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Sample records for 23s rrna mutations

  1. 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

  2. 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

  3. 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.

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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).

  15. 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

  16. 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

  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. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Change in antibiotic resistance of Helicobacter pylori strains and the effect of A2143G point mutation of 23S rRNA on the eradication of H. pylori in a single center of Korea.

    PubMed

    Hwang, Tae Jun; Kim, Nayoung; Kim, Hong Bin; Lee, Byoung Hwan; Nam, Ryoung Hee; Park, Ji Hyun; Lee, Mi Kyoung; Park, Young Soo; Lee, Dong Ho; Jung, Hyun Chae; Song, In Sung

    2010-09-01

    The current prevalence of primary antibiotic resistance of H. pylori is not known in Korea. This study was done to evaluate the prevalence of primary antibiotic resistance of H. pylori, and to evaluate the effect of point mutations of 23S rRNA on the rate of eradication of H. pylori. H. pylori were isolated from gastric mucosal biopsy specimens obtained from 222 Koreans. The susceptibilities of the H. pylori isolates to amoxicillin, clarithromycin, metronidazole, tetracycline, ciprofloxacin, and levofloxacin were examined using the agar dilution method. DNA sequencing was carried out to detect H. pylori 23S rRNA mutations. The resistance to clarithromycin, tetracycline, ciprofloxacin, and levofloxacin increased during the period of 2007 to 2009 compared with 2003 to 2005 (P<0.05). However, amoxicillin and metronidazole resistance slightly decreased. The rates of eradication were 95.5% for the clarithromycin-sensitive strains, which was higher than the 67.9% for the clarithromycin-resistant strains (P=0.001). By contrast, the eradication rate was 100% in patients with amoxicillin-resistant H. pylori. Among 26 clarithromyin-resistant strains, 6 (23%) had A2143G mutations, and all of the cases in which these mutations were present were not eradicated by proton pump inhibitor-based triple therapy (P=0.0004). By contrast, none of the 26 clarithromyin-sensitive strains had A2143G mutations. The T2183C and A2223G mutations were frequently found in the sensitive strains and in the resistant strains. Clarithromycin resistance of H. pylori, which determined the efficacy of H. pylori eradication of proton pump inhibitor triple regimen, was found to be increased in a single center study. A2143G was an important 23S rRNA mutation associated with clarithromycin resistance and affected the H. pylori eradication efficacy.

  5. 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.

  6. 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.

  7. DbpA: a DEAD box protein specifically activated by 23s rRNA.

    PubMed Central

    Fuller-Pace, F V; Nicol, S M; Reid, A D; Lane, D P

    1993-01-01

    The Escherichia coli protein DbpA is a member of the 'DEAD box' family of putative RNA-dependent ATPases and RNA helicases, so called because they share the highly conserved motif Asp-Glu-Ala-Asp, together with several other conserved elements. We have investigated DbpA expression under conditions where an endogenous promoter is used. In this context, translation initiation does not occur at the previously identified AUG, but at an upstream, in-frame GUG. Mutation of the GUG initiation codon to AUG virtually abolishes DbpA expression, suggesting an unusual translation initiation mechanism. Using an inducible overexpression plasmid, we have purified milligram quantities of DbpA to homogeneity and shown that the purified protein hydrolyses ATP in an RNA-dependent manner. This ATPase activity is interesting in that, unlike that of other DEAD box proteins investigated to date, it absolutely requires a specific bacterial RNA, which we have identified as 23S rRNA. This observation is particularly significant since DbpA will bind other RNAs and DNA, but will only hydrolyse ATP in the presence of 23S rRNA. Images PMID:8253085

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

    PubMed Central

    Douglas, S E; Doolittle, W F

    1984-01-01

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

  9. Mycoplasma bovis isolates from dairy calves in Japan have less susceptibility than a reference strain to all approved macrolides associated with a point mutation (G748A) combined with multiple species-specific nucleotide alterations in 23S rRNA.

    PubMed

    Sato, Toyotaka; Higuchi, Hidetoshi; Yokota, Shin-Ichi; Tamura, Yutaka

    2017-06-01

    Erythromycin, tylosin and tilmicosin are approved for use in cattle in Japan, the latter two being used to treat Mycoplasma bovis infection. In this study, 58 M. bovis isolates obtained from Japanese dairy calves all exhibited reduced susceptibility to these macrolides, this widespread reduced susceptibility being attributable to a few dominant lineages. All 58 isolates contained the G748A variant in both the rrl3 and rrl4 alleles of 23S rRNA, whereas a reference strain (PG45) did not. G748 localizes in the central loop of domain II (from C744 to A753) of 23S rRNA, which participates in binding to mycinose, a sugar residue present in both tylosin and tilmicosin. A number of in vitro-selected mutants derived from M. bovis PG45 showed reduced susceptibility to tylosin and tilmicosin and contained a nucleotide insertion within the central loop of domain II of rrl3 (U747-G748Ins_CU/GU or A743-U744Ins_UA), suggesting that mutations around G748 confer this reduced susceptibility phenotype. However, other Mycoplasma species containing G748A were susceptible to tylosin and tilmicosin. Sequence comparison with Escherichia coli revealed that M. bovis PG45 and isolates harbored five nucleotide alterations (U744C, G745A, U746C, A752C and A753G) in the central loop of domain II of 23S rRNA, whereas other Mycoplasma species lacked at least two of these five nucleotide alterations. It was therefore concluded that G748 mutations in combination with species-specific nucleotide alterations in the central loop of domain II of 23S rRNA are likely sufficient to reduce susceptibility of M. bovis to tylosin and tilmicosin. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

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

    PubMed Central

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

    1978-01-01

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

  11. 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

  12. The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

    PubMed Central

    Nicol, S M; Fuller-Pace, F V

    1995-01-01

    The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction. Images Fig. 1 Fig. 2 PMID:8524828

  13. Ketolide Antimicrobial Activity Persists after Disruption of Interactions with Domain II of 23S rRNA

    PubMed Central

    Novotny, Guy W.; Jakobsen, Lene; Andersen, Niels M.; Poehlsgaard, Jacob; Douthwaite, Stephen

    2004-01-01

    Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis. An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities. Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752. We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding. Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides. Mutations at position 748 confer no resistance. Substitution of guanine at A2058 gives rise to the MLSB (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs. However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA. In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin. Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin. The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance. PMID:15388419

  14. Methylation of 23S rRNA Nucleotide G748 by RlmAII Methyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

    PubMed Central

    Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

    2013-01-01

    Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAII to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAII renders S. pneumoniae TEL susceptible. PMID:23716046

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

    PubMed Central

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

    1994-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    PubMed

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

    1994-03-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  19. Interaction of tRNA with domain II of 23S rRNA.

    PubMed

    Hill, W E; Tassanakajohn, A; Tapprich, W E

    1990-08-27

    The interaction of tRNA with domain II of 23S rRNA in E. coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides. Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA. It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811. However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region. Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA.

  20. Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly

    PubMed Central

    Leppik, Margus; Liiv, Aivar

    2017-01-01

    Abstract Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly. PMID:28334881

  1. Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly.

    PubMed

    Leppik, Margus; Liiv, Aivar; Remme, Jaanus

    2017-06-02

    Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. [Value of detecting bacterial 16S and 23S rRNA in interface membrane in diagnosis of periprosthetic joint infection].

    PubMed

    An, Sen-Bo; Cai, Peng-de; Wang, Long; Hu, Yi-He

    2016-02-01

    To explore the value of detecting bacterial 16S rRNA with 23S rRNA in the diagnosis of periprosthetic joint infection (PJI). A prospective study was conducted among 67 patients with previous total hip arthroplasty (THA) undergoing a reoperation for infection (23 patients) or aseptic loosening (44 patients). Bacterial 16S rRNA and 23S rRNA in the interface membrane were detected by real-time PCR and their value in diagnosis of PJI was assessed. The 16S rRNA and 23S rRNA showed no significant difference in their power in the diagnosis of PJI. The detection of 16S rRNA/23S rRNA showed a higher sensitivity and a greater negative predictive value in PJI diagnosis than the detection of 16S rRNA+23S rRNA (95.7% vs 52.2%, P<0.01; 97.6% vs 79.6%, P=0.01). The specificity, positive predictive value, and accuracy of the 4 diagnostic strategies were not significantly different. The diagnostic power of 16S rRNA and 23S rRNA was similar in detecting PJI. Compared with the diagnostic strategy with 16S rRNA+23S rRNA, 16S rRNA/23S rRNA is more sensitive in detecting PJI.

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

    PubMed Central

    Jacq, B

    1981-01-01

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

  4. 23S rRNA as an a-Maz-ing new bacterial toxin target.

    PubMed

    Schifano, Jason M; Woychik, Nancy A

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a bacterium with the unique ability to persist for years or decades as a latent infection. This latent state, during which bacteria have a markedly altered physiology and are thought to be dormant, is crucial for the bacteria to survive the stressful environments it encounters in the human host. Importantly, M. tuberculosis cells in the dormant state are generally refractory to antibiotics, most of which target cellular processes occurring in actively replicating bacteria. The molecular switches that enable M. tuberculosis to slow or stop its replication and become dormant remain unknown. However, the slow growth and dormant state that are hallmarks of latent tuberculosis infection have striking parallels to the "quasi-dormant" state of Escherichia coli cells caused by the toxin components of chromosomal toxin-antitoxin (TA) modules. An unusually large number of TA modules in M. tuberculosis, including nine in the mazEF family, may contribute to initiating this latent state or to adapting to stress conditions in the host. Toward filling the gap in our understanding of the physiological role of TA modules in M. tuberculosis, we are interested in identifying their molecular mechanisms to better understand how toxins impart growth control. Our recent publication (1) uncovered a novel function of a MazF toxin in M. tuberculosis that had not been associated with any other MazF ortholog. This toxin, MazF-mt6, can disrupt protein synthesis by cleavage of 23S rRNA at a single location in an evolutionarily conserved five-base sequence in the ribosome active center.

  5. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  6. A conserved secondary structural motif in 23S rRNA defines the site of interaction of amicetin, a universal inhibitor of peptide bond formation.

    PubMed Central

    Leviev, I G; Rodriguez-Fonseca, C; Phan, H; Garrett, R A; Heilek, G; Noller, H F; Mankin, A S

    1994-01-01

    The binding site and probable site of action have been determined for the universal antibiotic amicetin which inhibits peptide bond formation. Evidence from in vivo mutants, site-directed mutations and chemical footprinting all implicate a highly conserved motif in the secondary structure of the 23S-like rRNA close to the central circle of domain V. We infer that this motif lies at, or close to, the catalytic site in the peptidyl transfer centre. The binding site of amicetin is the first of a group of functionally related hexose-cytosine inhibitors to be localized on the ribosome. Images PMID:8157007

  7. Macrolides and lincomycin susceptibility of Mycoplasma hyorhinis and variable mutation of domain II and V in 23S ribosomal RNA.

    PubMed

    Kobayashi, Hideki; Nakajima, Hiromi; Shimizu, Yuka; Eguchi, Masashi; Hata, Eiji; Yamamoto, Koshi

    2005-08-01

    A total of 151 strains of Mycoplasma hyorhinis isolated from porcine lung lesions (weaned pigs, n=71, and finishers, n=80) were investigated for their in vitro susceptibility to 10 antimicrobial agents. Thirty-one strains (28 from weaned pigs and 3 from finishers) showed resistance to 16-membered macrolide antibiotics and lincomycin. The prevalence of the 16-membered macrolide-resistant M. hyorhinis strain in weaned pigs from Japanese herds has approximately quadrupled in the past 10 years. Several of the 31 strains were examined for mutations in the 23S ribosomal RNA (rRNA). All field strains tested showed a transition of A to G at position 2059 of 23S rRNA-rendered Escherichia coli. On the other hand, individual tylosin- and lincomycin-resistant mutants of M. hyorhinis were selected in vitro from the susceptible type strain BTS7 by 3 to 9 serial passages in subinhibitory concentrations of each antibiotic. The 23S rRNA sequences of both tylosin and lincomycin-resistant mutants were compared with that of the radical BTS7 strain. The BTS7 mutant strain selected by tylosin showed the same transition as the field-isolated strains of A2059G. However, the transition selected in lincomycin showed mutations in domains II and V of 23S rRNA, G2597U, C2611U in domain V, and the addition of an adenine at the pentameric adenine loop in domain II. The strain selected by lincomycin showed an additional point mutation of A2062G selected by tylosin.

  8. Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin.

    PubMed

    Gupta, K N; Baranwal, V K; Haq, Q M R

    2012-03-01

    High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5-8 years old orchards of kinnow mandarin {Citrus reticulate Balanco ('King' × 'Willow mandarin')} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus ('Ca L. asiaticus') showing maximum identity of 95.9% with 'Ca L. asiaticus' from USA and Brazil in 16S rRNA. The study indicates definite association of 'Ca L. asiaticus' with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.

  9. Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA.

    PubMed

    Sergiev, P V; Bogdanov, A A; Dahlberg, A E; Dontsova, O

    2000-06-02

    The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation. Copyright 2000 Academic Press.

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

    PubMed Central

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

    2016-01-01

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

  11. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

    PubMed Central

    Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian

    2016-01-01

    Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance. PMID:27138552

  12. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

    PubMed

    Moazed, D; Noller, H F

    1991-05-01

    Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

  13. Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

    PubMed Central

    Moazed, D; Noller, H F

    1991-01-01

    Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state. Images PMID:2023922

  14. 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.

  15. 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

  16. 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

  17. Decreased Susceptibility to Macrolide-Lincosamide in Mycoplasma synoviae Is Associated with Mutations in 23S Ribosomal RNA.

    PubMed

    Lysnyansky, Inna; Gerchman, Irena; Flaminio, Barbara; Catania, Salvatore

    2015-12-01

    The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

  18. Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

    PubMed

    Christensen, H; Møller, P L; Vogensen, F K; Olsen, J E

    2000-01-01

    The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

  19. Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly.

    PubMed

    Arai, Taiga; Ishiguro, Kensuke; Kimura, Satoshi; Sakaguchi, Yuriko; Suzuki, Takeo; Suzuki, Tsutomu

    2015-08-25

    Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2'-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2'-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.

  20. Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

    PubMed Central

    Arai, Taiga; Ishiguro, Kensuke; Kimura, Satoshi; Sakaguchi, Yuriko; Suzuki, Takeo; Suzuki, Tsutomu

    2015-01-01

    Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2′-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2′-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly. PMID:26261349

  1. 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.

  2. Differentiation of Acinetobacter baumannii biotypes by amplification of 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Garcia, A; Montoya, R; Bello, H; Gonzalez, G; Dominguez, M; Zemelman, R

    1996-01-01

    Isolates of Acinetobacter baumannii (32 strains) from blood samples obtained from patients in five Chilean hospitals were identified and biotyped according to their phenotypic properties. They were also submitted to random amplified polymorphic DNA (RAPD) using eight randomly designed 10-mers and the core sequence of M13 phage (15-mers) as well as amplification of the spacer regions between 16S and 23S genes in the prokaryotic rRNA genetic loci. With some primers, RAPD discriminated between biotypes, whereas with others each isolate showed a particular profile. When amplification of spacer regions was performed, a clear correlation between patterns and biotypes was found. This last technique allowed correct biotyping of clinical isolates. Both genetic methods might be used for the identification of A. baumannii biotypes.

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

    PubMed Central

    2010-01-01

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

  4. Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD.

    PubMed

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

    2010-11-01

    Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K(M) for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the k(cat) values are 4.95 ± 1.10 min⁻¹ and 6.4 ± 1.3 min⁻¹, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K(M) value for substrate 50S subunits is 0.98 ± 0.18 μM and the k(cat) value is 1.97 ± 0.46 min⁻¹. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.

  5. Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

    PubMed Central

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

    2010-01-01

    Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem–loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent KM for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the kcat values are 4.95 ± 1.10 min−1 and 6.4 ± 1.3 min−1, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The KM value for substrate 50S subunits is 0.98 ± 0.18 μM and the kcat value is 1.97 ± 0.46 min−1. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits. PMID:20817755

  6. PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

    PubMed Central

    2010-01-01

    Background The genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species. Results In this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. Conclusion This rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations

  7. 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

  8. Mutations at three sites in the Escherichia coli 23S ribosomal RNA binding region for protein L11 cause UGA-specific suppression and conditional lethality.

    PubMed

    Murgola, E J; Xu, W; Arkov, A L

    1995-01-01

    A single nucleotide change, G to A, at nucleotide position 1093 of E. coli 23S ribosomal RNA was found to cause UGA-specific suppression (D.K. Jemiolo, F.T. Pagel and E.J. Murgola, Proc. Natl. Acad. Sci. USA, in press). To obtain new kinds of UGA-specific suppressors in 23S rRNA, we used segment-directed mutagenic PCR, and targeted first the 1405 nucleotide SnaBI/I-CeuI segment, which includes position 1093, of the rrnB operon cloned into a multicopy plasmid. The mutagenized fragments were subcloned into the plasmid vector and used to transform to ampicillin resistance (Ampr) a recipient strain containing a UGA mutation in trpA. The Ampr transformants were then screened for suppression of UGA. After purification, Trp+ transformants were tested for association of the suppressor phenotype first with the plasmid and then specifically with the SnaBI/I-CeuI fragment. In one screening, four different kinds of mutational change were found, all at three sites within a highly conserved hexanucleotide loop in domain II of 23S rRNA. This region is part of the site for binding of the large subunit protein L11, which has been shown to be involved in peptide chain termination in a specific way. All of the mutants (G1093A, G1093 delta, A1095 delta, and U1097 delta) suppress UGA mutations, but not UAA or UAG mutations, and all four types exhibit high-temperature conditional lethality when highly expressed. Several mechanisms can be suggested for the UGA-specific suppression exhibited by these mutants, including altered interaction with protein L11, Second-site mutations that overcome the conditional lethality of G1093A indicate that intramolecular interactions within 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

  9. An A to U transversion at position 1067 of 23 S rRNA from Escherichia coli impairs EF-Tu and EF-G function.

    PubMed

    Saarma, U; Remme, J; Ehrenberg, M; Bilgin, N

    1997-09-26

    Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (kcat/KM) for both EF-Tu and EF-G binding. In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are significantly increased by the A to U transversion. The U1067 mutation impairs EF-Tu function more than EF-G function. The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu.GDP release from the mutated ribosomes. There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition. With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu.

  10. Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure.

    PubMed

    Osorio, Carlos R; Collins, Matthew D; Romalde, Jesús L; Toranzo, Alicia E

    2005-02-01

    Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

  11. NMR characterisation of a highly conserved secondary structural RNA motif of Halobacterium halobium 23S rRNA.

    PubMed

    King, John; Shammas, Christos; Nareen, Misbah; Lelli, Moreno; Ramesh, Vasudevan

    2013-05-28

    The highly conserved 29-mer RNA motif corresponding to the peptidyl transferase central circle region of the domain V of Halobacterium halobium 23S rRNA has been characterised by multidimensional NMR spectroscopy. The NMR structure has a good all atom average RMSD of 1.28 Å and a stable A-form helical conformation. The NMR structure differs from the X-ray crystal structure of an analogous motif, contained within the Escherichia coli ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. Thus in the observed NMR structure, the predicted A7 to U30 base pair is not seen and a non-canonical U6 to U30 base pair was formed in its place. Similarly the predicted A9 to U26 base pair was also not observed and another non-canonical A9 to A27 base pair was formed. It was also seen from the conformational analysis that the steps near the bulges had the greatest deviation from the canonical Watson-Crick base pair step parameters. Despite these differences, the 29-mer structure provides a working model of the RNA within the ribosome in a more natural solution state than that observed in the intact ribosome crystal structures, particularly around the A27 residue. The NMR structure determination of the 29-mer RNA motif provides a solid foundation for determining the NMR structure of the RNA-amicetin complex in the next step. To extend the above study, a fully (13)C and (15)N isotopically labelled 37-mer RNA version of the Halobacterium halobium RNA sample has been characterised using ultra high field 1 GHz spectroscopy. The results have been used to demonstrate the advantages conferred by the use of a 1 GHz spectrometer frequency over 800 MHz in terms of superior sensitivity and greater spectral dispersion achieved in the spectrum of the RNA.

  12. The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Ching, A.; Stetter, K. O.; Woese, C. R.

    1991-01-01

    We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

  13. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOEpatents

    Bavykin, Sergei G.; Mirzabekova, legal representative, Natalia V.; Mirzabekov, deceased, Andrei D.

    2007-12-04

    The present invention relates to methods and compositions for using nucleotide sequence variations of 16S and 23S rRNA within the B. cereus group to discriminate a highly infectious bacterium B. anthracis from closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations and discriminate B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed samples, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  14. The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales

    NASA Technical Reports Server (NTRS)

    Burggraf, S.; Ching, A.; Stetter, K. O.; Woese, C. R.

    1991-01-01

    We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

  15. The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales.

    PubMed

    Burggraf, S; Ching, A; Stetter, K O; Woese, C R

    1991-01-01

    We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei. This is the first such sequence from a member of the Methanomicrobiales. Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles. Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so. Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

  16. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

    PubMed Central

    Kaminska, Katarzyna H.; Purta, Elzbieta; Hansen, Lykke H.; Bujnicki, Janusz M.; Vester, Birte; Long, Katherine S.

    2010-01-01

    The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity. PMID:20007606

  17. Detection of the new cosmopolitan genus Thermoleptolyngbya (Cyanobacteria, Leptolyngbyaceae) using the 16S rRNA gene and 16S-23S ITS region.

    PubMed

    Sciuto, Katia; Moro, Isabella

    2016-12-01

    Cyanobacteria are widespread prokaryotes that are able to live in extreme conditions such as thermal springs. Strains attributable to the genus Leptolyngbya are among the most common cyanobacteria sampled from thermal environments. Leptolyngbya is a character-poor taxon that was demonstrated to be polyphyletic based on molecular analyses. The recent joining of 16S rRNA gene phylogenies with 16S-23S ITS secondary structure analysis is a useful approach to detect new cryptic taxa and has led to the separation of new genera from Leptolyngbya and to the description of new species inside this genus and in other related groups. In this study, phylogenetic investigations based on both the 16S rRNA gene and the 16S-23S ITS region were performed alongside 16S rRNA and 16S-23S ITS secondary structure analyses on cyanobacteria of the family Leptolyngbyaceae. These analyses focused on filamentous strains sampled from thermal springs with a morphology ascribable to the genus Leptolyngbya. The phylogenetic reconstructions showed that the Leptolyngbya-like thermal strains grouped into a monophyletic lineage that was distinct from Leptolyngbya. The 16S-23S ITS secondary structure results supported the separation of this cluster. A new genus named Thermoleptolyngbya was erected to encompass these strains, and two species were described inside this new taxon: T. albertanoae and T. oregonensis. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Importance of tRNA interactions with 23S rRNA for peptide bond formation on the ribosome: studies with substrate analogs.

    PubMed

    Beringer, Malte; Rodnina, Marina V

    2007-07-01

    The major enzymatic activity of the ribosome is the catalysis of peptide bond formation. The active site -- the peptidyl transferase center -- is composed of ribosomal RNA (rRNA), and interactions between rRNA and the reactants, peptidyl-tRNA and aminoacyl-tRNA, are crucial for the reaction to proceed rapidly and efficiently. Here, we describe the influence of rRNA interactions with cytidine residues in A-site substrate analogs (C-puromycin or CC-puromycin), mimicking C74 and C75 of tRNA on the reaction. Base-pairing of C75 with G2553 of 23S rRNA accelerates peptide bond formation, presumably by stabilizing the peptidyl transferase center in its productive conformation. When C74 is also present in the substrate analog, the reaction is slowed down considerably, indicating a slow step in substrate binding to the active site, which limits the reaction rate. The tRNA-rRNA interactions lead to a robust reaction that is insensitive to pH changes or base substitutions in 23S rRNA at the active site of the ribosome.

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

    PubMed Central

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

    2009-01-01

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

  20. Methylation of 23S rRNA caused by tlrA (ermSF), a tylosin resistance determinant from Streptomyces fradiae.

    PubMed Central

    Zalacain, M; Cundliffe, E

    1989-01-01

    Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus). Images PMID:2753855

  1. 23S rRNA domain V, a fragment that can be specifically methylated in vitro by the ErmSF (TlrA) methyltransferase.

    PubMed Central

    Kovalic, D; Giannattasio, R B; Jin, H J; Weisblum, B

    1994-01-01

    The DNA sequence that encodes 23S rRNA domain V of Bacillus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics on Streptomyces fradiae NRRL 2702, the host from which it was cloned. Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics. Images PMID:7961463

  2. Discrimination of bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

    SciTech Connect

    Bavykin, S. G.; Mikhailovich, V. M.; Zakharyev, V. M.; Lysov, Y. P.; Kelly, J. J.; Alferov, O. S.; Jackman, J.; Stahl, D. A.; Mirzabekov, A. D.; Gavin, I. M.; Kukhtin, A. V.; Chandler, D.

    2008-01-30

    Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5 min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect

  3. RlmCD-mediated U747 methylation promotes efficient G748 methylation by methyltransferase RlmAII in 23S rRNA in Streptococcus pneumoniae; interplay between two rRNA methylations responsible for telithromycin susceptibility

    PubMed Central

    Shoji, Tatsuma; Takaya, Akiko; Sato, Yoshiharu; Kimura, Satoshi; Suzuki, Tsutomu; Yamamoto, Tomoko

    2015-01-01

    Adenine at position 752 in a loop of helix 35 from positions 745 to 752 in domain II of 23S rRNA is involved in binding to the ribosome of telithromycin (TEL), a member of ketolides. Methylation of guanine at position 748 by the intrinsic methyltransferase RlmAII enhances binding of telithromycin (TEL) to A752 in Streptococcus pneumoniae. We have found that another intrinsic methylation of the adjacent uridine at position 747 enhances G748 methylation by RlmAII, rendering TEL susceptibility. U747 and another nucleotide, U1939, were methylated by the dual-specific methyltransferase RlmCD encoded by SP_1029 in S. pneumoniae. Inactivation of RlmCD reduced N1-methylated level of G748 by RlmAII in vivo, leading to TEL resistance when the nucleotide A2058, located in domain V of 23S rRNA, was dimethylated by the dimethyltransferase Erm(B). In vitro methylation of rRNA showed that RlmAII activity was significantly enhanced by RlmCD-mediated pre-methylation of 23S rRNA. These results suggest that RlmCD-mediated U747 methylation promotes efficient G748 methylation by RlmAII, thereby facilitating TEL binding to the ribosome. PMID:26365244

  4. Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII

    PubMed Central

    Lebars, Isabelle; Yoshizawa, Satoko; Stenholm, Anne R.; Guittet, Eric; Douthwaite, Stephen; Fourmy, Dominique

    2003-01-01

    The bacterial rRNA methyltransferase RlmAII (formerly TlrB) contributes to resistance against tylosin-like 16-membered ring macrolide antibiotics. RlmAII was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmAII methylates 23S rRNA at nucleotide G748, which is situated in a stem–loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmAII is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G–A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmAII induces changes in NMR signals at specific nucleotides that are involved in the methyltransferase–RNA interaction. The conformation of hairpin 35 that interacts with RlmAII is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly. PMID:12514124

  5. Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    González, Angel; Mas, Albert

    2011-06-30

    The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.

  6. Crystal structure of RlmM, the 2′O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA

    PubMed Central

    Punekar, Avinash S.; Shepherd, Tyson R.; Liljeruhm, Josefine; Forster, Anthony C.; Selmer, Maria

    2012-01-01

    RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2′O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM–AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2′O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate. PMID:22923526

  7. Identification of Lactobacillus isolates from the gastrointestinal tract, silage, and yoghurt by 16S-23S rRNA gene intergenic spacer region sequence comparisons.

    PubMed

    Tannock, G W; Tilsala-Timisjarvi, A; Rodtong, S; Ng, J; Munro, K; Alatossava, T

    1999-09-01

    Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.

  8. Discrimination of Bacillus anthracis from closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microchips

    DOEpatents

    Bavykin, Sergei G.; Mirzabekov, Andrei D.

    2007-10-30

    The present invention is directed to a novel method of discriminating a highly infectious bacterium Bacillus anthracis from a group of closely related microorganisms. Sequence variations in the 16S and 23S rRNA of the B. cereus subgroup including B. anthracis are utilized to construct an array that can detect these sequence variations through selective hybridizations. The identification and analysis of these sequence variations enables positive discrimination of isolates of the B. cereus group that includes B. anthracis. Discrimination of single base differences in rRNA was achieved with a microchip during analysis of B. cereus group isolates from both single and in mixed probes, as well as identification of polymorphic sites. Successful use of a microchip to determine the appropriate subgroup classification using eight reference microorganisms from the B. cereus group as a study set, was demonstrated.

  9. Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains

    PubMed Central

    Molini, Barbara J.; Tantalo, Lauren C.; Sahi, Sharon K.; Rodriguez, Veronica I.; Brandt, Stephanie L.; Fernandez, Mark C.; Godornes, Charmie B.; Marra, Christina M.; Lukehart, Sheila A.

    2016-01-01

    Background High rates of 23S rDNA mutations implicated in macrolide resistance have been identified in Treponema pallidum samples from syphilis patients in many countries. Nonetheless, some clinicians have been reluctant to abandon azithromycin as a treatment for syphilis, citing the lack of a causal association between these mutations and clinical evidence of drug resistance. Although azithromycin resistance has been demonstrated in vivo for the historical Street 14 strain, no recent T. pallidum isolates have been tested. We used the well-established rabbit model of syphilis to determine the in vivo efficacy of azithromycin against 23S rDNA mutant strains collected in 2004 to 2005 from patients with syphilis in Seattle, Wash. Methods Groups of 9 rabbits were each infected with a strain containing 23S rDNA mutation A2058G (strains UW074B, UW189B, UW391B) or A2059G (strains UW228B, UW254B, and UW330B), or with 1 wild type strain (Chicago, Bal 3, and Mexico A). After documentation of infection, 3 animals per strain were treated with azithromycin, 3 were treated with benzathine penicillin G, and 3 served as untreated control groups. Treatment efficacy was documented by darkfield microscopic evidence of T. pallidum, serological response, and rabbit infectivity test. Results Azithromycin uniformly failed to cure rabbits infected with strains harboring either 23S rDNA mutation, although benzathine penicillin G was effective. Infections caused by wild type strains were successfully treated by either azithromycin or benzathine penicillin G. Conclusions A macrolide resistant phenotype was demonstrated for all strains harboring a 23S rDNA mutation, demonstrating that either A2058G or A2059G mutation confers in vivo drug resistance. PMID:27513385

  10. 23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

    PubMed

    Bae, Hee-Sung; Hou, Aixin

    2013-02-01

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

  11. Development of a signature probe targeting the 16S-23S rRNA internal transcribed spacer of a ruminal Ruminococcus flavefaciens isolate from reindeer.

    PubMed

    Præsteng, K E; Mackie, R I; Cann, I K O; Mathiesen, S D; Sundset, M A

    2011-03-01

    The cellulolytic Ruminococcus flavefaciens has previously been introduced into the ruminant rumen to increase microbial degradation of plant cell wall carbohydrates. The functional effect of an introduced bacterium depends on its ability to establish in the digestive tract, and signature probes can be used as a tool to track and quantify introduced strains. The purpose of this current study was to develop an oligonucleotide signature probe targeting the 16S-23S rRNA internal transcribed spacer (ITS) of a putative probiotic cellulolytic isolate (R. flavefaciens strain 8/94-32) from the rumen of reindeer (Rangifer tarandus tarandus). The 16S-23S rRNA gene ITS of three Ruminococcus strains; R. flavefaciens strain 8/94-32, R. flavefaciens FD-1 and Ruminococcus albus Ra-8, was investigated. The ITS region has been reported to vary more between closely related bacteria compared to the widely used 16S rRNA gene, and a high degree of sequence polymorphism was indeed detected between the three Ruminococcus strains studied. Based on observed sequence differences, two oligonucloetide probes, ITSRumi1 and ITSRumi2, targeting the ITS region of the R. flavefaciens isolate 8/94-32 were developed. Probe specificity was evaluated in dot blot hybridisations with R. flavefaciens isolate 8/94-32 and four other Ruminococcus-strains tested. The probe ITSRumi1 gave positive signals for the R. flavefaciens isolate 8/94-32 only, while probe ITSRumi2 gave positive signals for R. flavefaciens isolate 8/94-32 as well as for R. albus Ra-8. The result of hybridisations with the probe ITSRumi1 indicates that the probe is specific for the R. flavefaciens strain 8/94-32 amongst the four Ruminococcus-strains tested, and is promising for further studies using it as a signature probe for tracking this strain when re-introduced to the reindeer rumen.

  12. Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region.

    PubMed

    Yang, G; Benson, R; Pelish, T; Brown, E; Winchell, J M; Fields, B

    2010-03-01

    Although the majority of cases of Legionnaires' disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.

  13. Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences.

    PubMed

    Takahashi, Mai; Yukphan, Pattaraporn; Yamada, Yuzo; Suzuki, Ken-ichiro; Sakane, Takeshi; Nakagawa, Yasuyoshi

    2006-06-01

    Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.

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

    PubMed

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

    2006-08-01

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

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

    PubMed

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

    2012-10-16

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

  16. The two faces of the Escherichia coli 23 S rRNA sarcin/ricin domain: the structure at 1.11 A resolution.

    PubMed

    Correll, C C; Wool, I G; Munishkin, A

    1999-09-17

    The sarcin/ricin domain of 23 S - 28 S ribosomal RNA is essential for protein synthesis because it forms a critical part of the binding site for elongation factors. A crystal structure of an RNA of 27 nucleotides that mimics the domain in Escherichia coli 23 S rRNA was determined at 1.11 A resolution. The domain folds into a hairpin distorted by four non-canonical base-pairs and one base triple. The fold is stabilized by cross-strand and intra-stand stacking; no intramolecular stabilizing metal ions are observed. This is the first structure to reveal in great detail the geometry and the hydration of two common motifs that are conserved in this rRNA domain, a GAGA tetraloop and a G-bulged cross-strand A stack. Differences in the region connecting these motifs to the stem in the E. coli and in the rat sarcin/ricin domains may contribute to the species-specific binding of elongation factors. Correlation of nucleotide protection data with the structure indicates that the domain has two surfaces. One surface is accessible, lies primarily in the major groove, and is likely to bind the elongation factors. The second lies primarily in the minor groove, and is likely to be buried in the ribosome. This minor groove surface includes the Watson-Crick faces of the cytosine bases in the unusual A2654.C2666 and U2653.C2667 water-mediated base-pairs. Copyright 1999 Academic Press.

  17. Specific detection and identification of [Actinobacillus] muris by PCR using primers targeting the 16S-23S rRNA internal transcribed spacer regions.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

    2013-08-01

    [Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

    SciTech Connect

    Sunita,S.; Zhenxing, H.; Swaathi, J.; Cygler, M.; Matte, A.; Sivaraman, J.

    2006-01-01

    Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine ({psi}) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E. coli RluF at 2.6 Angstroms resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of {psi}-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

  19. Development of a multiplex PCR assay based on the 16S-23S rRNA internal transcribed spacer for the detection and identification of rodent Pasteurellaceae.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Bleich, André; Gougoula, Christina; Sager, Martin

    2013-11-01

    The rodents Pasteurellaceae have to be excluded from the specified pathogen free experimental animal facilities. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals just a few molecular based methods are available for their detection and identification. The aim of the present investigation was to develop a multiplex PCR assay allowing detection of all rodent Pasteurellaceae and identification of [Pasteurella] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl and [Actinobacillus] muris, as the most prevalent members of the group. For this, a Pasteurellaceae common forward primer located on the 16S rRNA gene was used in conjunction with four different reverse primers specific for [P.] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl, [A.] muris and a common reverse primer for all rodent Pasteurellaceae, all targeting the 16S-23S rRNA internal transcribed spacer sequences. The performance characteristics of the assay were tested against 125 Pasteurellaceae isolates belonging to eleven different species and including 34 strains of [P.] pneumotropica biotype Jawetz, 44 strains of [P.] pneumotropica biotype Heyl and 37 strains of [A.] muris. Additionally, eight other mouse associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA gene sequencing. This multiplex PCR represents the first molecular tool able to detect and differentiate in a single assay among the Pasteurellaceae found in laboratory mouse and may become a reliable alternative to the present diagnostic methods. © 2013.

  20. Sequence diversity in the 16S-23S intergenic spacer region (ISR) of the rRNA operons in representatives of the Escherichia coli ECOR collection.

    PubMed

    Antón, A I; Martínez-Murcia, A J; Rodríguez-Valera, F

    1998-07-01

    The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S-23S intergenic spacer region (ISR) for tRNA(Glu-2) and three (rrnA, D, and H) contain genes for tRNA(Ile-1) and tRNA(Ala-1B). To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

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

    PubMed Central

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

    2005-01-01

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

  2. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

    PubMed

    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  3. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

    PubMed Central

    Liu, Mingfu; Douthwaite, Stephen

    2002-01-01

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit. PMID:12417742

  4. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy.

    PubMed

    Liu, Mingfu; Douthwaite, Stephen

    2002-11-12

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit.

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

    PubMed Central

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

    1996-01-01

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

  6. Functional interaction between bases C1049 in domain II and G2751 in domain VI of 23S rRNA in Escherichia coli ribosomes

    PubMed Central

    Miyoshi, Tomohiro; Uchiumi, Toshio

    2008-01-01

    The factor-binding center within the Escherichia coli ribosome is comprised of two discrete domains of 23S rRNA: the GTPase-associated region (GAR) in domain II and the sarcin–ricin loop in domain VI. These two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. Current X-ray crystallography of the ribosome shows an interaction between C1049 in the GAR and G2751 in domain VI. We have confirmed this interaction by site-directed mutagenesis and chemical probing. Disruption of this base pair affected not only the chemical modification of some bases in domains II and VI and in helix H89 of domain V, but also ribosome function dependent on both EF-G and EF-Tu. Mutant ribosomes carrying the C1049 to G substitution, which show enhancement of chemical modification at G2751, were used to probe the interactions between the regions around 1049 and 2751. Binding of EF-G-GDP-fusidic acid, but not EF-G-GMP-PNP, to the ribosome protected G2751 from modification. The G2751 protection was also observed after tRNA binding to the ribosomal P and E sites. The results suggest that the interactions between the bases around 1049 and 2751 alter during different stages of the translation process. PMID:18252772

  7. Cleavage of the sarcin–ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

    PubMed Central

    García-Ortega, Lucía; Álvarez-García, Elisa; Gavilanes, José G.; Martínez-del-Pozo, Álvaro; Joseph, Simpson

    2010-01-01

    Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation. PMID:20215430

  8. 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.

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

    PubMed

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

    2003-05-28

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

  10. The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

    PubMed Central

    Golovina, Anna Y.; Dzama, Margarita M.; Osterman, Ilya A.; Sergiev, Petr V.; Serebryakova, Marina V.; Bogdanov, Alexey A.; Dontsova, Olga A.

    2012-01-01

    The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation. PMID:22847818

  11. Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

    PubMed

    Cremonesi, P; Zottola, T; Locatelli, C; Pollera, C; Castiglioni, B; Scaccabarozzi, L; Moroni, P

    2013-01-01

    Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    NASA Astrophysics Data System (ADS)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  13. Update of the All-Species Living Tree Project based on 16S and 23S rRNA sequence analyses.

    PubMed

    Yarza, Pablo; Ludwig, Wolfgang; Euzéby, Jean; Amann, Rudolf; Schleifer, Karl-Heinz; Glöckner, Frank Oliver; Rosselló-Móra, Ramon

    2010-10-01

    The "All-Species Living Tree Project" (LTP) provides the scientific community with a useful taxonomic tool consisting of a curated database of type strain sequences, a universal and optimized alignment and a single phylogenetic tree harboring all the type strains of the hitherto classified species. On the website http://www.arb-silva.de/projects/living-tree an update has been regularly maintained by including the 1301 new descriptions that have appeared in the validation and notification lists of the IJSEM journal. The topology of the 16S rRNA-based tree was validated with a detailed comparison against a collection of taxa-specific and broad-range trees made using different approaches, subsets of sequences and alignments. Seven percent of the classified species is still missing, as their type strains do not have a good quality SSU sequence. In addition, a new database of type strains for which adequate 23S rRNA entries existed in public repositories was built. Among the 8602 species with validly published names until February 2010, we were able to find good quality LSU representatives for 792 type strains, whereas around 91% of the complete catalogue still remains unsequenced. Despite the scarce representation of some groups in LSU databases, we have devised a highly optimized alignment and a reliable LSU tree in order to set up a stable phylogenetic starting point for taxonomic purposes. The current release corresponds to the fourth update of the project (LTPs102), and contains additional features which increase usability and compatibility. Use the contact address living-tree@arb-silva.de to provide additional input for the development of this taxonomic tool. Copyright © 2010 Elsevier GmbH. All rights reserved.

  14. The influence of base identity and base pairing on the function of the alpha-sarcin loop of 23S rRNA.

    PubMed Central

    O'Connor, M; Dahlberg, A E

    1996-01-01

    The alpha-sarcin loop of large subunit rRNAs is one of the sites of interaction of elongation factors with the ribosome, and the target of the cytotoxins alpha-sarcin and ricin. Using a genetic selection for increased frameshifting in a reporter gene, we have isolated a C --> U mutation at position 2666 in the alpha-sarcin loop. In the NMR-derived structure of the loop, bases equivalent to 2666 and 2654 are paired via a non-canonical base pairing interaction. Each of the three base substitutions at C2666 and A2654 was constructed by site-directed mutagenesis of a plasmid borne copy of the rrnB operon of Escherichia coli. Only the C2666 --> U and A2654 --> G mutations that resulted in the formation of canonical A-U and C-G base pairs respectively, increased the levels of stop codon readthrough and frameshifting. The effects of different base pair combinations at positions 2666 and 2654 on ribosome function were then tested by constructing and analyzing all possible base combinations at these sites. All A --> G base substitution mutations at position 2654 and C --> U substitutions at position 2666 increased the levels of translational errors. However, these effects were greatest when G2654 and U2666 had the potential to engage in standard Watson-Crick base pairing interactions. These data indicate that base identity as well as base pairing interactions are important for the function of this essential component of the large subunit rRNA. PMID:8758999

  15. Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

    PubMed Central

    Kirillov, S V; Porse, B T; Garrett, R A

    1999-01-01

    A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the

  16. The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG▿

    PubMed Central

    Raghavan, Rahul; Miller, Scott R.; Hicks, Linda D.; Minnick, Michael F.

    2007-01-01

    We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer. PMID:17644584

  17. Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms.

    PubMed

    Hochrein, Hubertus; Kirschning, Carsten J

    2013-03-01

    The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13.

  18. Diverse and Unique Picocyanobacteria in Chesapeake Bay, Revealed by 16S-23S rRNA Internal Transcribed Spacer Sequences†§

    PubMed Central

    Chen, Feng; Wang, Kui; Kan, Jinjun; Suzuki, Marcelino T.; Wommack, K. Eric

    2006-01-01

    rRNA internal transcribed spacer phylogeny showed that Chesapeake Bay is populated with diverse Synechococcus strains, including members of the poorly studied marine cluster B. Marine cluster B prevailed in the upper bay, while marine cluster A was common in the lower bay. Interestingly, marine cluster B Synechococcus included phycocyanin- and phycoerythrin-rich strains. PMID:16517680

  19. 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

  20. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

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

    ERIC Educational Resources Information Center

    Guan, Min-Xin

    2005-01-01

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

  2. 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…

  3. Use of PCR primers and probes based on the 23S rRNA and internal transcription spacer (ITS) gene sequence for the detection and enumerization of Lactobacillus acidophilus and Lactobacillus plantarum in feed supplements.

    PubMed

    Tsai, Cheng-Chih; Lai, Chieh-Hsien; Yu, Bi; Tsen, Hau-Yang

    2010-06-01

    Novel polymerase chain reaction (PCR) primers designed from the 16S-23S internal transcription spacer (ITS) rRNA and 23S rRNA genes, respectively, were used for the specific detection of Lactobacillus acidophilus and Lactobacillus plantarum. Molecular weights of the PCR products were 221 and 599 bp, respectively. Strains of L. acidophilus and L. plantarum obtained from the culture center, dairy products, infant stool and other samples, could be identified with these PCR primers. DNAs from other lactic acid bacteria (LAB) species including strains of Lactobacillus pentosus which was closely related to L. plantarum, and bacteria species other than LAB, would not generate the false positive results. When this PCR primer set was used for the detection of L. acidophilus and L. plantarum in feed supplement or feed starter samples, reliable results were obtained. Furthermore, when these L. acidophilus or L. plantarum specific primers were used as DNA probes for the colony hybridization of L. acidophilus and L. plantarum, the viable cells of these LAB species in culture and feed supplements or starter products could be identified and enumerized. The method described here thus offers a rapid and economic way to inspect and assure the quality of the feed supplements or fermentation starters.

  4. 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.

  5. Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

    PubMed Central

    Roth, Andreas; Reischl, Udo; Streubel, Anna; Naumann, Ludmila; Kroppenstedt, Reiner M.; Habicht, Marion; Fischer, Marga; Mauch, Harald

    2000-01-01

    A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S r

  6. Sequence specific detection of bacterial 23S ribosomal RNA by TLR13

    PubMed Central

    Li, Xiao-Dong; Chen, Zhijian J

    2012-01-01

    Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity. DOI: http://dx.doi.org/10.7554/eLife.00102.001 PMID:23110254

  7. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

  8. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

    PubMed Central

    Sawada, H; Takeuchi, T; Matsuda, I

    1997-01-01

    Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola. PMID:8979356

  9. 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.

  10. Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes

    PubMed Central

    Madico, Guillermo; Quinn, Thomas C.; Boman, Jens; Gaydos, Charlotte A.

    2000-01-01

    Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (κ, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples. PMID:10699002

  11. Updates on quick identification of acetic acid bacteria with a focus on the 16S-23S rRNA gene internal transcribed spacer and the analysis of cell proteins by MALDI-TOF mass spectrometry.

    PubMed

    Trček, Janja; Barja, François

    2015-03-02

    Acetic acid bacteria have attracted much attention over the past few years, due mainly to their metabolic traits that are of interest to the biotechnology industry. In addition, it turns out that their ecological habitats are almost unlimited since they have been found as symbionts in different insects and also as emerging opportunistic human pathogens. Very surprising is the finding that they colonize niches considered anaerobic, disproving the generalized statement that they are strict aerobes. Since they have taken on different biological roles in our environment, more and more people are charged with the task of identifying them. However, this turns out to be not always easy, especially if we are using phenotypic approaches for identification. A substantial step forward in making the identification of acetic acid bacteria easier was made possible using molecular biological methods, which have been extensively tested since 2000. However, some molecular methods require expensive machines and experienced staff, and moreover the level of their discrimination varies. All these factors must be considered when selecting the most appropriate approach for identifying acetic acid bacteria. With this objective in mind, this review article discusses the benefits and drawbacks of molecular biological methods for identification of acetic acid bacteria, with a focus on the 16S-23S rRNA gene ITS regions and the recently described alternative method for identification of acetic acid bacteria, MALDI-TOF MS.

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

    PubMed

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

    2013-12-27

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

  13. 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.

  14. Gateway role for rRNA precursors in ribosome assembly.

    PubMed

    Gutgsell, Nancy S; Jain, Chaitanya

    2012-12-01

    In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Identification of different subtypes of rapid growing Atypical Mycobacterium from water and soil sources: Using PCR-RFLP using hsp65 and rRNA 16s-23s genes.

    PubMed

    Varahram, Mohammad; Farnia, Parissa; Saif, Shima; Marashian, Mehran; Farnia, Poopak; Ghanavi, Jaladein; Velayati, Ali Akbar

    2016-12-01

    Nontuberculosis mycobacteria (NTM) are a diverse group of microorganisms that cause a variety of diseases in humans including skin, respiratory, and gastrointestinal tract infection. Generally, NTM are classified into two categories: rapid (<7days) and slow growing (>7days). In this study, we aimed to investigate NTM frequency and prevalence in environmental samples. Additionally, we tried to identify various subtypes of isolated rapid growing mycobacteria (RGM). Through a prospective descriptive cross-sectional study, water and soil samples were gathered from four neighboring towns around Tehran, the capital of Iran, at different geographic directions. Every 100m(2) of the studied areas gave one sample containing 6g of soil in 3-5cm depth deposited in 50mL sterile water as sampling media. After digestion and decontamination, DNA from culture-positive specimens (RGM) were extracted using phenol-chloroform methods. Then the molecular identification of species and subspecies were performed using 16s-23s rRNA and hsp65 gene. In total, 341 RGM were found, out of which 322 (94.4%) were identified and 20 (5.8%) could not be identified. The most frequent RGM was, Mycobacterium fortuitum (72; 22%), Mycobacterium senegalense (58; 17.7%), Mycobacterium parafortuitum (44; 13.4%) and Mycobacterium conceptionense type 1 (24; 7.2%), and Mycobacterium cheloni type 1 (20; 6.0%). As shown in Table 1, M. fortuitum had more subtypes (8), and the frequency of subtypes 1 (27.7%), 4 (16.6%), and 5 (13.8%) were higher. Among subtypes of M. senegalense, subtype 1 had a higher frequency (70.4%) in comparison to subtype 2 (29.5%). M. cheloni had just one subtype. Our results showed M. fortuitum as the most prominent strain isolated from environmental samples. The frequency was similar in different places, irrespective of climatic variations. Availability of various subtypes of M. fortuitum might indicate a large circulation of this RGM in soil and water of Iranian territory. This high

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

    PubMed

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

    1985-10-01

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

  1. RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.

    PubMed

    Volokhov, Dmitriy V; Simonyan, Vahan; Davidson, Maureen K; Chizhikov, Vladimir E

    2012-01-01

    Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in

  2. 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

  3. 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

  4. Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12.

    PubMed

    García-Martínez, J; Martínez-Murcia, A; Antón, A I; Rodríguez-Valera, F

    1996-11-01

    Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

  5. 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.

  6. 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.

  7. Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Trościańczyk, Aleksandra; Banach, Tomasz; Kowalski, Cezary

    2015-03-01

    Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals. © 2015 The Authors.

  8. Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance.

    PubMed

    Johnson, Steven R; Grad, Yonatan; Abrams, A Jeanine; Pettus, Kevin; Trees, David L

    2017-02-01

    The whole-genome sequences of 24 isolates of Neisseria gonorrhoeae with elevated minimum inhibitory concentrations (MICs) to azithromycin (≥2.0 µg/mL) were analyzed against a modified sequence derived from the whole-genome sequence of N. gonorrhoeae FA1090 to determine, by signal ratio, the number of mutant copies of the 23S rRNA gene and the copy number effect on 50S ribosome-mediated azithromycin resistance. Isolates that were predicted to contain four mutated copies were accurately identified compared with the results of direct sequencing. Fewer than four mutated copies gave less accurate results but were consistent with elevated MICs.

  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. [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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Edwardsiella ictaluri

    USDA-ARS?s Scientific Manuscript database

    Comparison of the 23S rRNA gene sequences of Edwardsiella tarda and Edwardsiella ictaluri confirmed a close phylogenetic relationship between these two fish pathogen species and a distant relation with the 'core' members of the Enterobacteriaceae family. Analysis of the rrl gene for 23S rRNA in E. i...

  18. Ribosomal Mutations in Arcanobacterium pyogenes Confer a Unique Spectrum of Macrolide Resistance

    PubMed Central

    Jost, B. Helen; Trinh, Hien T.; Songer, J. Glenn; Billington, Stephen J.

    2004-01-01

    Four macrolide-resistant Arcanobacterium pyogenes isolates contained A2058T, A2058G, or C2611G (Escherichia coli numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin. PMID:14982799

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

    PubMed Central

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

    1996-01-01

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

  20. Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)

    PubMed Central

    Moissenet, Didier; Bidet, Philippe; Garbarg-Chenon, Antoine; Arlet, Guillaume; Vu-Thien, Hoang

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNAIle and tRNAAla genes, which are identical to genes described for R. pickettii and R. solanacearum. PMID:11136807

  1. Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

    PubMed

    Moissenet, D; Bidet, P; Garbarg-Chenon, A; Arlet, G; Vu-Thien, H

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

  2. 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

  3. Rapid Emergence of Resistance to Linezolid and Mutator Phenotypes in Staphylococcus aureus Isolates from an Adult Cystic Fibrosis Patient

    PubMed Central

    Tazi, Asmaa; Chapron, Jeanne; Touak, Gerald; Longo, Magalie; Hubert, Dominique; Collobert, Gislène; Dusser, Daniel; Poyart, Claire

    2013-01-01

    Linezolid has emerged as an important therapeutic option for the treatment of Staphylococcus aureus in patients with cystic fibrosis. We report the rapid emergence, upon treatment with linezolid, of linezolid-resistant S. aureus clinical isolates through the accumulation of resistance-associated 23S rRNA mutations, together with acquisition of an altered mutator phenotype. PMID:23917314

  4. 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.

  5. 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.

  6. 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.

  7. Phylogenetic Sequence Variations in Bacterial rRNA Affect Species-Specific Susceptibility to Drugs Targeting Protein Synthesis▿‡

    PubMed Central

    Akshay, Subramanian; Bertea, Mihai; Hobbie, Sven N.; Oettinghaus, Björn; Shcherbakov, Dimitri; Böttger, Erik C.; Akbergenov, Rashid

    2011-01-01

    Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket. PMID:21730122

  8. 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.

  9. 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

  10. 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

  11. 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

  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. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

    PubMed

    Vester, B; Garrett, R A

    1988-11-01

    The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site. The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible. In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol. All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories. The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface.

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

    PubMed Central

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

    2010-01-01

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

  16. Reconstruction of ancestral 16S rRNA reveals mutation bias in the evolution of optimal growth temperature in the Thermotogae phylum.

    PubMed

    Green, Anna G; Swithers, Kristen S; Gogarten, Jan F; Gogarten, Johann Peter

    2013-11-01

    Optimal growth temperature is a complex trait involving many cellular components, and its physiology is not yet fully understood. Evolution of continuous characters, such as optimal growth temperature, is often modeled as a one-dimensional random walk, but such a model may be an oversimplification given the complex processes underlying the evolution of continuous characters. Recent articles have used ancestral sequence reconstruction to infer the optimal growth temperature of ancient organisms from the guanine and cytosine content of the stem regions of ribosomal RNA, allowing inferences about the evolution of optimal growth temperature. Here, we investigate the optimal growth temperature of the bacterial phylum Thermotogae. Ancestral sequence reconstruction using a nonhomogeneous model was used to reconstruct the stem guanine and cytosine content of 16S rRNA sequences. We compare this sequence reconstruction method with other ancestral character reconstruction methods, and show that sequence reconstruction generates smaller confidence intervals and different ancestral values than other reconstruction methods. Unbiased random walk simulation indicates that the lower temperature members of the Thermotogales have been under directional selection; however, when a simulation is performed that takes possible mutations into account, it is the high temperature lineages that are, in fact, under directional selection. We find that the evolution of Thermotogales optimal growth temperatures is best fit by a biased random walk model. These findings suggest that it may be easier to evolve from a high optimal growth temperature to a lower one than vice versa.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA.

    PubMed

    Zweib, C; Dahlberg, A E

    1984-09-25

    Three different small deletions were produced at a single Pvu 2 restriction site in E. coli 23S rDNA of plasmid pKK 3535 using exonuclease Bal 31. The deletions were located around position 1760 in 23S rRNA and were characterized by DNA sequencing as well as by direct fingerprinting and S1-mapping of the rRNA. Two of the mutant plasmids, Pvu 2-32 and Pvu 2-33, greatly reduced the growth rate of transformed cells while the third mutant, Pvu 2-14 grew as fast as cells containing the wild-type plasmid pKK 3535. All three mutant 23S rRNAs were incorporated into 50S-like particles and were even found in 70S ribosomes and polysomes in vivo. The conformation of mutant 23S rRNA in 50S subunits was probed with a double-strand specific RNase from cobra venom. These analyses revealed changes in the accessibility of cleavage sites near the deletions around position 1760 and in the area around position 800 in all three mutant rRNAs. We suggest, that an altered conformation of the rRNAs at the site of the deletion is responsible for the slow growth of cells containing mutant plasmids Pvu 2-32 and Pvu 2-33.

  5. Structural and functional analysis of Escherichia coli ribosomes containing small deletions around position 1760 in the 23S ribosomal RNA.

    PubMed Central

    Zweib, C; Dahlberg, A E

    1984-01-01

    Three different small deletions were produced at a single Pvu 2 restriction site in E. coli 23S rDNA of plasmid pKK 3535 using exonuclease Bal 31. The deletions were located around position 1760 in 23S rRNA and were characterized by DNA sequencing as well as by direct fingerprinting and S1-mapping of the rRNA. Two of the mutant plasmids, Pvu 2-32 and Pvu 2-33, greatly reduced the growth rate of transformed cells while the third mutant, Pvu 2-14 grew as fast as cells containing the wild-type plasmid pKK 3535. All three mutant 23S rRNAs were incorporated into 50S-like particles and were even found in 70S ribosomes and polysomes in vivo. The conformation of mutant 23S rRNA in 50S subunits was probed with a double-strand specific RNase from cobra venom. These analyses revealed changes in the accessibility of cleavage sites near the deletions around position 1760 and in the area around position 800 in all three mutant rRNAs. We suggest, that an altered conformation of the rRNAs at the site of the deletion is responsible for the slow growth of cells containing mutant plasmids Pvu 2-32 and Pvu 2-33. Images PMID:6091057

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

    PubMed

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

    2012-06-01

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

  7. Paenibacillus larvae 16S-23S rDNA intergenic transcribed spacer (ITS) regions: DNA fingerprinting and characterization.

    PubMed

    Dingman, Douglas W

    2012-07-01

    Paenibacillus larvae is the causative agent of American foulbrood in honey bee (Apis mellifera) larvae. PCR amplification of the 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) regions, and agarose gel electrophoresis of the amplified DNA, was performed using genomic DNA collected from 134 P. larvae strains isolated in Connecticut, six Northern Regional Research Laboratory stock strains, four strains isolated in Argentina, and one strain isolated in Chile. Following electrophoresis of amplified DNA, all isolates exhibited a common migratory profile (i.e., ITS-PCR fingerprint pattern) of six DNA bands. This profile represented a unique ITS-PCR DNA fingerprint that was useful as a fast, simple, and accurate procedure for identification of P. larvae. Digestion of ITS-PCR amplified DNA, using mung bean nuclease prior to electrophoresis, characterized only three of the six electrophoresis bands as homoduplex DNA and indicating three true ITS regions. These three ITS regions, DNA migratory band sizes of 915, 1010, and 1474 bp, signify a minimum of three types of rrn operons within P. larvae. DNA sequence analysis of ITS region DNA, using P. larvae NRRL B-3553, identified the 3' terminal nucleotides of the 16S rRNA gene, 5' terminal nucleotides of the 23S rRNA gene, and the complete DNA sequences of the 5S rRNA, tRNA(ala), and tRNA(ile) genes. Gene organization within the three rrn operon types was 16S-23S, 16S-tRNA(ala)-23S, and l6S-5S-tRNA(ile)-tRNA(ala)-23S and these operons were named rrnA, rrnF, and rrnG, respectively. The 23S rRNA gene was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to be present as seven copies. This was suggestive of seven rrn operon copies within the P. larvae genome. Investigation of the 16S-23S rDNA regions of this bacterium has aided the development of a diagnostic procedure and has helped genomic mapping investigations via characterization of the ITS regions. Copyright © 2012 Elsevier Inc

  8. C/D box sRNA-guided 2'-O-methylation patterns of archaeal rRNA molecules.

    PubMed

    Dennis, Patrick P; Tripp, Vanessa; Lui, Lauren; Lowe, Todd; Randau, Lennart

    2015-08-22

    In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures. We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences. This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.

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

    SciTech Connect

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

    2006-02-10

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

  10. MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains.

    PubMed

    Alonso, Rodrigo; Mateo, Estibaliz; Churruca, Estibaliz; Martinez, Irati; Girbau, Cecilia; Fernández-Astorga, Aurora

    2005-10-01

    Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.

  11. Defining 5S rRNA structure space: point mutation data can be used to predict the phenotype of multichange variants.

    PubMed

    Nayar, Madhavi; Fox, George E

    2011-09-01

    A portion of the 5S ribosomal RNA (rRNA) structure space in the vicinity of the Vibrio proteolyticus 5S rRNA sequence is explored in detail with the intention of establishing principles that will allow a priori prediction of which sequences would be valid members of a particular RNA structure space. Four hundred and one sequence variants differing from the V. proteolyticus 5S rRNA wild-type sequence in 1-7 positions were characterized using an in vivo assay system. Most significantly, it was found that in general, the phenotypic effects of single changes were independent of the phenotypic effect of a second change. As a result, it was possible to use the new data in conjunction with results from prior studies of the same RNA to develop "truth tables" to predict which multiple change variants would be functional and which would be nonfunctional. The actual phenotype of 93.8% of the multichange variants studied was consistent with the predictions made using truth tables thereby providing for perhaps the first time an upper limit estimate of how frequent unexpected interactions are. It was also observed that single changes at positions involved in secondary structure were no more likely to be invalid than changes in other regions. In particular, internal changes in long standard stems were in fact almost always tolerated. Changes at positions that were hypervariable in the context of an alignment of related sequences were, as expected, usually found to be valid. However, the potential validity of changes that were idiosyncratic to a single lineage of related sequences when placed in the V. proteolyticus 5S rRNA context was unpredictable.

  12. 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.

  13. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

    PubMed

    Stackebrandt, E; Witt, D; Kemmerling, C; Kroppenstedt, R; Liesack, W

    1991-05-01

    The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.

  14. Mutation of EMG1 causing Bowen–Conradi syndrome results in reduced cell proliferation rates concomitant with G2/M arrest and 18S rRNA processing delay

    PubMed Central

    Armistead, Joy; Hemming, Richard; Patel, Nehal; Triggs-Raine, Barbara

    2014-01-01

    Bowen–Conradi syndrome (BCS) is a lethal autosomal recessive disorder caused by a D86G substitution in the protein, Essential for Mitotic Growth 1 (EMG1). EMG1 is essential for 18S rRNA maturation and 40S ribosome biogenesis in yeast, but no studies of its role in ribosome biogenesis have been done in mammals. To assess the effect of the EMG1 mutation on cell growth and ribosomal biogenesis in humans, we employed BCS patient cells. The D86G substitution did not interfere with EMG1 nucleolar localization. In BCS patient lymphoblasts, cells accumulated in G2/M, resulting in reduced proliferation rates; however, patient fibroblasts showed normal proliferation. The rate of 18S rRNA processing was consistently delayed in patient cells, although this did not lead to a difference in the levels of 40S ribosomes, or a change in protein synthesis rates. These results demonstrate that as in yeast, EMG1 in mammals has a role in ribosome biogenesis. The obvious phenotype in lymphoblasts compared to fibroblasts suggests a greater need for EMG1 in rapidly dividing cells. Tissue-specific effects have been seen in other ribosomal biogenesis disorders, and it seems likely that the impact of EMG1 deficiency would be larger in the rapidly proliferating cells of the developing embryo. PMID:26676230

  15. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

    PubMed Central

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-01-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  16. Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes.

    PubMed Central

    Srivastava, A K; Schlessinger, D

    1988-01-01

    In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated. Images PMID:3050989

  17. Two New Point Mutations at A2062 Associated with Resistance to 16-Membered Macrolide Antibiotics in Mutant Strains of Mycoplasma hominis

    PubMed Central

    Furneri, Pio Maria; Rappazzo, Giancarlo; Musumarra, Maria Pia; Di Pietro, Patrizia; Catania, Lucrezia S.; Roccasalva, Lucia Silvana

    2001-01-01

    We describe two mutants of Mycoplasma hominis PG-21 which show resistance to 16-membered macrolides but susceptibility to lincosamides, obtained by in vitro exposure to increasing doses of josamycin. The 23S rRNA gene showed that each had a mutation (A2062G and A2062T) corresponding to nucleotide 2062 in Escherichia coli, which was associated with the acquired phenotype. PMID:11557502

  18. Corynebacterium glutamicum RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5' maturation of 5S rRNA.

    PubMed

    Maeda, Tomoya; Wachi, Masaaki

    2012-02-01

    Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the -1 site of the 5' end of 5S rRNA. However, 3' maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5' maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.

  19. Surveillance of Helicobacter pylori Antibiotic Susceptibility in Indonesia: Different Resistance Types among Regions and with Novel Genetic Mutations

    PubMed Central

    Miftahussurur, Muhammad; Syam, Ari Fahrial; Nusi, Iswan Abbas; Makmun, Dadang; Waskito, Langgeng Agung; Zein, Lukman Hakim; Akil, Fardah; Uwan, Willy Brodus; Simanjuntak, David; Wibawa, I Dewa Nyoman; Waleleng, Jimmy Bradley; Saudale, Alexander Michael Joseph; Yusuf, Fauzi; Mustika, Syifa; Adi, Pangestu; Maimunah, Ummi; Maulahela, Hasan; Rezkitha, Yudith Annisa Ayu; Subsomwong, Phawinee; Nasronudin; Rahardjo, Dadik; Suzuki, Rumiko; Akada, Junko; Yamaoka, Yoshio

    2016-01-01

    Information regarding Helicobacter pylori antibiotic resistance in Indonesia was previously inadequate. We assessed antibiotic susceptibility for H. pylori in Indonesia, and determined the association between virulence genes or genetic mutations and antibiotic resistance. We recruited 849 dyspeptic patients who underwent endoscopy in 11 cities in Indonesia. E-test was used to determine the minimum inhibitory concentration of five antibiotics. PCR-based sequencing assessed mutations in 23S rRNA, rdxA, gyrA, gyrB, and virulence genes. Next generation sequencing was used to obtain full-length sequences of 23S rRNA, infB, and rpl22. We cultured 77 strains and identified 9.1% with clarithromycin resistance. Low prevalence was also found for amoxicillin and tetracycline resistance (5.2% and 2.6%, respectively). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were demonstrated. Strains isolated from Sumatera Island had significantly higher metronidazole resistance than those from other locations. Metronidazole resistant strains had highly distributed rdxA amino acid substitutions and the 23S rRNA A2143G mutation was associated with clarithromycin resistance (42.9%). However, one strain with the highest MIC value had a novel mutation in rpl22 without an A2143G mutation. Mutation at Asn-87 and/or Asp-91 of gyrA was associated with levofloxacin-resistance and was related to gyrB mutations. In conclusions, although this is a pilot study for a larger survey, our current data show that Indonesian strains had the high prevalence of metronidazole and levofloxacin resistance with low prevalence of clarithromycin, amoxicillin, and tetracycline resistance. Nevertheless, clarithromycin- or metronidazole-based triple therapy should be administered with caution in some regions of Indonesia. PMID:27906990

  20. FGF23-S129F mutant bypasses ER/Golgi to the circulation of hyperphosphatemic familial tumoral calcinosis patients.

    PubMed

    Shawar, Said M; Ramadan, Ahmad R; Ali, Bassam R; Alghamdi, Manal A; John, Anne; Hudaib, Ferial M

    2016-12-01

    FGF23 is essential for the homeostasis of phosphate, and vitamin D. Loss-of-function mutations in this hormone cause hyperphosphatemic familial tumoral calcinosis (HFTC). Earlier reports suggested that intact FGF23 from loss of function mutants such as FGF23/S129F (iFGF23/S129F) is retained intracellularly while the carboxy-terminal fragment is secreted. We sought to investigate the fate of iFGF23/S129F mutant hormone in vivo and in vitro. Five patients clinically diagnosed with HFTC and confirmed by DNA sequencing to carry the c.386 C>T; p.S129F mutation in the homozygous state were studied. Healthy and heterozygous individuals were used as controls in the study. Using ELISA assays, we showed that iFGF23/S129F was 2-5 folds higher in patients' plasma, compared to heterozygous or healthy controls. Importantly, the mutant hormone could not be detected in the patients' sera. However, using proteinase inhibition profiling, we found that a serum metalloproteinase degraded the iFGF23/S129F explaining our failure to detect it in sera. The serum metalloproteinase degrades the WT and the mutant at different rates. Also, confocal microscopy imaging using wild-type (WT) FGF23 or FGF23/S129F mutant in transiently transfected HEK293 and HeLa cells showed weak staining of the Golgi complex with some vesicular staining resembling the ER. Additionally, FGF23 variants (FGF23/WT, FGF23/S129F, FGF23/S71G, and FGF23/R176Q) from stably transfected HEK293 cells secreted high levels into a serum-free medium that can be detected by ELISA and Western blot. Our results suggest that iFGF23/S129F mutant bypasses the ER/Golgi quality control system to the circulation of HFTC patients by an unknown pathway. Finally, we hypothesize that either the mutant hormone is unable to bind α-Klotho-FGFR1c, or it binds the dyad receptor with low affinity and, therefore, incapable of initiating maximal intracellular signaling. Our findings raise the potential use of the WT hormone in therapies of some

  1. Analysis of 16S-23S rRNA internal transcribed spacer regions in Pasteurellaceae isolated from laboratory rodents.

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Christensen, Henrik; Sager, Martin

    2012-09-01

    The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. 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.

  3. Helix 69 of E. coli 23S ribosomal RNA as a peptide nucleic acid target.

    PubMed

    Kulik, Marta; Markowska-Zagrajek, Agnieszka; Wojciechowska, Monika; Grzela, Renata; Wituła, Tomasz; Trylska, Joanna

    2017-04-07

    A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines () and U1915 with 3-methyl-. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA. We also verified the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which turned out to be in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.

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

    PubMed

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

    1995-01-01

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

  5. Mini-III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis.

    PubMed

    Redko, Yulia; Bechhofer, David H; Condon, Ciarán

    2008-06-01

    The late steps of both 16S and 5S ribosomal RNA maturation in the Gram-positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram-negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5' and 3' extremities of B. subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini-III contains an RNase III-like catalytic domain, but curiously lacks the double-stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well-known members of this family of enzymes. Cells lacking Mini-III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini-III functions much more efficiently on precursor 50S ribosomal subunits than naked pre-23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini-III recognizes and cleaves double-stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.

  6. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila

    PubMed Central

    Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-01-01

    ABSTRACT Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila. Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings. PMID:28069647

  7. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

    PubMed

    Descours, Ghislaine; Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-03-01

    Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

  8. GENETIC MUTATIONS AFFECTING THE FIRST LINE ERADICATION THERAPY OF Helicobacter pylori-INFECTED EGYPTIAN PATIENTS

    PubMed Central

    RAMZY, Iman; ELGAREM, Hassan; HAMZA, Iman; GHAITH, Doaa; ELBAZ, Tamer; ELHOSARY, Waleed; MOSTAFA, Gehan; ELZAHRY, Mohammad A. Mohey Eldin

    2016-01-01

    SUMMARY Introduction: Several genetic mutations affect the first-line triple therapy for Helicobacter pylori. We aimed to study the most common genetic mutations affecting the metronidazole and clarithromycin therapy for H. pylori-infected Egyptian patients. Patients and Methods: In our study, we included 100 successive dyspeptic patients scheduled for diagnosis through upper gastroscopy at Cairo's University Hospital, Egypt. Gastric biopsies were tested for the presence of H. pylori by detection of the 16S rRNA gene. Positive biopsies were further studied for the presence of the rdxA gene deletion by Polymerase Chain Reaction (PCR), while clarithromycin resistance was investigated by the presence of nucleotide substitutions within H. pylori 23S rRNA V domain using MboII and BsaI to carry out a Restricted Fragment Length Polymorphism (RFLP) assay. Results: Among 70 H. pylori positive biopsies, the rdxA gene deletion was detected in 44/70 (62.9%) samples, while predominance of the A2142G mutations within the H. pylori 23S rRNA V domain was evidenced in 39/70 (55.7%) of the positive H. pylori cases. No statistically significant difference was found between the presence of gene mutations and different factors such as patients 'age, gender, geographic distribution, symptoms and endoscopic findings. Conclusion: Infection with mutated H. pylori strains is considerably high, a finding that imposes care in the use of the triple therapy to treat H. pylori in Egypt, since the guidelines recommend to abandon the standard triple therapy when the primary clarithromycin resistance rate is over 20%1. PMID:27982354

  9. Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA.

    PubMed

    Olmedo, Gabriela; Guzmán, Plinio

    2008-06-01

    Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.

  10. Bacterial Load in Daily Urine Samples of Patients Infected with Mycoplasma genitalium, Mutation Analysis, and Response to Treatment

    PubMed Central

    Nordbø, S. A.; Pukstad, B.

    2016-01-01

    Objective. Increasing macrolide resistant strains of Mycoplasma genitalium is a challenge, and to differentiate between treatment failure and reinfection a timely test of cure (TOC) is warranted. The aim of this study was to evaluate the best time for TOC after five days' treatment of Mycoplasma genitalium infection with azithromycin. Methods. Nineteen patients with positive PCR for Mycoplasma genitalium in urine provided urine samples daily for 2 weeks and on days 21, 28, and 35. Samples were tested by a commercial qPCR and by sequencing of the 23S rRNA gene. Results. Eight patients with a wild type of Mycoplasma genitalium responded successfully within four days after treatment initiation. Eleven patients had a mutation in the 23S rRNA gene. These samples exhibited high variations in bacterial load, and some patients tested negative at several time points during the observation period. Conclusions. Day-to-day fluctuations in the mutation samples allow for false negative TOC during the first 5 weeks after start of treatment. Due to increasing macrolide resistance of Mycoplasma genitalium, pretreatment mutation analysis is recommended. When a wild type is verified, TOC performed one week after initiation of treatment is suggested. PMID:27829780

  11. Mutational and Transcriptomic Changes Involved in the Development of Macrolide Resistance in Campylobacter jejuni

    PubMed Central

    Hao, Haihong; Yuan, Zonghui; Shen, Zhangqi; Han, Jing; Sahin, Orhan; Liu, Peng

    2013-01-01

    Macrolide antibiotics are important for clinical treatment of infections caused by Campylobacter jejuni. Development of resistance to this class of antibiotics in Campylobacter is a complex process, and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure of C. jejuni to escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing, and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome of C. jejuni were examined by real-time reverse transcription-PCR, immunoblotting, and DNA microarray analysis. Multiple types of mutations in ribosomal proteins L4 and L22 occurred early during stepwise selection. On the contrary, the mutations in the 23S rRNA gene, mediating high resistance to macrolides, were observed only in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediately resistant mutants, and the magnitude of upregulation declined with the occurrence of mutations in the 23S rRNA gene. DNA microarray analysis revealed the differential expression of 265 genes, most of which occurred in the intermediate mutant, including the upregulation of genes encoding ribosomal proteins and the downregulation of genes involved in energy metabolism and motility. These results indicate (i) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and (ii) that the development of macrolide resistance affects the pathways important for physiology and metabolism in C. jejuni, providing an explanation for the reduced fitness of macrolide-resistant Campylobacter. PMID:23274667

  12. Restriction Profiling of 23S Microheterogenic Ribosomal Repeats for Detection and Characterizing of E. coli and Their Clonal, Pathogenic, and Phylogroups

    PubMed Central

    Jayasree Rajagopalan Nair, Parvathi

    2015-01-01

    Correlating ribosomal microheterogenicity with unique restriction profiles can prove to be an efficacious and cost-effective approach compared with sequencing for microbial identification. An attempt to peruse restriction profiling of 23S ribosomal assemblage was ventured; digestion patterns with Bfa I discriminated E. coli from its colony morphovars, while Hae III profiles assisted in establishing distinct clonal groups. Among the gene pool of 399 ribosomal sequences extrapolated from 57 E. coli genomes, varying degree of predominance (I > III > IV > II) of Hae III pattern was observed. This was also corroborated in samples collected from clinical, commensal, and environmental origin. K-12 and its descendants showed type I pattern whereas E. coli-B and its descendants exhibited type IV, both of these patterns being exclusively present in E. coli. A near-possible association between phylogroups and Hae III profiles with presumable correlation between the clonal groups and different pathovars was established. The generic nature, conservation, and barcode gap of 23S rRNA gene make it an ideal choice and substitute to 16S rRNA gene, the most preferred region for molecular diagnostics in bacteria. PMID:26885397

  13. Molecular characterization of Streptococcus suis strains by 16S–23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis

    PubMed Central

    Le Devendec, Laëtitia; Gottschalk, Marcelo; Kobisch, Marylène

    2006-01-01

    Abstract We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S–23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S–23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations. PMID:16639941

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

    PubMed

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

    2001-02-01

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

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

    PubMed

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

    2002-11-01

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

  16. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

    PubMed Central

    Occhialini, A; Urdaci, M; Doucet-Populaire, F; Bébéar, C M; Lamouliatte, H; Mégraud, F

    1997-01-01

    Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes. PMID:9420046

  17. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

    PubMed

    Occhialini, A; Urdaci, M; Doucet-Populaire, F; Bébéar, C M; Lamouliatte, H; Mégraud, F

    1997-12-01

    Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.

  18. Identification of dairy lactic acid bacteria by tRNAAla-23S rDNA-RFLP.

    PubMed

    Mancini, Andrea; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gatti, Monica

    2012-12-01

    The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.

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

    PubMed

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

    2015-03-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Organization, Structure, and Variability of the rRNA Operon of the Whipple's Disease Bacterium (Tropheryma whippelii)

    PubMed Central

    Maiwald, Matthias; von Herbay, Axel; Lepp, Paul W.; Relman, David A.

    2000-01-01

    Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete, Tropheryma whippelii. We determined a nearly complete rRNA operon sequence of T. whippelii from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features. PMID:10809715

  2. [Application of molecular chaperones to soluble expression of e23sFv/His fusion proteins].

    PubMed

    Chang, Jing; Ouyang, Qing; Du, Xiao; Yang, Angang; Zhao, Jing; Yan, Bo

    2015-09-01

    To explore the feasibility of applying molecular chaperones to the soluble expression of e23sFv/His fusion proteins. The molecular chaperone plasmid pGro7 or pKJE7 was transformed into BL21 (DE3) competent cells together with the prokaryotic expression vector harboring His-tagged e23sFv. The soluble expression of e23sFv/His proteins was induced at 16 °C. The yield and antigen-binding activity of the soluble products were compared with those of the insoluble products conventionally purified from inclusion bodies. Both the overall yield and the purification ratio of soluble e23sFv/His proteins were relatively lower. The binding affinity of the soluble products to immobilized HER2 was not superior to that of the insoluble products from inclusion bodies. The molecular chaperone plasmids pGro7 and pKJE7 partially facilitate the soluble expression of e23sFv/His proteins, but both the yield and the purification ratio are still limited.

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

    PubMed Central

    Ogasawara, N; Moriya, S; Yoshikawa, H

    1983-01-01

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

  4. Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

    PubMed Central

    Siibak, Triinu; Remme, Jaanus

    2010-01-01

    Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly. PMID:20719918

  5. Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified.

    PubMed

    Siibak, Triinu; Remme, Jaanus

    2010-10-01

    Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.

  6. Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences.

    PubMed

    Sasaki, Y; Yamamoto, K; Kojima, A; Tetsuka, Y; Norimatsu, M; Tamura, Y

    2000-12-01

    Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.

  7. Determining RNA quality for NextGen sequencing: some exceptions to the gold standard rule of 23S to 16S rRNA ratio

    USDA-ARS?s Scientific Manuscript database

    Using next-generation-sequencing technology to assess entire transcriptomes requires high quality starting RNA. Currently, RNA quality is routinely judged using automated microfluidic gel electrophoresis platforms and associated algorithms. Here we report that such automated methods generate false-n...

  8. Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains.

    PubMed

    Haga, S; Hirano, Y; Murayama, O; Millar, B C; Moore, J E; Matsuda, M

    2003-01-01

    To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

  9. Prevalence of macrolide and fluoroquinolone resistance-mediating mutations in Mycoplasma genitalium in five cities in Russia and Estonia.

    PubMed

    Shipitsyna, Elena; Rumyantseva, Tatiana; Golparian, Daniel; Khayrullina, Guzel; Lagos, Amaya C; Edelstein, Inna; Joers, Kai; Jensen, Jörgen S; Savicheva, Alevtina; Rudneva, Natalia; Sukhanova, Larisa; Kozlov, Roman; Guschin, Alexander; Unemo, Magnus

    2017-01-01

    Resistance in the sexually transmitted bacterium Mycoplasma genitalium to all recommended therapeutic antimicrobials have rapidly emerged. However, to date, internationally reported resistance surveillance data for M. genitalium strains circulating in Eastern Europe are entirely lacking. The aim of this study was to estimate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in four cities in Russia and one in Estonia, 2013-2016. Consecutive urogenital samples found positive for M. genitalium during diagnostic testing were retrospectively analyzed for resistance-associated mutations in the 23S rRNA and parC genes using pyrosequencing and conventional Sanger sequencing, respectively. In total, 867 M. genitalium positive samples from 2013-2016 were analyzed. Macrolide resistance-associated mutations were detected in 4.6% of the samples from Russia (0.7-6.8% in different cities) and in 10% of the samples from Estonia. The mutations A2059G and A2058G were highly predominating in both Russia and Estonia, accounting together for 90.9% of the cases positive for nucleotide substitutions in the 23S rRNA gene. The rates of possible fluoroquinolone resistance-associated mutations were 6.2% in Russia (2.5-7.6% in different cities) and 5% in Estonia. The mutations S83I and S83N were the most frequent ones in Russia (24.4% each), whereas D87N highly predominated in Estonia (83.3% of all fluoroquinolone resistance-associated mutations). Approximately 1% of the samples in both countries harbored both macrolide and possible fluoroquinolone resistance-associated mutations, with A2058G and S83I being the most frequent combination (37.5%). The prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium was 4.6% and 6.2%, respectively, in Russia, and 10% and 5%, respectively, in Estonia. Despite the relatively low rates of macrolide and fluoroquinolone resistance in these countries, antimicrobial resistance

  10. Specific features of 5S rRNA structure - its interactions with macromolecules and possible functions.

    PubMed

    Smirnov, A V; Entelis, N S; Krasheninnikov, I A; Martin, R; Tarassov, I A

    2008-12-01

    Small non-coding RNAs are today a topic of great interest for molecular biologists because they can be regarded as relicts of a hypothetical "RNA world" which, apparently, preceded the modern stage of organic evolution on Earth. The small molecule of 5S rRNA (approximately 120 nucleotides) is a component of large ribosomal subunits of all living beings (5S rRNAs are not found only in mitoribosomes of fungi and metazoans). This molecule interacts with various protein factors and 23S (28S) rRNA. This review contains the accumulated data to date concerning 5S rRNA structure, interactions with other biological macromolecules, intracellular traffic, and functions in the cell.

  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. Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

    PubMed Central

    Wullings, B. A.; Van Beuningen, A. R.; Janse, J. D.; Akkermans, A. D. L.

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  13. Regarding the Charmed-Strange Member of the 23S1 Meson State

    PubMed Central

    Feng, Xue-Chao; Chen, Jing

    2013-01-01

    By employing the mass relations derived from the mass matrix and Regge trajectory, we investigate the masses of charmed and charmed-strange members of the 23S1 meson. The masses are compared with the values predicted by other theoretical approaches and experimental data. The results may be useful for the discovery of the unobserved meson and the determination of the quantum number of the newly discovered states. PMID:24250272

  14. Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci.

    PubMed

    Naimi, A; Beck, G; Monique, M; Lefèbvre, G; Branlanti, C

    1999-02-01

    The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.

  15. Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains

    PubMed Central

    Sulyok, Kinga M.; Kreizinger, Zsuzsa; Wehmann, Enikő; Lysnyansky, Inna; Bányai, Krisztián; Marton, Szilvia; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Jánosi, Szilárd; Nagy, Sára Ágnes

    2016-01-01

    ABSTRACT The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis. For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 μg/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains. PMID:27895010

  16. Higher-order structure of rRNA

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  17. Higher-order structure of rRNA

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  18. rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data

    PubMed Central

    Li, Yi; Yang, Rui-Heng; Jiang, Lan; Hu, Xiao-Di; Wu, Zu-Jian; Yao, Yi-Jian

    2017-01-01

    The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes. PMID:28637809

  19. rRNA Pseudogenes in Filamentous Ascomycetes as Revealed by Genome Data.

    PubMed

    Li, Yi; Yang, Rui-Heng; Jiang, Lan; Hu, Xiao-Di; Wu, Zu-Jian; Yao, Yi-Jian

    2017-08-07

    The nuclear ribosomal DNA (rDNA) is considered as a paradigm of concerted evolution. Components of the rDNA tandem repeats (45S) are widely used in phylogenetic studies of different organisms and the internal transcribed spacer (ITS) region was recently selected as a fungal DNA bar code. However, rRNA pseudogenes, as one kind of escape from concerted evolution, were reported in a wide range of organisms, especially in plants and animals. Moreover, large numbers of 5S rRNA pseudogenes were identified in several filamentous ascomycetes. To study whether rDNA evolves in a strict concerted manner and test whether rRNA pseudogenes exist in more species of ascomycetes, intragenomic rDNA polymorphisms were analyzed using whole genome sequences. Divergent rDNA paralogs were found to coexist within a single genome in seven filamentous ascomycetes examined. A great number of paralogs were identified as pseudogenes according to the mutation and secondary structure analyses. Phylogenetic analyses of the three rRNA coding regions of the 45S rDNA repeats, i.e., 18S, 5.8S, and 28S, revealed an interspecies clustering pattern of those different rDNA paralogs. The identified rRNA pseudogenic sequences were validated using specific primers designed. Mutation analyses revealed that the repeat-induced point (RIP) mutation was probably responsible for the formation of those rRNA pseudogenes. Copyright © 2017 Li et al.

  20. Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine.

    PubMed

    Kouvela, Ekaterini C; Gerbanas, George V; Xaplanteri, Maria A; Petropoulos, Alexandros D; Dinos, George P; Kalpaxis, Dimitrios L

    2007-01-01

    5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N1-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA(Phe) at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent 'loosening' of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.

  1. Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.

    PubMed

    Burman, Luke G; Mauro, Vincent P

    2012-09-01

    Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈ 15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5' external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

  2. Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system

    PubMed Central

    Burman, Luke G.; Mauro, Vincent P.

    2012-01-01

    Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5′ external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology. PMID:22718970

  3. Mutations Outside the Anisomycin-Binding Site Can Make Ribosomes Drug-Resistant

    SciTech Connect

    Blaha,G.; Gurel, G.; Schroeder, S.; Moore, P.; Steitz, T.

    2008-01-01

    Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.

  4. Structure-activity relationships of diverse oxazolidinones for linezolid-resistant Staphylococcus aureus strains possessing the cfr methyltransferase gene or ribosomal mutations.

    PubMed

    Locke, Jeffrey B; Finn, John; Hilgers, Mark; Morales, Gracia; Rahawi, Shahad; G C, Kedar; Picazo, Juan José; Im, Weonbin; Shaw, Karen Joy; Stein, Jeffrey L

    2010-12-01

    Staphylococcal resistance to linezolid (LZD) is mediated through ribosomal mutations (23S rRNA or ribosomal proteins L3 and L4) or through methylation of 23S rRNA by the horizontally transferred Cfr methyltransferase. To investigate the structural basis for oxazolidinone activity against LZD-resistant (LZD(r)) strains, we compared structurally diverse, clinically relevant oxazolidinones, including LZD, radezolid (RX-1741), TR-700 (torezolid), and a set of TR-700 analogs (including novel CD-rings and various A-ring C-5 substituents), against a panel of laboratory-derived and clinical LZD(r) Staphylococcus aureus strains possessing a variety of resistance mechanisms. Potency against all strains was correlated with optimization of C- and D-rings, which interact with more highly conserved regions of the peptidyl transferase center binding site. Activity against cfr strains was retained with either hydroxymethyl or 1,2,3-triazole C-5 groups but was reduced by 2- to 8-fold in compounds with acetamide substituents. LZD, which possesses a C-5 acetamide group and lacks a D-ring substituent, demonstrated the lowest potency against all strains tested, particularly against cfr strains. These data reveal key features contributing to oxazolidinone activity and highlight structural tradeoffs between potency against susceptible strains and potency against strains with various resistance mechanisms.

  5. Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli.

    PubMed

    Zaman, Sephorah; Fitzpatrick, Megan; Lindahl, Lasse; Zengel, Janice

    2007-11-01

    L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated 'tentacles' that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.

  6. High rate of A2142G point mutation associated with clarithromycin resistance among Iranian Helicobacter pylori clinical isolates.

    PubMed

    Khashei, Reza; Dara, Mahintaj; Bazargani, Abdollah; Bagheri Lankarani, Kamran; Taghavi, Alireza; Moeini, Maryam; Dehghani, Behzad; Sohrabi, Maryam

    2016-09-01

    This study aimed to investigate the clarithromycin resistance and its associated molecular mechanisms among Helicobacter pylori isolates from dyspeptic patients in Shiraz, Iran. From January to May 2014, 100 H. pylori strains were isolated from patients with gastroduodenal disorders. The resistance to clarithromycin was quantitatively evaluated, using Epsilometer (E-test) method. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on all the isolates to detect A2143G and A2142G mutations in 23S rRNA gene. The H. pylori isolation rate was found to be 31.4%. E-test showed that 20% of isolates were resistant to clarithromycin (MIC ≥ 1 mg/L). MIC of clarithromycin ranged between 0.016 and 24 mg/L. Findings of PCR-RFLP showed that the A2142G was the most (90%) frequently point mutation, followed by the A2143G (10%). No statistically significant difference was found between H. pylori clarithromycin resistance point mutations and patients' gender or age. To the best of our knowledge, this is the first report of high frequency of A2142G point mutation in Iran and probably in other regions of the world. Considering the increasing trend of H. pylori resistance to clarithromycin due to these mutations, it is crucial to investigate the new therapeutic approaches against H. pylori infection.

  7. Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli

    PubMed Central

    Zaman, Sephorah; Fitzpatrick, Megan; Lindahl, Lasse; Zengel, Janice

    2007-01-01

    L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated ‘tentacles’ that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance. PMID:17956547

  8. 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.

  9. 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

  10. RISSC: a novel database for ribosomal 16S–23S RNA genes spacer regions

    PubMed Central

    García-Martínez, Jesús; Bescós, Ignacio; Rodríguez-Sala, Jesús Javier; Rodríguez-Valera, Francisco

    2001-01-01

    A novel database, under the acronym RISSC (Ribosomal Intergenic Spacer Sequence Collection), has been created. It compiles more than 1600 entries of edited DNA sequence data from the 16S–23S ribosomal spacers present in most prokaryotes and organelles (e.g. mitochondria and chloroplasts) and is accessible through the Internet (http://ulises.umh.es/RISSC), where systematic searches for specific words can be conducted, as well as BLAST-type sequence searches. Additionally, a characteristic feature of this region, the presence/absence and nature of tRNA genes within the spacer, is included in all the entries, even when not previously indicated in the original database. All these combined features could provide a useful documen­tation tool for studies on evolution, identification, typing and strain characterization, among others. PMID:11125084

  11. Behavior of 23S metastable state He atoms in low-temperature recombining plasmas

    NASA Astrophysics Data System (ADS)

    Kajita, Shin; Tsujihara, Tadashi; Aramaki, Mitsutoshi; van der Meiden, Hennie; Oshima, Hiroshi; Ohno, Noriyasu; Tanaka, Hirohiko; Yasuhara, Ryo; Akiyama, Tsuyoshi; Fujii, Keisuke; Shikama, Taiichi

    2017-07-01

    We measured the electron density and temperature using laser Thomson scattering and metastable state (23S) of He atoms by laser absorption spectroscopy in the detached recombining plasmas in the divertor simulator NAGDIS-II. Using the measured electron density and temperature combined with the particle trajectory trace simulation, we discussed the behavior of the metastable state He atoms based on comparisons with the experimental results. It is shown that the metastable state atoms are mainly produced in the peripheral region of the plasma column, where the temperature is lower than the central part, and diffused in the vacuum vessel. It was shown that the 0D model is not valid and the transport of the metastable states is to be taken into account for the population distribution of He atoms in the detached plasmas.

  12. Effects of (22S,23S)-Homobrassinolide and Related Compounds on Membrane Potential and Transport of Egeria Leaf Cells.

    PubMed

    Dahse, I; Sack, H; Bernstein, M; Petzold, U; Müller, E; Vorbrodt, H M; Adam, G

    1990-07-01

    (22S,23S)-Homobrassinolide was tested for its effect on the electric cell potential, proton extrusion, ferricyanide reduction, and amino acid and sucrose uptake of leaves of Egeria densa Planchon. In the light, (22S,23S)-homobrassinolide and its derivative, 2alpha-3alpha-dihydroxy-5alpha-stigmast-22-en-6-one, were similar to each other and similar to fusicoccin in causing hyperpolarization and proton extrusion, whereas stigmasterol was less effective. In darkness, the three sterols showed comparable effects. (22S,23S)-Homobrassinolide slightly stimulated ferricyanide reduction and promoted uptake of sucrose and alpha-aminoisobutyric acid. The results are compatible with a stimulation of an electrogenic proton pump mechanism at the plasmalemma by (22S,23S)-homobrassinolide.

  13. Effects of (22S,23S)-Homobrassinolide and Related Compounds on Membrane Potential and Transport of Egeria Leaf Cells

    PubMed Central

    Dahse, Ingo; Sack, Holger; Bernstein, Matthias; Petzold, Uwe; Müller, Eberhard; Vorbrodt, Hans Matthias; Adam, Günter

    1990-01-01

    (22S,23S)-Homobrassinolide was tested for its effect on the electric cell potential, proton extrusion, ferricyanide reduction, and amino acid and sucrose uptake of leaves of Egeria densa Planchon. In the light, (22S,23S)-homobrassinolide and its derivative, 2α-3α-dihydroxy-5α-stigmast-22-en-6-one, were similar to each other and similar to fusicoccin in causing hyperpolarization and proton extrusion, whereas stigmasterol was less effective. In darkness, the three sterols showed comparable effects. (22S,23S)-Homobrassinolide slightly stimulated ferricyanide reduction and promoted uptake of sucrose and α-aminoisobutyric acid. The results are compatible with a stimulation of an electrogenic proton pump mechanism at the plasmalemma by (22S,23S)-homobrassinolide. PMID:16667589

  14. Occurrence of Mutations in the Antimicrobial Target Genes Related to Levofloxacin, Clarithromycin, and Amoxicillin Resistance in Helicobacter pylori Isolates from Buenos Aires City.

    PubMed

    Zerbetto De Palma, Gerardo; Mendiondo, Nicolas; Wonaga, Andrés; Viola, Luis; Ibarra, Daniela; Campitelli, Esteban; Salim, Nicolas; Corti, Rodolfo; Goldman, Cinthia; Catalano, Mariana

    2017-04-01

    Domain V of 23S rRNA, gyrA and gyrB Quinolones Resistance-Determining Region (QRDR), and pbp-1A gene point mutations were investigated in Helicobacter pylori-resistant isolates from three centres of Buenos Aires. Minimal inhibitory concentrations (MICs) were performed in 197 isolates from 52 H. pylori-positive naive patients by agar dilution method. Point mutations were achieved by amplification and sequencing of the target genes, and their association with resistance was determined by natural transformation assays. Resistance rates were as follows: metronidazole 28.8%, clarithromycin (CLA) 26.9%, levofloxacin (LEV) 32.7%, and amoxicillin (AMX) 7.6%. Nearly one-third of patients carried multidrug-resistant isolates. A2143G or A2142G in domain V of 23S-rRNA was found in all isolates showing high level of resistance to CLA (MIC >2 mg/L), accounting for 76.0% (38/50) of those with the resistant phenotype. The mutations A2267G or T1861C carried by 8/12 isolates with MIC 1-2 mg/L (low level) did not confer resistance by transformation. Substitutions at GyrA position 87 or 91, mainly N87K and D91G, were found in 92.8% (52/56) of the LEV-resistant isolates: 48 isolates with MIC 4-64 mg/L and 4/8 isolates with MIC 2 mg/L. The remaining four harboured K133N, also present in susceptible isolates. None of the substitutions in GyrB demonstrated to confer resistance. Transformation proved that PBP-1A N562Y and/or T556S substitutions confer the AMX resistance in our isolates, showing an additive effect. In conclusion, the usually reported mutations related to CLA, LEV, and AMX resistance were found in our isolates. However, low-level CLA resistance seems not to be due to mutations in Domain V of 23S rRNA gene.

  15. Occurrence of Mutations in the Antimicrobial Target Genes Related to Levofloxacin, Clarithromycin, and Amoxicillin Resistance in Helicobacter pylori Isolates from Buenos Aires City

    PubMed Central

    Zerbetto De Palma, Gerardo; Mendiondo, Nicolas; Wonaga, Andrés; Viola, Luis; Ibarra, Daniela; Campitelli, Esteban; Salim, Nicolas; Corti, Rodolfo; Goldman, Cinthia

    2017-01-01

    Domain V of 23S rRNA, gyrA and gyrB Quinolones Resistance-Determining Region (QRDR), and pbp-1A gene point mutations were investigated in Helicobacter pylori-resistant isolates from three centres of Buenos Aires. Minimal inhibitory concentrations (MICs) were performed in 197 isolates from 52 H. pylori-positive naive patients by agar dilution method. Point mutations were achieved by amplification and sequencing of the target genes, and their association with resistance was determined by natural transformation assays. Resistance rates were as follows: metronidazole 28.8%, clarithromycin (CLA) 26.9%, levofloxacin (LEV) 32.7%, and amoxicillin (AMX) 7.6%. Nearly one-third of patients carried multidrug-resistant isolates. A2143G or A2142G in domain V of 23S-rRNA was found in all isolates showing high level of resistance to CLA (MIC >2 mg/L), accounting for 76.0% (38/50) of those with the resistant phenotype. The mutations A2267G or T1861C carried by 8/12 isolates with MIC 1–2 mg/L (low level) did not confer resistance by transformation. Substitutions at GyrA position 87 or 91, mainly N87K and D91G, were found in 92.8% (52/56) of the LEV-resistant isolates: 48 isolates with MIC 4–64 mg/L and 4/8 isolates with MIC 2 mg/L. The remaining four harboured K133N, also present in susceptible isolates. None of the substitutions in GyrB demonstrated to confer resistance. Transformation proved that PBP-1A N562Y and/or T556S substitutions confer the AMX resistance in our isolates, showing an additive effect. In conclusion, the usually reported mutations related to CLA, LEV, and AMX resistance were found in our isolates. However, low-level CLA resistance seems not to be due to mutations in Domain V of 23S rRNA gene. PMID:27391421

  16. Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro

    PubMed Central

    Sharwood, Robert E.; Hotto, Amber M.; Bollenbach, Thomas J.; Stern, David B.

    2011-01-01

    Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3′-to-5′ exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNAArg, raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S–AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1. PMID:21148395

  17. Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro.

    PubMed

    Sharwood, Robert E; Hotto, Amber M; Bollenbach, Thomas J; Stern, David B

    2011-02-01

    Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3'-to-5' exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNA(Arg), raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S-AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2009-08-01

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

  1. 5S rRNA and ribosome.

    PubMed

    Gongadze, G M

    2011-12-01

    5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

  2. Inter- and intraspecific genomic variability of the 16S-23S intergenic spacer regions (ISR) in representatives of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans.

    PubMed

    Ni, Yong-Qing; Yang, Yuan; Bao, Jing-Ting; He, Kai-Yu; Li, Hong-Yu

    2007-05-01

    The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.

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

    PubMed

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

    2005-11-25

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

  4. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    PubMed

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  5. The weak measurement process and the weak value of spin for metastable helium 23S1

    NASA Astrophysics Data System (ADS)

    Monachello, Vincenzo; Barker, Peter; Flack, Robert; Hiley, Basil

    2016-05-01

    An experiment is being designed and constructed in order to measure the weak value of spin for an atomic system. The principle of the ``weak measurement'' process was first proposed by Aharonov, Albert and Vaidman, and describes a scenario in which a system is weakly coupled to a pointer between well-defined pre- and post-selected states. This experiment will utilise a pulsed supersonic beam of spin-1 metastable Helium (He*) atoms in the 23S1 state. The spin of the pre-selected He* atoms will be weakly coupled to its centre-of-mass. During its flight, the atomic beam will be prepared in a desired quantum state and travel through two inhomogeneous magnets (weak and strong) which both comprise the ``weak measurement'' process. The deviation of the post-selected ms = + 1 state as measured using a micro-channel plate, phosphor screen and CCD camera setup will allow for the determination of the weak value of spin. This poster will report on the methods used and the experimental realisation.

  6. Yeast 18 S rRNA Is Directly Involved in the Ribosomal Response to Stringent AUG Selection during Translation Initiation*

    PubMed Central

    Nemoto, Naoki; Singh, Chingakham Ranjit; Udagawa, Tsuyoshi; Wang, Suzhi; Thorson, Elizabeth; Winter, Zachery; Ohira, Takahiro; Ii, Miki; Valášek, Leoš; Brown, Susan J.; Asano, Katsura

    2010-01-01

    In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNAiMet, show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits. PMID:20699223

  7. Mutation in the L3 Ribosomal Protein Could Be Associated with Risk of Selection of High-Level Linezolid-Resistant Staphylococcus epidermidis Strains.

    PubMed

    Rouard, Caroline; Aslangul, Elisabeth; Rivière, Alexandre; Deback, Claire; Butel, Marie-José; Doucet-Populaire, Florence; Bourgeois-Nicolaos, Nadège

    2017-06-01

    Linezolid (LZD) has arisen as an alternative treatment in diabetic foot osteitis due to staphylococci. LZD resistance selection is difficult and involved various molecular mechanisms. As a better knowledge of those mechanisms could be beneficial for pathogenic strains' screening, we simulated in vitro the spontaneous mutagenesis process that leads to LZD-resistant strains from two Staphylococcus epidermidis strains responsible for monomicrobial diabetic foot osteitis. LZD high resistance was selected for both strains, with the same timeline of mutation appearance. Mutation in L3 protein (G152D) occurred first and quickly, but did not cause phenotypically detectable resistance or fitness cost. It was later followed by different 23S rRNA mutations (G2505A, G2447T), leading this time to detectable resistance (minimum inhibitory concentration [MIC] ≥8 mg/L). This phenomenon underlies the difficulty of resistance selection in coagulase-negative staphylococci (CoNS). This study is the first description of G2505A mutation in CoNS. Various phenotypical impacts were observed depending on strain and mutation: (i) fitness cost of G2505A and G2447T mutations; (ii) loss of erythromycin resistance concomitantly with L3 mutation selection; (iii) correlation between number of mutated rrl copies and LZD resistance level for G2447T. In conclusion, the risk of selection of high-level LZD-resistant S. epidermidis strains is weak, but does exist. It could probably appear in case of long-term treatment and be favored in the case of a pre-existing mutation in L3 ribosomal protein. Thus, broad screening conditions for pathogenic strains should probably be considered.

  8. Differential assembly of 16S rRNA domains during 30S subunit formation.

    PubMed

    Xu, Zhili; Culver, Gloria M

    2010-10-01

    Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.

  9. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

    PubMed

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-09-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

  10. Binding site for Xenopus ribosomal protein L5 and accompanying structural changes in 5S rRNA.

    PubMed

    Scripture, J Benjamin; Huber, Paul W

    2011-05-10

    The structure of the eukaryotic L5-5S rRNA complex was investigated in protection and interference experiments and is compared with the corresponding structure (L18-5S rRNA) in the Haloarcula marismortui 50S subunit. In close correspondence with the archaeal structure, the contact sites for the eukaryotic ribosomal protein are located primarily in helix III and loop C and secondarily in loop A and helix V. While the former is unique to L5, the latter is also a critical contact site for transcription factor IIIA (TFIIIA), accounting for the mutually exclusive binding of these two proteins to 5S RNA. The binding of L5 causes structural changes in loops B and C that expose nucleotides that contact the Xenopus L11 ortholog in H. marismortui. This induced change in the structure of the RNA reveals the origins of the cooperative binding to 5S rRNA that has been observed for the bacterial counterparts of these proteins. The native structure of helix IV and loop D antagonizes binding of L5, indicating that this region of the RNA is dynamic and also influenced by the protein. Examination of the crystal structures of Thermus thermophilus ribosomes in the pre- and post-translocation states identified changes in loop D and in the surrounding region of 23S rRNA that support the proposal that 5S rRNA acts to transmit information between different functional domains of the large subunit.

  11. METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

    PubMed

    Bengtsson-Palme, Johan; Hartmann, Martin; Eriksson, Karl Martin; Pal, Chandan; Thorell, Kaisa; Larsson, Dan Göran Joakim; Nilsson, Rolf Henrik

    2015-11-01

    The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The METAXA software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to METAXA - METAXA2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of METAXA2 was compared to other commonly used taxonomic classifiers, showing that METAXA2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. METAXA2 is freely available from http://microbiology.se/software/metaxa2/.

  12. RRNA and dnaK relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legume trees in Costa Rica.

    PubMed

    Parker, Matthew A

    2004-05-01

    Enzyme electrophoresis and sequencing of rRNA and dnaK genes revealed high genetic diversity among root nodule bacteria from the Costa Rican trees Andira inermis, Dalbergia retusa, Platymiscium pinnatum (Papilionoideae tribe Dalbergieae) and Lonchocarpus atropurpureus (Papilionoideae tribe Millettieae). A total of 21 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 36 isolates analyzed, and no ETs were shared in common by isolates from different legume hosts. However, three of the ETs from D. retusa were identical to Bradyrhizobium sp. isolates detected in prior studies of several other legume genera in both Costa Rica and Panama. Nearly full-length 16S rRNA sequences and partial 23S rRNA sequences confirmed that two isolates from D. retusa were highly similar or identical to Bradyrhizobium strains isolated from the legumes Erythrina and Clitoria (Papilionoideae tribe Phaseoleae) in Panama. rRNA sequences for five isolates from L. atropurpureus, P. pinnatum and A. inermis were not closely related to any currently known strains from Central America or elsewhere, but had affinities to the reference strains Bradyrhizobium japonicum USDA 110 (three isolates) or to B. elkanii USDA 76 (two isolates). A phylogenetic tree for 21 Bradyrhizobium strains based on 603 bp of the dnaK gene showed several significant conflicts with the rRNA tree, suggesting that genealogical relationships may have been altered by lateral gene transfer events.

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

    PubMed Central

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

    2001-01-01

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

  14. Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

    PubMed Central

    Toth, I. K.; Avrova, A. O.; Hyman, L. J.

    2001-01-01

    Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica. PMID:11526007

  15. 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

  16. Mutations in the 50S ribosomal subunit of Brachyspira hyodysenteriae associated with altered minimum inhibitory concentrations of pleuromutilins.

    PubMed

    Hillen, Sonja; Willems, Hermann; Herbst, Werner; Rohde, Judith; Reiner, Gerald

    2014-08-06

    Brachyspira hyodysenteriae, the causative agent of swine dysentery, is responsible for severe mucohaemorrhagic colitis with considerable financial loss to worldwide swine production. Antimicrobial resistance against macrolides and lincosamides is widespread and the mechanisms are well known. Currently, the most common treatment for swine dysentery is the use of pleuromutilins and resistance to these drugs also is increasingly being reported. Although resistance mechanisms against pleuromutilins are less clear than for other drugs, they seem to involve alterations of the peptidyl transferase centre (PTC), including ribosomal RNA and the ribosomal protein L3. The present study was conducted to examine molecular mechanisms of resistance on a representative set of B. hyodysenteriae field strains with different resistance patterns. In total, we identified 24 single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and genes of the ribosomal proteins L3, L4, L2 and L22. The SNP in the ribosomal protein gene L3 at position 443 led to an amino acid substitution of asparagine (Asn) by serine (Ser) at position 148, significantly associated with MICs for pleuromutilins. Based on this SNP a correct assignment of 71% of the strains with respect to a threshold of >0.625 μg tiamulin/ml was reached. Unexpectedly low MICs in some of the Asn-strains were explained by a second SNP at position 2535 of the 23S rRNA. Our results clearly show the associations between MICs for pleuromutilins and mutations in their binding site. A complete list of SNPs that influence MICs of B. hyodysenteriae strains is needed to enable the interpretation of future molecular susceptibility testing. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Measurements of helium 23S metastable atom density in low-pressure glow discharge plasmas by self-absorption spectroscopy of HeI 23S-23P transition

    NASA Astrophysics Data System (ADS)

    Shikama, Taiichi; Ogane, Shuhei; Ishii, Hidekazu; Iida, Yohei; Hasuo, Masahiro

    2014-08-01

    The helium 23S metastable atom densities are experimentally evaluated by self-absorption spectroscopy of the HeI 23S-23P transition spectra in two kinds of cylindrical glow discharge plasmas, which have different radii and are operated under different pressures of 300 and 20 Pa. The spectra are measured by using an interference spectroscopy system with a wavelength resolution of about 60 pm, and the relative intensities of the fine structure transitions are analyzed. It is found that the method is in principle applicable to plasmas with the pressure up to about the atmospheric pressure and electron density on the order of up to 1022 m-3. For a plasma with an absorption length of 10 mm and a spatially uniform temperature of 300 K, the method is sensitive to the metastable atom density roughly from 1016 to 1019 m-3.

  18. A Single Acetylation of 18 S rRNA Is Essential for Biogenesis of the Small Ribosomal Subunit in Saccharomyces cerevisiae*

    PubMed Central

    Ito, Satoshi; Akamatsu, Yu; Noma, Akiko; Kimura, Satoshi; Miyauchi, Kenjyo; Ikeuchi, Yoshiho; Suzuki, Takeo; Suzuki, Tsutomu

    2014-01-01

    Biogenesis of eukaryotic ribosome is a complex event involving a number of non-ribosomal factors. During assembly of the ribosome, rRNAs are post-transcriptionally modified by 2′-O-methylation, pseudouridylation, and several base-specific modifications, which are collectively involved in fine-tuning translational fidelity and/or modulating ribosome assembly. By mass-spectrometric analysis, we demonstrated that N4-acetylcytidine (ac4C) is present at position 1773 in the 18 S rRNA of Saccharomyces cerevisiae. In addition, we found an essential gene, KRE33 (human homolog, NAT10), that we renamed RRA1 (ribosomal RNA cytidine acetyltransferase 1) encoding an RNA acetyltransferase responsible for ac4C1773 formation. Using recombinant Rra1p, we could successfully reconstitute ac4C1773 in a model rRNA fragment in the presence of both acetyl-CoA and ATP as substrates. Upon depletion of Rra1p, the 23 S precursor of 18 S rRNA was accumulated significantly, which resulted in complete loss of 18 S rRNA and small ribosomal subunit (40 S), suggesting that ac4C1773 formation catalyzed by Rra1p plays a critical role in processing of the 23 S precursor to yield 18 S rRNA. When nuclear acetyl-CoA was depleted by inactivation of acetyl-CoA synthetase 2 (ACS2), we observed temporal accumulation of the 23 S precursor, indicating that Rra1p modulates biogenesis of 40 S subunit by sensing nuclear acetyl-CoA concentration. PMID:25086048

  19. Phylogenetic diversity based on rrs, atpD, recA genes and 16S-23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru.

    PubMed

    Santillana, Nery; Ramírez-Bahena, Martha Helena; García-Fraile, Paula; Velázquez, Encarna; Zúñiga, Doris

    2008-03-01

    In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132(T) and Rhizobium etli CFN42(T) (=USDA 9032(T)), respectively. The analysis of the 16S-23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132(T) and CFN42(T). These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.

  20. 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

  1. 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

  2. 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.

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

    PubMed Central

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

    1998-01-01

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

  4. Recombineering with tolC as a Selectable/Counter-selectable Marker: remodeling the rRNA Operons of Escherichia coli

    PubMed Central

    DeVito, Joseph A.

    2008-01-01

    This work describes the novel use of tolC as a selectable/counter-selectable marker for the facile modification of DNA in Escherichia coli. Expression of TolC (an outer membrane protein) confers relative resistance to toxic small molecules, while its absence renders the cell tolerant to colicin E1. These features, coupled with the λredgam recombination system, allow for selection of tolC insertions/deletions anywhere on the E. coli chromosome or on plasmid DNA. This methodology obviates the need for minimal growth media, specialized wash protocols and the lengthy incubation times required by other published recombineering methods. As a rigorous test of the TolC selection system, six out of seven 23S rRNA genes were consecutively and seamlessly removed from the E. coli chromosome without affecting expression of neighboring genes within the complex rrn operons. The resulting plasmid-free strain retains one 23S rRNA gene (rrlC) in its natural location on the chromosome and is the first mutant of its kind. These new rRNA mutants will be useful in the study of rRNA gene regulation and ribosome function. Given its high efficiency, low background and facility in rich media, tolC selection is a broadly applicable method for the modification of DNA by recombineering. PMID:18084036

  5. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA.

    PubMed

    Moazed, D; Noller, H F

    1990-01-05

    Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.

  6. Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

    PubMed

    Henn, Arnon; Cao, Wenxiang; Licciardello, Nicholas; Heitkamp, Sara E; Hackney, David D; De La Cruz, Enrique M

    2010-03-02

    DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

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

    PubMed

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

    1999-04-01

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

  8. Eukaryotic 5S rRNA biogenesis.

    PubMed

    Ciganda, Martin; Williams, Noreen

    2011-01-01

    The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. Copyright © 2011 John Wiley & Sons, Ltd.

  9. Eukaryotic 5S rRNA biogenesis

    PubMed Central

    Ciganda, Martin; Williams, Noreen

    2012-01-01

    The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. PMID:21957041

  10. Calculation and Comparative Analysis of the IR Spectra of Homobrassinolide and (22S,23S)-Homobrassinolide

    NASA Astrophysics Data System (ADS)

    Andrianov, V. M.; Korolevich, M. V.

    2015-09-01

    Normal vibrational frequencies and absolute IR band intensities of the biologically active steroid phytohormones homobrassinolide and (22S,23S)-homobrassinolide were calculated in the framework of an original approach that combined classical analysis of normal modes using molecular mechanics with quantum-chemical estimation of the absolute intensities. IR absorption bands were interpreted based on a comparison of the experimental and theoretical absorption spectra. The impact of structural differences in the side chains of these molecules on the formation of their IR spectra in the region 1500-950 cm -1 was estimated.

  11. Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions.

    PubMed

    López, Victoria; Gil, Rosario; Vicente Carbonell, José; Navarro, Alfonso

    2002-04-01

    We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication. Copyright 2002 John Wiley & Sons, Ltd.

  12. Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae.

    PubMed

    Yang, Jun; Sharma, Sunny; Kötter, Peter; Entian, Karl-Dieter

    2015-02-27

    Methylation of ribose sugars at the 2'-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2'-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5' central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D' box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae

    PubMed Central

    Yang, Jun; Sharma, Sunny; Kötter, Peter; Entian, Karl-Dieter

    2015-01-01

    Methylation of ribose sugars at the 2′-OH group is one of the major chemical modifications in rRNA, and is catalyzed by snoRNA directed C/D box snoRNPs. Previous biochemical and computational analyses of the C/D box snoRNAs have identified and mapped a large number of 2′-OH ribose methylations in rRNAs. In the present study, we systematically analyzed ribose methylations of 18S rRNA in Saccharomyces cerevisiae, using mung bean nuclease protection assay and RP-HPLC. Unexpectedly, we identified a hitherto unknown ribose methylation at position G562 in the helix 18 of 5′ central domain of yeast 18S rRNA. Furthermore, we identified snR40 as being responsible to guide snoRNP complex to catalyze G562 ribose methylation, which makes it only second snoRNA known so far to target three ribose methylation sites: Gm562, Gm1271 in 18S rRNA, and Um898 in 25S rRNA. Our sequence and mutational analysis of snR40 revealed that snR40 uses the same D′ box and methylation guide sequence for both Gm562 and Gm1271 methylation. With the identification of Gm562 and its corresponding snoRNA, complete set of ribose methylations of 18S rRNA and their corresponding snoRNAs have finally been established opening great prospects to understand the physiological function of these modifications. PMID:25653162

  14. 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.

  15. Molecular dynamics simulations of sarcin–ricin rRNA motif

    PubMed Central

    Špačková, Nad'a; Šponer, Jiří

    2006-01-01

    Explicit solvent molecular dynamics (MD) simulations were carried out for sarcin–ricin domain (SRD) motifs from 23S (Escherichia coli) and 28S (rat) rRNAs. The SRD motif consists of GAGA tetraloop, G-bulged cross-strand A-stack, flexible region and duplex part. Detailed analysis of the overall dynamics, base pairing, hydration, cation binding and other SRD features is presented. The SRD is surprisingly static in multiple 25 ns long simulations and lacks any non-local motions, with root mean square deviation (r.m.s.d.) values between averaged MD and high-resolution X-ray structures of 1–1.4 Å. Modest dynamics is observed in the tetraloop, namely, rotation of adenine in its apex and subtle reversible shift of the tetraloop with respect to the adjacent base pair. The deformed flexible region in low-resolution rat X-ray structure is repaired by simulations. The simulations reveal few backbone flips, which do not affect positions of bases and do not indicate a force field imbalance. Non-Watson–Crick base pairs are rigid and mediated by long-residency water molecules while there are several modest cation-binding sites around SRD. In summary, SRD is an unusually stiff rRNA building block. Its intrinsic structural and dynamical signatures seen in simulations are strikingly distinct from other rRNA motifs such as Loop E and Kink-turns. PMID:16456030

  16. RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

    PubMed Central

    Tang, Thean Hock; Rozhdestvensky, Timofey S.; d’Orval, Béatrice Clouet; Bortolin, Marie-Line; Huber, Harald; Charpentier, Bruno; Branlant, Christiane; Bachellerie, Jean-Pierre; Brosius, Jürgen; Hüttenhofer, Alexander

    2002-01-01

    The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA. PMID:11842103

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

    PubMed Central

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

    2013-01-01

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

  18. Mycoplasma genitalium infection: current treatment options, therapeutic failure, and resistance-associated mutations

    PubMed Central

    Couldwell, Deborah L; Lewis, David A

    2015-01-01

    Mycoplasma genitalium is an important cause of non-gonococcal urethritis, cervicitis, and related upper genital tract infections. The efficacy of doxycycline, used extensively to treat non-gonococcal urethritis in the past, is relatively poor for M. genitalium infection; azithromycin has been the preferred treatment for several years. Research on the efficacy of azithromycin has primarily focused on the 1 g single-dose regimen, but some studies have also evaluated higher doses and longer courses, particularly the extended 1.5 g regimen. This extended regimen is thought to be more efficacious than the 1 g single-dose regimen, although the regimens have not been directly compared in clinical trials. Azithromycin treatment failure was first reported in Australia and has subsequently been documented in several continents. Recent reports indicate an upward trend in the prevalence of macrolide-resistant M. genitalium infections (transmitted resistance), and cases of induced resistance following azithromycin therapy have also been documented. Emergence of antimicrobial-resistant M. genitalium, driven by suboptimal macrolide dosage, now threatens the continued provision of effective and convenient treatments. Advances in techniques to detect resistance mutations in DNA extracts have facilitated correlation of clinical outcomes with genotypic resistance. A strong and consistent association exists between presence of 23S rRNA gene mutations and azithromycin treatment failure. Fluoroquinolones such as moxifloxacin, gatifloxacin, and sitafloxacin remain highly active against most macrolide-resistant M. genitalium. However, the first clinical cases of moxifloxacin treatment failure, due to bacteria with coexistent macrolide-associated and fluoroquinolone-associated resistance mutations, were recently published by Australian investigators. Pristinamycin and solithromycin may be of clinical benefit for such multidrug-resistant infections. Further clinical studies are required to

  19. Increased 5S rRNA oxidation in Alzheimer's disease.

    PubMed

    Ding, Qunxing; Zhu, Haiyan; Zhang, Bing; Soriano, Augusto; Burns, Roxanne; Markesbery, William R

    2012-01-01

    It is widely accepted that oxidative stress is involved in neurodegenerative disorders such as Alzheimer's disease (AD). Ribosomal RNA (rRNA) is one of the most abundant molecules in most cells and is affected by oxidative stress in the human brain. Previous data have indicated that total rRNA levels were decreased in the brains of subjects with AD and mild cognitive impairment concomitant with an increase in rRNA oxidation. In addition, level of 5S rRNA, one of the essential components of the ribosome complex, was significantly lower in the inferior parietal lobule (IP) brain area of subjects with AD compared with control subjects. To further evaluate the alteration of 5S rRNA in neurodegenerative human brains, multiple brain regions from both AD and age-matched control subjects were used in this study, including IP, superior and middle temporal gyro, temporal pole, and cerebellum. Different molecular pools including 5S rRNA integrated into ribosome complexes, free 5S rRNA, cytoplasmic 5S rRNA, and nuclear 5S rRNA were studied. Free 5S rRNA levels were significantly decreased in the temporal pole region of AD subjects and the oxidation of ribosome-integrated and free 5S rRNA was significantly increased in multiple brain regions in AD subjects compared with controls. Moreover, a greater amount of oxidized 5S rRNA was detected in the cytoplasm and nucleus of AD subjects compared with controls. These results suggest that the increased oxidation of 5S rRNA, especially the oxidation of free 5S rRNA, may be involved in the neurodegeneration observed in AD.

  20. The role of 23S ribosomal RNA residue A2451 in peptide bond synthesis revealed by atomic mutagenesis.

    PubMed

    Lang, Kathrin; Erlacher, Matthias; Wilson, Daniel N; Micura, Ronald; Polacek, Norbert

    2008-05-01

    Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 2'-hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical characteristics by replacing its 2'-hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-2'-hydroxyl directly hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C2'-endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 3'- to 2'-oxygen unfeasible, thus promoting effective peptide bond synthesis.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2014-12-01

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

  3. Unusual features of the sequences of copies of the 16S-23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events.

    PubMed

    Maslunka, Christopher; Gürtler, Volker; Seviour, Robert

    2015-02-01

    The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

  4. Insertions or Deletions (Indels) in the rrn 16S-23S rRNA Gene Internal Transcribed Spacer Region (ITS) Compromise the Typing and Identification of Strains within the Acinetobacter calcoaceticus-baumannii (Acb) Complex and Closely Related Members

    PubMed Central

    Maslunka, Christopher; Gifford, Bianca; Tucci, Joseph; Gürtler, Volker; Seviour, Robert J.

    2014-01-01

    To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2–13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species. PMID:25141005

  5. An RNA molecular switch: Intrinsic flexibility of 23S rRNA Helices 40 and 68 5’-UAA/5’-GAN internal loops studied by molecular dynamics methods

    PubMed Central

    Réblová, Kamila; Střelcová, Zora; Kulhánek, Petr; Beššeová, Ivana; Mathews, David H.; Nostrand, Keith Van; Yildirim, Ilyas; Turner, Douglas H.; Šponer, Jiří

    2010-01-01

    Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5’-UAA/5’-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na+ vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational transition towards the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water mediated and alternates with the canonical A–U base pair, which is not indicated by the NMR data. Completion of full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts. PMID:21132104

  6. 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

  7. The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

    PubMed Central

    Long, Katherine S.; Poehlsgaard, Jacob; Kehrenberg, Corinna; Schwarz, Stefan; Vester, Birte

    2006-01-01

    A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones. PMID:16801432

  8. Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Clustered Mutations in qPCR

    PubMed Central

    Tan, Lit Yeen; Walker, Samantha Michelle; Lonergan, Tina; Lima, Nicole Elizabeth; Todd, Alison Velyian

    2017-01-01

    Background Whilst qPCR provides an extremely powerful tool for genetic analysis, some applications such as multiplexing variant alleles (eg SNPs, point mutations or deletions), remain challenging using current primer/probe systems. The novel design features of PlexPrimers allow sensitive, multiplexed analysis of variant alleles even when these are tightly clustered. Method PlexPrimers were combined with PlexZymes in qPCR assays for the detection of SNPs in human absorption, distribution, metabolism, and excretion (ADME) genes; clustered mutations in the 23S rRNA gene which confer antibiotic resistance to Mycoplasma genitalium; and deletions within the human epidermal growth factor receptor (EGFR) gene. Results The combination of PlexPrimers and PlexZymes allowed robust multiplexing of targets which resulted in 100% concordance with results obtained using hydrolysis probe kits for 14 SNPs in the ADME genes. A 7-plex qPCR assay targeting M. genitalium, 5 clustered mutations associated with macrolide resistance and an internal control, allowed efficient amplification of all targets, with all 5 mutations detected in a single channel. Finally, the strategy was employed to analyse common EGFR mutants with high sensitivity, detecting deletions present at only 0.01%. Conclusion PlexPrime is a novel technology for the detection of genetic variants. Unlike previous strategies, the combination of PlexPrimers with PlexZymes enables both allele-specific detection and allele-specific amplification in qPCR. The study demonstrated highly sensitive and specific detection of mutations and SNPs, and superior multiplexing capacity. The ability to multiplex clustered genetic variants reduces the time to result providing more actionable information. PMID:28114309

  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. Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.

    PubMed

    Nummi, Maaret; Mannonen, Laura; Puolakkainen, Mirja

    2015-01-01

    The aim of this study was to improve detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in clinical specimens by developing a multiplex real-time PCR assay that includes identification of macrolide-resistant M. pneumoniae. Novel assays targeting a M. pneumoniae conserved hypothetical protein gene, M. pneumoniae 23S rRNA gene mutations associated with macrolide resistance and human β-globin gene (an endogenous internal control) were designed and combined with a previously published C. pneumoniae PCR targeting ompA gene. The resulting quadraplex PCR was validated with a panel of clinical specimens supplemented with external quality assessment specimens, simulated specimens and various bacterial and viral strains. The obtained results were compared to those obtained by reference PCRs or confirmed by sequencing (typing of macrolide resistance). The novel multiplex PCR assay was in 100 % agreement with reference PCRs. Four M. pneumoniae strains with macrolide resistance-associated mutations were identified among 42 strains, which comprises 9.5 % of the study material. Amplification of an internal control excluded sample-derived inhibition possibly leading to false-negative reporting. In conclusion, we have developed a resources conserving multiplex real-time PCR assay for simultaneous detection of M. pneumoniae, C. pneumoniae and the most common mutations leading to macrolide resistance in M. pneumoniae. The assay is a widely useful tool for detection of these respiratory pathogens and will also shed light on the occurrence of macrolide resistance in M. pneumoniae.

  11. Characterisation of clinical meticillin-resistant Staphylococcus epidermidis demonstrating high levels of linezolid resistance (>256 μg/ml) resulting from transmissible and mutational mechanisms.

    PubMed

    Gabriel, Emma M; Fitzgibbon, Siobhan; Clair, James; Coffey, Aidan; O'Mahony, Jim M

    2015-07-01

    Staphylococcus epidermidis (S. epidermidis), one of the leading etiological agents of nosocomial infections poses a significant economic burden globally. Introduced in 2000, linezolid (LZD) has become an important antibiotic, used in nearly seventy countries worldwide to treat infections caused by Gram-positive pathogens such as meticillin-resistant Staphylococcus and Streptococcus species along with vancomycin-resistant enterococci. Resistance to LZD in clinical settings remains rare. Here, we report the emergence of meticillin resistant S. epidermidis (MRSE) clinical isolates from two voluntary general acute hospitals exhibiting higher than typically reported levels of LZD resistance (MIC>256 μg/ml). The MRSE ST-2 clone isolated from eight patients (2010-2011) not only possessed resistance-conferring mutations such as G2576T in domain V of 23S rRNA gene (as determined by HRM-PCR analysis) and R172C substitution in the ribosomal protein L3, but also carried the cfr gene (the only known transmissible mechanism of LZD resistance). All isolates possessed several key biofilm-associated genes (such as icaA, icaD, aap and atlE) and resistance to multiple clinically significant antibiotics was recorded. This study reports the earliest incidence (2010) of clinical MRSE in the Republic of Ireland demonstrating multiple LZD resistance mechanisms both mutational and potentially transmissible, and characterises this emerging resistance from a molecular perspective.

  12. A new layer of rRNA regulation by small interference RNAs and the nuclear RNAi pathway.

    PubMed

    Zhou, Xufei; Chen, Xiangyang; Wang, Yun; Feng, Xuezhu; Guang, Shouhong

    2017-06-22

    Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process remain poorly understood. For a long time, small rRNA sequences have been widely treated as non-specific degradation products and neglected as garbage sequences. Recently, we identified a new class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNA through the nuclear RNAi pathway in C. elegans. risiRNAs exhibit sequence characteristics similar to 22G RNA while complement to 18S and 26S rRNA. risiRNAs elicit the translocation of the nuclear Argonaute protein NRDE-3 from the cytoplasm to nucleus and nucleolus, in which the risiRNA/NRDE complex binds to pre-rRNA and silences rRNA expression. Interestingly, when C. elegans is exposed to environmental stimuli, such as cold shock and ultraviolet illumination, risiRNAs accumulate and further turn on the nuclear RNAi-mediated gene silencing pathway. risiRNA may act in a quality control mechanism of rRNA homeostasis. When the exoribonuclease SUSI-1(ceDis3L2) is mutated, risiRNAs are dramatically increased. In this Point of View article, we will summarize our understanding of the small antisense ribosomal siRNAs in a variety of organisms, especially C. elegans, and their possible roles in the quality control mechanism of rRNA homeostasis.

  13. 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

  14. 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

  15. Delayed rRNA Processing Results in Significant Ribosome Biogenesis and Functional Defects

    PubMed Central

    Meskauskas, Arturas; Baxter, Jennifer L.; Carr, Edward A.; Yasenchak, Jason; Gallagher, Jennifer E. G.; Baserga, Susan J.; Dinman, Jonathan D.

    2003-01-01

    mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed −1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at −1 frameshift signals, promoting increased programmed −1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis. PMID:12588980

  16. rRNA transcription rate in Escherichia coli.

    PubMed Central

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

    1991-01-01

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

  17. 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.

  18. Resurrection of an ancestral 5S rRNA.

    PubMed

    Lu, Qing; Fox, George E

    2011-07-22

    In addition to providing phylogenetic relationships, tree making procedures such as parsimony and maximum likelihood can make specific predictions of actual historical sequences. Resurrection of such sequences can be used to understand early events in evolution. In the case of RNA, the nature of parsimony is such that when applied to multiple RNA sequences it typically predicts ancestral sequences that satisfy the base pairing constraints associated with secondary structure. The case for such sequences being actual ancestors is greatly improved, if they can be shown to be biologically functional. A unique common ancestral sequence of 28 Vibrio 5S ribosomal RNA sequences predicted by parsimony was resurrected and found to be functional in the context of the E. coli cellular environment. The functionality of various point variants and intermediates that were constructed as part of the resurrection were examined in detail. When separately introduced the changes at single stranded positions and individual double variants at base-paired positions were also viable. An additional double variant was examined at a different base-paired position and it was also valid. The results show that at least in the case of the 5S rRNAs considered here, ancestors predicted by parsimony are likely to be realistic when the prediction is not overly influenced by single outliers. It is especially noteworthy that the phenotype of the predicted ancestors could be anticipated as a cumulative consequence of the phenotypes of the individual variants that comprised them. Thus, point mutation data is potentially useful in evaluating the reasonableness of ancestral sequences predicted by parsimony or other methods. The results also suggest that in the absence of significant tertiary structure constraints double variants that preserve pairing in stem regions will typically be accepted. Overall, the results suggest that it will be feasible to resurrect additional meaningful 5S rRNA ancestors as well

  19. Downregulation of rRNA transcription triggers cell differentiation.

    PubMed

    Hayashi, Yuki; Kuroda, Takao; Kishimoto, Hiroyuki; Wang, Changshan; Iwama, Atsushi; Kimura, Keiji

    2014-01-01

    Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

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

    PubMed Central

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

    2000-01-01

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

  1. Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA.

    PubMed

    Hatamoto, Masashi; Nakai, Kazufumi; Ohashi, Akiyoshi; Imachi, Hiroyuki

    2009-10-01

    Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.

  2. Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis.

    PubMed

    Drygin, Denis; Siddiqui-Jain, Adam; O'Brien, Sean; Schwaebe, Michael; Lin, Amy; Bliesath, Josh; Ho, Caroline B; Proffitt, Chris; Trent, Katy; Whitten, Jeffrey P; Lim, John K C; Von Hoff, Daniel; Anderes, Kenna; Rice, William G

    2009-10-01

    Hallmark deregulated signaling in cancer cells drives excessive ribosome biogenesis within the nucleolus, which elicits unbridled cell growth and proliferation. The rate-limiting step of ribosome biogenesis is synthesis of rRNA (building blocks of ribosomes) by RNA Polymerase I (Pol I). Numerous kinase pathways and products of proto-oncogenes can up-regulate Pol I, whereas tumor suppressor proteins can inhibit rRNA synthesis. In tumorigenesis, activating mutations in certain cancer-associated kinases and loss-of-function mutations in tumor suppressors lead to deregulated signaling that stimulates Pol I transcription with resultant increases in ribosome biogenesis, protein synthesis, cell growth, and proliferation. Certain anticancer therapeutics, such as cisplatin and 5-fluorouracil, reportedly exert, at least partially, their activity through disruption of ribosome biogenesis, yet many prime targets for anticancer drugs within the ribosome synthetic machinery of the nucleolus remain largely unexploited. Herein, we describe CX-3543, a small molecule nucleolus-targeting agent that selectively disrupts nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting Pol I transcription and inducing apoptosis in cancer cells. CX-3543 is the first G-quadruplex interactive agent to enter human clinical trials, and it is currently under evaluation against carcinoid/neuroendocrine tumors in a phase II clinical trial.

  3. Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants.

    PubMed Central

    Gaal, T; Barkei, J; Dickson, R R; deBoer, H A; deHaseth, P L; Alavi, H; Gourse, R L

    1989-01-01

    Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription. PMID:2527844

  4. Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

    PubMed Central

    Matsuda, Daiki; Mauro, Vincent P.

    2014-01-01

    Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example—translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson–Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA–rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine–Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction. PMID:25313046

  5. Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo.

    PubMed

    Matsuda, Daiki; Mauro, Vincent P

    2014-10-28

    Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example--translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine-Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.

  6. Bacterial 16S rRNA gene analysis revealed that bacteria related to Arcobacter spp. constitute an abundant and common component of the oyster microbiota (Tiostrea chilensis).

    PubMed

    Romero, J; García-Varela, M; Laclette, J P; Espejo, R T

    2002-11-01

    To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.

  7. Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli.

    PubMed

    Johnson, Y A; Nagpal, M; Krahmer, M T; Fox, K F; Fox, A

    2000-05-01

    Assessment of 16S-23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5' terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S-23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.

  8. Bases in 16S rRNA Important for Subunit Association, tRNA binding, and Translocation

    PubMed Central

    Shi, Xinying; Chiu, Katie; Ghosh, Srikanta; Joseph, Simpson

    2009-01-01

    Ribosomes are the cellular machinery responsible for protein synthesis. A well-orchestrated step in the elongation cycle of protein synthesis is the precise translocation of the tRNA-mRNA complex within the ribosome. Here we report the application of a new in vitro modification-interference method for the identification of bases in 16S rRNA that are essential for translocation. Our results suggest that conserved bases U56, U723, A1306, A1319, and A1468 in 16S rRNA are important for translocation. These five bases were deleted or mutated in order to study their role in translation. Depending on the type of mutation, we observed inhibition of growth rate, subunit association, tRNA binding and/or translocation. Interestingly, deletion of U56 or A1319 or mutation of A1319 to C showed a lethal phenotype and were defective in protein synthesis in vitro. Further analysis showed that deletion of U56 or A1319 caused defects in 30S subunit assembly, subunit association and tRNA binding. In contrast, A1319C mutation showed no defects in subunit association; however, the extent of tRNA binding and translocation was significantly reduced. These results show that conserved bases located as far away as 100 Å from the tRNA binding sites can be important for translation. PMID:19545171

  9. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria

    PubMed Central

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J.; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC. PMID:27749897

  10. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

    PubMed

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

  11. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides

    PubMed Central

    Almutairi, Mashal M.; Park, Sung Ryeol; Rose, Simon; Hansen, Douglas A.; Vázquez-Laslop, Nora; Douthwaite, Stephen; Sherman, David H.; Mankin, Alexander S.

    2015-01-01

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism. PMID:26438831

  12. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides.

    PubMed

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon; Hansen, Douglas A; Vázquez-Laslop, Nora; Douthwaite, Stephen; Sherman, David H; Mankin, Alexander S

    2015-10-20

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

  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. Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p.

    PubMed

    Buchhaupt, Markus; Meyer, Britta; Kötter, Peter; Entian, Karl-Dieter

    2006-09-01

    The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Deltasnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Deltanep1 growth defect. SnR57 mediates 2'-O-ribose-methylation of G(1570) in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553-1577 of the 18S rRNA, which includes G(1570), the site of snR57-dependent 18S rRNA methylation. From protein-protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.

  15. 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.

  16. Characterization of Mycobacterium leprae Genotypes in China—Identification of a New Polymorphism C251T in the 16S rRNA Gene

    PubMed Central

    You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China. PMID:26196543

  17. Concurrent titration and determination of antibiotic resistance in ureaplasma species with identification of novel point mutations in genes associated with resistance.

    PubMed

    Beeton, Michael L; Chalker, Victoria J; Maxwell, Nicola C; Kotecha, Sailesh; Spiller, O Brad

    2009-05-01

    Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.

  18. Collision-energy-resolved penning ionization electron spectroscopy of HCOOH, CH3COOH, and HCOOCH3 by collision with He*(2(3)S) metastable atoms.

    PubMed

    Borodin, Andriy; Yamazaki, Masakazu; Kishimoto, Naoki; Ohno, Koichi

    2005-06-02

    Penning ionization of formic acid (HCOOH), acetic acid (CH3COOH), and methyl formate (HCOOCH3) upon collision with metastable He*(2(3)S) atoms was studied by collision-energy/electron-energy-resolved two-dimensional Penning ionization electron spectroscopy (2D-PIES). Anisotropy of interaction between the target molecule and He*(2(3)S) was investigated based on the collision energy dependence of partial ionization cross sections (CEDPICS) obtained from 2D-PIES as well as ab initio molecular orbital calculations for the access of a metastable atom to the target molecule. For the interaction potential calculations, a Li atom was used in place of He*(2(3)S) metastable atom because of its well-known similarity in interaction with targets. The results indicate that in the studied collision energy range the attractive potential localizes around the oxygen atoms and that the potential well at the carbonyl oxygen atom is at least twice as much as that at the hydroxyl oxygen. Moreover we can notice that attractive potential is highly anisotropic. Repulsive interactions can be found around carbon atoms and the methyl group.

  19. Shewanella sp. O23S as a Driving Agent of a System Utilizing Dissimilatory Arsenate-Reducing Bacteria Responsible for Self-Cleaning of Water Contaminated with Arsenic

    PubMed Central

    Drewniak, Lukasz; Stasiuk, Robert; Uhrynowski, Witold; Sklodowska, Aleksandra

    2015-01-01

    The purpose of this study was a detailed characterization of Shewanella sp. O23S, a strain involved in arsenic transformation in ancient gold mine waters contaminated with arsenic and other heavy metals. Physiological analysis of Shewanella sp. O23S showed that it is a facultative anaerobe, capable of growth using arsenate, thiosulfate, nitrate, iron or manganite as a terminal electron acceptor, and lactate or citrate as an electron donor. The strain can grow under anaerobic conditions and utilize arsenate in the respiratory process in a broad range of temperatures (10–37 °C), pH (4–8), salinity (0%–2%), and the presence of heavy metals (Cd, Co, Cr, Cu, Mn, Mo, Se, V and Zn). Under reductive conditions this strain can simultaneously use arsenate and thiosulfate as electron acceptors and produce yellow arsenic (III) sulfide (As2S3) precipitate. Simulation of As-removal from water containing arsenate (2.5 mM) and thiosulfate (5 mM) showed 82.5% efficiency after 21 days of incubation at room temperature. Based on the obtained results, we have proposed a model of a microbially mediated system for self-cleaning of mine waters contaminated with arsenic, in which Shewanella sp. O23S is the main driving agent. PMID:26121297

  20. 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

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

    PubMed Central

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

    2016-01-01

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

  2. A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

    PubMed Central

    Baßler, Jochen; Paternoga, Helge; Holdermann, Iris; Thoms, Matthias; Granneman, Sander; Barrio-Garcia, Clara; Nyarko, Afua; Stier, Gunter; Clark, Sarah A.; Schraivogel, Daniel; Kallas, Martina; Beckmann, Roland; Tollervey, David

    2014-01-01

    Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation. PMID:25404745

  3. Functional Specialization of Domains Tandemly Duplicated Witin 16S rRNA Methyltransferase RsmC

    SciTech Connect

    Sunita,S.; Purta, E.; Durawa, M.; Tkaczuk, K.; Swaathi, J.; Bujnicki, J.; Sivaraman, J.

    2007-01-01

    RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Angstroms resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

  4. [Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates].

    PubMed

    Nagano, Makoto; Ichimura, Sadahiro; Ito, Nobuko; Tomii, Takayuki; Kazumi, Yuko; Takei, Katsuaki; Abe, Chiyoji; Sugawara, Isamu

    2008-07-01

    The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.

  5. An archaeal RNA binding protein, FAU-1, is a novel ribonuclease related to rRNA stability in Pyrococcus and Thermococcus.

    PubMed

    Ikeda, Yoshiki; Okada, Yasuhiro; Sato, Asako; Kanai, Tamotsu; Tomita, Masaru; Atomi, Haruyuki; Kanai, Akio

    2017-10-04

    Ribosome biogenesis and turnover are processes necessary for cell viability and proliferation, and many kinds of proteins are known to regulate these processes. However, many questions still remain, especially in the Archaea. Generally, several ribonucleases are required to process precursor rRNAs to their mature forms, and to degrade rRNAs for quality control. Here, we found that FAU-1, which is known to be an RNA binding protein, possesses an RNase activity against precursor 5S rRNA derived from P. furiosus and T. kodakarensis in the order Thermococcales in vitro. An in vitro analysis revealed that UA sequences in the upstream of 5S rRNA were preferentially degraded by addition of FAU-1. Moreover, a fau-1 gene deletion mutant of T. kodakarensis showed a delay of exponential phase, reduction of maximum cell number and significant changes in the nucleotide sequence lengths of its 5S, 16S, and 23S rRNAs in early exponential phase. Our results suggest that FAU-1 is a potential RNase involved in rRNA stability through maturation and/or degradation processes.

  6. Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase.

    PubMed

    Freihofer, Pietro; Akbergenov, Rashid; Teo, Youjin; Juskeviciene, Reda; Andersson, Dan I; Böttger, Erik C

    2016-12-01

    Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TlyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TlyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.

  7. Imprint of Ancient Evolution on rRNA Folding.

    PubMed

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

    2016-08-23

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

  8. Control of rRNA transcription in Escherichia coli.

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Impact of azithromycin resistance mutations on the virulence and fitness of Chlamydia caviae in guinea pigs.

    PubMed

    Binet, Rachel; Bowlin, Anne K; Maurelli, Anthony T; Rank, Roger G

    2010-03-01

    Azithromycin (AZM) is a major drug used in the treatment and prophylaxis of infections caused by Chlamydia, yet no significant clinical resistance has been reported for these obligate intracellular bacteria. Nevertheless, spontaneous AZM resistance (Azm(r)) arose in vitro at frequencies ranging from 3 x 10(-8) to 8 x 10(-10) for clonal isolates of Chlamydia caviae, which is a natural pathogen of guinea pigs. Sequencing of the unique 23S rRNA gene copy in 44 independent Azm(r) isolates identified single mutations at position A(2058) or A(2059) (Escherichia coli numbering system). While SP(6)AZ(1) (A(2058)C) and SP(6)AZ(2) (A(2059)C) Azm(r) mutants showed growth defects in cell culture and were less pathogenic in the guinea pig ocular infection model than in the parent SP(6), the three isogenic C. caviae isolates grew equally well in the animal. On the other hand, coinoculation of the C. caviae parent strain with one of the Azm(r) strains was detrimental for the mutant strain. This apparent lack of association between pathology and bacterial load in vivo showed that virulence of the two Azm(r) mutants of C. caviae was attenuated. While chlamydial growth in vitro reflects the ability of the bacteria to multiply in permissive cells, survival in the host is a balance between cellular multiplication and clearance by the host immune system. The obligate intracellular nature of Chlamydia may therefore limit emergence of resistance in vivo due to the strength of the immune response induced by the wild-type antibiotic-sensitive bacteria at the time of antibiotic treatment.

  11. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    PubMed

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  12. hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0.

    PubMed

    Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

    2015-01-01

    Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5'-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site.

  13. hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0

    PubMed Central

    Tomecki, Rafal; Labno, Anna; Drazkowska, Karolina; Cysewski, Dominik; Dziembowski, Andrzej

    2015-01-01

    Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5′-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site. PMID:26237581

  14. Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

    PubMed Central

    Challand, Martin R.; Salvadori, Enrico; Driesener, Rebecca C.; Kay, Christopher W. M.; Roach, Peter L.; Spencer, James

    2013-01-01

    The ‘radical S-adenosyl-L-methionine (AdoMet)’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage. PMID:23861844

  15. 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.

  16. Detection of two Bartonella tamiae-like sequences in Amblyomma americanum (Acari: Ixodidae) using 16S-23S intergenic spacer region-specific primers.

    PubMed

    Billeter, Sarah A; Miller, Melissa K; Breitschwerdt, Edward B; Levy, Michael G

    2008-01-01

    Four hundred and sixty-six questing Amblyomma americanum (L.) (Acari: Ixodidae) from Carolina County, VA, and 98 questing A. americanum from Chatham County, NC, were screened by polymerase chain reaction (PCR) for the Bartonella 16S-23S intergenic spacer region. Two amplicons, approximately 270-280 bp, were detected in two ticks from Virginia. Based upon PCR and sequencing, an adult male and adult female tick harbored DNA sequences closely related to Bartonella tamiae (DQ395180). Bartonella DNA was not detected in A. americanum from North Carolina. Potential transmission of Bartonella spp. by A. americanum should be the focus of future experimental studies.

  17. Development of a PCR assay based on the 16S-23S rDNA internal transcribed spacer for identification of strictly anaerobic bacterium Zymophilus.

    PubMed

    Felsberg, Jurgen; Jelínková, Markéta; Kubizniaková, Petra; Matoulková, Dagmar

    2015-06-01

    PCR-primers were designed for identification of strictly anaerobic bacteria of the genus Zymophilus based on genus-specific sequences of the 16S-23S rDNA internal transcribed spacer region. The specificity of the primers was tested against 37 brewery-related non-target microorganisms that could potentially occur in the same brewery specimens. None DNA was amplified from any of the non-Zymophilus strains tested including genera from the same family (Pectinatus, Megasphaera, Selenomonas), showing thus 100% specificity. PCR assay developed in this study allows an extension of the spectra of detected beer spoilage microorganisms in brewery laboratories.

  18. 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.

  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. Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNA(Ala) gene segments in Campylobacter lari.

    PubMed

    Hayashi, K; Tazumi, A; Nakanishi, S; Nakajima, T; Matsubara, K; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

    2012-06-01

    Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

  1. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides

    PubMed Central

    Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S.; McBee, Megan E.; Dedon, Peter C.

    2015-01-01

    A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. PMID:25539917

  2. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

    PubMed

    Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

    2015-03-11

    A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells.

    PubMed

    Jack, Karen; Bellodi, Cristian; Landry, Dori M; Niederer, Rachel O; Meskauskas, Arturas; Musalgaonkar, Sharmishtha; Kopmar, Noam; Krasnykh, Olya; Dean, Alison M; Thompson, Sunnie R; Ruggero, Davide; Dinman, Jonathan D

    2011-11-18

    How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation.

    PubMed

    Montellese, Christian; Montel-Lehry, Nathalie; Henras, Anthony K; Kutay, Ulrike; Gleizes, Pierre-Emmanuel; O'Donohue, Marie-Françoise

    2017-04-10

    The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Poly(A)-specific ribonuclease is a nuclear ribosome biogenesis factor involved in human 18S rRNA maturation

    PubMed Central

    Montellese, Christian; Montel-Lehry, Nathalie; Henras, Anthony K.; Kutay, Ulrike

    2017-01-01

    Abstract The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN. PMID:28402503

  6. On the mechanism underlying (23S)-25-dehydro-1alpha(OH)-vitamin D3-26,23-lactone antagonism of hVDRwt gene activation and its switch to a superagonist.

    PubMed

    Mizwicki, Mathew T; Bula, Craig M; Mahinthichaichan, Paween; Henry, Helen L; Ishizuka, Seiichi; Norman, Anthony W

    2009-12-25

    (23S)-25-Dehydro-1alpha(OH)-vitamin D(3)-26,23-lactone (MK) is an antagonist of the 1alpha,25(OH)(2)-vitamin D(3) (1,25D)/human nuclear vitamin D receptor (hVDR) transcription initiation complex, where the activation helix (i.e. helix-12) is closed. To study the mode of antagonism of MK an hVDR mutant library was designed to alter the free molecular volume in the region of the hVDR ligand binding pocket occupied by the ligand side-chain atoms (i.e. proximal to helix-12). The 1,25D-hVDR structure-function studies demonstrate that 1) van der Waals contacts between helix-12 residues Leu-414 and Val-418 and 1,25D enhance the stability of the closed helix-12 conformer and 2) removal of the side-chain H-bonds to His-305(F) and/or His-397(F) have no effect on 1,25D transactivation, even though they reduce the binding affinity of 1,25D. The MK structure-function results demonstrate that the His-305, Leu-404, Leu-414, and Val-418 mutations, which increase the free volume of the hVDR ligand binding pocket, significantly enhance MK antagonist potency. Surprisingly, the H305F and H305F/H397F mutations turn MK into a VDR superagonist (EC(50) approximately 0.05 nm) but do not concomitantly alter MK binding affinity. Molecular modeling studies demonstrate that MK antagonism stems from its side chain energetically preferring a pose in the VDR ligand binding pocket where its terminal C26-methylene atom is far removed from helix-12. MK superagonism results from an energetically favored increase in interaction between Leu-404/Val-418 and C26, resulting in an increase in the stability and population of the closed, helix-12 conformer. Finally, the results/model generated, coupled with application of a VDR ensemble allosterics model, provide an understanding for the species specificity of MK.

  7. 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.

  8. Amplification of the 16S-23S rDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum.

    PubMed

    Sasaki, Y; Yamamoto, K; Amimoto, K; Kojima, A; Ogikubo, Y; Norimatsu, M; Ogata, H; Tamura, Y

    2001-12-01

    Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.

  9. Use of specific primers based on the 16S-23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples.

    PubMed

    Tsai, Cheng-Chih; Lai, Chieh-Hsien; Yu, Bi; Tsen, Hau-Yang

    2008-10-01

    Effective methods for the identification and enumeration of lactic acid producing bacteria (LAB) cells are important for the quality control and assurance of probiotic products. In this study, we designed a polymerase chain reaction (PCR) primer set from the sequence in 16S-23S internal transcribed spacer (ITS) region and used it for the specific detection of Bifidobacterium adolescentis, one of the Bifidobacterium species used in probiotics. Specificity of the PCR primers, i.e., bits-1/bits-2, was assured by assay strains of B. adolescentis, other Bifidobacterium species, and strains of non-Bifidobacterium spp. Coupled with the use of a known primer set specific for Bifidobacterium species, Bifidobacterium strains and B. adolescentis could be identified from LAB strains in fermented dairy products and human fecal samples.

  10. 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

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

    PubMed

    Dingman, Douglas W

    2009-01-01

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

  12. Applicability of the 16S-23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil.

    PubMed

    Baudoin, E; Couillerot, O; Spaepen, S; Moënne-Loccoz, Y; Nazaret, S

    2010-01-01

    To assess the applicability of the 16S-23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400-550 bp) from Azospirillum strains but also from certain non-Azospirillum strains in vitro, therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (10(2)-10(8) CFU g(-1) soil) was obtained. The PCR primers targeting 16S-23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil. Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.

  13. Further evidence for preoperative chemoradiotherapy and transanal endoscopic surgery (TEM) in T2-3s,N0,M0 rectal cancer.

    PubMed

    Pericay, C; Serra-Aracil, X; Ocaña-Rojas, J; Mora-López, L; Dotor, E; Casalots, A; Pisa, A; Saigí, E

    2016-07-01

    Preoperative chemoradiotherapy and local excision via transanal endoscopic surgery (TEM) in T2-3s,N0,M0 rectal cancer achieve promising results in selected patients. We describe our long-term follow-up experience with this combination, and evaluate complete clinical and pathological responses, local recurrence and overall survival. The prospective observational follow-up study carried out since 2007. Out of 476 consecutive patients treated with TEM, we selected those with adenocarcinoma of low or moderate grade of differentiation, clinical stages T2-superficial T3,N0,M0, who refused radical surgery. Preoperative chemoradiotherapy comprised 5-fluorouracil or capecitabine combined with radiotherapy at a dose of 50.4 Gy. TEM was performed after 8 weeks. Complications were recorded and long-term follow-up was conducted. Fifteen patients undergoing preoperative chemoradiotherapy and TEM (median age 76 years, 95 % CI 70.3-80.4, and median follow-up 38 months, 95 % CI 20-44) were studied. No local recurrence was observed, and only one patient (6.7 %) presented systemic relapse. The overall survival was 76 %. Complete clinical response was achieved in seven patients (46.7 %) and complete pathological response in four (26.7 %). With regard to toxicity associated with neoadjuvant treatment, four patients (26.7 %) developed grade 3 adverse effects; no grade 4 or 5 adverse effects were observed. There was no postoperative mortality. The results of our study, with a response rate of 26.7 % and without local relapse, support the treatment of T2-3s,N0,M0 of rectal cancer with preoperative chemoradiotherapy and local excision (TEM).

  14. Identification of 23-(S)-2-Amino-3-Phenylpropanoyl-Silybin as an Antiviral Agent for Influenza A Virus Infection In Vitro and In Vivo

    PubMed Central

    Dai, Jian-Ping; Wu, Li-Qi; Li, Rui; Zhao, Xiang-Feng; Wan, Qian-Ying; Chen, Xiao-Xuan; Li, Wei-Zhong

    2013-01-01

    It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection. PMID:23836164

  15. 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...

  16. 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.

  17. Clinical Characteristics, Treatment Outcomes, and Resistance Mutations Associated with Macrolide-Resistant Mycobacterium avium Complex Lung Disease.

    PubMed

    Moon, Seong Mi; Park, Hye Yun; Kim, Su-Young; Jhun, Byung Woo; Lee, Hyun; Jeon, Kyeongman; Kim, Dae Hun; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Kim, Hong Kwan; Choi, Yong Soo; Kim, Jhingook; Lee, Seung-Heon; Kim, Chang Ki; Shin, Sung Jae; Daley, Charles L; Koh, Won-Jung

    2016-11-01

    Macrolide antibiotics are key components of the multidrug treatment regimen for treating lung disease (LD) due to Mycobacterium avium complex (MAC). Despite the emergence of macrolide resistance, limited data are available on macrolide-resistant MAC-LD. This study evaluated the clinical features and treatment outcomes of patients with macrolide-resistant MAC-LD and the molecular characteristics of the macrolide-resistant isolates. A retrospective review of the medical records of 34 patients with macrolide-resistant MAC-LD who were diagnosed between January 2002 and December 2014 was performed, along with genetic analysis of 28 clinical isolates. Nineteen (56%) patients had the fibrocavitary form of MAC-LD, and 15 (44%) had the nodular bronchiectatic form. M. intracellulare was the etiologic organism in 21 (62%) patients. Approximately two-thirds (22/34 [65%]) of the patients had been treated with currently recommended multidrug regimens that included macrolide, ethambutol, and rifamycin prior to the emergence of macrolide resistance, and none had been treated with macrolide monotherapy. The median duration of treatment after the detection of macrolide resistance was 23.0 months (interquartile range, 16.8 to 45.3 months). Treatment outcomes were poor after the development of macrolide resistance, with favorable treatment outcomes achieved in only five (15%) patients, including two patients who underwent surgical resection. One-, 3-, and 5-year mortality rates were 9, 24, and 47%, respectively. Molecular analysis of 28 clinical isolates revealed that 96% (27/28) had point mutations at position 2058 or 2059 of the 23S rRNA gene. Our analyses indicate that more effective therapy is needed to treat macrolide-resistant MAC-LD and prevent its development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Model-Free RNA Sequence and Structure Alignment Informed by SHAPE Probing Reveals a Conserved Alternate Secondary Structure for 16S rRNA.

    PubMed

    Lavender, Christopher A; Lorenz, Ronny; Zhang, Ge; Tamayo, Rita; Hofacker, Ivo L; Weeks, Kevin M

    2015-05-01

    Discovery and characterization of functional RNA structures remains challenging due to deficiencies in de novo secondary structure modeling. Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms--the eubacteria E. coli and C. difficile and the archeon H. volcanii--could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery.

  19. Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

    PubMed Central

    Ralph, D; McClelland, M; Welsh, J; Baranton, G; Perolat, P

    1993-01-01

    Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains. Images PMID:8094390

  20. Model-Free RNA Sequence and Structure Alignment Informed by SHAPE Probing Reveals a Conserved Alternate Secondary Structure for 16S rRNA

    PubMed Central

    Lavender, Christopher A.; Lorenz, Ronny; Zhang, Ge; Tamayo, Rita; Hofacker, Ivo L.; Weeks, Kevin M.

    2015-01-01

    Discovery and characterization of functional RNA structures remains challenging due to deficiencies in de novo secondary structure modeling. Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms – the eubacteria E. coli and C. difficile and the archeon H. volcanii – could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery. PMID:25992778

  1. 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

  2. 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.

  3. Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana.

    PubMed

    Lange, Heike; Holec, Sarah; Cognat, Valérie; Pieuchot, Laurent; Le Ret, Monique; Canaday, Jean; Gagliardi, Dominique

    2008-05-01

    Yeast Rrp6p and its human counterpart, PM/Scl100, are exosome-associated proteins involved in the degradation of aberrant transcripts and processing of precursors to stable RNAs, such as the 5.8S rRNA, snRNAs, and snoRNAs. The activity of yeast Rrp6p is stimulated by the polyadenylation of its RNA substrates. We identified three RRP6-like proteins in Arabidopsis thaliana: AtRRP6L3 is restricted to the cytoplasm, whereas AtRRP6L1 and -2 have different intranuclear localizations. Both nuclear RRP6L proteins are functional, since AtRRP6L1 complements the temperature-sensitive phenotype of a yeast rrp6Delta strain and mutation of AtRRP6L2 leads to accumulation of an rRNA maturation by-product. This by-product corresponds to the excised 5' part of the 18S-5.8S-25S rRNA precursor and accumulates as a polyadenylated transcript, suggesting that RRP6L2 is involved in poly(A)-mediated RNA degradation in plant nuclei. Interestingly, the rRNA maturation by-product is a substrate of AtRRP6L2 but not of AtRRP6L1. This result and the distinctive subcellular distribution of AtRRP6L1 to -3 indicate a specialization of RRP6-like proteins in Arabidopsis.

  4. 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.

  5. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

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

    2013-01-01

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

  6. An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

    PubMed Central

    Weitnauer, Gabriele; Gaisser, Sibylle; Trefzer, Axel; Stockert, Sigrid; Westrich, Lucy; Quiros, Luis M.; Mendez, Carmen; Salas, Jose A.; Bechthold, Andreas

    2001-01-01

    Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA. PMID:11181344

  7. Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination.

    PubMed

    Korza, George; Setlow, Barbara; Rao, Lei; Li, Qiao; Setlow, Peter

    2016-12-15

    rRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium Incubation of spores of these two species for ∼20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10- to 30-fold, to ≤0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination.IMPORTANCE A recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486-3495, 2015, doi:http://dx.doi.org/10.1016/j.molcel.2014.12.019) suggested that protein synthesis is essential for early steps in the germination of dormant spores of Bacillus subtilis If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores of Bacillus megaterium or B. subtilis, and these

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

    SciTech Connect

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

    1988-02-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. The rRNA evolution and procaryotic phylogeny

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1986-01-01

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

  12. The rRNA evolution and procaryotic phylogeny

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1986-01-01

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

  13. Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group”

    PubMed Central

    Daffonchio, Daniele; Cherif, Ameur; Borin, Sara

    2000-01-01

    Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus. PMID:11097928

  14. A Bartonella vinsonii berkhoffii typing scheme based upon 16S-23S ITS and Pap31 sequences from dog, coyote, gray fox, and human isolates.

    PubMed

    Maggi, Ricardo G; Chomel, Bruno; Hegarty, Barbara C; Henn, Jennifer; Breitschwerdt, Edward B

    2006-04-01

    Since the isolation of Bartonella vinsonii subspecies berkhoffii from a dog with endocarditis in 1993, this organism has emerged as an important pathogen in dogs and as an emerging pathogen in people. Current evidence indicates that coyotes, dogs and gray foxes potentially serve as reservoir hosts. Based upon sequence differences within the 16S-23S ITS region and Pap31 gene, we propose a classification scheme that divides B. vinsonii subsp. berkhoffii isolates into four distinct types. Two conserved sequences, of 37 and 18 bp, respectively, are differentially present within the ITS region of each of the four B. vinsonii subsp. berkhoffii types. To date, B. vinsonii berkhoffii types I, II, and III have been identified in the US, type III in Europe and type IV in Canada. Based upon the proposed genotyping scheme, the geographic distribution of B. vinsonii berkhoffii types needs to be more thoroughly delineated in future molecular epidemiological studies involving Bartonella infection in coyotes, dogs, gray foxes, human beings and potentially other animals or in arthropod vectors. Strain typing may help to better define the reservoir potential, carriership patterns, modes of transmission, and geographic distribution for each B. vinsonii berkhoffii type.

  15. Internal and external relationships of the Cnidaria: implications of primary and predicted secondary structure of the 5'-end of the 23S-like rDNA.

    PubMed

    Odorico, D M; Miller, D J

    1997-01-22

    Since both internal (class-level) and external relationships of the Cnidaria remain unclear on the basis of analyses of 18S and (partial) 16S rDNA sequence data, we examined the informativeness of the 5'-end of the 23S-like rDNA. Here we describe analyses of both primary and predicted secondary structure data for this region from the ctenophore Bolinopsis sp., the placozoan Trichoplax adhaerens, the sponge Hymeniacidon heliophila, and representatives of all four cnidarian classes. Primary sequence analyses clearly resolved the Cnidaria from other lower Metazoa, supported sister group relationships between the Scyphozoa and Cubozoa and between the Ctenophora and the Placozoa, and confirmed the basal status of the Anthozoa within the Cnidaria. Additionally, in the ctenophore, placozoan and sponge, non-canonical base pairing is required to maintain the secondary structure of the B12 region, whereas amongst the Cnidaria this is not the case. Although the phylogenetic significance of this molecular character is unclear, our analyses do not support the close relationship between Cnidaria and Placozoa suggested by previous studies.

  16. Internal and external relationships of the Cnidaria: implications of primary and predicted secondary structure of the 5'-end of the 23S-like rDNA.

    PubMed Central

    Odorico, D M; Miller, D J

    1997-01-01

    Since both internal (class-level) and external relationships of the Cnidaria remain unclear on the basis of analyses of 18S and (partial) 16S rDNA sequence data, we examined the informativeness of the 5'-end of the 23S-like rDNA. Here we describe analyses of both primary and predicted secondary structure data for this region from the ctenophore Bolinopsis sp., the placozoan Trichoplax adhaerens, the sponge Hymeniacidon heliophila, and representatives of all four cnidarian classes. Primary sequence analyses clearly resolved the Cnidaria from other lower Metazoa, supported sister group relationships between the Scyphozoa and Cubozoa and between the Ctenophora and the Placozoa, and confirmed the basal status of the Anthozoa within the Cnidaria. Additionally, in the ctenophore, placozoan and sponge, non-canonical base pairing is required to maintain the secondary structure of the B12 region, whereas amongst the Cnidaria this is not the case. Although the phylogenetic significance of this molecular character is unclear, our analyses do not support the close relationship between Cnidaria and Placozoa suggested by previous studies. PMID:9061962

  17. Posttranscriptional down-regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency.

    PubMed

    Badhai, Jitendra; Fröjmark, Anne-Sophie; Razzaghian, Hamid Reza; Davey, Edward; Schuster, Jens; Dahl, Niklas

    2009-06-18

    Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.

  18. Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

    PubMed Central

    Sharrock, R A; Gourse, R L; Nomura, M

    1985-01-01

    The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage lambda. By analyzing pulse-labeled RNA with an RNA.DNA filter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoter-proximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage lambda system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears that E. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons. PMID:3161080

  19. Mutation and the environment

    SciTech Connect

    Mendelsohn, M.L. ); Albertini, R.J. )

    1990-01-01

    This book is covered under the following topics: Somatic Mutation: Animal Model; Somatic Mutation: Human; Heritable Mutation: Animal Model; Heritable Mutation: Approaches to Human Induction Rates; Heritable Mutation: Human Risk; Epidemiology: Population Studies on Genotoxicity; and Epidemiology: Workplace Studies of Genotoxicity.

  20. 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

  1. 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.

  2. Mutation rates and mutational loads in man

    SciTech Connect

    Cavalli-Sforza, L.L.

    1984-01-01

    The following areas of research are discussed: (1) the study of human mutation rates; (2) geography of human genes and its relevance to mutation; (3) sociocultural studies correlated with population genetics; (4) consanguineous marriages; and (5) surnames. (ACR)

  3. RRP20, a component of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae

    PubMed Central

    Senapin, Saengchan; Desmond Clark-Walker, G.; Jie Chen, Xin; Séraphin, Bertrand; Daugeron, Marie-Claire

    2003-01-01

    A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20. PMID:12736301

  4. RRP20, a component of the 90S preribosome, is required for pre-18S rRNA processing in Saccharomyces cerevisiae.

    PubMed

    Senapin, Saengchan; Clark-Walker, G Desmond; Chen, Xin Jie; Séraphin, Bertrand; Daugeron, Marie-Claire

    2003-05-15

    A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A1 and A2, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.

  5. 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.

  6. Species identification through mitochondrial rRNA genetic analysis

    PubMed Central

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

    2014-01-01

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

  7. Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China.

    PubMed

    Gou, Huitian; Guan, Guiquan; Ma, Miling; Liu, Aihong; Liu, Zhijie; Ren, Qiaoyun; Li, Youquan; Yang, Jifei; Chen, Ze; Yin, Hong; Luo, Jianxun

    2013-04-01

    Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2006-01-01

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

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

    PubMed

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

    2016-04-01

    A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii. Copyright © 2016 Kakumanu et al.

  11. Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

    PubMed Central

    Mankin, A S; Teterina, N L; Rubtsov, P M; Baratova, L A; Kagramanova, V K

    1984-01-01

    The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium. PMID:6089119

  12. 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.

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

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1985-01-01

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

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

    PubMed Central

    Borovjagin, Anton V.; Gerbi, Susan A.

    2001-01-01

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

  15. Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

    PubMed

    Babitzke, P; Granger, L; Olszewski, J; Kushner, S R

    1993-01-01

    RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.

  16. Characteristic archaebacterial 16S rRNA oligonucleotides

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  17. Characteristic archaebacterial 16S rRNA oligonucleotides.

    PubMed

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

    1986-01-01

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

  18. Hepatic rRNA transcription regulates high-fat-diet-induced obesity.

    PubMed

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Tokunaga, Shinji; Waku, Tsuyoshi; Han, Song-Iee; Iwasaki, Naoya; Mikogai, Aya; Yasuzawa-Tanaka, Kayoko; Kishimoto, Hiroyuki; Hiyoshi, Hiromi; Nakajima, Yuka; Araki, Toshiyuki; Kimura, Keiji; Yanagisawa, Junn; Murayama, Akiko

    2014-05-08

    Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Evaluating rRNA as an indicator of microbial activity in environmental communities: limitations and uses

    PubMed Central

    Blazewicz, Steven J; Barnard, Romain L; Daly, Rebecca A; Firestone, Mary K

    2013-01-01

    Microbes exist in a range of metabolic states (for example, dormant, active and growing) and analysis of ribosomal RNA (rRNA) is frequently employed to identify the ‘active' fraction of microbes in environmental samples. While rRNA analyses are no longer commonly used to quantify a population's growth rate in mixed communities, due to rRNA concentration not scaling linearly with growth rate uniformly across taxa, rRNA analyses are still frequently used toward the more conservative goal of identifying populations that are currently active in a mixed community. Yet, evidence indicates that the general use of rRNA as a reliable indicator of metabolic state in microbial assemblages has serious limitations. This report highlights the complex and often contradictory relationships between rRNA, growth and activity. Potential mechanisms for confounding rRNA patterns are discussed, including differences in life histories, life strategies and non-growth activities. Ways in which rRNA data can be used for useful characterization of microbial assemblages are presented, along with questions to be addressed in future studies. PMID:23823491

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

    PubMed

    Kiparisov, Sergey; Petrov, Alexey; Meskauskas, Arturas; Sergiev, Petr V; Dontsova, Olga A; Dinman, Jonathan D

    2005-10-01

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

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

    PubMed

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

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

  2. 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

  3. rRNA suppressor of a eukaryotic translation initiation factor 5B/initiation factor 2 mutant reveals a binding site for translational GTPases on the small ribosomal subunit.

    PubMed

    Shin, Byung-Sik; Kim, Joo-Ran; Acker, Michael G; Maher, Kathryn N; Lorsch, Jon R; Dever, Thomas E

    2009-02-01

    The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

  4. The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from its Structure in Crystals of 50S Ribosomal Subunits

    PubMed Central

    Shankar, Neelaabh; Kennedy, Scott D.; Chen, Gang; Krugh, Thomas R.; Turner, Douglas H.

    2014-01-01

    Internal loops play an important role in structure and folding of RNA and in RNA recognition by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5'1009CUAAG10133'3'1168GAAGC11645' and 5'998CUAAG10023'3'1157GAAGC11535' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex, 5'1GGCUAAGAC93'3'18CCGAAGCUG105' The internal loop forms a different structure in solution than in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson–Crick UA pair. PMID:17002278

  5. Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection.

    PubMed

    Hiraishi, Hiroyuki; Shin, Byung-Sik; Udagawa, Tsuyoshi; Nemoto, Naoki; Chowdhury, Wasimul; Graham, Jymie; Cox, Christian; Reid, Megan; Brown, Susan J; Asano, Katsura

    2013-09-01

    In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

  6. Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes and phylogenetic comparison with rRNA genes.

    PubMed

    Pilhofer, Martin; Rappl, Kristina; Eckl, Christina; Bauer, Andreas Peter; Ludwig, Wolfgang; Schleifer, Karl-Heinz; Petroni, Giulio

    2008-05-01

    In the past, studies on the relationships of the bacterial phyla Planctomycetes, Chlamydiae, Lentisphaerae, and Verrucomicrobia using different phylogenetic markers have been controversial. Investigations based on 16S rRNA sequence analyses suggested a relationship of the four phyla, showing the branching order Planctomycetes, Chlamydiae, Verrucomicrobia/Lentisphaerae. Phylogenetic analyses of 23S rRNA genes in this study also support a monophyletic grouping and their branching order--this grouping is significant for understanding cell division, since the major bacterial cell division protein FtsZ is absent from members of two of the phyla Chlamydiae and Planctomycetes. In Verrucomicrobia, knowledge about cell division is mainly restricted to the recent report of ftsZ in the closely related genera Prosthecobacter and Verrucomicrobium. In this study, genes of the conserved division and cell wall (dcw) cluster (ddl, ftsQ, ftsA, and ftsZ) were characterized in all verrucomicrobial subdivisions (1 to 4) with cultivable representatives (1 to 4). Sequence analyses and transcriptional analyses in Verrucomicrobia and genome data analyses in Lentisphaerae suggested that cell division is based on FtsZ in all verrucomicrobial subdivisions and possibly also in the sister phylum Lentisphaerae. Comprehensive sequence analyses of available genome data for representatives of Verrucomicrobia, Lentisphaerae, Chlamydiae, and Planctomycetes strongly indicate that their last common ancestor possessed a conserved, ancestral type of dcw gene cluster and an FtsZ-based cell division mechanism. This implies that Planctomycetes and Chlamydiae may have shifted independently to a non-FtsZ-based cell division mechanism after their separate branchings from their last common ancestor with Verrucomicrobia.

  7. Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification.

    PubMed

    Buchhaupt, Markus; Sharma, Sunny; Kellner, Stefanie; Oswald, Stefanie; Paetzold, Melanie; Peifer, Christian; Watzinger, Peter; Schrader, Jens; Helm, Mark; Entian, Karl-Dieter

    2014-01-01

    Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

  8. Partial Methylation at Am100 in 18S rRNA of Baker's Yeast Reveals Ribosome Heterogeneity on the Level of Eukaryotic rRNA Modification

    PubMed Central

    Kellner, Stefanie; Oswald, Stefanie; Paetzold, Melanie; Peifer, Christian; Watzinger, Peter; Schrader, Jens; Helm, Mark; Entian, Karl-Dieter

    2014-01-01

    Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2′-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2′-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process. PMID:24586927

  9. 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.

  10. 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

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

    PubMed

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

    2002-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2001-01-01

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

  14. 'View From A Bridge': A New Perspective on Eukaryotic rRNA Base Modification.

    PubMed

    Sharma, Sunny; Lafontaine, Denis L J

    2015-10-01

    Eukaryotic rRNA are modified frequently, although the diversity of modifications is low: in yeast rRNA, there are only 12 different types out of a possible natural repertoire exceeding 112. All nine rRNA base methyltransferases (MTases) and one acetyltransferase have recently been identified in budding yeast, and several instances of crosstalk between rRNA, tRNA, and mRNA modifications are emerging. Although the machinery has largely been identified, the functions of most rRNA modifications remain to be established. Remarkably, a eukaryote-specific bridge, comprising a single ribosomal protein (RP) from the large subunit (LSU), contacts four rRNA base modifications across the ribosomal subunit interface, potentially probing for their presence. We hypothesize in this article that long-range allosteric communication involving rRNA modifications is taking place between the two subunits during translation or, perhaps, the late stages of ribosome assembly. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. rRNA regulation during growth and under stringent conditions in Staphylococcus aureus.

    PubMed

    Kästle, Benjamin; Geiger, Tobias; Gratani, Fabio Lino; Reisinger, Rudolf; Goerke, Christiane; Borisova, Marina; Mayer, Christoph; Wolz, Christiane

    2015-11-01

    The control of rRNA synthesis and, thereby, translation is vital for adapting to changing environmental conditions. The decrease of rRNA is a common feature of the stringent response, which is elicited by the rapid synthesis of (p)ppGpp. Here we analysed the properties and regulation of one representative rRNA operon of Staphylococcus aureus under stringent conditions and during growth. The promoters, P1 and P2, are severely downregulated at low intracellular guanosine triphosphate (GTP) concentrations either imposed by stringent conditions or in a guanine auxotroph guaBA mutant. In a (p)ppGpp(0) strain, the GTP level increased under stringent conditions, and rRNA transcription was upregulated. The correlation of the intracellular GTP levels and rRNA promoter activity could be linked to GTP nucleotides in the initiation region of both promoters at positions between +1 and +4. This indicates that not only transcriptional initiation, but also the first steps of elongation, requires high concentrations of free nucleotides. However, the severe downregulation of rRNA in post-exponential growth phase is independent of (p)ppGpp, the composition of the initiation region and the intracellular nucleotide pool. In summary, rRNA transcription in S. aureus is only partially and presumably indirectly controlled by (p)ppGpp. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Mechanisms of viral mutation.

    PubMed

    Sanjuán, Rafael; Domingo-Calap, Pilar

    2016-12-01

    The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

  17. CF Mutation Panel

    MedlinePlus

    ... AACC products and services. Advertising & Sponsorship: Policy | Opportunities CF Gene Mutations Testing Share this page: Was this page helpful? Also known as: Cystic Fibrosis Genotyping; CF DNA Analysis; CF Gene Mutation Panel; ...

  18. The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

    PubMed Central

    Sardana, Richa; White, Joshua P.; Johnson, Arlen W.

    2013-01-01

    Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome. PMID:23604635

  19. The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP.

    PubMed

    Sardana, Richa; White, Joshua P; Johnson, Arlen W

    2013-06-01

    Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

  20. 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

  1. Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.

    PubMed

    Polikanov, Yury S; Melnikov, Sergey V; Söll, Dieter; Steitz, Thomas A

    2015-04-01

    We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

  2. 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.

  3. Magnetic and electrical properties of Fe{sub 0.9}Ag{sub 0.1}In{sub 2.3}S{sub 4.4} single crystals

    SciTech Connect

    Bodnar, I. V.; Trukhanov, S. V.; Barugu, T. H.

    2015-10-15

    The magnetic and electrical properties of the Fe{sub 0.9}Ag{sub 0.1}In{sub 2.3}S{sub 4.4} single crystal are studied in the temperature range 4–300 K and in magnetic fields of 0–14 T. It is established that the sample under study is paramagnetic. In the ground state, short-range-order correlations typical of a spin glass with a freezing temperature of 10 K are detected. The magnetic ordering temperature is 15 K. The sample is a semiconductor with a resistivity of 3.5 kΩ cm at room temperature. For the Fe{sub 0.9}Ag{sub 0.1}In{sub 2.3}S{sub 4.4} single crystal, a mechanism for the formation of magnetic and electrical states is suggested.

  4. Prevalence of mitochondrial DNA mutations in sporadic patients with nonsyndromic sensorineural hearing loss.

    PubMed

    Jiang, Hua; Chen, Jia; Li, Ying; Lin, Peng-Fang; He, Jian-Guo; Yang, Bei-Bei

    2016-01-01

    Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(®) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNA(Ser(UCN)) gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNA(Ser(UCN)) gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  5. Deciphering the spectrum of somatic mutations in the entire mitochondrial DNA genome.

    PubMed

    Chen, X Z; Fang, Y; Shi, Y H; Cui, J H; Li, L Y; Xu, Y C; Ling, B

    2015-04-30

    The mitochondrion is a crucial intracellular organelle responsible for regulating cellular energy metabolism, producing free radicals, initiating and executing the apoptotic pathways. Previous studies have shown that somatic mutations in mitochondrial DNA are associated with various tumors, which may be involved during carcinogenesis and tumor progressi