GENETIC ANALYSIS OF PEPTIDE HORMONE ACTION

我们将研究多肽的分子机制 激素调节培养细胞中特定基因的转录 哺乳动物细胞系。许多实验室的生物化学研究 已经确定了这些激素在细胞内的第一步 反应途径，但导致调控的后续步骤 转录情况仍不得而知。我们将结合使用 分子生物学和体细胞遗传学来定义步骤 以及这些通路的组成部分。推动者和监管机构 激素反应基因序列(催乳素和SV40 早期)将与Eco gpt的结构基因融合，因此 产生荷尔蒙反应的可选择标记基因。这些 杂交基因将被引入对激素敏感的基因 培养的细胞系(分别为GH3和HepG2) 受多种激素和诱导剂(TRH、EGF、 催乳素用佛波酯和催乳素用钙 SV40)。选择性培养基可用于选择或 对gpt基因的表达产生抑制作用 缺乏诱导者。我们将为细胞变种选择 缺乏正常的荷尔蒙反应，然后描述这些 基因和生化上的变异。这些相同的反应 细胞增殖的调控涉及多种途径；事实上 目前已知的大多数癌基因产品被认为是 成为这些途径的组成部分。因此，生化细节 这些途径中的一条对于理解 癌症。

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.