IMPROVING THE REPLICATION AND SEGREGATION OF YAC CLONES

使用酵母人工染色体（YAC）作为克隆载体， 人类基因组的分析受到高频率的严重阻碍， 克隆缺口和不稳定的YAC， YAC不稳定的原因。 然而，我们认为重组是 由DNA断裂引发，DNA断裂是由于 染色体复制或分离。 的存在或不存在 偶然模仿功能性酵母的人类插入序列 染色体因素会导致这些缺陷。 特别是， 人类基因组中富含GC的基因区域， 预期在可用作复制的序列中存在缺陷 在酵母中的起源染色体的不完全复制 有丝分裂会产生断裂的分子。 这一问题将进一步加剧 由于复制叉屏障或元件的偶然存在， 延迟起源激活直到5阶段的后期，偶然序列 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。断裂事件导致删除和重排 最终通过重组而稳定下来 消除 重组不会消除基本问题。 识别和 消除断裂的原因是必要的。 我们建议开发简单的人类基因组DNA的质粒分析，使用 酵母的功能测试，以检测起源缺陷以及 存在偶然的着丝粒，复制叉障碍和晚期 起始决定因素这些试验将用于评估 这些障碍对稳定的人YAC克隆的贡献通过（i） 筛选人X或粘粒克隆以确定每个克隆的频率。 人类基因组中的YAC克隆障碍类型。 我们将使用随机 克隆和那些来自基因丰富的GC isochore。(2)测试这些 人类基因组特定区域的不同YAC克隆障碍 作为稳定的YACS是不可克隆的，但作为lambda或 粘粒克隆 我们建议，然后测试方法，以减轻YAC的不稳定性 这些偶然因素的存在或缺失所带来的问题。 通过构建第二个YAC文库，很容易解决着丝粒问题 使用缺乏着丝粒的载体臂。大多数其他问题可以 通过使用具有减少的序列的酵母菌株来减轻 特异性的起源识别，从而创造更多的起源， 人体插入物。 许多候选酵母菌株已经存在。

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.