AMPLIFICATION OF LARGE ARTIFICIAL CHROMOSOMES

被引:73
作者
SMITH, DR [1 ]
SMYTH, AP [1 ]
MOIR, DT [1 ]
机构
[1] COLLABORAT RES INC,DEPT YEAST MOLEC BIOL,BEDFORD,MA 01730
关键词
cloning vectors; conditional centromere; thymidine kinase; yeast;
D O I
10.1073/pnas.87.21.8242
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Yeast artificial chromosome cloning is an attractive technology for genomic mapping studies because very large DNA segments can be readily propagated. However, detailed analyses often require the extensive application of blotting-hybridization techniques because artificial chromosomes are normally present at only one copy per haploid genome. We have developed a cloning vector and host strain that alleviate this problem by permitting copy number amplification of artificial chromosomes. The vector includes a conditional centromere that can be turned on or off by changing the carbon source. Strong selective pressure for extra copies of the artificial chromosome can be applied by selecting for the expression of a heterologous thymidine kinase gene. When this system was used, artificial chromosomes ranging from about 100 to 600 kilobases in size were readily amplified 10- to 20-fold. The selective conditions did not induce obvious rearrangements in any of the clones tested. Reactivation of the centromere in amplified artificial chromosome clones resulted in stable maintenance of an elevated copy number for 20 generations. Applications of copy number control to various aspects of artificial chromosome analysis are addressed.
引用
收藏
页码:8242 / 8246
页数:5
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