MECHANISM OF POLY (A) SITE UTILIZATION

基因表达的调控是分子生物学的中心主题 生物学。现在已经发现，真核细胞利用许多 减弱信使RNA水平的机制(Darnell，1982； 奈文斯，1983；1984)。在这些机制中，有一种是聚腺苷酸化- -合成和稳定活性信使核糖核酸所需的步骤。 它已经表明，有一个基因的要求 聚(A)裂解位点下游的序列(McDevitt et 1984年；吉尔和普罗德福特，1984年；萨多夫斯基和阿尔文，1984年； McLauchlan等人，1985年；Hart等人，1985 a) 已知的保守的AAUAAA序列(ProudFoot和Brownlee， 1976年)。我已经证明了这个序列可能不同，也就是不同 序列可以交换(Hart等人，1985a)，并且 序列在体外反应中被识别(Hart等人，1985b)。 我现在计划将识别和识别的核提取物分离出来 处理主要成绩单，确定涉及的组件 在此过程中，并将中间络合物从In中分离出来 体外反应。通过确定特定的相互作用 分离因子和已知的Poly(A)位点序列突变， 可以确定利用聚(A)部位的机制，以及 这将向我们展示可用于监管的步骤。朝向那个方向 最后，我还计划使用缺失分析来定位序列 调节特定聚(A)位点所需的( 二氢叶酸还原酶(DHFR)基因，Leys和Kellems 1981； 考夫曼和夏普，1983)。随着监管信号和 Poly(A)机制和组件，我计划确定监管 DHFR聚(A)位点的影响因素。这一知识将提供 基因调控步骤的具体例子可能是至关重要的 用于组织分化、发育以及可能的致癌作用。

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.