HELICOBACTER PYLORI PLASMIDS--IMPACT ON STRAIN DIVERSITY

描述(改编自申请者摘要)：本项目 涉及幽门螺杆菌的微生物，幽门螺杆菌是 胃十二指肠疾病。大多数早期对幽门螺杆菌的研究是 临床/组织学观察，直到最近才有实验室 开始了分子研究。很少有研究提到可能的生物学 幽门螺杆菌质粒DNA的作用，特别是在少数族裔人群中。这 是申请人调查的重点。 幽门螺杆菌的三个质粒pHPM179、pHPM180和pHPM186在 大小，但共享大量的标识，尤其是在 可编码Theta-type复制的Rep蛋白的ORF。两者都有 PHPM179和PHPM180包含具有一定序列相似性的DNA序列 对染色体致病基因：PHPM179含有一短(44个碱基) 该区域与染色体DNA和 新近报道的致病岛(PAI)；PHPM180含有两个232 与已知的102个核苷酸具有序列同源性的碱基直接重复序列 PAI的cagA基因内的序列；PHPM179还携带 转座子的来源未知；PHPM186携带两个H。 幽门螺杆菌特异插入序列、IS605以及6kb的染色体 序列。所有这些观察结果都很有可能导致 质粒在幽门螺杆菌的致病过程中也起着重要作用。 直接或作为病原水平传播的载体 基因。 长期目标是使用分子技术来研究 幽门螺杆菌中的质粒DNA。这些拟议的研究是对 并提供必要的信息以了解 幽门螺杆菌质粒DNA在致病机制中的作用 具体目标1：检验许多大的幽门螺杆菌质粒 含有染色体DNA。确定哪条染色体上存在DNA 以及IS605是否总是存在。具体目标2： 研究质粒整合和切除的程度和性质 从染色体上。假说是一些质粒会整合到 并从染色体上切除，从而获得染色体序列。 切除可能会导致染色体的缺失。特定目标 3：研究pBPM179转座子DNA的范围和性质 232马力的直达重复。这些序列的其他出现 将在染色体和质粒DNA中寻找。具体目标4： 目的：研究幽门螺杆菌REPA基因的性质。假设是这样的 REPA蛋白对大肠杆菌的复制有毒害作用。质粒将会是 构造为允许分析这种效果并允许进行提纯 用于体外研究的REPA蛋白。

DESCRIPTION (Adapted from the Applicant's Abstract): This project involves the microorganism Helicobacter pylori, a causal agent of gastroduodenal disease. Most early studies of H. pylori were clinical/histological observations and only recently have laboratories begun molecular investigations. Few studies mention possible biological roles of H. pylori plasmid DNA, especially in minority populations. This is the focus of the applicant's investigations. Three H. pylori plasmids,pHPM179, pHPM180, and pHPM186 are different in size but share a significant amount of identity, especially within an ORF that could encode a Rep protein for theta-type replication. Both pHPM179 and pHPM180 contain DNA sequences with some sequence identity to chromosomal pathogenicity genes: pHPM179 contains a short (44 bp) region that is identical to the junction between chromosomal DNA and the pathogenicity island (PAI) recently described; pHPM180 contains two 232 bp direct repeats that have some sequence identity with a known 102 bp sequence within the cagA gene of the PAI; pHPM179 also carries a transposon of unknown origin; and pHPM186 carries two copies of the H. pylori-specific insertion sequence, IS605 as well as 6 kb of chromosomal sequence. All of these observations lead to the strong possibility that plasmids play a significant role in H. pylori pathogenesis, either directly or as vehicles for horizontal transmission of pathogenesis genes. The long term goal is to use molecular techniques to study the role of plasmid DNA in H. pylori. These proposed studies are an extension of the above observations and provide information necessary to understand the role of H. pylori plasmid DNA in pathogenesis. Specific Aim 1: Test the hypothesis that many large H. pylori plasmids contain chromosomal DNA. Determine which chromosomal DNA is present on plasmids and whether IS605 is always present. Specific Aim 2: Investigate the extent and nature of plasmid integration and excision from the chromosome. The hypothesis is that some plasmids integrate into and excise from the chromosome, thus acquiring chromosomal sequences. Excision may lead to deletion formation in the chromosome. Specific Aim 3: Investigate the extent and nature of the pBPM179 transposon DNA and the 232 hp direct repeats. Additional occurrences of these sequences will be sought in both chromosomal and plasmid DNA. Specific Aim 4: Investigate the nature of H.pylori plasmid repA. The hypothesis is that RepA protein has toxic effects on E. coli replication. Plasmids will be constructed to allow analysis of this effect and to allow purification of RepA protein for in vitro studies.