Construction of mammalian artificial chromosomes using YAC

使用 YAC 构建哺乳动物人工染色体

• 批准号: 08044200
• 负责人: OKAZAKI Tuneko
• 金额: $ 1.73万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044200/
• 关键词: cetromere; teloreme; alphoid DNA; YAC; MAC; CENP-B; CENP-B box

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 intormation on the essential DNA structure is available until now. The purpose of this work is to clone functional human centromere DNA using technology of the yeast artificial chromosome (YAC). We have found previously two megabase-sized domains of alphoid DNA,alpha21-I and alpha21-II,in the human chromosome 21. The alpha21-I contained many CENP-B boxes, binding sites of CENP-B,at high and regular intervals, while the alpha21-II contained very few, if any. We have cloned into YAC 100 kb alphoid DNA derived from alpha21-Iand alpha21-II domain using a recombination minus yeast strain. In collaboration with Dr.Cooke, we constructed YAC arm vectors containing human telomere sequences and selectable markers for mammalian cells and both arms of alphoid YAC were replaced with these arms by recombination in vivo. We transformed a cultured human cell line with these human telomere and alphoid containing YAC and obtained transformants with both YACs. FISH analyzes of the transformed cell lines showed that transformants of alpha21-I YAC contained a stably maintained mammalian artificial chromosome but those of alpha21-II YAC was integrated at telomere or centromere. These results indicated that the essential centromere DNA sequence resides in alpha21-I region.

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中着丝粒结构的主要决定因素，但迄今为止，对DNA的基本结构还没有确定的信息。本工作的目的是利用酵母人工染色体（YAC）技术克隆具有功能的人着丝粒DNA。我们先前已经在人类21号染色体中发现了两个兆碱基大小的α DNA结构域，α 21-I和α 21-II。alpha 21-I含有许多CENP-B盒，CENP-B的结合位点，以高和规则的间隔，而alpha 21-II含有很少，如果有的话。我们用重组减酵母菌株将来自α 21-I和α 21-II结构域的100 kb α DNA克隆到YAC中。与Cooke博士合作，我们构建了含有人端粒序列和哺乳动物细胞选择性标记的YAC臂载体，并通过体内重组用这些臂替换α YAC的两臂。我们用这些含有人端粒和α的YAC转化培养的人细胞系，并获得具有两种YAC的转化体。对转化细胞系的FISH分析表明，α 21-I YAC的转化体含有稳定维持的哺乳动物人工染色体，而α 21-II YAC的转化体整合在端粒或着丝粒处。这些结果表明，必需的着丝粒DNA序列位于α 21-I区。

项目成果

K.Yoda: "Site specific base deletions in human α-satellite monomer DNAs are associated with regularly distributed CENP-B boxes." Chromosome Res.(in press). (1997)

K. Yoda：“人类 α-卫星单体 DNA 中的位点特异性碱基缺失与规则分布的 CENP-B 盒相关。（正在出版）。”

岡崎恒子: "セントロメアの構造" 蛋白質・核酸・酵素. 41・15. 74-84 (1996)

冈崎常子：“着丝粒的结构”蛋白质、核酸和酶 41・15（1996）。

K.Yoda, T.Nakamura, H.Masumoto, N.Suzuki, K.Kitagawa, M.Nakano, A.Shinjo and T.OKazaki: "Centromere protein B of African Green Monkey cells ; Genestructure, cellular expression and centromeric localization." Mol.Cell.Biol.16. 5169-5177 (1996)

K.Yoda、T.Nakamura、H.Masumoto、N.Suzuki、K.Kitakawa、M.Nakano、A.Shinjo 和 T.OKazaki：“非洲绿猴细胞的着丝粒蛋白 B；基因结构、细胞表达和着丝粒定位。

K.Yoda T.Okazaki: "site specific base deletions in human alpha-satellite monomer DNAs are associated with regularly distributed CENP-B boxes." Chromosome Res.(in press). (1997)

K.Yoda T.Okazaki：“人类 α-卫星单体 DNA 中的位点特异性碱基缺失与规则分布的 CENP-B 盒相关。”

K.Yoda: "Site specific base deletions in human α-satellite monomer DNAs are associated with regularly distributed CEDNP-B boxes." Chromosome Res.(in press). (1997)

K. Yoda：“人类 α-卫星单体 DNA 中的位点特异性碱基缺失与规则分布的 CEDNP-B 盒相关。”（正在出版）。