Complete nucleotide sequence of the 26S rRNA gene of Physarum polycephalum: its significance in gene evolution.

The complete nucleotide sequences of the 5.8S and 26S rRNA genes of Physarum polycephalum and the transcribed spacer between them were determined. Comparison of the sequences with those of the Escherichia coli 23S rRNA and yeast 26S rRNA genes showed that there are 16 highly homologous regions in the sequences of Physarum and E. coli and that eukaryotes have some eukaryote-specific extra sequences. The sequence immediately following the 5.8S-like region of E. coli 23S rRNA was found to be highly homologous to the 5' terminus of Physarum 26S rRNA, indicating that the eukaryote-specific 5.8S rRNA gene is derived from the 5'-terminal region of the prokaryote large rRNA gene.     

Mitochondrial DNA of chloramphenicol-resistant mouse cells contains a single nucleotide change in the region encoding the 3' end of the large ribosomal RNA.

The complete DNA sequence of the rRNA genes of mouse L cell mtDNA provides a basis for the examination of the nucleotide sequence of this region in a mutant mouse cell line that is resistant to chloramphenicol, a known inhibitor of mitochondrial protein synthesis. Resistance to chloramphenicol (CAPr) is conferred by a cytoplasmic determinant that is linked to mtDNA restriction endonuclease site polymorphisms. We have determined the sequence of a 212-nucleotide region of mtDNA from a CAPr mouse cell line that encodes a portion of the 1582-nucleotide large rRNA. This sequence is located 107-318 nucleotides from the 5' end of the heavy strand coding sequence, which corresponds to the 3' end of the rRNA. There is a single nucleotide difference in the large rRNA gene from CAPr cells, an A-to-G transition 243 nucleotides from the 5' end of the coding sequence. This single transition is located within a region of 10 nucleotides tht is otherwise completely homologous to human and yeast mitochondrial large rRNAs and Escherichia coli 23S rRNA and is positioned immediately adjacent to a single nucleotide transversion known to occur in a yeast CAPr mutant. This characterization of a mammalian mitochondrial mutant at the nucleotide level directly demonstrates that a mutant phenotype may result from a single mtDNA nucleotide change in an animal cell.     

Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.     

常见致病菌23S rRNA基因片段序列分析及其应用初探

目的通过临床常见致病菌23S rRNA基因序列差异的分析，探索建立可同时检测或鉴定多种细菌的分子生物学方法。方法对常见致病菌23S rRNA基因的一个多变区序列进行聚合酶链反应（PCR）扩增和测序分析，根据序列差异设计相应引物和探针，并采用PCR-凝胶电泳分析和PCR-反向杂交技术检测或鉴定临床常见致病菌。结果1.革兰阳性菌和革兰阴性菌间的23S rRNA基因序列存在较大差异，通过PCR扩增和凝胶电泳分析能快速区分待测菌株为革兰阳性菌抑或革兰阴性菌。2.不同菌种间亦存在一定差异，可通过PCR-反向杂交技术将细菌鉴定到种。结论.临床常见致病菌的23S rRNA基因之间存在可供细菌鉴定的序列差异，据此可望建立各种具有快速，灵敏，准确特点的分子生物学方法，为细菌感染的快速病原诊断提供客观依据。     

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

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.     

某高级中学结核病爆发分离菌株的实验室鉴定

目的鉴定从某中学结核病爆发分离菌株的种属。方法对2006年5—8月,辽宁省某高级中学发生的10例痰检阳性病例中的3份痰标本分离的菌株经表型鉴定方法;传统生化方法;扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型;16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析进行鉴定。结果临床分离株经生化方法初步鉴定1份为结核分枝杆菌复合群和2份为非结核分枝杆菌,随后经表型鉴定和扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型,说明属于结核分枝杆菌复合群的菌株为结核分枝杆菌北京基因型现代株,MIRU基因型为223325173533。经16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析说明属于非结核分枝杆菌的菌株为猪分枝杆菌。结论本次从结核病爆发累及病人的标本中分离出结核分枝杆菌北京基因型现代株和猪分枝杆菌,猪分枝杆菌在暴发流行中的意义尚需进一步研究。     

Molecular approaches for identification and characterization of lactic acid bacteria

The last few years have produced a revolution in the development of very sensitive, rapid, automated, molecular detection methods for a variety of various species of lactic acid bacteria (LAB) associated with food and dairy products. Nowadays many such strains of LAB are considered probiotics. The genome-based methods are useful in identifying bacteria as a complementary or alternative tool to phenotypical methods. Over the years, identification methodologies using primers that target different sequences, such as the 16S ribosomal RNA (rRNA)-encoding gene, the 16S-23S rRNA intergenic spacer region, the 23S rRNA-encoding, recA and ldhD genes; randomly amplified polymorphic DNA, restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, amplification rDNA restriction analysis, restriction enzyme analysis, rRNA, pulse field gel electrophoresis and amplification fragment length polymorphism have played a significant role in probiotic bacteriology. Hence, the aim of this review is to provide an overview of some rapid and reliable polymerase chain reaction-based molecular methods used for identifying and differentiating closely related species and strains of LAB associated with food and industry.     

Characterization of a programmed alteration in an 18S ribosomal gene that accompanies the experimental induction of drug resistance in Schistosoma mansoni.

Stable resistance to the anthelmintic hycanthone can be produced in the human blood fluke Schistosoma mansoni by exposing immature parasites in mice to the drug. Within a single generation, genomic rearrangements, detected as rRNA-encoding DNA restriction fragment length polymorphisms (RFLPs), accompany the appearance of resistance in this model. One of these RFLPs, an approximately 3.6-kilobase BamHI fragment, was shown previously to associate consistently with resistance in independent generations of the JHU strain of S. mansoni. To characterize the genetic changes responsible for this RFLP, the fragment was cloned and sequenced. A comparison of the cloned fragment with a normal 18S rRNA gene demonstrated that the drug resistance-associated RFLP fragment arises through the addition of 732 base pairs into an 18S rRNA gene, 134 base pairs downstream of the junction of the intergenic spacer and the mature 18S rRNA gene. The mutation is nonrandom, targets one, or a few only, of the 100 or so copies of the ribosomal genes, and may represent the incomplete duplication of the gene since the inserted element is identical in sequence to the region contiguous to it. The sequence spanning the junction of the insertion and the original 18S rRNA gene was used as a specific primer for the BamHI RFLP in PCR experiments. The analysis conclusively demonstrated that the mutation is induced rather than selected by the drug since the junctional sequence was not detectable in the drug-sensitive parent population of schistosomes. In addition, analysis of four, independently derived, resistant lines indicated that the same region of the gene was mutated each time. Together, these data demonstrate that reproducible changes are induced during the acquisition of resistance in schistosomes and suggest that the resistant phenotype is induced rather than selected from preexisting forms.     

Clarithromycin resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia

OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA. METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively. RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains. CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.     

Linezolid resistance in Staphylococcus aureus: characterization and stability of resistant phenotype

Linezolid is an important therapeutic option for treatment of infections caused by glycopeptide- and beta-lactam-resistant gram-positive organisms. Linezolid resistance is caused by mutations within the domain V region of the 23S ribosomal RNA (rRNA) gene, which is present in multiple copies in most bacteria. Among clinical Staphylococcus aureus isolates, there has been only 1 reported case of linezolid resistance. In the present study, this isolate was further characterized by determination of the number of mutant 23S rRNA copies, assessment of the stability of the resistant phenotype, and comparison of its growth characteristics with those of linezolid-susceptible S. aureus. All 5 copies of the 23S rRNA gene contained a G2576U mutation in the domain V region. After serial passage on antibiotic-free medium, the isolate maintained resistance to high concentrations of linezolid. Compared with 2 linezolid-susceptible S. aureus isolates, the linezolid-resistant S. aureus isolate demonstrated no significant differences in in vitro growth characteristics.     

粪便基因型检测幽门螺杆菌对克拉霉素耐药性的研究

背景：幽门螺杆菌（Hp）耐药情况日趋严重,选择快速、敏感、价廉的分子生物学技术对Hp耐药进行检测具有重要的临床意义。目的：评价检测粪便Hp基因突变对诊断克拉霉素耐药的有效性,并探讨cagA基因与耐药的相关性。方法：纳入74例13C-尿素呼气试验阳性患者,采集其新鲜粪便标本,提取粪便DNA,采用巢式PCR法扩增Hp23SrRNA,采用PCR—RFLP法检测限制性内切酶BbsI、BceAI、BsaI对23SrRNA扩增产物的酶切情况,采用PCR法扩增cagA基因。结果：74例患者的粪便标本中,60例扩增出Hp23SrRNA367bp片段,其中17例可被BsaI酶切,60例均未被BbsI、BceAI酶切。cagA阳性、阴性表达者的23SrRNA突变率相比差异无统计学意义（P〉0．05）。结论：通过粪便基因型检测Hp对克拉霉素耐药是快速、简便的方法。江苏地区Hp对克拉霉素的耐药机制主要为23SrRNAA2143G突变。cagA基因与Hp对克拉霉素耐药不相关。     

Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.

The complete nucleotide sequence of the 16S RNA gene from the rrnB cistron of Escherichia coli has been determined by using three rapid DNA sequencing methods. Nearly all of the structure has been confirmed by two to six independent sequence determinations on both DNA strands. The length of the 16S rRNA chain inferred from the DNA sequence is 1541 nucleotides, in close agreement with previous estimates. We note discrepancies between this sequence and the most recent version of it reported from direct RNA sequencing [Ehresmann, C., Stiegler, P., Carbon, P. & Ebel, J.P. (1977) FEBS Lett. 84, 337-341]. A few of these may be explained by heterogeneity among 16S rRNA sequences from different cistrons. No nucleotide sequences were found in the 16S rRNA gene that cannot be reconciled with RNase digestion products of mature 16S rRNA.     

应用16S-23S rRNA基因区间对败血症常见菌的鉴定

目的：建立检测不同菌种细菌的16S－23S rNA基因区间的特异图谱。方法：应用聚合酶链反应（PCR），限制性内切酶片段长度多态性分析（RFLP），分子克隆及测序技术，对临床常见的代表20个属26个种的标准菌株及相应的临床分离菌株共61株进行PCR扩增，同时对临床标本进行培养并与PCR－RFLP比较，探讨其在临床应用中的价值。结果：26株不同的标准菌株行PCR扩增后，分别出现1条带，2条带，3条带及多条带的不同DNA图谱，其敏感性为2．5CFU，与人类基因组DNA，真菌及病毒无交叉反应，其中14种菌经PCR扩增即可区分，另10种经Hinf I或AluI酶切后才能区分，肺炎克雷伯菌与坚韧肠球菌间的差异在第779位碱基上不同，Xma Ⅲ酶能进行区别，临床42例血培养15例阳性，阳性率35．7％；而PCR阳性27例，阳性率达64．3％，其阳性率明显高于血培养（P＜0．01），6例脑脊液标本中1例培养为表皮葡萄球菌，其PCR也阳性，2例培养阴性标本，其PCR也阳性，经图谱分析为葡萄球菌，1例培养为新型隐球菌的脑脊液标本PCR检测为阴性，另2例PCR及培养均阴性。结论：建立了PCR加RFLP技术快速检测细菌16S－23S rRNA基因区间的方法，进一步为临床细菌感染的病原诊断提供了新的科学依据。     

The bacteriorhodopsin gene

The bacteriorhodopsin gene has been identified in a 5.3-kilobase restriction endonuclease fragment isolated from Halobacterium halobium DNA, using a cloned cDNA fragment as the probe. Of the 1229 nucleotides whose sequence was determined in the genomic fragment, 786 correspond to the structural gene of bacteriorhodopsin, 360 are upstream from the initiator methionine codon, and 83 are downstream from the COOH terminus. The bacteriorhodopsin gene codes for a precursor sequence of 13 amino acids at the NH2 terminus, 248 amino acids that are present in the mature protein and an additional aspartic acid at the COOH terminus. This determination of the DNA sequence for an archaebacterial gene reveals that the standard genetic code is used; however, there is a marked preference for either G or C in the third codon position. The gene does not contain any intervening sequences and no prokaryotic promoter can be identified in the region immediately upstream from the structural gene. The bacteriorhodopsin mRNA contains at the 5' terminus only three nucleotides beyond the initiating AUG codon and this terminus can form a hairpin structure. Immediately downstream from this structure there is a sequence complementary to the 3' terminus of H. halobium 16S rRNA.     

分支杆菌临床分离株的16S-23S rRNA基因转录间隔区的序列分析

目的　建立鉴定分支杆菌分离株的 16S - 2 3SrDNA转录间隔区 (internaltranscribedspacer ,ITS)序列分析的方法 ,用该方法对临床分离株进行鉴定。方法　对从重庆某医院分离的 2 9株分支杆菌菌株分别进行了PCR扩增 ,得到它们的16S - 2 3SrDNAITS片段 ,并测定所得到片段的DNA序列。用DNA分析软件将所获得的序列与GenBank的分支杆菌序列相比较 ,计算种间相似性 ,并用序列构建分支杆菌菌株的系统发育树 ,对分离株进行鉴定。结果　在ITS序列分析中 ,有 10株的序列分别与结核分支杆菌复合群 (Mycobacteriumtuberculosiscomplex ,MTC)、戈登分支杆菌 ,脓肿分支杆菌的序列完全一致。依据完全一致的序列可以准确鉴定这些临床分离株为相应的种 ;4株 16SrDNA序列分析不能准确鉴定的分离株中其中 3株(M 4、M 5、M 12 )鉴定为戈登分支杆菌 ,1株 (M 2 3)鉴定为脓肿分支杆菌。部分分离株的的ITS序列在GenBank序列检索中无完全一致的匹配序列 ,这些不同的序列之间的相似程度为 2 7.1%～ 99.2 %。用临床分离株的ITS序列与其最相似的已知分支杆菌的ITS序列构建的系统发育树 ,菌株分群与 16SrDNA序列构建的系统发育树的分群基本一致。结论　分支杆菌的16S - 2 3SrDNAITS序列比 16SrDNA序列有更大的多样性 ,该序列分析可作为     

An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum.

The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.     

日本血吸虫组织蛋白酶L1基因的编码区全序列分析及克隆

目的 分析日本血吸虫组织蛋白酶L1(SjCL1)基因编码区的完整序列,并定向克隆到真核表达质粒pcD-NA3中。 方法 从日本血吸虫成虫提取总RNA,进行反向巢式RT-PCR,T载体克隆后测序。PCR扩增SjCL1基因的编码区序列,并将扩增产物克隆到pcDNA3质粒的BamHI和Xhol位点上。结果 通过反向巢式RT-PCR扩增出332 bp SjCL1基因5’端序列,测序后与报道的SiCL1基因部分序列拼接,可得到一个编码317个氨基酸的完整编码区序列。PCR特异性扩增出SjCL1编码区基因序列,其大小约为1 kb。经酶切、PCR鉴定和测序表明所构建的质粒pcDNA-SjCL1中含有所扩增的基因序列。 结论 构建了含SjCL1基因的编码区序列的真核表达质粒pcDNA-SjCL1。