SITE-SPECIFIC MUTAGENESIS OF CLONED GENES

本文提出的研究目标是改进研究方法。 基于一种共同策略的定点突变-- 在圆形上的限定位置处生成短的单股间隙 DNA分子，然后利用这样的空隙作为靶标进行高效 体外诱变反应。这项研究将涉及改进 用于在特定位置引入镍并将其转化为 这种特定地点的缺口形成了很短的缺口。此外，一种新的诱变剂 反应，通过刺激DNA聚合酶来诱导突变 单链修复(误修复)过程中的核苷酸错结合 将对差距进行广泛评估。最初，这种反应将是 依次应用于质粒pBR322上的几个独特的限制酶切位点 优化诱变效率，分析诱变模式 诱发碱基替换突变。一旦这些属性被 使用此方法定义的定点突变将用于 对两个克隆的基因进行分析。首先，导致氨基酸的突变 TEM型β-内酰胺酶信号肽的替换将为 构建，以及它们对这种酶跨 将对细菌细胞膜进行研究。第二，一个长期的突变 将对酿酒酵母的肌动蛋白基因进行分析。 该项目将涉及开发有效的方法 用构建的突变等位基因替换染色体肌动蛋白基因 体外培养。除了为特定站点提供改进的方法之外 突变应该适用于任何克隆的DNA片段，这 可以预期，研究将在生物化学方面产生新的信息 原核生物的蛋白质分泌。此外，曾经有一批 酵母肌动蛋白基因的条件致死突变等位基因已经被 得到了许多涉及分子生物学的基本问题 肌动蛋白--在维持细胞结构和正常生理中的作用 真核细胞--应该负责。

The research here proposed has as its objective the improvement of methods of site-specific mutagenesis which are based on a common strategy--the generation of a short, single-stranded gap at a defined site on a circular DNA molecule, followed by the use of such a gap as a target for efficient in vitro mutagenic reactions. This research will involve refinement of the enzymatic reactions used to introduce nicks at unique sites and to convert such site-specific nicks into short gaps. In addition, a new mutagenic reaction, which induces mutations by stimulating DNA polymerase to misincorporate nucleotides during repair (misrepair) of single-stranded gaps, will be extensively evaluated. Initially, this reaction will be applied to several unique restriction sites on the plasmid pBR322 in order to optimize the efficiency of mutagenesis and to analyze the pattern of induced base substitution mutations. Once these properties have been defined, site-specific mutagenesis with this method will be used in the analysis of two cloned genes. First, mutations causing amino acid substitutions in the signal peptide of the TEM beta-lactamase will be constructed, and their effects on the transport of this enzyme across the bacterial cell membrane will be studied. Secondly, a long-term mutational analysis of the actin gene of Saccharomyces cerevisiae will be conducted. This project will involve the development of methods for efficiently replacing the chromosomal actin gene with mutant alleles constructed in vitro. In addition to providing improved methods for site-specific mutagenesis which should be applicable to any cloned segment of DNA, this research can be expected to yield new information on the biochemistry of protein secretion in prokaryotes. Furthermore, once a collection of conditional lethal mutant alleles of the yeast actin gene have been obtained, many basic questions dealing with the molecular biology of actin--its roles in maintaining cell structure and normal physiology in eukaryotic cells--should be answerable.