TRANSFORMATION RELATED GENE EXPRESSION IN SV40 INFECTED KERATINOCYTES

描述(改编自申请)：本提案调查 诱导转化过程中相关基因的表达 人角质形成细胞感染致癌病毒SV40。 此前，调查人员已经证明，感染病毒的人 长期培养的角质形成细胞(晚期转化体) 对象之前的单元格的转换属性。 这一危机期标志着永久性细胞系的建立。这个 本提案中描述的实验的目标是详细说明更改 在基因表达上，出现在早期阶段与晚期阶段 转型。在一组实验中，与小说转型相关的 晚传代SV40转化基因文库中的基因 人角质形成细胞的克隆和鉴定：(1)克隆 用正常人PolA+RNA制备的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 表达文库在转染细胞后的保留 生长期的免疫印迹和免疫沉淀将用于 研究两种途径的中间产物随时间的变化 参与细胞生长的调节，ras依赖的信号 细胞周期蛋白/细胞周期蛋白依赖性激酶(CDK)与信号转导途径 细胞周期的调节元件。一种建筑，它将 地塞米松调控下SV40早期基因的表达 可诱导启动子将用于确定基因表达的变化 维持对SV40T抗原表达的依赖，并 确定病毒无关的基因表达变化可能会出现在 长期的文化。

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.