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

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.     

Molecular cloning and comparative sequence analyses of rRNA operons in Streptomyces nodosus ATCC 14899.

The genome of Streptomyces nodosus contains six ribosomal RNA (rRNA) operons. Four of the rRNA operons; rrnB, rrnD, rrnE and rrnF were cloned. We have completely sequenced all four operons, including a region 750 base pairs (bp) upstream of the 16S rRNA gene. The three rRNA genes present in each operon were closely linked in the order 16S-23S-5S. A sequence comparison of the four operons showed more than 99% sequence similarity between the corresponding 16S and 23S rRNA genes, and more than 97% similarity between 5S rRNA genes. The sequence differences observed between 23S rRNA genes appeared to be localized in two specific regions. Substantial sequence differences were found in the region upstream of the 16S rRNA gene as well as in the internal transcribed spacers. No tRNA gene was found in the 16S-23S spacer regions.     

Variability of ribosomal RNA genes in Rauwolfia species: parallelism between tissue culture-induced rearrangements and interspecies polymorphism

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝 （Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝(宽翅菘蓝,Isatis violascens Bunge)的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝（Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝（宽翅菘蓝,Isatis violascens Bunge）的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

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

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.     

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.     

Unusual ribosomal RNA gene organization in copepods of the genus Calanus

Ribosomal RNA genes in the nuclear genomes of eukaryotes are generally found in tandemly repeated units encoding 18 S, 5.8 S and 28 S rRNA (in that order). 5 S rRNA genes typically lie outside these units, most often in tandem clusters coding exclusively for 5 S rRNA. Inclusion of 5 S genes within the 18 S-5.8 S-28 S repeat unit is known only for certain protozoa and fungi. Here we report that, in the copepod Calanus finmarchicus, single 5 S genes are included within many or all of the 18 S-5.8 S-28 S repeat units. Sequence analyses of regions cloned from two of these repeat units show that they indeed include 5 S genes (which are distal to 28 S genes) and that these are transcribed from opposite strands.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.     

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

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.     

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.     

高黎贡山旱冬瓜Frankia的IGS PCR-RFLP分析

在云南省高黎贡山自然保护区海拔1310～2400m的范围内,采集30个旱冬瓜根瘤样品,直接从根瘤中提取Frankia DNA,对其,nifD-nifK基因间隔区(intergenic spacer,IGS)和16S-23S rDNA IGS进行PCR—RFLP分析．结果表明,nifD-nifK IGS的PCR产物长度差异很大,经HaeⅢ和Afa I双酶切后,得到15种酶切带型,检测到多种基因型的菌株同时与同一株宿主植物共生;16S-23S rDNA IGS的PCR产物长度相似,酶切后亦区分出15种酶切带型．通过对两个基因间隔区的PCR-RFLP联合分析,发现高黎贡山旱冬瓜Frankia存在20种基因型．     

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.     

Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon.

We amplified the 16S-23S rRNA intergenic spacer region of Acholeplasma laidlawii PG8 by polymerase chain reaction (PCR) and obtained two specific PCR products in different sizes. We have sequenced both PCR products and found that one of them has sequence homologous to the spacer tRNA genes in Bacillus subtilis. This is the first evidence of tRNA genes between the 16S-23S rRNA intergenic spacer regions in members of the class Mollicutes.