Integrity of human YACs during propagation in recombination-deficient yeast strains.
Integrity of human YACs during propagation in recombination-deficient yeast strains.
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人类 YAC 在重组缺陷酵母菌株中繁殖过程中的完整性。
DOI：
10.1006/geno.1998.5727
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Larionov,V
中科院分区：
文献类型：
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作者：
Kouprina,N;Nikolaishvili,N;Graves,J;Koriabine,M;Resnick,MA;Larionov,V
研究了几种具有重组/修复基因缺陷的等基因菌株（RAD1、RAD50、RAD51、RAD52、RAD54和rad55）准确繁殖多种含有人类DNA插入物的线状和圆形酵母人工染色体（YACs）的能力。为了评估YAC的稳定性，用ade2 - pbr - ura3cassette对人DNA插入物进行内部标记。在含有5-氟糖酸的培养基上对Ura -克隆进行筛选后，发现了以下类型的YAC缺失：(i)由端粒序列同源重组引起的缺失；（ii）内部缺失，假定是由常见DNA重复序列（如aluandlinesequences）之间的重组而发生的；以及（iii）缺失导致部分YAC臂的丧失。rad52host菌株，而不是其他重组缺陷菌株，使所有类型的YAC缺失率降低了25- 400倍。我们还开发并测试了具有条件rad52基因的kar1菌株，该菌株允许将YAC从任何宿主转移到重组缺乏的背景中。这些菌株为稳定YACs提供了有效的工具，并有助于对YACs进行额外的重组修饰。
Several isogenic strains with defects in recombination/repair genes (RAD1, RAD50, RAD51, RAD52, RAD54,andRAD55) were examined for their ability to propagate accurately a variety of linear and circular yeast artificial chromosomes (YACs) containing human DNA inserts. To assess YAC stability, the human DNA inserts were internally marked by anADE2-pBR-URA3cassette. Following selection for Ura−clones on 5-fluoroorotic acid containing medium, the following types of YAC deletions were identified: (i) those caused by homologous recombination with a telomericpBRsequence; (ii) internal deletions, presumed to occur by recombination between commonly occurring DNA repeats such asAluandLINEsequences; and (iii) deletions leading to loss of part of a YAC arm.rad52host strains, but not other recombination-deficient strains, decreased the rate of all types of YAC deletions 25- to 400-fold. We have also developed and testedkar1strains with a conditionalRAD52gene that allow transfer of a YAC from any host into a recombination-deficient background. These strains provide an efficient tool for stabilization of YACs and are useful for allowing additional recombinational modification of YACs.