MECHANISM OF POLY (A) SITE UTILIZATION

聚 (A) 场地利用机制

• 批准号: 3877640
• 负责人: RONALD HART
• 金额: --
• 依托单位: RUTGERS THE STATE UNIV OF NJ NEWARK
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3877640
• 关键词: carcinogenesis; cell differentiation; dihydrofolate reductase; eukaryote; gene expression; genetic regulation; genetic transcription; growth /development; hormone regulation /control mechanism; messenger RNA; polyadenylate

The regulation of gene expression is a central theme of molecular biology. It has now been found that eukaryotic cells utilize many mechanisms to attenuate messenger RNA levels (Darnell, 1982; Nevins, 1983; 1984). Among these mechanisms is polyadenylation- -a step required for synthesis and stabilization of active mRNA. It has already shown that there is a requirement for genetic sequences downstream of the poly(A) cleavage site (McDevitt et al., 1984; Gil and Proudfoot, 1984; Sadofsky and Alwine, 1984; McLauchlan et al., 1985; Hart et al., 1985a) in addition to the known, conserved AAUAAA sequence (Proudfoot and Brownlee, 1976). I have shown that this sequence may vary, that different sequences can be exchanged (Hart et al., 1985a), and that the sequence is recognized in an in vitro reaction (Hart et al., 1985b). I now plan to fractionate the nuclear extract that recognizes and processes the primary transcript, determine components involved in this process, and isolate intermediate complexes from the in vitro reaction. By determining specific interactions between isolate factors and known poly(A) site sequence mutations, the mechanism of utilization of a poly(A) site can be determined, and this will show us the steps available for regulation. Towards that end, I also plan to use deletion analysis to locate the sequences required for regulation of a specific poly(A) site (the dihydrofolate reductase (DHFR) gene, Leys and Kellems 1981; Kaufman and Sharp, 1983). With the regulatory signals and the poly(A) mechanism and components, I plan to identify regulatory factors for the DHFR poly(A) site. This knowledge will provide specific examples for a gene regulation step that may be crucial for tissue differentiation, development, and likely, carcinogenesis.

基因表达的调控是分子生物学的一个中心主题， 生物学 现在已经发现，真核细胞利用许多 降低信使RNA水平的机制（Darnell，1982; Nevins，1983; 1984）。 其中一种机制是多聚腺苷酸化- - 合成和稳定活性mRNA所需的步骤。 它已经表明，有一个遗传的要求， poly（A）切割位点下游的序列（McDevitt et 例如，1984; Gil和Proudfoot，1984; Sadofsky和Alwine，1984; McLauchlan等人，1985;哈特等人，（1985年） 已知的保守AAUAAA序列（Proudfoot和Brownlee， 1976年）。 我已经证明了这个序列可能会有所不同， 序列可以交换（哈特等人，1985年（a）， 序列在体外反应中被识别（哈特等人，1985年b）。 我现在计划破碎核提取物， 处理原始转录本，确定涉及的组件 在这一过程中，并从中间体中分离出中间体复合物， 体外反应 通过确定 分离因子和已知的poly（A）位点序列突变， 可以确定多聚（A）位点的利用机制，并且 这将向我们展示可供监管的步骤。 为此 最后，我还计划使用缺失分析来定位序列 所需的规管特定聚（甲）酸位点（ 二氢叶酸还原酶（DHFR）基因，Leys和Kellems 1981; 考夫曼和夏普，1983年）。 随着监管信号和 聚（A）机制和组件，我计划确定监管 DHFR poly（A）位点的因子。 这些知识将提供 基因调控步骤的具体例子可能是至关重要的 组织分化、发育和可能的致癌作用。