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Cloning of functional human centromere DNA by YAC technology

YAC技术克隆功能性人类着丝粒DNA

基本信息

批准号：
07044191
负责人：
OKAZAKI Tuneko
金额：
$ 1.92万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044191/
关键词：
centromere telomere artificial chromosome YAC alphoid DNA CENP-B CENP-B box

项目摘要

The purpose of this work is to clone functional human centromeric DNA using the technology of the yeast artificial chromosome (YAC).Centromere is the chromosomal domain essential to the segregation of eukaryotic chromosomes. Although DNA is believed to be the primary determinant of the functional centromere structure in mammalian cells, no conclusive information on the essential DNA structure is available until now. To solve this problem, we previously analyzed long range organization of the centromeric satellite DNA of the human chromosome 21, in specific emphasis on the distribution of alphoid DNA containing the recognition sequence of CENP-B,the CENP-B box. We have found two megabase-sized domains of alphoid DNA (alpha21-I and alpha21-II) in the chromosome 21 : alpha21-I,contained many CENP-B boxes at regular intervals and alpha21-II containing very few, if any, CENP-B boxes. In this international collaboration with Dr.P.Hieter, we performed the cloning into YAC of 100 kb size DNA from the alpha21-I and alpha21-II region of the chromosome 21. We found that these alphoid YAC clones were stably maintained in the recombination minus yeast strains. We then introduced the human telomere sequences into the two vector arms of the alphoid YAC clones so that these YAC clones could be maintained in the mammalian cells as mammalian artificial chromosomes (MAC), if they contained functional centromere DNA.We introduced resulting clones into a human cell line by microinjection and lipofection. We have obtained a few transformants with both alpha21-I YAC and alpha21-II YAC.We are currently analyzing intranuclear state of incorporated alphoid YACs.

本工作的目的是利用酵母人工染色体（YAC）技术克隆功能性人类着丝粒DNA。着丝粒是真核生物染色体分离所必需的染色体结构域。虽然DNA被认为是哺乳动物细胞功能着丝粒结构的主要决定因素，但迄今为止还没有关于基本DNA结构的结论性信息。为了解决这个问题，我们之前分析了人类21号染色体的着丝粒卫星DNA的远程组织，特别强调了包含CENP-B识别序列的阿尔法体DNA的分布，即CENP-B盒。我们在21号染色体上发现了两个巨型酶大小的阿尔法DNA结构域（alpha21-I和alpha21-II）： alpha21-I定期包含许多CENP-B盒子，而alpha21-II包含很少（如果有的话）CENP-B盒子。在与p。随后，我们将21号染色体alpha21-I和alpha21-II区100 kb大小的DNA克隆到YAC中。我们发现这些阿尔法型的YAC克隆在重组后的酵母菌株中稳定地维持着。然后，我们将人类端粒序列引入到alpha型YAC克隆的两个载体臂中，如果这些YAC克隆含有功能性着丝粒DNA，则这些YAC克隆可以作为哺乳动物人工染色体（MAC）在哺乳动物细胞中维持。我们通过显微注射和脂肪转染将得到的克隆导入人类细胞系。我们已经获得了几个同时具有alpha21-I和alpha21-II YAC的变形子。我们目前正在分析合并蛋白YACs的核内状态。