CLONING AND CHARACTERIZATION OF CYTOSOLIC PROTEIN TYROSINE KINASE

胞浆蛋白酪氨酸激酶的克隆和表征

• 批准号: 6301682
• 负责人: J. Michael Pierce
• 金额: $ 6.34万
• 依托单位: TEXAS A&M UNIVERSITY-KINGSVILLE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301682
• 关键词: Escherichia coli; Shigella; affinity chromatography; bacillary dysentery; biological signal transduction; colorimetry; cytoplasm; enzyme activity; enzyme linked immunosorbent assay; enzyme substrate; expression cloning; gene expression; genetic screening; host organism interaction; laboratory mouse; laboratory rabbit; microorganism growth; molecular cloning; monoclonal antibody; nucleic acid sequence; polymerase chain reaction; protein tyrosine kinase; protein tyrosine phosphatase; site directed mutagenesis; virulence; western blottings

Shigella species cause bacillary dysentery characterized by bloody and mucous laden stool. One of the hallmarks of Shigella infection is the invasion of intestinal mucosal cells where the bacteria grow intracellularly and spread from host cell to adjacent host cell. A wide variety of virulence factors have been described and shown to be involved in various stages of the infection process. The expression and activity of many of the virulence genes in Shigella are regulated by temperature and/or osmolarity. However, the details of the regulatory pathways for many of the virulence genes have not been thoroughly elucidated. Activation of protein kinases and transcriptional regulatory proteins are events associated with signaling pathways of eukaryotic and prokaryotic cells. Tyrosine phosphorylated proteins have been recently demonstrated in a number of bacterial species including Shigella. Furthermore, protein tyrosine kinase activity has been observed in Shigella flexneri. We have evidence for a protein tyrosine kinase activity associated with the membrane of S. flexneri and its possible role in the ability of these bacteria to invade epithelial cells. Targets of this tyrosine protein kinase and other intracellular tyrosine protein kinases have yet to be identified. The overall goal of this proposal is to clone and characterize genes coding for cytosolic tyrosine protein kinases and substrates of tyrosine protein kinases. We will try to determine if these kinase(s) form a signaling network with the membrane associated tyrosine protein kinase or with other molecules. Furthermore, studies will be initiated to define the roles of these genomes in S. flexneri physiology and in invasion. We will accomplish these goals by identifying clones that react with antibodies to phosphotyrosine. The gene products of these clones will be localized within the cell. The genes will be sequenced and expressed in E. coli for the purpose of antibody production. These antibodies will be used to characterize further the intracellular location of the gene products. Null mutants will be constructed by two separate methods and will be transferred into invasive strains of Shigella to test whether or not these gen products are required for pathogenicity.

志贺氏菌属引起细菌性痢疾， 粘液粪便。志贺氏菌感染的特征之一是 侵入细菌生长的肠粘膜细胞 并从宿主细胞扩散到相邻宿主细胞。广泛 已经描述了多种毒力因子，并显示其参与 在感染过程的不同阶段。的表达和活性 志贺菌的许多毒力基因受温度的调节 和/或摩尔渗透压浓度。然而，监管途径的细节， 许多毒力基因尚未完全阐明。 蛋白激酶和转录调节蛋白的激活是 与真核生物和原核生物信号通路相关的事件 细胞酪氨酸磷酸化蛋白质最近已被证明在 包括志贺氏菌在内的多种细菌。此外，蛋白质 在弗氏志贺菌中观察到酪氨酸激酶活性。我们有 蛋白酪氨酸激酶活性的证据与 S.弗氏及其在这些能力中可能发挥的作用 细菌侵入上皮细胞。这种酪氨酸蛋白的靶点 激酶和其它细胞内酪氨酸蛋白激酶尚未被 鉴定本提案的总体目标是克隆和表征 编码胞质酪氨酸蛋白激酶和底物的基因 酪氨酸蛋白激酶。我们将尝试确定这些激酶是否 与膜相关酪氨酸蛋白形成信号网络 激酶或其它分子。此外，还将开展研究， 定义这些基因组在S.弗氏生理学和 入侵我们将通过识别克隆体来实现这些目标， 用磷酸酪氨酸抗体这些克隆体的基因产物 被定位在细胞内。这些基因将被测序并在 E.大肠杆菌，用于抗体生产。这些抗体将 用于进一步表征基因的细胞内定位 产品.将通过两种不同的方法构建突变体， 将被转移到志贺氏菌的入侵菌株，以测试是否或 致病性不需要这些基因产物。