IDENTIFICATION OF HELICOBACTER PYLORI VIRULENCE GENES

描述(改编自申请者摘要)：慢性幽门螺杆菌 感染胃粘膜，与人类的发病机制有关 胃炎、消化性溃疡和胃癌。这份提案概述了一个 以功能基因组学为基础的方法旨在鉴定和表征H. 幽门螺杆菌毒力基因与人类胃病相关。建议数 实验将提供幽门螺杆菌基因组的功能图谱， 补充两个完全测序的幽门螺杆菌基因组的完整序列。一个 方法称为“动力学监测逆转录酶启动的聚合酶链式反应”； (KRT-PCR)是实验计划的基础。这种方法是发展起来的 在首席调查员的实验室中，并允许高吞吐量 文字记录定量。提出了全基因组转录图谱的研究方法。 幽门螺杆菌在某些基准州。生长期、pH值变化、 铁和人上皮细胞对幽门螺杆菌基因的表达情况进行分析。 这些生长条件以及CAG内的基因敲除突变 致病岛和特定转录因子基因内，将 测试它们对幽门螺杆菌转录组的影响。候选毒力 从基因组序列中识别的基因包括那些与分子有关的基因 拟态，内毒素生物合成，外膜蛋白，候选“相变” 基因，以及通过比较鉴定的数百个幽门螺杆菌特异基因 幽门螺杆菌基因组序列与其他致病性和非致病性幽门螺杆菌的比较 细菌。幽门螺杆菌转录组将被组织成操纵子。操纵子 结构和配位表达谱应揭示潜在功能 对于未知的ORF。幽门螺杆菌在体内的基因表达谱将 是用非人类灵长类模型获得的。在以下情况下获得的成绩单档案 各种体外条件将被组织到关系数据集和 分析以揭示在幽门螺杆菌中起作用的转录路径和网络。

DESCRIPTION (Adapted from the Applicant's Abstract): H. pylori chronically infects gastric mucosa and is implicated in the pathogenesis of human gastritis, peptic ulcer disease, and gastric cancer. This proposal outlines a functional genomics-based approach designed to identify and characterize H. pylori virulence genes associated with human gastric disease. The proposed experiments will provide a functional map of the H. pylori genome which complements the complete sequences of two fully sequenced H. pylori genomes. A method termed "kinetically monitored reverse transcriptase-initiated PCR"; (kRT-PCR) forms the basis for the experimental plan. This method was developed in the laboratory of the principal investigator, and permits high throughput transcript quantitation. Genome-wide transcript profiling is proposed for H. pylori in certain benchmark states. The effects of growth phase, pH shift, iron, and human epithelial cells on H. pylori gene expression will be analyzed. These growth conditions, as well as knockout mutations within the cag pathogenicity island and within specific transcription factor genes, will be tested for their effects on the H. pylori transcriptome. Candidate virulence genes identified from the genome sequence include those involved in molecular mimicry, LPS biosynthesis, outer membrane proteins, candidate "phase variation" genes, and several hundred H. pylori-specific genes identified by comparisons of H. pylori genome sequences with those of other pathogenic and non-pathogenic bacteria. The H. pylori transcriptome will be organized into operons. Operon structures and coordinate expression profiles should reveal potential functions for unknown ORFs. Gene expression profiles for H. pylori in vivo will be obtained using a non-human primate model. Transcript profiles obtained under a variety of in vitro conditions will be organized into relational data sets and analyzed to reveal transcriptional paths and networks operative in H. pylori.