GENETICS, DNA CHEMISTRY AND PHAGE PHI X 174

遗传学、DNA 化学和噬菌体 PHI X 174

• 批准号: 3480489
• 负责人: Clyde A. Hutchison
• 金额: $ 22.79万
• 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 1992-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3480489
• 关键词: Adenoviridae; DNA; Escherichia coli; affinity chromatography; bacteriophage phi X174; chemical synthesis; clone cells; density gradient ultracentrifugation; electrofocusing; endonuclease; flow cytometry; gel electrophoresis; gene expression; gene mutation; genetic library; genetic manipulation; genetic mapping; genome; immunochemistry; major histocompatibility complex; microinjections; microorganism classification; mouse mammary tumor virus; mutagens; nucleic acid sequence; oligonucleotides; plaque assay; point mutation; radiotracer; regulatory gene; tissue /cell culture; virus DNA; 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.

该申请提出设计改进的定向方法 诱变，并应用这些方法来定义监管 DNA 中的序列。 我们将开发一种新的生产方法 “完整的突变体库”，可以分离所有突变体 区域内可能出现的单碱基取代突变 脱氧核糖核酸。 目标序列经过轻微修改后合成 通常的自动化合成程序，其中链是 通过添加核苷从 3' 端开始逐步构建 亚磷酰胺单体单元。 在合成之前，每个 四个单体储库分别“掺杂”了少量 其他三个中，产生足够的污染水平 每个合成分子产生少量突变。 合成后，诱变序列通过以下方式扩增 生物克隆，产生包含所有突变体的文库 原始序列可能的点替换突变。 它 通过随机测序直接识别突变体是实用的 与图书馆隔离。 我们正在探索突变体的表型 在我们已经构建的三个库中 特征：1)糖皮质激素反应元件(GRE) 小鼠乳腺肿瘤病毒，2) 中的增强子样元件 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-2DP α I 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。 我们建议构建基因 E 的完整突变体库 和噬菌体 PhiX174 的 J。 该项目还将用于评估 完整化学合成的可行性和实用性 PhiX174 基因组。 我们建议使用来自特定 DNA 序列的突变 原核来源作为体内测定的“穿梭靶标” 哺乳动物的诱变。 我们的目标是开发一个相对 快速且经济的突变率测量方法 在整个动物中。 噬菌体 PhiX174 基因组，携带突变 具有确定的序列，已稳定引入小鼠 L 细胞 在组织培养中，我们可以通过以下方式在大肠杆菌中拯救它： 原生质球的转染。 我们建议使用该系统 测量 PhiX174 突变的回复率 L细胞的诱变处理。 我们建议使用DNA 显微注射产生携带整合拷贝的小鼠 PhiX174 基因组，用于整个动物诱变测定。