Genomic variability in field populations of Schistosoma mansoni in Brazil as detected with a ribosomal gene probe.

A cloned fragment of the ribosomal gene of Schistosoma mansoni, pSM 389, which contains part of the small rRNA gene plus a portion of the nontranscribed intergenic spacer, was used in Southern hybridization analyses to investigate genomic variation in natural populations of S. mansoni in Brazil. Genomic DNAs were isolated from schistosomes from infected patients (some of whom did not respond to antischistosomal chemotherapy), and from snails from disparate geographic locations in Brazil. Restriction fragment length polymorphisms (RFLPs) were evident in Southern blot hybridizations of these schistosome DNAs, and the RFLPs indicated that the genomic profiles of a number of Brazilian strains were more similar to each other than they were to parasites from two laboratory reference strains of Puerto Rican origin. In addition, the Brazilian isolates could generally be separated from each other based on these RFLPs. Isolates from the southeastern state of Minas Gerais were more similar to each other than they were to parasites isolated in the northeastern states of Alagoas and Pernambuco. Variation was evident among individual worms from some of the isolates, and these individual variations contributed to the complex RFLP patterns that were characteristic for particular isolates. The variation within a natural population isolated directly from snails at Ressaca, Belo Horizonte, may be more marked than that exhibited by more established strains maintained in the laboratory for numerous generations.     

Genotyping of sand fly species in Peruvian Andes where leishmaniasis is endemic

Genotyping of sand fly species circulating in Peru was established on the basis of PCR-restriction fragment length polymorphisms (RFLPs) of the 18S ribosomal RNA (rRNA) gene. The sequences of 18S rRNA gene fragments from 12 Lutzomyia and 1 Warileya species were determined and their RFLP-patterns were analyzed. Consequently, RFLP analysis with the restriction enzyme AfaI and then HapII or KpnI, followed by XspI successfully differentiated them. Intraspecific genetic diversity affecting RFLP-patterns was not detected in the specimens collected from 24 areas of 8 departments. The genotyping was applied to the surveillance of sand flies collected from Andean areas where leishmaniasis is endemic, and its usability was verified. The present method promises to be a powerful tool for the classification and surveillance of sand flies circulating in Peru.     

幽门螺杆菌对克拉霉素耐药的分子机制研究

目的：研究幽门螺杆菌（Hp)对克拉霉素耐的分子机制。方法：用E－test进行克拉霉素药敏试验,选取治疗前敏感、治疗后耐药的配对菌株及原发耐药Hp菌株进行研究;应用随机扩增多态性DNA（RAPD）分析,确定治疗前后菌株的同一性;用PCR－限制性片段长度多态性（RFLP）分析探讨克拉霉素耐药机制。结果9株克拉霉素耐药菌23SrRNA基因功能区V PCR扩增片段,8株被BsaI酶切,9株均未被BbsI酶切,提示8株在2144位点有A→G突变。结论上海地区大多数克拉霉素耐药Hp菌株存在23SrRNA基因功能区V2144位点A→G突变。     

淮南地区幽门螺杆菌对克拉霉素耐药性及其耐药分子机制的研究

目的探讨淮南地区幽门螺杆菌(Helimbacter pylori,Hp)对克拉霉素耐药情况及其耐药分子机制。方法用E-test和琼脂稀释法进行克拉霉素药敏试验,提取Hp基因组DNA,PCR扩增Hp 23S rRNA基因,并用PCR-限制性片段长度多态性(RFLP)分析克拉霉素耐药机制。结果淮南地区克拉霉素耐药率为9.15%(27/141),克拉霉素耐药与性别、年龄无关。PCR从Hp基因组DNA中扩增出425bp的Hp 23S rRNA基因,PCR-RFLP检测,27株耐药菌株均可被Bbsl酶切成332、93bp两个片段,未检测到被BsaⅠ酶切的耐药菌株。结论淮南地区克拉霉素耐药率较高,耐药菌株存在23S rRNA基因功能区V2143位点A-G突变。     

Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as BamHI fragments.

Over 95% of the herpes simplex virus type 1 (strain F) DNA sequences have been cloned as BamHI fragments in the pBR322 plasmid. With one exception, all of the cloned fragments have the same electrophoretic mobilities and restriction enzyme cleavage sites as do the authentic fragments derived from the BamHI digests of the viral genome. The exception is the BamHI B fragment mapping at the right end of L component in the prototype arrangement of the DNA. Thus, a small deletion mapping near the left end of the fragment was present in two independently derived plasmids. Included in the collection of plasmids are several clones containing DNA sequences that span the junction between the L and S components of the virus DNA. Several plasmids containing the junction fragment were found to be sufficiently stable to permit the preparation of large amounts of the DNA fragment for fine-structure mapping of the restriction enzyme cleavage sites. Preliminary studies on one cloned fragment (BamHI G) have shown that it is biologically active in marker rescue of a temperature-sensitive mutation and in transfer of a plaque morphology marker.     

Mitochondrial DNA and nuclear DNA from normal rat liver have a common sequence.

Although Pst I does not cut the circular mitochondrial genome of the rat, BamHI generates from this genome two unequal fragments of DNA. Each of these fragments was cloned in pBR322. Nuclear DNA was digested from rat liver singly or doubly with Pst I and BamHI, and it was demonstrated that nuclear DNA shared a common sequence with the larger mitochondrial DNA BamHI fragment. The cloned larger mitochondrial DNA fragment was further subdivided with HindIII into four pieces that were labeled and then used to probe the double-digested nuclear DNA. The hybridization data showed that the common sequence is less than 3 kilobase pairs long and lies within the part of the mitochondrial genome containing the D-loop and a portion of the rRNA genes. It therefore appears that, as in lower eukaryotes, there are shared sequences between the nuclear and mitochondrial genomes in mammals.     

CagA＋及VacA＋幽门螺杆菌对克拉霉素的耐药性突变分析

目的分析CagA＋及VacA＋的幽门螺杆菌（Hp）对克拉霉素耐药与23S rRNA基因点突变的关系。方法采集右江民族医学院附属医院2006~2008年确诊为Hp感染患者的胃窦部黏膜样本进行Hp分离培养和鉴定,PCR扩增CagA＋及VacA＋基因,E-test进行药敏实验,PCR方法扩增23S rRNA基因,基因测序检测克拉霉素耐药菌株的点突变。结果对克拉霉素耐药的CagA＋及VacA＋Hp菌株均存在23S rRNA基因v功能区第2144位和第2143位A-G突变,而敏感菌株没有发现该位点突变。结论Hp对克拉霉素耐药的23S rRNA基因A2143G、A2144G点发生突变,与基因分型无关。     

日本血吸虫SjPGK基因克隆和序列分析

目的　克隆日本血吸虫磷酸甘油酸激酶 (Sj PGK)编码基因片段 ,分析其核苷酸序列。方法　根据曼氏血吸虫磷酸甘油酸激酶 (Sm PGK) c DNA序列设计并合成一对引物 ,以日本血吸虫成虫总 RNA为模板 ,采用逆转录 -聚合酶链反应 (RT- PCR)特异性扩增 Sj PGK基因片段 ,将其克隆入p MD18- T载体中 ,经双酶切分析和 PCR鉴定 ,将阳性克隆进行脱氧核糖核酸序列测定 ;运用BL AST程序 ,将测序结果及其推导的编码氨基酸序列与 NCBI数据库在核苷酸水平和氨基酸水平进行同源性比较。结果　 RT- PCR特异性扩增出一条长约 830 bp的条带 ;重组质粒的双酶切和以其脱氧核糖核酸为模板的 PCR均可见一条与 RT- PCR产物相同的条带 ;脱氧核糖核酸序列测定和分析结果表明 :Sj PGK基因片段长为 830 bp,与 Sm PGK的核苷酸同源性为 85 % ,分值为 6 72 ;氨基酸同源性为 94 % ,分值为 4 73。结论　成功地克隆了 Sj PGK编码基因片段 ,为寻找日本血吸虫新的抗感染疫苗候选分子奠定基础     

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.     

Mapping the mitochondrial DNA of Zea mays: Ribosomal gene localization

We have located the 18S and 26S ribosomal genes on a 32.2-kilobase pair (kb) restriction map of Zea mays mitochondrial DNA. In a BamHI restriction digest of mitochondrial DNA, band 4 carries all of the 26S gene whereas band 2 carries the 18S gene sequence. We have cloned and mapped bands 2 and 4 and show that they are contiguous in the genome. The 26S sequence is at one end of the 13.7-kb fragment 4, immediately adjacent to the junction with fragment 2. The 18S sequence is located at the far end of the 17.5-kb fragment 2, about 15 kb away from the 26S gene. A second region of 18S sequence homology is found on band 40. This region contains sequences that cross-hybridize with those in band 2. The nature of this apparent sequence repetition is unclear.     

Restriction fragment length polymorphisms for the renin gene in Dahl rats

Genomic DNAs from inbred Dahl salt-hypertension sensitive (S) and inbred Dahl salt-hypertension resistant (R) rats were examined for restriction fragment length polymorphisms (RFLPs) using a rat renin cDNA probe. Eight of the 28 restriction enzymes used yielded polymorphic fragments between S and R strains. This suggests a major structural difference in or near the renin gene of Dahl rats. Some, but not all, of the polymorphisms are compatible with an insertion/deletion mutation of about 1.1 kb in size. The ratio of renin gene copy numbers for S to R was found to be 1, showing that the RFLPs are not likely to be due to gene duplication.     

Phospholipase C genes display restriction fragment length polymorphisms between the genomes of normotensive and hypertensive rats.

The genomic loci of four distinct phospholipase C genes (PLC-beta, PLC-gamma I, PLC-delta and PLC-gamma II) were examined for restriction fragment length polymorphisms (RFLPs) between the genomes of three normotensive [Sprague-Dawley, Donryu and Wistar-Kyoto (WKY)] and two closely related hypertensive [spontaneously hypertensive (SHR) and SHR stroke-prone (SHR-SP)] rat strains. The RFLPs observed between SHR and WKY were classified into three types. Type I RFLPs are those observed at 4.3 kilobase (kb) and 1.9 kb by AvaI digestion for PLC-gamma probe and at 1.9 kb by AccI digestion for PLC-beta probe, where RFLP banding patterns are conserved in two hypertensive (SHR and SHR-SP) and one normotensive (Sprague-Dawley) strains. Type II RFLPs are those observed by AccI, BamHI, EcoRI and PstI digestions for PLC-beta probe, where RFLP pattern observed in SHR is shared by one normotensive (Sprague-Dawley) strain but not by SHR-SP, WKY or Donryu rats. Type III RFLPs are those detected at 6.3 kb band by Bg/II digestion for PLC-beta probe and at 1.0 kb by BamHI digestion for PLC-gamma II probe, where RFLP pattern observed in SHR is shared by two normotensive rats other than WKY. No RFLP was found for PLC-gamma I probe after testing 13 restriction enzymes. Since PLC plays a pivotal role in regulating the intracellular calcium concentration and the intracellular signal transduction, these RFLPs may offer a valuable tool for the analysis of genomic predisposition for hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)     

幽门螺杆菌基因多态性与克拉霉素耐药性关系的研究

目的研究幽门螺杆菌（Helicobacter pylori,Hp）对克拉霉素耐药情况及与23S rRNA基因点突变的关系。方法因上消化道症状进行胃镜检查的189例患者获得胃活检组织,微需氧培养得到坳,提取11例敏感菌和和19例耐药菌的DNA,对23S rRNA基因进行PCR扩增,再对敏感菌和耐药菌的23S rRNA基因进行全基因测序对比和生物信息学分析。结果Hp菌株对克拉霉素的耐药率是29．2％;19个对克拉霉素耐药的却菌株中17株出现23S rRNA基因突变,各种突变的比例分别为A→G36．8％、G→A21．5％、C→T15．8％、A→C10．5％和T→C5．3％。11例敏感株及2例耐药株均未发现23S rRNA基因突变。结论克拉霉素耐药的却菌株比较常见,23SrRNA基因的多个不同位点突变均与跏对克拉霉素耐药有关,而A—G和G—A突变是主要的形式。     

Mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea

OBJECTIVE: Identification of the presence and drug resistance of Mycobacterium leprae is key to the diagnosis and treatment of leprosy in non-endemic country like Korea. The aim of this study was to screen the drug target DNA such as folP, rpoB, gyr, and 23S rRNA of drug resistance strain of M. leprae. PATIENTS AND METHODS: Sequences of those genes were analyzed for the 104 bacterial index positive cases out of 171 leprosy patients in Korea using touchdown PCR, single stranded conformational polymorphism. RESULTS: Twenty (19.2%) cases have shown the mutations in folP gene of dapsone-resistant M. leprae in which three (2.89%) cases were mutations in two genes, folP and rpoB, of multidrugs resistant strains to dapsone and rifampin, and two (1.92%) cases in folP and gyr genes of resistance to dapsone and oflaxacin, respectively. Besides double mutation for folP gene was one case (0.96%) and for rpoB gene one case, respectively. There was no mutant isolates in 23S rRNA gene against clarithromycin. CONCLUSIONS: This result should leads to a better understanding of the status of multidrug resistant leprosy in Korea and may assist in the rapid diagnosis of drug resistant M. leprae and the choice of the appropriate treatment regimens.     

类鼻疽假单胞菌核糖分型

目的　用rRNA基因 (rDNA)的限制性片段长度多态性 (RFLPs)将同一血清型类鼻疽菌株进行基因分型。方法　应用PCR技术 ,从大肠杆菌中分别扩增出 16S、2 3S　rDNA作探针。对不同来源的 9株血清Ⅰ型类鼻疽菌染色体DNA进行酶切 ,通过Southern杂交 ,获得各菌株rDNA限制图谱。结果　BamHI、PstI、ApaI和EcoRI单酶切的rDNA指纹图分别显示 6、3、3和 3个不同的核糖型 (ribotypes) ,尤以BamHI区别效果最为显著。综合四种限制酶图谱 ,计算出各菌株间相似度 ,并绘制其系统关系图。结论　在类鼻疽菌仅根据不耐热抗原的有无分血清Ⅰ和Ⅱ型的情况下 ,核糖分型 (Ribotyping)无疑是对血清分型的一个有力补充 ,提供了一种敏感性高、重复性强的分子流行病学调查工具。可用于菌株遗传本质微小差异上的进一步区分     

Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure

BACKGROUND: It was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori. AIMS: To study the incidence and mechanism of development of clarithromycin resistance by analysing these mutations. SUBJECTS: Eighty two H pylori positive patients who had an endoscopic examination and no history of treatment with macrolide antibiotics. METHODS: Clarithromycin resistance was screened for by polymerase chain reaction-restriction fragment length polymorphism of the 23S rRNA gene coupled with antibiotic susceptibility testing. In clinical isolates with mutations or resistance, mutations in individual colonies were analysed by direct sequencing. RESULTS: Of the 79 amplicons (DNA fragments amplified by polymerase chain reaction), Alw26I and MboII digestion disclosed the mutation in four (5%) and one (1%) respectively. However, the Alw26I cleavage was incomplete in two of the four amplicons, as was the MboII cleavage. Individual colony analysis of the isolates with incomplete cleavage patterns showed the presence of both wild type and mutated strains in the 23S rRNA genes. CONCLUSIONS: Both clarithromycin sensitive and resistant strains colonised in some patients with no history of exposure to macrolides. The results suggest that resistant strains may not be formed but selected by clarithromycin administration.     

广东省吸毒人群新发HIV-1感染pol基因多态性和耐药分析

目的研究广东省部分吸毒人群2007年新发现的艾滋病病毒Ⅰ型（HIV-1）感染者pol基因的多态性耐药突变。方法选取2007年吸毒人群中新发现的HIV感染者63例,从血浆中提取病毒RNA,运用一步法逆转录聚合酶链反应（RT—PCR）和套式聚合酶链反应（Nested—PCR）扩增HIV pol基因段,并对扩增的目的片段进行测序,将测序结果与斯坦福大学耐药数据库比对,获得耐药和基因多态性相关信息。结果在63例HIV感染者中,有效扩增并且测序成功49例。检出耐药者2例,占4．1％,分别为低度和潜在耐药,耐药突变类型分别为V179D和Q151LQ。pol基因耐药突变的发生率为38．7％,主要突变类型为：L33I、A71V、A71T、T69S。其中T69S为主要的耐药突变位点,占耐药突变的33％。在不同的基因亚型中,pot基因蛋白酶区和逆转录酶区的突变的多态差异较大。结论广东省吸毒人群2007年新发现HIV感染者耐药发生率仍处于较低水平,为4．1％,但耐药突变的发生率较高。     

Suppressor of yeast mitochondrial ochre mutations that maps in or near the 15S ribosomal RNA gene of mtDNA.

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho- strains of yeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSU1-carrying rho- clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho- does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.     

日本血吸虫黏蛋白样蛋白部分基因的扩增及测序

目的　体外扩增日本血吸虫中国大陆株黏蛋白样蛋白(SjMLP)抗原的部分基因序列。　方法　利用PCGENE软件查找SmMLP的抗原决定簇;特定寡核苷酸引物的设计与合成;Trizol抽提日本血吸虫成虫总RNA,逆转录聚合酶链反应(RTPCR)扩增目的基因,测序后与SmMLP进行同源性分析。　结果　RT PCR特异性扩增出SjMLP编码区基因序列,其片段大小为756bp,测序结果与SmMLP具有高度同源性。　结论　RTPCR扩增的SjMLP抗原编码区基因序列与预期相符合。