these requirements differentially. Dnah11avc4 did not disrupt SHF Hh signaling and caused AVSDs only concurrently with heterotaxy, a left/right axis abnormality. In contrast, Mks1avc6 disrupted SHF Hh signaling and caused AVSDs without heterotaxy. We performed unbiased whole-genome SHF transcriptional profiling and found that cilia motility genes were not expressed in the SHF whereas cilia structural and signaling genes were highly expressed. SHF cilia gene expression predicted the phenotypic concordance between AVSDs and heterotaxy in mice and humans with cilia gene mutations. A two-step model of cilia action accurately predicted the AVSD/heterotaxyu phenotypic expression pattern caused by cilia gene mutations. We speculate that cilia gene mutations contribute to both syndromic and non-syndromic AVSDs in humans and provide a model that predicts the phenotypic consequences of specific cilia gene mutations. PMID:27340223

  12. 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.

  13. 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

  14. Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations.

    PubMed

    Sampieri, Katia; Hadjistilianou, Theodora; Mari, Francesca; Speciale, Caterina; Mencarelli, Maria Antonietta; Cetta, Francesco; Manoukian, Siranoush; Peissel, Bernard; Giachino, Daniela; Pasini, Barbara; Acquaviva, Antonio; Caporossi, Aldo; Frezzotti, Renato; Renieri, Alessandra; Bruttini, Mirella

    2006-01-01

    Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

  15. Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

    PubMed

    Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

    2009-03-01

    Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

  16. Bioinformatic Analysis of GJB2 Gene Missense Mutations.

    PubMed

    Yilmaz, Akin

    2015-04-01

    Gap junction beta 2 (GJB2) gene is the most commonly mutated connexin gene in patients with autosomal recessive and dominant hearing loss. According to Ensembl (release 74) database, 1347 sequence variations are reported in the GJB2 gene and about 13.5% of them are categorized as missense SNPs or nonsynonymous variant. Because of the high incidence of GJB2 mutations in hearing loss patients, revealing the molecular effect of GJB2 mutations on protein structure may also provide clear point of view regarding the molecular etiology of deafness. Hence, the aim of this study is to analyze structural and functional consequences of all known GJB2 missense variations to the Cx26 protein by applying multiple bioinformatics methods. Two-hundred and eleven nonsynonymous variants were collected from Ensembl release 74, Leiden Open Variation Database (LOVD) and The Human Gene Mutation Database (HGMD). A number of bioinformatic tools were utilized for predicting the effect of GJB2 missense mutations at the sequence, structural, and functional levels. Some of the mutations were found to locate highly conserved regions and have structural and functional properties. Moreover, GJB2 mutations were also found to affect Cx26 protein at the molecular level via loss or gain of disorder, catalytic site, and post-translational modifications, including methylation, glycosylation, and ubiquitination. Findings, presented here, demonstrated the application of bioinformatic algorithms to predict the effects of mutations causing hearing impairment. I expect, this type of analysis will serve as a start point for future experimental evaluation of the GJB2 gene mutations and it will also be helpful in evaluating other deafness-related gene mutations.

  17. Founder mutations in BRCA1 and BRCA2 genes.

    PubMed

    Ferla, R; Calò, V; Cascio, S; Rinaldi, G; Badalamenti, G; Carreca, I; Surmacz, E; Colucci, G; Bazan, V; Russo, A

    2007-06-01

    BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.

  18. Variable expressivity and mutation databases: The androgen receptor gene mutations database.

    PubMed

    Gottlieb, B; Beitel, L K; Trifiro, M A

    2001-05-01

    For over 50 years genetics has presumed that variations in phenotypic expression have, for the most part, been the result of alterations in genotype. The importance and value of mutation databases has been based on the premise that the same gene or allelic variation in a specific gene that has been proven to determine a specific phenotype, will always produce the same phenotype. However, recent evidence has shown that so called "simple" Mendelian disorders or monogenic traits are often far from simple, exhibiting phenotypic variation (variable expressivity) that cannot be explained solely by a gene or allelic alteration. The AR gene mutations database now lists 25 cases where different degrees of androgen insensitivity are caused by identical mutations in the androgen receptor gene. In five of these cases the phenotypic variability is due to somatic mosaicism, that is, somatic mutations that occur in only certain cells of androgen-sensitive tissue. Recently, a number of other cases of variable expressivity have also been linked to somatic mosaicism. The impact of variable expressivity due to somatic mutations and mosaicism on mutation databases is discussed. In particular, the effect of an organism exhibiting genetic heterogeneity within its tissues, and the possibility of an organism's genotype changing over its lifetime, are considered to have important implications for mutation databases in the future. Copyright 2001 Wiley-Liss, Inc.

  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. 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

  1. Recessive truncating titin gene, TTN, mutations presenting as centronuclear myopathy

    PubMed Central

    Ceyhan-Birsoy, Ozge; Agrawal, Pankaj B.; Hidalgo, Carlos; Schmitz-Abe, Klaus; DeChene, Elizabeth T.; Swanson, Lindsay C.; Soemedi, Rachel; Vasli, Nasim; Iannaccone, Susan T.; Shieh, Perry B.; Shur, Natasha; Dennison, Jane M.; Lawlor, Michael W.; Laporte, Jocelyn; Markianos, Kyriacos; Fairbrother, William G.; Granzier, Henk

    2013-01-01

    Objective: To identify causative genes for centronuclear myopathies (CNM), a heterogeneous group of rare inherited muscle disorders that often present in infancy or early life with weakness and hypotonia, using next-generation sequencing of whole exomes and genomes. Methods: Whole-exome or -genome sequencing was performed in a cohort of 29 unrelated patients with clinicopathologic diagnoses of CNM or related myopathy depleted for cases with mutations of MTM1, DNM2, and BIN1. Immunofluorescence analyses on muscle biopsies, splicing assays, and gel electrophoresis of patient muscle proteins were performed to determine the molecular consequences of mutations of interest. Results: Autosomal recessive compound heterozygous truncating mutations of the titin gene, TTN, were identified in 5 individuals. Biochemical analyses demonstrated increased titin degradation and truncated titin proteins in patient muscles, establishing the impact of the mutations. Conclusions: Our study identifies truncating TTN mutations as a cause of congenital myopathy that is reported as CNM. Unlike the classic CNM genes that are all involved in excitation-contraction coupling at the triad, TTN encodes the giant sarcomeric protein titin, which forms a myofibrillar backbone for the components of the contractile machinery. This study expands the phenotypic spectrum associated with TTN mutations and indicates that TTN mutation analysis should be considered in cases of possible CNM without mutations in the classic CNM genes. PMID:23975875

  2. 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

  3. Simulation of gene evolution under directional mutational pressure

    NASA Astrophysics Data System (ADS)

    Dudkiewicz, Małgorzata; Mackiewicz, Paweł; Kowalczuk, Maria; Mackiewicz, Dorota; Nowicka, Aleksandra; Polak, Natalia; Smolarczyk, Kamila; Banaszak, Joanna; R. Dudek, Mirosław; Cebrat, Stanisław

    2004-05-01

    The two main mechanisms generating the genetic diversity, mutation and recombination, have random character but they are biased which has an effect on the generation of asymmetry in the bacterial chromosome structure and in the protein coding sequences. Thus, like in a case of two chiral molecules-the two possible orientations of a gene in relation to the topology of a chromosome are not equivalent. Assuming that the sequence of a gene may oscillate only between certain limits of its structural composition means that the gene could be forced out of these limits by the directional mutation pressure, in the course of evolution. The probability of the event depends on the time the gene stays under the same mutation pressure. Inversion of the gene changes the directional mutational pressure to the reciprocal one and hence it changes the distance of the gene to its lower and upper bound of the structural tolerance. Using Monte Carlo methods we were able to simulate the evolution of genes under experimentally found mutational pressure, assuming simple mechanisms of selection. We found that the mutation and recombination should work in accordance to lower their negative effects on the function of the products of coding sequences.

  4. [Obesity based on mutation of genes involved in energy balance].

    PubMed

    Hainerová, I

    2007-01-01

    Within the last decade an intensive research led to an identification of several genes which are involved in a regulation of energy balance. In most cases, carriers of these gene mutations do not exhibit further characteristic phenotypic features except for a severe obesity. Obesity based on mutation of one gene product is called monogenic obesity. Mutations in genes for leptin, leptin receptor, proopiomelanocortin, prohormone convertase 1, melanocortin 4 and 3 receptor disrupt the physiological humoral signalization between peripheral signals and the hypothalamic centres of satiety and hunger. Defects of all above mentioned genes lead to phenotype of abnormal eating behaviour followed by a development of severe early-onset obesity. Mutations of melanocortin 4 receptor gene represent the most common cause of monogenic obesity because they are detected in almost 6 % children with early-onset severe obesity. Mutations of the other genes involved in energy homeostasis are very rare. Although these mutations are sporadic we assume that further research of monogenic forms of obesity might lead to our understanding of physiology and pathophysiology of regulation of the energy homeostasis and eating behaviour. Additionally, they may open new approach to the management of eating behaviour and to the treatment of obesity.

  5. 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

  6. Molecular analysis of mutations in the human HPRT gene.

    PubMed

    Keohavong, Phouthone; Xi, Liqiang; Grant, Stephen G

    2014-01-01

    The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

  7. 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.

  8. 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.

  9. 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.

  10. 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)

  11. Clinical resistance to erythromycin and clindamycin in cutaneous propionibacteria isolated from acne patients is associated with mutations in 23S rRNA.

    PubMed Central

    Ross, J I; Eady, E A; Cove, J H; Jones, C E; Ratyal, A H; Miller, Y W; Vyakrnam, S; Cunliffe, W J

    1997-01-01

    The genetic basis of erythromycin resistance in cutaneous propionibacteria was determined by comparing the nucleotide sequences of the peptidyl transferase region in the 23S rRNAs from 9 susceptible and 26 resistant clinical isolates as well as 4 laboratory-selected erythromycin-resistant mutants of a susceptible strain. In 13 isolates and the 4 laboratory mutants, cross-resistance to macrolides, lincosamides, and B-type streptogramins was associated with an A-->G transition at a position cognate with Escherichia coli 23S rRNA base 2058. These strains were resistant to > or = 512 microg of erythromycin per ml. Two other mutations were identified, an A-->G transition at base 2059 in seven strains, associated with high-level resistance to all macrolides, and a G-->A transition at base 2057 in six strains, associated with low-level resistance to erythromycin. These mutations correspond to three of four phenotypic classes previously identified by using MIC determinations. PMID:9145890

  12. Microarray-based mutation detection in the dystrophin gene.

    PubMed

    Hegde, Madhuri R; Chin, Ephrem L H; Mulle, Jennifer G; Okou, David T; Warren, Stephen T; Zwick, Michael E

    2008-09-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.

  13. Cancer Susceptibility Gene Mutations in Individuals With Colorectal Cancer.

    PubMed

    Yurgelun, Matthew B; Kulke, Matthew H; Fuchs, Charles S; Allen, Brian A; Uno, Hajime; Hornick, Jason L; Ukaegbu, Chinedu I; Brais, Lauren K; McNamara, Philip G; Mayer, Robert J; Schrag, Deborah; Meyerhardt, Jeffrey A; Ng, Kimmie; Kidd, John; Singh, Nanda; Hartman, Anne-Renee; Wenstrup, Richard J; Syngal, Sapna

    2017-04-01

    Purpose Hereditary factors play an important role in colorectal cancer (CRC) risk, yet the prevalence of germline cancer susceptibility gene mutations in patients with CRC unselected for high-risk features (eg, early age at diagnosis, personal/family history of cancer or polyps, tumor microsatellite instability [MSI], mismatch repair [MMR] deficiency) is unknown. Patients and Methods We recruited 1,058 participants who received CRC care in a clinic-based setting without preselection for age at diagnosis, personal/family history, or MSI/MMR results. All participants underwent germline testing for mutations in 25 genes associated with inherited cancer risk. Each gene was categorized as high penetrance or moderate penetrance on the basis of published estimates of the lifetime cancer risks conferred by pathogenic germline mutations in that gene. Results One hundred five (9.9%; 95% CI, 8.2% to 11.9%) of 1,058 participants carried one or more pathogenic mutations, including 33 (3.1%) with Lynch syndrome (LS). Twenty-eight (96.6%) of 29 available LS CRCs demonstrated abnormal MSI/MMR results. Seventy-four (7.0%) of 1,058 participants carried non-LS gene mutations, including 23 (2.2%) with mutations in high-penetrance genes (five APC, three biallelic MUTYH, 11 BRCA1/2, two PALB2, one CDKN2A, and one TP53), 15 of whom lacked clinical histories suggestive of their underlying mutation. Thirty-eight (3.6%) participants had moderate-penetrance CRC risk gene mutations (19 monoallelic MUTYH, 17 APC*I1307K, two CHEK2). Neither proband age at CRC diagnosis, family history of CRC, nor personal history of other cancers significantly predicted the presence of pathogenic mutations in non-LS genes. Conclusion Germline cancer susceptibility gene mutations are carried by 9.9% of patients with CRC. MSI/MMR testing reliably identifies LS probands, although 7.0% of patients with CRC carry non-LS mutations, including 1.0% with BRCA1/2 mutations.

  14. 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

  15. 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

  16. 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.

  17. Convergence in pigmentation at multiple levels: mutations, genes and function

    PubMed Central

    Manceau, Marie; Domingues, Vera S.; Linnen, Catherine R.; Rosenblum, Erica Bree; Hoekstra, Hopi E.

    2010-01-01

    Convergence—the independent evolution of the same trait by two or more taxa—has long been of interest to evolutionary biologists, but only recently has the molecular basis of phenotypic convergence been identified. Here, we highlight studies of rapid evolution of cryptic coloration in vertebrates to demonstrate that phenotypic convergence can occur at multiple levels: mutations, genes and gene function. We first show that different genes can be responsible for convergent phenotypes even among closely related populations, for example, in the pale beach mice inhabiting Florida's Gulf and Atlantic coasts. By contrast, the exact same mutation can create similar phenotypes in distantly related species such as mice and mammoths. Next, we show that different mutations in the same gene need not be functionally equivalent to produce similar phenotypes. For example, separate mutations produce divergent protein function but convergent pale coloration in two lizard species. Similarly, mutations that alter the expression of a gene in different ways can, nevertheless, result in similar phenotypes, as demonstrated by sister species of deer mice. Together these studies underscore the importance of identifying not only the genes, but also the precise mutations and their effects on protein function, that contribute to adaptation and highlight how convergence can occur at different genetic levels. PMID:20643733

  18. Hereditary angioedema with a mutation in the plasminogen gene.

    PubMed

    Bork, K; Wulff, K; Steinmüller-Magin, L; Braenne, I; Staubach-Renz, P; Witzke, G; Hardt, J

    2017-08-10

    Hereditary angioedema (HAE) with normal C1-INH (HAEnCI) may be linked to specific mutations in the coagulation factor 12 (FXII) gene (HAE-FXII) or functional mutations in other genes that are still unknown. We sought to identify and characterize a hitherto unknown type of HAE with normal C1-INH and without mutation in the F12 gene. The study comprised analysis of whole-exome sequencing, Sanger sequencing, and clinical data of patients. We detected a mutation in the plasminogen (PLG) gene in patients with HAEnCI. The mutation c.9886A>G was located in exon 9 leading to the missense mutation p.Lys330Glu (K330E) in the kringle 3 domain of the PLG protein. The mutation was identified by next-generation sequencing in 14 patients with HAEnCI belonging to 4 of 7 families. Family studies revealed that this type of HAE was transmitted as an autosomal dominant trait. The PLG gene mutation was present in all studied symptomatic patients and was also found in 9 of 38 index patients from 38 further families with HAEnCI. Most patients had swelling of face/lips (78.3%) and tongue (78.3%). A total of 331 of all 3.795 tongue swellings (8.7%) were associated with dyspnea, voice changes, and imminent asphyxiation. Two women died by asphyxiation due to a tongue swelling. Hereditary angioedema with a mutation in the PLG gene is a novel type of HAE. It is associated with a high risk of tongue swellings. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  19. Mutations That Alter the Transmission of Chloroplast Genes in Chlamydomonas

    PubMed Central

    Sager, Ruth; Ramanis, Zenta

    1974-01-01

    Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt- greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt- (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt+, acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes. PMID:4531010

  20. 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.

  1. 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...

  2. 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...

  3. Pancreatic adenocarcinomas frequently show p53 gene mutations.

    PubMed Central

    Scarpa, A.; Capelli, P.; Mukai, K.; Zamboni, G.; Oda, T.; Iacono, C.; Hirohashi, S.

    1993-01-01

    Thirty-four pancreatic adenocarcinomas were studied for the presence of p53 gene mutations by the single-strand conformation polymorphism method and by direct sequencing of PCR-amplified fragments. p53 protein expression was immunohistochemically evaluated using monoclonal PAb1801 and polyclonal CM1 antibodies. Mutations were detected in 14 cases. The transitions were six G to A and two A to G; the transversions were one C to G and two A to C; the remaining three were frameshift mutations. Immunostaining results were identical with both antibodies. Nuclear immunohistochemical p53-positive cells were found in nine p53 mutated cases and in 12 cases in which no mutation was detected. In most of these latter cases only a minority of cancer cells showed immunohistochemical positivity. Twenty-nine cases, including all p53 mutated cancers, were known to contain codon 12 Ki-ras gene mutations. Also in the light of the demonstrated cooperation of ras and p53 gene alterations in the transformation of cultured cells, our data suggest that p53 mutation is one of the genetic defects that may have a role in the pathogenesis of a proportion of pancreatic cancers. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8494051

  4. [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.

  5. Dihydropteroate Synthase Gene Mutations in Pneumocystis and Sulfa Resistance

    PubMed Central

    Crothers, Kristina; Atzori, Chiara; Benfield, Thomas; Miller, Robert; Rabodonirina, Meja; Helweg-Larsen, Jannik

    2004-01-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about development of resistant organisms. The inability to culture human Pneumocystis, Pneumocystis jirovecii, in a standardized culture system prevents routine susceptibility testing and detection of drug resistance. In other microorganisms, sulfa drug resistance has resulted from specific point mutations in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone. PMID:15504256

  6. De novo mutation in the NOTCH3 gene causing CADASIL.

    PubMed

    Stojanov, Dragan; Grozdanović, Danijela; Petrović, Sladjana; Benedeto-Stojanov, Daniela; Stefanović, Ivan; Stojanović, Nebojša; Ilić, Dušica N

    2014-02-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is one of the most common hereditary forms of stroke, and migraine with aura, mood disorders and dementia. CADASIL is caused by mutations of the NOTCH3 gene. This mutation is inherited as an autosomal dominant trait. Most individuals with CADASIL have a parent with the disorder. In extremely rare cases, CADASIL may occur due to a spontaneous genetic mutation that occurs for unknown reasons (de novo mutation). We report a new case of patient with de novo mutation of the NOTCH3 gene and a condition strongly suggestive of CADASIL (migraine, stroke, and white matter abnormalities), except that this patient did not have any first-degree relatives with similar symptoms.

  7. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance.

    PubMed

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara; Benfield, Thomas; Miller, Robert; Rabodonirina, Meja; Helweg-Larsen, Jannik

    2004-10-01

    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about development of resistant organisms. The inability to culture human Pneumocystis, Pneumocystis jirovecii, in a standardized culture system prevents routine susceptibility testing and detection of drug resistance. In other microorganisms, sulfa drug resistance has resulted from specific point mutations in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone.

  8. 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.

  9. 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.

  10. 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

  11. [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.

  12. 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

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

    PubMed

    Matsunaga, Kengo; Kubota, Kengo; Harada, Hideki

    2014-01-01

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

  14. 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

  15. 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.

  16. ATM Gene Mutation Detection Techniques and Functional Analysis.

    PubMed

    Rieunier, Guillaume; D'Enghien, Catherine Dubois; Fievet, Alice; Bellanger, Dorine; Stoppa-Lyonnet, Dominique; Stern, Marc-Henri

    2017-01-01

    Ataxia Telangiectasia (A-T) is caused by biallelic inactivation of the Ataxia Telangiectasia Mutated (ATM) gene, due to nonsense or missense mutations, small insertions/deletions (indels), splicing alterations, and large genomic rearrangements. After establishing A-T clinical diagnosis, a molecular confirmation is needed, based on the detection of one of these loss-of-function mutations in at least one allele. In most cases, the pathogenicity of the detected mutations is sufficient to make a definitive diagnosis. More rarely, mutations of unknown consequences are identified and direct biological analyses are required to establish their pathogenic characters. In such cases, complementary analyses of ATM expression, localization, and activity allow fine characterization of these mutations and facilitate A-T diagnosis. Here, we present genetic and biochemical protocols currently used in the laboratory that have proven to be highly accurate, reproducible, and quantitative. We also provide additional discussion on the critical points of the techniques presented here.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Preservation of duplicate genes by complementary, degenerative mutations.

    PubMed Central

    Force, A; Lynch, M; Pickett, F B; Amores, A; Yan, Y L; Postlethwait, J

    1999-01-01

    The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between

  10. PFAPA and 12 Common MEFV Gene Mutations Our Clinical Experience

    PubMed Central

    Salehzadeh, Farhad; Vahedi, Maryam; Hosseini-Asl, Saeid; Jahangiri, Sepideh; Habibzadeh, Shahram; Hosseini-Khotbesara, Mahsa

    2014-01-01

    Objective: Marshall Syndrome or PFAPA is an inflammatory periodic disease characterized by periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis. Although PFAPA is an auto inflammatory disease, it doesn't have genetic basis such as other periodic fevers. This study evaluates the 12 common MEFV gene mutations in patients with PFAPA syndrome. Methods: 21 patients with PFAPA syndrome who had diagnostic criteria were enrolled in this study and 12 common MEFV gene mutations i.e. P369S, F479L, M680I (G/C), M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H, E148Q evaluated. All the patients were screened for MEFV gene mutations by a reverse hybridization assay (FMF Strip Assay, Vienna lab, Vienna, Austria) according to the instructions provided by the manufacturer. Findings : The age of patients was between 6 months to 14 years, and 15 were males. Seven patients had heterozygote and one had compound heterozygote (K695R, V725A) mutation. There were 4 alleles M694V, 3 alleles V726A, 1 allele E148Q and 1 allele K694R. No significant difference existed between mutated patients with non-mutated in symptoms like aphthous and stomatitis, duration of attacks, episodes of fever and response to treatment. Gaslini score test was not helpful to predict the probability of gene mutations. Conclusion: About 30 percent of patients had MEFV gene mutations but these mutations did not play a main role in presentation of PFAPA symptoms. PMID:25793047

  11. Microarray-based mutation detection in the dystrophin gene

    PubMed Central

    Hegde, Madhuri R.; Chin, Ephrem L.H.; Mulle, Jennifer G.; Okou, David T.; Warren, Stephen T.; Zwick, Michael E.

    2008-01-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans > 2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6–10% of males with either DMD or BMD. The remaining 30–35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, MLPA, DOVAM-S, and SCAIP; however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution CGH microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin. PMID:18663755

  12. PFAPA and 12 Common MEFV Gene Mutations Our Clinical Experience.

    PubMed

    Salehzadeh, Farhad; Vahedi, Maryam; Hosseini-Asl, Saeid; Jahangiri, Sepideh; Habibzadeh, Shahram; Hosseini-Khotbesara, Mahsa

    2014-02-01

    Marshall Syndrome or PFAPA is an inflammatory periodic disease characterized by periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis. Although PFAPA is an auto inflammatory disease, it doesn't have genetic basis such as other periodic fevers. This study evaluates the 12 common MEFV gene mutations in patients with PFAPA syndrome. 21 patients with PFAPA syndrome who had diagnostic criteria were enrolled in this study and 12 common MEFV gene mutations i.e. P369S, F479L, M680I (G/C), M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H, E148Q evaluated. All the patients were screened for MEFV gene mutations by a reverse hybridization assay (FMF Strip Assay, Vienna lab, Vienna, Austria) according to the instructions provided by the manufacturer. Findings : The age of patients was between 6 months to 14 years, and 15 were males. Seven patients had heterozygote and one had compound heterozygote (K695R, V725A) mutation. There were 4 alleles M694V, 3 alleles V726A, 1 allele E148Q and 1 allele K694R. No significant difference existed between mutated patients with non-mutated in symptoms like aphthous and stomatitis, duration of attacks, episodes of fever and response to treatment. Gaslini score test was not helpful to predict the probability of gene mutations. About 30 percent of patients had MEFV gene mutations but these mutations did not play a main role in presentation of PFAPA symptoms.

  13. Noninvasive detection of filaggrin gene mutations using Raman spectroscopy.

    PubMed

    González, Francisco J; Valdes-Rodríguez, Rodrigo; Ramírez-Elías, Miguel G; Castillo-Martínez, Claudio; Saavedra-Alanis, Victor M; Moncada, Benjamín

    2011-12-01

    Knowledge of the existence of filaggrin (FLG) gene mutations might be helpful for a subclassification of patients with atopic dermatitis (AD) which can be used to introduce individualized treatments. In this work the filaggrin content in the skin is assessed using Raman spectroscopy and the results are compared to FLG genotyping of Mexican-mestizo patients. Results showed that the 2282del4 and R501X mutations present in the European population but absent in people of Asian or African descent are also present in the Mexican-mestizo population. The results also showed that patients with filaggrin gene mutations presented lower filaggrin concentrations measured using the vector correlation of their skin Raman spectra and a fixed spectrum of pure human recombinant filaggrin, these results indicate that Raman spectroscopy may be used as a noninvasive tool to detect FLG gene mutations.

  14. Noninvasive detection of filaggrin gene mutations using Raman spectroscopy

    PubMed Central

    González, Francisco J.; Valdes-Rodríguez, Rodrigo; Ramírez-Elías, Miguel G.; Castillo-Martínez, Claudio; Saavedra-Alanis, Victor M.; Moncada, Benjamín

    2011-01-01

    Knowledge of the existence of filaggrin (FLG) gene mutations might be helpful for a subclassification of patients with atopic dermatitis (AD) which can be used to introduce individualized treatments. In this work the filaggrin content in the skin is assessed using Raman spectroscopy and the results are compared to FLG genotyping of Mexican-mestizo patients. Results showed that the 2282del4 and R501X mutations present in the European population but absent in people of Asian or African descent are also present in the Mexican-mestizo population. The results also showed that patients with filaggrin gene mutations presented lower filaggrin concentrations measured using the vector correlation of their skin Raman spectra and a fixed spectrum of pure human recombinant filaggrin, these results indicate that Raman spectroscopy may be used as a noninvasive tool to detect FLG gene mutations. PMID:22162825

  15. Mutation in δ-Sg Gene in Familial Dilated Cardiomyopathy

    PubMed Central

    Asadi, Marzieh; Foo, Roger; Salehi, Ahmad Reza; Salehi, Rasoul; Samienasab, Mohammad Reza

    2017-01-01

    Background: Mutations in different genes including dystrophin-associated glycoprotein complex caused familial dilated cardiomyopathy which is a genetically heterogeneous disease. The δ-SG gene contains nine exons spanning a 433-kb region of genomic DNA. It encodes a 35-kDa, singlepass, and type II transmembrane glycoprotein. Materials and Methods: In this study for the first time in Iran we screened 6 patients of a large family that they had positive family history of MI or sudden death by next generation sequencing method. Results: By employing NGS method we found missense mutation (p.R97Q) of δ-SG gene in 2 of 6 patients. Conclusions: The missense mutation (p.R97Q) in familial DCM patients is reported for the first time in Iranian patients with cardiac disease. Although this mutation is already known in other populations in Iran, it is not reported before. PMID:28401079

  16. Mutations in MTP gene in abeta- and hypobeta-lipoproteinemia.

    PubMed

    Di Leo, Enza; Lancellotti, Sandra; Penacchioni, Junia Y; Cefalù, Angelo B; Averna, Maurizio; Pisciotta, L; Bertolini, Stefano; Calandra, Sebastiano; Gabelli, Carlo; Tarugi, Patrizia

    2005-06-01

    Familial hypobetalipoproteinemia (FHBL) and abetalipoproteinemia (ABL) are inherited disorders of apolipoprotein B (apo B)-containing lipoproteins that result from mutations in apo B and microsomal triglyceride transfer protein (MTP) genes, respectively. Here we report three patients with severe deficiency of plasma low-density lipoprotein (LDL) and apo B. Two of them (probands F.A. and P.E.) had clinical and biochemical phenotype consistent with ABL. Proband F.A. was homozygous for a minute deletion/insertion (c.1228delCCCinsT) in exon 9 of MTP gene predicted to cause a truncated MTP protein of 412 amino acids. Proband P. E. was heterozygous for a mutation in intron 9 (IVS9-1G>A), previously reported in an ABL patient. We failed to find the second pathogenic mutation in MTP gene of this patient. No mutations were found in apo B gene. The third proband (D.F.) had a less severe lipoprotein phenotype which was similar to that of heterozygous FHBL and appeared to be inherited as a co-dominant trait. However, he had no mutations in apo B gene. He was found to be a compound heterozygote for two missense mutations (D384A and G661A), involving highly conserved regions of MTP. Since this proband was also homozygous for varepsilon2 allele of apolipoprotein E (apo E), it is likely that his hypobetalipoproteinemia derives from a combined effect of a mild MTP deficiency and homozygosity for apo E2 isoform.

  17. 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

  18. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  19. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  20. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  1. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  2. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  3. 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.

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

    PubMed

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

    2015-01-01

    Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology,