DESIGN AND CONSTRUCTION OF HUMAN ARTIFICIAL CHROMOSOME VECTORS

人类人工染色体载体的设计和构建

• 批准号: 6162551
• 负责人: P P LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 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/6162551
• 关键词: biotechnology; chromosomes; fusion gene; gene expression; gene targeting; genetic library; genetic manipulation; genetic markers; nucleic acid sequence; regulatory gene; technology /technique development; tissue /cell culture; transfection /expression vector

In our effort to generate a suitable yeast host for human artificial chromosomes, we have modified the template region of the S. cerevisiae telomerase RNA gene (TLC1) to produce (TTAGGG)n, the human telomere sequence. Yeast strains with the tlc1-human allele are viable with no growth retardation, and express the humanized gene at a level comparable to wildtype. Southern hybridization and sequencing have demonstrated the addition of TTAGGG repeats to the ends of the endogenous yeast chromosomes and a yeast artificial chromosome. These strains will be useful for human artifical chromosome assembly and propagation, and will provide an important tool for studying the function of both yeast and human telomerases and their regulation by telomere binding proteins. The significance of these findings is three-fold: (1) this is the first report of one species producing functional telomeres of a different species, especially considering that the human telomere sequence is quite different from that of the yeast; (2) our data strongly suggest that the irregularity of the yeast telomere sequence, (TG1-3)n is determined entirely by the template and not by an intrinsic mechanistic nature of the protein component of the telomerase; and (3) these haploid strains could potentially serve as ideal hosts for the assembly and propagation of human artificial chromosomes since human telomere sequences will be added and maintained during the process. We will introduce our human artificial chromosome constructs into the yeast strains with the humanized telomerase template, propagate, and purify the construct DNA. The DNA will then be transfected into human cells in culture and its ability to form artificial chromosomes will be compared with constructs propagated in wild type yeast cells. In addition, the yeast strains with humanized telomerase template will be used to study the regulation of telomere synthesis and stabilization by telomere binding proteins.

在我们努力为人类人工合成合适的酵母宿主的过程中 染色体，我们已经修饰了酿酒酵母的模板区 端粒酶RNA基因(TLC1)产生(TTAGGG)n，人端粒 序列。携带tlc1-人类等位基因的酵母菌株在没有 生长迟缓，并在可比的水平上表达人源化基因 变成野人。Southern杂交和测序已经证明 在内源酵母末端添加TTAGGG重复序列 染色体和酵母人工染色体。这些菌株将是 有助于人类人工染色体的组装和繁殖，并将 为研究酵母和酵母的功能提供了重要的工具 人类端粒酶及其端粒结合蛋白的调控。这个 这些发现的意义有三个方面：(1)这是第一次 一种产生不同功能端粒的物种的报告 物种，特别是考虑到人类端粒序列 与酵母菌不同；(2)我们的数据强烈表明 测定了酵母端粒序列(TG1-3)n的不规则性 完全通过模板，而不是通过 端粒酶的蛋白质成分；以及(3)这些单倍体菌株 可以潜在地用作组装和传播的理想主机 因为人类端粒序列将是 在此过程中添加和维护。我们将介绍我们的人类 人工染色体被构建到酵母菌株中 人源化端粒酶模板，扩增和纯化构建的DNA。 然后，DNA将被导入培养中的人类细胞，其 形成人造染色体的能力将与构建进行比较 在野生型酵母细胞中繁殖。此外，酵母菌与 人源化端粒酶模板将用于研究端粒酶的调节 端粒结合蛋白对端粒的合成和稳定作用。