TRANSFORMATION RELATED GENE EXPRESSION IN SV40 INFECTED KERATINOCYTES

描述（改编自应用）：本提案调查 诱导转化过程相关基因的表达， 人角质形成细胞感染致癌病毒，SV 40。 此前，研究人员已经证明， 长期培养的角质形成细胞（晚期转化体）表现出 这些特性将它们与之前的细胞区分开来。 危机时期，标志着永久细胞系的建立。的 本提案中所述实验的目标是详细说明 在基因表达中，出现在早期与晚期阶段， 转型在一组实验中，小说转换相关 来自来自晚期传代的SV 40转化的 人角质形成细胞将通过以下方式克隆和鉴定：（1）集落 使用cDNA探针进行杂交，所述cDNA探针由来自正常人的polyA+ RNA制成。 并转化角质形成细胞以选择携带转化的克隆- 相关的cDNA，和（2）通过克隆回cDNA序列， 保留在用cDNA表达文库转染的细胞中， 将使用蛋白质印迹和免疫沉淀法来 研究两种途径中间产物随时间的变化 参与调节细胞生长，ras依赖信号 细胞周期蛋白/细胞周期蛋白依赖性激酶（cdk） 细胞周期的调控元件。一种结构， 在地塞米松的控制下SV 40早期基因的表达， 诱导型启动子将用于确定基因表达的变化 维持对SV 40 T抗原表达的依赖性， 鉴定出的不依赖于病毒的基因表达变化可能出现在 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.