GENETICS AND DNA CHEMISTRY OF PHAGE PHI X-174

噬菌体 PHI X-174 的遗传学和 DNA 化学

• 批准号: 3124483
• 负责人: Clyde A. Hutchison
• 金额: $ 19.67万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-03-01 至 1987-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124483
• 关键词: DNA; Escherichia coli; bacteriophage phi X174; density gradient ultracentrifugation; endonuclease; gel electrophoresis; genetic manipulation; genetic mapping; genome; nucleic acid sequence; oligonucleotides; radiotracer; tissue /cell culture; virus DNA; virus genetics

This application proposes to devise improved methods for site-directed mutagenesis, and to apply these methods to define regulatory sequences in DNA. We propose to refine oligonucleotide directed mutagenesis, and to explore methods for isolation of mutagenic oligonucleotides from naturally occurring DNAs, as an alternative to synthesis by chemical or enzymatic procedures. We will explore the use of N-hydroxy dCTP as a site-specific mutagen, and will search for other modified nucleotides which are very ambiguously incorporated. As specific applications of these methods we will mutagenize the gene E ribosome binding site of phage PhiX174, and will also mutagenize promoters for human RNA polymerase III occurring on several different sequenced DNAs. We will also map and compare numerous examples of pol III promoters which occur fortuitously on sequenced DNA molecules. These studies could lead to general methods for the precise determination of regulatory sequences in DNA. We propose to use mutations of defined DNA sequence to develop assays for in vivo mutagenesis. Prokaryotic genes carrying mutations of defined sequence will be introduced into mammalian cells in tissue culture. We will attempt to measure mutation rates following reintroduction of the DNA into a bacterial system. As a long-term goal we would like to produce mice-carrying mutant genes of defined sequence whose mutation rates could be measured in E. coli. This could perhaps provide a relatively rapid and economical method for measurement of mutation rates in whole animals.

本申请提出设计改进的方法用于定点 诱变，并应用这些方法来定义调控序列， DNA. 我们建议改进寡核苷酸定向诱变， 探索从自然界中分离诱变寡核苷酸的方法 存在的DNA，作为化学或酶促合成的替代方法， 程序. 我们将探索使用N-羟基dCTP作为位点特异性 诱变剂，并将寻找其他修饰的核苷酸， 模糊地合并。 作为这些方法的具体应用， 将诱变噬菌体PhiX 174的基因E核糖体结合位点，并将 还诱变了存在于几个细胞上的人RNA聚合酶III的启动子， 不同的DNA序列 我们还将绘制和比较许多例子 pol III启动子偶然出现在测序的DNA分子上。 这些研究可能导致精确测定的一般方法 DNA中的调节序列。 我们建议使用特定DNA的突变 序列以开发用于体内诱变的测定。 原核基因 携带确定序列的突变将被引入哺乳动物中， 组织培养中的细胞。 我们将尝试测量突变率 在将DNA重新引入细菌系统之后。 作为 我们的长期目标是生产携带突变基因的老鼠， 定义的序列，其突变率可以在E.杆菌 这 也许可以提供一种相对快速和经济的方法， 测量整个动物的突变率。