MUTATIONAL ANALYSIS OF PROTEIN STRUCTURE/FUNCTION

蛋白质结构/功能的突变分析

• 批准号: 2650010
• 负责人: Clyde A. Hutchison
• 金额: $ 23.07万
• 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: 1977-09-30 至 1999-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2650010
• 关键词: RNA directed DNA polymerase; X ray crystallography; enzyme activity; gene mutation; genetic library; genetic regulatory element; human immunodeficiency virus 1; intermolecular interaction; microorganism culture; nucleic acid metabolism; nucleic acid sequence; nucleic acids; protein metabolism; site directed mutagenesis; virus genetics

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.

此应用程序建议设计改进的方法，用于定向 突变，并应用这些方法来定义调控 DNA中的序列。我们将开发一种新的生产方法 “完整的突变体文库”，它允许分离所有 在一个区域内可能的单碱基替换突变 DNA只需稍加修饰即可合成靶序列 通常的自动合成程序，其中链是 通过添加核苷从3‘端逐步构建 亚磷酰胺单体单元。在合成之前，每个 在四个单体储存库中分别掺入少量的 在其他三种中，产生足够的污染水平 每个合成的分子都会产生少量的突变。 在合成后，诱变序列通过以下方式扩增 生物克隆，以产生包含所有 原始序列可能的点替换突变。它 通过随机测序直接鉴定突变体是可行的 从文库中分离出来。我们正在探索突变体的表型 在我们已经建造的三个图书馆中， 其特征在于：1)其糖皮质激素反应元件 小鼠乳腺肿瘤病毒，2)一个增强子样元件在 酵母的TYL转座子，以及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-2DPαI结构域的突变文库 来自小鼠主要组织相容性复合体的基因。 我们建议构建完整的E基因突变文库 和噬菌体phiX174的J。该项目还将用于评估 一种完整的化学合成的可行性和有用性 PhiX174基因组。 我们建议使用定义的DNA序列的突变来自 以原核源为“穿梭靶点”进行体内检测 哺乳动物的诱变作用。我们的目标是发展一种相对 一种快速经济的突变率测定方法 在整个动物身上。噬菌体phiX174基因组，携带突变 已稳定地导入小鼠L细胞 在组织培养中，我们能够通过以下方式在大肠杆菌中拯救它 转染球形体。我们建议使用这一系统来 测量以下phiX174突变的返回率 L细胞的诱变处理。我们建议使用DNA 显微注射产生携带整合拷贝的小鼠 PhiX174基因组，用于整个动物的诱变分析。