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.

使用酵母人工染色体（YAC）作为克隆和 人类基因组的分析受到高频的严重阻碍 克隆间隙和不稳定的YAC，通常将重组称为 YAC不稳定的原因。 但是，我们建议重组是 由DNA断裂引发的，由于缺陷而发生 染色体复制或隔离。 存在或不存在 人类插入的序列可幸运地模仿功能性酵母 染色体元素将导致这些缺陷。 尤其， 在基因中丰富的人类基因组的富含GC的区域是 预计将缺乏可以用作复制的序列 酵母中染色体不完整复制的起源 有丝分裂会产生断裂的分子。 这个问题将被加剧 通过偶然存在的复制叉障碍或元素 延迟原点激活直到5相中的后期，序列是偶然的 被酵母认可为Centromeres 有丝分裂中断。破损事件导致删除和重排 最终通过重组稳定。 消除 重组不会消除基本问题。 识别和 消除休息的原因至关重要。 我们建议使用人类基因组DNA开发简单的质粒测定 酵母中的功能测试，以检测起源不足以及 有偶然的centromeres，复制叉障碍和迟到 启动决定因素。这些测定将用于评估 这些障碍对（i）对稳定的人类克隆的贡献 筛选人X或宇宙克隆以确定每个克隆的频率 人类基因组中YAC克隆屏障的类型。 我们将使用随机 克隆和来自富基因的GC等形线的克隆。 （2）测试这些 人类基因组特定区域的不同YAC克隆屏障 作为稳定的YAC，但作为lambda或 宇宙克隆。 我们建议然后测试减轻YAC不稳定的方法 这些偶然元素的不存在或存在带来的问题。 通过制作第二个YAC库可以轻松解决中心粒问题 使用矢量臂缺乏中心粒。其他大多数问题都可以 通过使用降低序列的酵母菌菌株来缓解 原点识别的特异性，从而在 人插入。 许多候选酵母菌菌株已经存在。