SITE-SPECIFIC MUTAGENESIS OF CLONED GENES

克隆基因的位点特异性诱变

• 批准号: 3284725
• 负责人: DAVID Robert SHORTLE
• 金额: $ 19.69万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1990-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3284725
• 关键词: DNA repair; Saccharomyces; aminoacid biosynthesis; circular DNA; gene expression; genetic manipulation; membrane permeability; molecular cloning; mutant; nuclease; nucleic acid sequence; plasmids

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.

这里提出的研究的目标是改进方法 基于一种共同的策略-- 在环上的限定位点处产生短的单链缺口 DNA分子，随后使用这样的缺口作为靶标进行高效的 体外致突变反应。 这项研究将涉及细化的 酶促反应用于在独特位点引入切口并转化 将这种位点特异性的切口切割成短的间隙。 此外，一种新的诱变剂 反应，通过刺激DNA聚合酶， 在单链的修复（错误修复）期间错误掺入核苷酸 差距，将得到广泛的评价。 最初，这种反应将是 应用于质粒pBR 322上的几个独特的限制性位点， 以优化诱变的效率并分析突变的模式。 诱导碱基置换突变。 一旦这些资产被 使用该方法进行的定义的、定点突变将用于 分析两个克隆基因。 首先，突变导致氨基酸 TEM β-内酰胺酶的信号肽中的取代将是 构建的，以及它们对这种酶跨膜转运的影响。 将研究细菌细胞膜。 第二，长期变异 将进行酿酒酵母肌动蛋白基因的分析。 该项目将涉及制定有效的方法， 用构建的突变等位基因替换染色体肌动蛋白基因 体外 除了提供改进的方法， 诱变，这应该适用于任何克隆的DNA片段，这 研究可以预期产生新的信息的生物化学 原核生物的蛋白质分泌。 此外，一旦集合 酵母肌动蛋白基因的条件致死突变等位基因已经被 获得，许多基本问题处理的分子生物学的 肌动蛋白--它在维持细胞结构和正常生理中的作用， 真核细胞应该是有责任的。