The function and role of telomeric palindrom sequence newly found in tobacco chromosomes

烟草染色体中新发现的端粒回文序列的功能和作用

基本信息

批准号：
06804046
负责人：
YOSHIDA Kazuo
金额：
$ 1.09万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

The increasing DNA and linearlization of genomes (chromosome formation) are fundamental basis for divergence of eukaryotes from prokaryotes during evolution. The telomeres and telomerase are indispensable for eukaryotic linear chromosomes. Unfortunately, little is known the molecular structure of telomeres in plants. In this research project, we have found and sequenced novel RETS (restriction enzymes-resistant high molecular weight telomeric DNA sequences) in tabacco chromosomes. By using RETS as a probe, we characterized the terminal structure of tobacco chromosomal ends. Furthermore, Southern analysis clearly indicates that RETS are spread out all over the tobacco chromosomes. From these results, we have presented a working hypothesis that RETS offer a seed for telomere formation in the repair of chromosomal damages. In order to know functions of telomere, we constructed the first mobilizable YAC (yeast artificial chromosome) in which oriT (origin of transfer) is inserted between two telomeres. This plasmid was successfully transferred from Escherichia coli bacterium into Saccharomyces cerevisae yeast with the help of mob (mobilization) and tra (transfer) genes on a helper plasmid. However, the transconjugant yeasts had not linear YACs but circular YACs although OriT between telomeres was nicked during trans-kingdom conjugation. This indicates that 5'end of linearlized YAC might be blocked or anchored by something like mob protein moiety which locates in a conjugation tube.

DNA 的增加和基因组的线性化（染色体形成）是进化过程中真核生物与原核生物分化的基本基础。端粒和端粒酶是真核线性染色体不可缺少的。不幸的是，人们对植物端粒的分子结构知之甚少。在这个研究项目中，我们在烟草染色体中发现了新的 RETS（限制性内切酶抗性高分子量端粒 DNA 序列）并对其进行了测序。通过使用 RETS 作为探针，我们表征了烟草染色体末端的末端结构。此外，Southern 分析清楚地表明 RETS 分布在整个烟草染色体上。根据这些结果，我们提出了一个可行的假设，即 RETS 为修复染色体损伤提供了端粒形成的种子。为了了解端粒的功能，我们构建了第一个可移动的YAC（酵母人工染色体），其中oriT（转移起点）插入到两个端粒之间。借助辅助质粒上的 mob（动员）和 tra（转移）基因，该质粒成功地从大肠杆菌转移到酿酒酵母中。然而，转接合酵母具有的不是线性 YAC，而是环状 YAC，尽管端粒之间的 OriT 在跨界接合过程中被切口。这表明线性化 YAC 的 5' 端可能被位于接合管中的 mob 蛋白部分等物质阻断或锚定。