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: 3755484
• 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/3755484
• 关键词: artificial chromosomes; chromosome deletion; fungal genetics; gene rearrangement; genetic library; genetic mapping; genetic recombination; genome; human genetic material tag; molecular cloning

The cloning of large chromosomal fragments is required for the physical mapping and sequencing of large genomes, particularly those of various mammals including humans. Systems developed in yeast that are based on artificial chromosome vectors (YACs) have provided the means for isolating the requisite large DNAs. It is essential for the accurate chracterization and manipulation of genomic material that the DNA within YACs be accurate, that there not be cloning artifacts and that the YACs be stable within yeast. However, two major categories of errors have been shown to arise during the development and the maintenance of human YAC libraries. It is estimated that 10 to 60% of clones in existing libraries represent co-cloned DNA sequences. A second category of errors resulting from internal rearrangements and deletions in YACs also limits the utility of YAC systems. Many deletions in YACs containing human DNA could arise during transformation by recombination between repeated DNAs that may be diverged. We are examining the contribution of yeast transformation to YAC rearrangements. We have shown that mitotically stable YACs can exhibit frequent internal deletions upon retransformation. Our observations with YACs are comparable to results with plasmids containing homologous and diverged repeat DNAs. Recombination during transformation of covalently closed circular plasmids was over 50-fold more frequent than during mitotic growth. When present in the repeats, unique single-strand breaks that are ligatable lead to a high level of recombination between diverged and identical repeats. Based on the results with model plasmids, we propose that internal deletions in YACs observed in retransformation experiments are due to recombination induced by nicks in the YAC DNA. While it remains to be determined, recombination could occur between Alu's. A high level of transformation-associated deletions in the human YACs as well as in plasmids was reduced when the host was a recombination-deficient strain deleted for the RAD52 gene.

克隆大的染色体片段是物理上 大型基因组的定位和测序，特别是各种 哺乳动物，包括人类。 在酵母中开发的系统基于 人工染色体载体（YAC）提供了 分离出必需的大型DNA 这是至关重要的准确 对基因组物质进行表征和操作， YAC是准确的，没有克隆文物，YAC 在酵母中保持稳定。 然而，有两大类错误 在人类的发展和维持过程中， YAC图书馆。 据估计，10%至60%的克隆在现有的 文库代表共克隆的DNA序列。 第二类错误 由于YAC的内部重排和删除， YAC系统的实用性。 含有人DNA的YAC中的许多缺失 在转化过程中，通过重复DNA之间的重组 这可能是分歧的。 我们正在研究酵母的作用 转型为YAC重组。 我们已经证明， 稳定的YAC可以表现出频繁的内部缺失， 再转化 我们对YAC的观察结果与 用含有同源和分歧重复DNA的质粒。 共价闭合环转变过程中的结晶 质粒比有丝分裂生长期间多50倍以上。 当 存在于重复序列中，可连接的独特单链断裂 导致了分歧和相同之间高水平重组 重复。 基于模型质粒的结果，我们提出， 在再转化实验中观察到的YAC中的内部缺失是 这是由于YAC DNA中的切口诱导的重组。 这个问题仍然没有 要确定的是，Alu之间可能发生重组。 高水平 在人类YAC中的转化相关缺失，以及在 当宿主为重组缺陷型菌株时， RAD52基因缺失。