GENETIC MANIPULATION OF YEAST ARTIFICIAL CHROMOSOMES

分离和保存大片段DNA的能力 酵母中的人工染色体(YAC)开创了人类染色体研究的新时代 对大基因组的分析。这项技术的进步将是至关重要的 为实现为人类基因组提出的目标 主动权。在生成和分析数据方面的改进 人类DNA文库将大大促进会议的进展 目标是用连续有序的克隆数组表示整个 人类基因组，基因组测序的起点。这 Proposal利用酵母生物学的几个有用特征来 推进这些目标，特别是极其有效的途径 同源重组。我们建议开发一组YAC向量 这将允许对YAC进行几种不同类型的基因操作 包括使用遗传策略进行染色体行走。步行将会是 通过在酵母中将给定的YAC克隆与YAC文库交配来完成 在用适当的基因标记载体制成的酵母中。殖民地 含有YAC对的将被测试是否有能力通过 同源重组，允许确定那些含有YAC的 重叠的基因组DNA片段。 一种新的体内定向克隆特定基因的方法 本课程将探索基因组DNA的特定片段。这一策略被称为 “DNA捕获”利用酵母菌修复DNA缺口的能力 使用完全未切割的基因组的位置遥远但基因相连的探针 DNA作为其修复模板。连接到YAC载体上的探针是 与未切割的染色体DNA一起通过 转化，然后选择稳定遗传的YAC 记号笔。这一策略也将被用于探索非酶 利用重复DNA序列构建基因组YAC文库 而不是独一无二的序列作为DNA捕获的探针。 一种同源YACs的遗传鉴定策略 将开发出独特的非重复探头。这种方法将利用 诱导的双链断裂促进同源重组 激活YAC克隆单臂上缺陷基因表达的DNA。 这种技术的一个变种，“cdna捕获”，也可以用来 识别文库中与特定YAC同源的所有cDNA， 在人类基因组发展的稍后阶段出现的需求 主动权。 Cre-lox介导的位点特异性重组在基因工程中的应用 酵母菌的研究有两个目的。第一，有效地清除 将探索来自YAC的CENS和可选标记，以促进 在此之前设计的同源重组的遗传选择 求婚。IBIS将需要开发一个针对 鲑鱼网站。其次，通过特定地点的方式将青年会循环 我们将考察重组作为改变拓扑状态的工具 从酵母染色体中分离纯化YAC DNA DNAYAC的环化也将被用来产生分子 它可以在大肠杆菌中成功复制，第二种方法有助于 YAC DNA的分离。

The ability to isolate and maintain large fragments of DNA as artificial chromosomes in yeast (YACs) has ushered in a new era in the analysis of large genomes. Advances in this technology will be critical to the accomplishment of the goals put forth for the Human Genome Initiative. Improvements in the areas of generation and analysis of the libraries of human DNA will greatly facilitate progress towards meeting the goal of a contiguous ordered array of clones representing the entire human genome, the starting point for sequencing the genome. This proposal takes advantage of several useful features of yeast biology to advance these goals, in particular the extremely efficient pathway for homologous recombination. We propose to develop a set of YAC vectors that will allow several different types of genetic manipulation of YACs including chromosome walking using a genetic strategy. Walking will be accomplished by mating of a given YAC clone in yeast to a library of YACs in yeast made with suitable genetically marked vectors. Colonies containing pairs of YACs will be tested for the ability to undergo homologous recombination, allowing determination of those YACs containing overlapping stretches of genomic DNA. A novel in vivo method for the directed cloning of particular defined segments of genomic DNA will be explored. This strategy called "DNA capture" utilizes the ability of yeast to gap repair DNA between two distantly located but genetically linked probes using total uncut genomic DNA as its repair template. The probes, attached to YAC vectors, are introduced into the yeast cells along with uncut chromosomal DNA via transformation, followed by selection for stable inheritance of the YAC markers. This strategy will also be used to explore the non-enzymatic production of genomic YAC libraries by using repetitive DNA sequences instead of unique sequences as probes for the DNA capture. A strategy for the genetic identification of YACs homologous to unique non-repetitive probes win be developed. This method will utilize homologous recombination enhanced by induced double-stranded breaks in DNA to activate expression of a defective gene on one arm of a YAC clone. A variation of this technique, " cDNA capture", can also be employed to identify all cDNAs in a library that are homologous to a particular YAC, a need that will arise later in the development of the Human Genome Initiative. The use of cre-lox-mediated site-specific recombination in yeast will be investigated for two purposes. First, efficient removal of CENs and selectable markers from YACs will be explored to facilitate the genetic selections for homologous recombination designed earlier in this proposal. Ibis will be entail developing a negative selection against lox sites. Secondly, circularization of YACs via site-specific recombination will be examined as a tool to alter the topological state of YACs to facilitate purification of YAC DNA away from yeast chromosomal DNA. Circularization of YACs will also be used to generate molecules that can successfully replicate in E. coli, a second method facilitating the isolation of YAC DNA.