TRANSFORMATION RELATED GENE EXPRESSION IN SV40 INFECTED KERATINOCYTES

SV40感染的角质形成细胞中转化相关基因的表达

• 批准号: 6425012
• 负责人: MARK L STEINBERG
• 金额: $ 2.57万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6425012
• 关键词: biological signal transduction; cell growth regulation; cell transformation; cyclin dependent kinase; cytogenetics; gene expression; human tissue; immunoprecipitation; keratinocyte; molecular cloning; regulatory gene; simian virus 40; tissue /cell culture; virus genetics; virus infection mechanism; western blottings

Description (Adapted from Application): This proposal investigates expression of genes related to the process of transformation induced in human keratinocytes by infection with the oncogenic virus, SV40. Previously the investigator has demonstrated that viral-infected keratinocytes in long-term culture (late stage transformants) exhibit transformed properties which distinguish them from cells prior to the crisis period that marks the establishment of permanent cell lines. The goal of the experiments described in this proposal is to detail changes in gene expression, which arise at the early versus late stages of transformation. In one set of experiments, novel-transformation related genes from cDNA libraries derived from late passaged SV40-transformed human keratinocytes will be cloned and identified by: (1) colony hybridization using a cDNA probe made from the polyA+ RNAs from normal and transformed keratinocytes to select clones carrying transformation- related cDNAs, and (2) by cloning back cDNA sequences which have been retained in cells transfected with a cDNA expression library after periods of growth Western blots and immunoprecipitation will be used to study time-dependent changes in the intermediates of two pathways involved in the regulation of cell growth, the ras-dependent signal transduction pathway and the cyclin/cyclin-dependent kinase (cdk) regulatory elements of the cell cycle. A construct which places expression of the SV40 early genes under the control of a dexamethasone- inducible promoter will be used to determine changes in gene expression that maintain dependence on expression of the SV40 T antigen and to identify viral independent changes in gene expression may emerge after long-term culture.

描述（改编自申请）：本提案调查 与诱导的转化过程相关的基因的表达 人类角质形成细胞被致癌病毒 SV40 感染。 此前，研究人员已经证明，病毒感染者 长期培养中的角质形成细胞（晚期转化体）表现出 转化后的特性将它们与之前的细胞区分开来 标志着永久细胞系建立的危机时期。这 本提案中描述的实验的目标是详细说明变化 基因表达，发生在早期与晚期 转变。在一组实验中，与小说转换相关的 来自晚期传代SV40转化的cDNA文库的基因 人类角质形成细胞将通过以下方式进行克隆和鉴定： (1) 集落 使用由正常人的多聚 A+ RNA 制成的 cDNA 探针进行杂交 并转化角质形成细胞以选择携带转化的克隆- 相关的 cDNA，以及 (2) 通过克隆回已被 保留在转染 cDNA 表达文库的细胞中 生长期的蛋白质印迹和免疫沉淀将用于 研究两个途径的中间体随时间的变化 ras依赖性信号参与细胞生长的调节 转导途径和细胞周期蛋白/细胞周期蛋白依赖性激酶 (cdk) 细胞周期的调节元件。一个结构，放置 SV40早期基因在地塞米松控制下的表达 诱导型启动子将用于确定基因表达的变化 维持对 SV40 T 抗原表达的依赖性并 确定病毒独立后可能出现的基因表达变化 长期文化。