构建覆盖三个基因组当量的酵母人工染色体（YAC）克隆文库，并分离出包含编码肿瘤坏死因子受体β的人类基因的YAC。

Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor beta.

作者信息

Deloukas P, Aigner L, Kania M, van Loon A P

机构信息

Pharmaceutical Research Department, F. Hoffman-La Roche Ltd., Basel, Switzerland.

出版信息

Gene. 1992 Jan 15;110(2):189-95. doi: 10.1016/0378-1119(92)90647-8.

DOI:10.1016/0378-1119(92)90647-8

Abstract

Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20,000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-R beta. These clones cover about 200 kb and have 170 kb in common. TNF-R beta is 9.3 kb long and contains two introns within the protein-coding sequence.

摘要

利用人类爱泼斯坦 - 巴尔病毒转化的B淋巴细胞系构建了酵母人工染色体（YAC）文库，其总共覆盖约三个单倍体基因组当量。构建了两个克隆文库：一个是使用pYAC4载体的20000个克隆，平均插入片段为350 kb；另一个是使用pJS89和pJS91载体的9850个克隆，平均插入片段为180 kb。将pYAC4与pJS89和pJS91进行直接比较，结果表明pYAC4是最合适的克隆载体。还构建了两个部分文库，分别是人内皮细胞DNA和上皮HEp2细胞DNA的平均插入片段大小为220 kb的文库，每个文库覆盖单倍体基因组的10%。开发了一种用于分离单个YAC克隆的快速三步PCR筛选程序，并用于鉴定两个编码TNF - Rβ的克隆。这些克隆覆盖约200 kb，共有170 kb。TNF - Rβ长9.3 kb，在蛋白质编码序列内含有两个内含子。