GENETIC ANALYSIS OF PEPTIDE HORMONE ACTION

我们将研究多肽分子机制， 激素调节培养细胞中特定基因的转录， 哺乳动物细胞系。 许多实验室的生化研究 已经确定了这些激素的第一个细胞内步骤 反应途径，但后来的步骤，导致调节 转录仍然未知。 我们将结合使用 分子生物学和体细胞遗传学来定义步骤 和这些途径的组成部分。 启动子和调控 生殖应答基因（催乳素和SV 40）的序列 早期）将融合到Eco gpt的结构基因上， 从而产生了免疫应答的可选择标记基因。 这些 杂交基因将被引入到具有免疫应答的 培养的细胞系（分别为GH 3和HepG 2），其中它们可以 受多种激素和诱导剂（TRH，EGF， 佛波醇酯和钙催乳素;佛波醇酯 SV40）。 选择性培养基可用于选择或 针对GPT基因的表达， 缺乏诱导剂。 我们将选择细胞变体， 缺乏正常的激素反应， 基因和生化上的变异 同样的反应 参与细胞增殖的调节;事实上， 大多数目前已知的癌基因产物被认为 成为这些途径的组成部分。 因此，生物化学的细节 这些途径对于理解 癌

We will investigate the molecular mechanisms by which peptide hormones regulate the transcription of specific genes in cultured mammalian cell lines. Biochemical studies in many laboratories have determined the first intracellular steps in these hormone response pathways, but the later steps that lead to regulation of transcription remain unknown. We will use a combination of molecular biology and somatic cell genetics to define the steps and components of these pathways. Promoters and regulatory sequences of hormonally responsive genes (prolactin and SV40 early) will be fused to the structural gene for Eco gpt, thus creating hormonally responsive selectable marker genes. These hybrid genes will be introduced into hormonally responsive cultured cell lines (GH3 and HepG2, respectively) where they can be regulated by a variety of hormones and inducers (TRH, EGF, phorbol esters, and calcium for prolactin; phorbol esters for SV40). Selective culture media can be used to select for or against the expression of the gpt gene either in the presence or the absence of inducers. We will select for cell variants that lack the normal hormone responses and then characterize these variants genetically and biochemically. These same response pathways are involved in regulation of cell proliferation; in fact most of the currently known oncogene products are believed to be components of these pathways. Thus, the biochemical details of these pathways are essential for understanding the nature of cancer.