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: 3777554
• 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/3777554
• 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 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.

生理上需要克隆大的染色体片段