MUTATIONAL ANALYSIS OF PROTEIN STRUCTURE/FUNCTION

本申请提出设计改进的方法，用于定向 诱变，并应用这些方法来定义调控 DNA序列。 我们将开发一种新的生产方法 “完整的突变体库”，它允许分离所有 在一个区域内可能的单碱基置换突变 DNA. 通过轻微的修饰合成了目标序列 通常的自动化合成程序，其中链是 从3'末端通过添加核苷逐步构建 亚磷酰胺单体单元。 在合成之前， 四个单体储库“掺杂”有少量的每一个 其他三个，以产生足够的污染水平， 以使每个合成分子产生少量突变。 合成后，通过PCR扩增诱变序列。 生物克隆，以产生包含所有 原始序列的可能的点置换突变。 它 通过随机测序直接鉴定突变体是可行的 从图书馆分离。 我们正在探索突变体的表型 在我们已经建成的三个图书馆里， 其特征在于：1）糖皮质激素反应元件（GRE）， 小鼠乳腺肿瘤病毒，2）增强子样元件， Tyl转座子，和3）RNA聚合酶III启动子 腺病毒的VAI基因。 我们建议建造和 分析H-2DP的α I结构域中的突变文库， 小鼠主要组织相容性复合体的基因。 我们建议构建完整的突变体库基因E 和噬菌体PhiX 174的J。 该项目还将评估 一个完整的化学合成的可行性和实用性， PhiX 174基因组 我们建议使用从一个特定的DNA序列的突变， 原核生物来源作为“穿梭靶”进行体内测定 哺乳动物的突变。 我们的目标是发展一个相对 一种快速经济的突变率测定方法 整个动物。 携带突变的噬菌体PhiX 174基因组 已稳定地导入小鼠L细胞 在组织培养中，我们能够在E.杆菌 原生质球的转染。 我们建议利用这一系统， 测量PhiX 174突变的回复率， L细胞的诱变处理。 我们建议使用DNA 显微注射，以产生携带整合拷贝的 PhiX 174基因组，用于全动物诱变试验。

This application proposes to devise improved methods for directed mutagenesis, and to apply these methods to define regulatory sequences in DNA. We will exploit a new method for producing "complete mutant libraries", which allows the isolation of all possible single base substitution mutations within a region of DNA. The target sequence is synthesized by a slight modification of the usual automated synthetic procedure, in which chains are built stepwise from the 3' end by the addition of nucleoside phosphoramidite monomeric units. Before synthesis each of the four monomer reservoirs is "doped" with a small amount of each of the other three, to produce a level of contamination sufficient to produce a small number of mutations per molecule synthesized. Following synthesis, the mutagenized sequences are amplified by biological cloning, to produce a mutant library containing all possible point substitution mutations of the original sequence. It is practical to identify mutants directly by sequencing random isolates from the library. We are exploring phenotypes of mutants in three libraries which we have already constructed and characterized: 1) the glucocorticoid response element (GRE) of mouse mammary tumor virus, 2) an enhancer-like element in the Tyl transposon of yeast, and 3) the RNA polymerase III promoter of the VAI gene of adenovirus. We propose to construct and analyze a library of mutations in the alpha I domain of the H-2DP gene from the major histocompatibility complex of the mouse. We propose to construct complete mutant libraries for genes E and J of phage PhiX174. This project will also serve to evaluate the feasibility and usefulness of a complete chemical synthesis of the PhiX174 genome. We propose to use mutations of defined DNA sequences from a prokaryotic source as a "shuttle target" to assay in vivo mutagenesis in mammals. Our objective is to develop a relatively rapid and economical method for measurement of mutation rates in whole animals. The phage PhiX174 genome, carrying mutations of defined sequence, has been stably introduced into mouse L cells in tissue culture, and we are able to rescue it in E. coli by transfection of spheroplasts. We propose to use this system to measure reversion rates of PhiX174 mutations following mutagenic treatment of the L cells. We propose to use DNA microinjection to produce mice carrying integrated copies of the PhiX174 genome, for use in whole animal mutagenesis assays.