IMPROVING THE REPLICATION AND SEGREGATION OF YAC CLONES

改善 YAC 克隆的复制和分离

• 批准号: 2519135
• 负责人: BONITA J BREWER
• 金额: $ 17.37万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519135
• 关键词: DNA replication; DNA replication origin; artificial chromosomes; chromosome aberrations; chromosome movement; gene frequency; genetic library; genetic mapping; genetic recombination; genetic regulatory element; genetic techniques; genome; human genetic material tag; molecular cloning; nucleic acid hybridization; nucleic acid repetitive sequence; plasmids; transfection /expression vector

The use of yeast artificial chromosomes (YACs) as vectors for cloning and analysis of the human genome is seriously hampered by high frequencies of cloning gaps and unstable YACs, Recombination is often cited as the cause of YAC instability. However, we propose that the recombination is initiated by DNA breaks that occur as a consequence of defects in chromosome replication or segregation. The presence or absence of sequences in human inserts that fortuitously mimic functional yeast chromosomal elements would contribute to these defects. In particular, GC-rich regions of the human genome that are abundant in genes are expected to be deficient in sequences that can be used as replication origins in yeast Incomplete replication of chromosomes at the time of mitosis will generate broken molecules. This problem will be exacerbated by the fortuitous presence of replication fork barriers or elements that delay origin activation until late in the 5-phase, Sequences fortuitously recognized as centromeres by yeast will create dicentric YACs that also break at mitosis. Breakage events lead to deletions and rearrangements that are eventually stabilized by recombination. Eliminating recombination will not eliminate the basic problem. Identifying and eliminating the cause of the breaks are essential. We propose to develop simple plasmid assays of human genomic DNA, using functional tests in yeast, to detect origin deficiency as well as the presence of fortuitous centromeres, replication fork barriers and late initiation determinants. These assays will be used to assess the contribution of these impediments to stable human YAC cloning by (i) Screening human X or cosmid clones to determine the frequency of each type of YAC cloning barrier in the human genome. We will use random clones and those from gene-rich GC isochores. (2) Testing for these different YAC cloning barriers in specific regions of the human genome that have been unclonable as stable YACS but which exist as lambda or cosmid clones. We propose to then test approaches for alleviating the YAC instability problems posed by the absence or presence of these fortuitous elements. The centromere problem is easily solved by making a second YAC library using vector arms lacking a centromere. Most of the other problems can be alleviated by using yeast strains that have a reduced sequence specificity for origin recognition, thereby creating more origins in the human insert. Many candidate yeast strains already exist.

利用酵母人工染色体(YAC)作为载体进行克隆和 人类基因组的分析受到高频的严重阻碍 在克隆差距和不稳定的YAC中，重组经常被引用为 YAC不稳定的原因。然而，我们认为重组是 由DNA断裂引发，这些DNA断裂是由于 染色体复制或分离。存在或不存在 人类插入片段中偶然模仿功能性酵母的序列 染色体元素会导致这些缺陷。特别是， 人类基因组中基因丰富的GC富集区有 预计在可用作复制的序列中存在缺陷 起源于酵母菌当时染色体的不完全复制 有丝分裂会产生破碎的分子。这个问题将进一步恶化。 由于复制分叉障碍或元素的偶然存在， 将原点激活推迟到5-阶段的后期，偶然地进行序列测定 被酵母识别为着丝粒的YAC将产生双着丝粒YAC 在有丝分裂时断裂。破损事件导致删除和重新安排 最终通过重组而稳定下来。消除 重组不会消除根本问题。识别和 消除断裂的原因是至关重要的。 我们建议开发简单的人类基因组DNA的质粒法，使用 酵母中的功能测试，以检测来源缺陷以及 存在偶然的着丝粒、复制分叉障碍和迟发 启动决定因素。这些检测将被用来评估 这些障碍对稳定的人YAC克隆的贡献(I) 筛选人类X或粘粒克隆以确定每个克隆的频率 人类基因组中YAC克隆障碍的类型。我们将使用随机 克隆和来自富含基因的GC等值线的克隆。(2)测试这些项目 人类基因组特定区域的不同YAC克隆障碍 已经不能作为稳定的YAC克隆，但以lambda或 宇宙飞船的克隆人。 我们建议然后测试缓解YAC不稳定的方法 因这些偶然因素的缺失或存在而引起的问题。 通过建立第二个YAC文库，着丝粒问题很容易得到解决 使用缺少着丝粒的载体臂。大多数其他问题都可以 通过使用序列减少的酵母菌株来缓解 原产地识别的专一性，从而在 人体插入物。许多候选酵母菌株已经存在。