HUMAN GENOME PROJECT--ARTIFICIAL CHROMOSOME STABILITY AND MAPPING IN YEAST

人类基因组计划--酵母人工染色体稳定性和图谱

• 批准号: 3841141
• 负责人: M A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3841141
• 关键词: DNA repair; DNA replication; DNA replication origin; artificial chromosomes; chemical stability; fungal genetics; genetic mapping; genetic recombination; genome; human genetic material tag; molecular cloning; nucleic acid repetitive sequence

The mapping and sequencing of the human genome is a world-wide effort with many anticipated health benefits. A cornerstone of the Human Genome Project (HGP) is the use of yeast artificial chromosome (YAC) vectors for the cloning of large chromosome fragments. It is essential for the HGP that human DNA within YACs be stable within yeast. A major source of artefacts as we have shown arises from the repetitive nature of the DNA. These are potential substrates for co-cloning events and transformation- associated and mitotic recombination since recombination between diverged DNAs has been demonstrated in yeast. Some YACs exhibit considerable internal recombinational repair. We have suggested that the extent of repeats, even if diverged, may be indicated by the sensitivity of YACs to ionizing radiation induced loss and that the survival of YACs is dictated by the amount of recombinational repair between the diverged DNAs (currently under study). Small repeats that surround large inverted repeats (such as Alu's) may be sites of excision, as we have shown for Tn5 DNA in yeast. Problems in replication may also contribute significantly to YAC instability. Two potential sources of replication problems are, 1) insufficient functional origins of replication in the long tracts of mammalian DNA, and 2) possible deletions of sequences flanked by inverted repeats, which have been shown to facilitate replication bypass. We have shown that specific replication mutatants can enhance recombination between large repeats in chromosomes and can stimulate up to 100-fold excision involving small repeats in Tn5. The goals of the project are, 1) improvement of cloning methods and recipient strains, 2) improvement of YAC stability utilizing replication and recombination mutants and gene products, and 3) development of in vivo fragmentation systems for the precise physical mapping of sequences of interest within YACs. We are also merging YAC cloning systems with mammalian viral cloning systems for the easy transfer of large fragments to human cells.

人类基因组的测绘和测序是世界范围内的一项努力， 许多人预计会给健康带来好处。人类基因组的基石 项目(HGP)是使用酵母人工染色体(YAC)载体 大染色体片段的克隆。对于人类基因组计划来说是必不可少的 YAC中的人类DNA在酵母中是稳定的。一个主要的来源 正如我们所展示的那样，人工制品来自DNA的重复性质。 这些是共克隆事件和转化的潜在底物- 分叉间重组后的伴生和有丝分裂重组 DNA已在酵母中得到证实。一些YAC表现出相当大的 内部重组修复。我们已经提出， 重复，即使有分歧，也可以通过YAC对 电离辐射引起的损耗和YAC的生存是决定的 根据不同DNA之间的重组修复量 (目前正在研究中)。围绕着大反转的小重复 重复序列(如Alu)可能是切除的部位，就像我们对Tn5所展示的那样 酵母中的DNA。 复制中的问题也可能对YAC有很大影响 不稳定。复制问题的两个潜在来源是：1) 在长距离复制中，复制的功能起源不足 哺乳动物DNA，以及2)侧翼倒置的序列的可能缺失 重复，这已被证明有助于复制绕过。我们有 研究表明，特定的复制突变体可以促进重组 染色体中的大重复，可以刺激高达100倍的切除 涉及Tn5的小重复。 该项目的目标是：1)改进克隆方法和 受体菌株，2)利用复制提高YAC的稳定性 重组突变体和基因产物；3)体内研究进展 用于精确物理映射序列的分段系统 在青年会内的利益。我们还将YAC克隆系统与 哺乳动物病毒克隆系统可方便地将大片段转移到 人类细胞。