GENETICS AND DNA CHEMISTRY OF PHAGE PHI X-174

此应用程序建议设计改进的站点定向方法 突变，并应用这些方法来定义调控序列在 DNA我们建议改进寡核苷酸定向突变，并 天然诱变寡核苷酸分离方法的探索 脱氧核糖核酸，作为化学或酶法合成的替代品 程序。我们将探索使用N-羟基dCTP作为站点特定的 诱变剂，并将寻找其他非常 模棱两可地合并。作为这些方法的具体应用，我们 将突变噬菌体phiX174的E基因核糖体结合部位，并将 还诱变人RNA聚合酶III的启动子 不同的DNA序列。我们还将绘制和比较大量的例子 偶然出现在测序的DNA分子上的PolIII启动子。 这些研究可能导致精确测定的一般方法。 DNA中的调控序列。我们建议使用已定义DNA的突变 序列以开发体内诱变的检测方法。原核基因 携带已定义序列的突变将被引入哺乳动物 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。作为一名 我们的长期目标是培育出携带突变基因的小鼠 确定的序列，其突变率可以在大肠杆菌中测量。这 或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。

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.