A novel site for streptomycin resistance in the “530 loop” of chloroplast 16S ribosomal RNA

The chloroplast gene for 16S rRNA was cloned from two maternally inherited streptomycin-resistant mutants ofNicotiana differing in degree of resistance at the whole plant and isolated chloroplast level. A single-nucleotide change in the 16S rRNA gene was detected for each mutant: a C to T transition at nucleotide 860 (Escherichia coli coordinate C₉₁₂) which is an often mutated site, and a novel transition of C to T at nucleotide 472 (E. coli coordinate C₅₂₅). The novel mutation is located in the phylogenetically conserved 530 loop.     

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment

Mutations in the mitochondrial DNA are one of the most important causes of sensorineural hearing loss, especially in the 12S ribosomal RNA (rRNA) gene. We have analyzed the mtDNA 12S rRNA gene in a cohort of 443 families with hearing impairment, and have identified the A1555G mutation in 69 unrelated cases. A1555G is not a fully penetrant change, since only 63% of subjects with this change have developed hearing impairment. In addition, only 22% of the 183 A1555G deaf subjects were treated with aminoglycosides. Two novel nucleotide changes (T1291C and T1243C) were identified. T1243C was found in five deafness cases and one control sample. Mutation T1291C was detected in all maternally related individuals of a pedigree and in none of 95 control samples. Conservation analysis and comparison of the 12S rRNA structure with the 16S rRNA of Escherichia coli showed that the T at nucleotide 1243 and A at nucleotide 1555 are conserved positions. Prediction of RNA secondary structure showed changes in all 12S rRNA variants, the most severe being for T1291C. The reported data confirm the high prevalence of mutation A1555G in deafness cases and the major role of the 12S rRNA gene in hearing. The two novel changes reported here might have different contributions as deafness-related variants. T1291C fulfills the criteria of a disease-causing change. As in the case of mutation A1555G, the underlying phenotype of T1291C is not homogeneous for all family members, providing evidence for the implication of environmental and/or additional genetic factors.     

16S Classifier: A Tool for Fast and Accurate Taxonomic Classification of 16S rRNA Hypervariable Regions in Metagenomic Datasets

The diversity of microbial species in a metagenomic study is commonly assessed using 16S rRNA gene sequencing. With the rapid developments in genome sequencing technologies, the focus has shifted towards the sequencing of hypervariable regions of 16S rRNA gene instead of full length gene sequencing. Therefore, 16S Classifier is developed using a machine learning method, Random Forest, for faster and accurate taxonomic classification of short hypervariable regions of 16S rRNA sequence. It displayed precision values of up to 0.91 on training datasets and the precision values of up to 0.98 on the test dataset. On real metagenomic datasets, it showed up to 99.7% accuracy at the phylum level and up to 99.0% accuracy at the genus level. 16S Classifier is available freely at http://metagenomics.iiserb.ac.in/16Sclassifier and http://metabiosys.iiserb.ac.in/16Sclassifier.     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。     

Sequence and characterisation of a ribosomal RNA operon fromAgrobacterium vitis

One of the four ribosomal RNA operons (rrnA) from theAgrobacterium vitis vitopine strain S4 was sequenced.rrnA is most closely related to therrn operons ofBradyrhizobium japonicum andRhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case ofR. sphaeroides. The 16S rRNA sequence of S4 differs from theA. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3 ends of the three otherrrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3 ends of the four repeats and defines two groups:rrnA/rrnB andrrnC/rrnD.     

Genetic interrelationships of proteolyticClostridium botulinum types A,B, and F and other members of theClostridium botulinum complex as revealed by small-subunit rRNA gene sequences

The phylogenetic interrelationships of members of theClostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolyticC. botulinum types A, B, and F, andC. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D andC. novyi type A, and iv) type G andC. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the species complex on the basis of phenotypic criteria.     

Arrangement of the Sense and Stop Codons of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking with Oligonucleotide Derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3-flanking sense or stop codon with a perfluoroarylazido group at G or U were used to study the positioning of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, tRNA^Phe cognate to UCC was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 and nucleotide A1825 in helix 44 close to the 3 end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem–loop fragment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in the ribosomal A site of various organisms.     

A Three Nucleotide Signature Sequence in Small Subunit rRNA Divides Human Giardia in Two Different Genotypes

ABSTRACT. The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis , obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5'end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are "signature" sequences, which divide Giardia isolates into two different groups.     

Diversity of 16S rRNA Genes within Individual Prokaryotic Genomes

Analysis of intragenomic variation of 16S rRNA genes is a unique approach to examining the concept of ribosomal constraints on rRNA genes; the degree of variation is an important parameter to consider for estimation of the diversity of a complex microbiome in the recently initiated Human Microbiome Project (http://nihroadmap.nih.gov/hmp). The current GenBank database has a collection of 883 prokaryotic genomes representing 568 unique species, of which 425 species contained 2 to 15 copies of 16S rRNA genes per genome (2.22 ± 0.81). Sequence diversity among the 16S rRNA genes in a genome was found in 235 species (from 0.06% to 20.38%; 0.55% ± 1.46%). Compared with the 16S rRNA-based threshold for operational definition of species (1 to 1.3% diversity), the diversity was borderline (between 1% and 1.3%) in 10 species and >1.3% in 14 species. The diversified 16S rRNA genes in Haloarcula marismortui (diversity, 5.63%) and Thermoanaerobacter tengcongensis (6.70%) were highly conserved at the 2° structure level, while the diversified gene in B. afzelii (20.38%) appears to be a pseudogene. The diversified genes in the remaining 21 species were also conserved, except for a truncated 16S rRNA gene in “Candidatus Protochlamydia amoebophila.” Thus, this survey of intragenomic diversity of 16S rRNA genes provides strong evidence supporting the theory of ribosomal constraint. Taxonomic classification using the 16S rRNA-based operational threshold could misclassify a number of species into more than one species, leading to an overestimation of the diversity of a complex microbiome. This phenomenon is especially seen in 7 bacterial species associated with the human microbiome or diseases.rRNA genes are widely used for estimation of evolutionary history and taxonomic assignment of individual organisms (14, 26, 50-52). The choice of rRNA genes as optimal tools for such purposes is based on both observations and assumptions of ribosomal conservation (13, 50). rRNA genes are essential components of the ribosome, which consists of >50 proteins and three classes of RNA molecules; precise spatial relationships may be essential for assembly of functional ribosomes, constraining rRNA genes from drastic change (9, 13). In bacteria, the three rRNA genes are organized into a gene cluster which is expressed as single operon, which may be present in multiple copies in the genome. In organisms with multiple rRNA gene operons, the gene sequences tend to evolve in concert. It is generally believed that copies of rRNA genes within an organism are subject to a homogenization process through homologous recombination, also known as gene conversion (18), a form of concerted evolution that maintains their fit within the ribosome. The homogenization process may involve short domains without affecting the entire sequence of each gene (8).However, significant differences between copies of rRNA genes in single organisms, albeit few, have been discovered in all three domains of life and in all three classes of rRNA genes. The amphibian Xenopus laevis and the loach Misgurnus fossilis have two types of 5S rRNA genes that are specific to either somatic or oocyte ribosomes (30, 48). The parasite Plasmodium berghei contains two types of 18S rRNA genes that differ at 3.5% of the nucleotide positions and are life cycle stage specific (17). The metazoan Dugesia mediterranea possesses two types of 18S rRNA genes with 8% dissimilarity (6). The archaeon Haloarcula marismortui contains two distinct types of 16S rRNA genes that differ by 5% (32, 33). In the domain Bacteria, the actinomycete Thermobispora bispora contains two types of 16S rRNA genes that differ by 6.4% (47). Copies of the 16S rRNA genes and 23S rRNA genes of the actinomycete Thermospora chromogena differ by approximately 6 and 10%, respectively (54). Paralogous copies of rRNA genes with different sequences may have functionally distinct roles.Divergent evolution between rRNA genes in the same genome may corrupt the record of evolutionary history and obscure the true identity of an organism. Substantial variation, if it occurs, may lead to the artificial classification of an organism into more than one species. For a cultivable organism, this problem can be resolved by cloning rRNA genes from a pure culture of the organism to identify the degree of variation. However, most environmental surveys and the recently initiated Human Microbiome Project (HMP) (http://nihroadmap.nih.gov/hmp/) (34) use cultivation-independent techniques to examine microbiomes that contain mixed species. In the case of the HMP, it is hoped that this approach may identify some idiopathic diseases that are caused by alterations in the microbiome in humans. In this type of study, it may be impossible to trace all rRNA genes observed back to their original host. For example, in the phylum TM7, multiple 16S rRNA gene sequences have been reported (21), but it is not known whether they belong to multiple species or to the same bacterium with a high degree of intragenomic variation among rRNA gene paralogs. Due to the limited number of microorganisms for which nucleotide sequences are available for all copies of the rRNA genes, intragenomic variation among 16S rRNA genes, and the likelihood of pyrosequencing errors (25, 40), the potential to overestimate the diversity of a microbiome exists.Coenye et al. analyzed 55 bacterial genomes and found the intragenomic heterogeneity between multiple 16S rRNA genes in these genomes was below the common threshold (1 to 1.3%) for distinguishing species (44) and was unlikely to have a profound effect on the classification of taxa (10). The analysis of 76 whole genomes by Acinas et al. revealed the extreme diversity (11.6%) of 16S rRNA genes in Thermoanaerobacter tengcongensis (2). These early analyses of intragenomic variation of 16S rRNA genes were limited to a small number of available whole genomes. With the increasing number of whole microbial genomes available from the National Center for Biotechnology Information (NCBI), the extent of diversity among the paralogous 16S rRNA genes within single organisms can now be more thoroughly assessed. In the present study, we (i) addressed the theory of 16S rRNA conservation by systematic evaluation of intragenomic diversity of 16S rRNA sequences in completely sequenced prokaryotic genomes to assess its effect on the accuracy of 16S rRNA-based molecular taxonomy and (ii) examined whether previously observed ribosomal constraints on conservation of 2° structures are uniformly applicable at the intragenomic level.     

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

应用TDI-FP技术分析宫颈癌组织HPV16 E7基因A647G点突变

模板指导的末端碱基掺入反应结合荧光偏振检测技术(template direct dye-terminator incorporation with fluorescence- polarization,TDI-FP) 是SNP检测新技术. 应用TDI-FP方法分析中国陕西HPV16阳性宫颈组织HPV16 E7基因第647位核苷酸A→G热点突变(即A647G),首先在HPV16阳性的91例宫颈癌及49例正常/宫颈炎妇女宫颈DNA标本中,PCR扩增含647位点在内的HPV16 E7部分基因, 然后将紧邻647位点5′端的寡核苷酸探针与PCR产物内的模板杂交,并延伸一个与647位点碱基互补的荧光标记碱基：TAMRA-ddTTP或R110-ddCTP. 用荧光偏振仪读取荧光偏振 (FP) 值,根据升高的相应FP值判断647位点碱基. 结果表明,宫颈组织HPV16 E7 A647G的总体检出率为35.71% (50/140). 宫颈癌组的A→G突变率为42.86% (39/91),显著高于正常/宫颈炎组22.45% (11/49) 的突变率 (x2 = 5.778, P = 0.016),两组间的OR值为2.59 (95% CI = 1.17~5.71). 提示TDI-FP 可用于HPV有意义点突变的分析;我国陕西地区妇女HPV 16 A647G突变率及其对宫颈癌的警示性与其他地区相比有明显差异,该地区携带此突变病毒株的妇女患宫颈癌的风险可能较高     

Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 microg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 microg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.     

Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

A new polymorphism in exon 7 of the cystic fibrosis transmembrane regulator (CFTR) gene

Here we describe a new polymorphism, located in exon 7 of the cystic fibrosis transmembrane regulator (CFTR) gene at nucleotide position 1104 (CG), detected by single-strand conformational polymorphism (SSCP) analysis.