PROLACTIN--TRANSCRIPTION FACTOR REGULATION

这个项目是为了确定DNA序列， 催乳素对基因表达的转录刺激， 分离和克隆负责这些的DNA结合蛋白 行动 催乳素是一个不同的家庭成员的调节 proteins. 催乳素主要参与妊娠和哺乳， 人类女性，并已牵连到一些乳腺和前列腺 癌的 这个家庭的其他成员在成长中很重要 调节、代谢调节和发育。 本项目的第一个目的是测量转录调控 嵌合启动子/报告基因在细胞培养物中的表达。 发起人将 源自鸽子的催乳素刺激的cp 35基因 克罗萨克 该基因的结构和调控已经被广泛地 调查和初步研究表明，它含有序列， 特异性结合核蛋白 该发起人还支持 体外转录 从催乳素细胞核中提取的蛋白质- 受刺激的组织刺激启动子在无细胞环境中的活性， 系统 第二个目标将是突变映射监管 通过缺失、接头扫描和位点- 定向诱变 为了鉴定参与观察到的细胞核蛋白质， 转录效应，cDNA克隆将进行第三个目标。 蛋白质将通过寡核苷酸筛选来鉴定， 表达文库和/或亲和纯化和肽测序。 该项目的第四和第五个目标将是解决荷尔蒙 目的3中克隆的转录因子的调节。 首先我们将 通过北方和原位杂交测量因子的mRNA水平 杂交方法 第二，我们将产生抗体， 转录因子肽或重组融合蛋白，然后 通过蛋白质印迹法评估因子的蛋白质水平。 这些实验对于理解 多肽激素对基因表达的转录调控。 我们可以很有信心地预测， 以前未鉴定的转录调节因子在这些 实验 这种信心是基于大量的 初步数据。 鸽子的基因模型对于 这些实验是因为催乳素引起的特异性 基因表达的差异性变化， 生物化学和分子生物学系统的可及性 study.

This project is to identify DNA sequences which are responsible for transcriptional stimulation of gene expression by prolactin and to isolate and clone the DNA binding proteins responsible for those actions. Prolactin is a member of a diverse family of regulatory proteins. Prolactin is primarily involved in pregnancy and lactation in human females and has been implicated in some breast and prostatic cancers. Other members of this family are important in growth regulation, metabolic regulation and development. The first aim of this project is to measure transcriptional regulation of chimeric promoter/reporter genes in cell cultures. The promoter will be derived from the prolactin-stimulated cp35 gene of the pigeon cropsac. This gene's structure and regulation have been extensively investigated and preliminary studies show that it contains sequences which specifically bind nuclear proteins. This promoter also supports in vitro transcription. Proteins extracted from nuclei of prolactin- stimulated tissues stimulate the promoter's activity in a cell-free system. The second aim will be to mutationally map the regulatory sequences in the cp35 promoter by deletion, linker scanning and site- directed mutagenesis. In order to identify the nuclear proteins involved in the observed transcriptional effects, cDNA cloning will be done for the third aim. Proteins will be identified by oligonucleotide screening of an expression library and/or affinity purification and peptide sequencing. The fourth and fifth aims of the project will be to address the hormonal regulation of the transcription factors cloned in aim 3. First we will measure the levels of mRNA for the factors(s) by Northern and in situ hybridization. Second we will produce antibodies to either transcription factor peptides or recombinant fusion proteins and then assess protein levels for the factor(s) by Western blotting. These experiments are to be of critical importance to understanding the transcriptional regulation of gene expression by polypeptide hormones. We can predict with a high degree of confidence that we will discover previously unidentified transcription regulatory factor(s) in these experiments. This confidence is based upon a substantial amount of preliminary data. The pigeon gene model is exceptionally productive for these experiments because of the specificity with which prolactin causes differentiative changes in gene expression and because of the accessibility of the system for biochemical and molecular biological study.