A MODEL SYSTEM TO ASSESS THE INTEGRITY OF MAMMALIAN YACS DURING TRANSFORMATION AND PROPAGATION IN YEAST
A MODEL SYSTEM TO ASSESS THE INTEGRITY OF MAMMALIAN YACS DURING TRANSFORMATION AND PROPAGATION IN YEAST
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DOI：
10.1006/geno.1994.1218
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发表时间：
1994-05-01
期刊：
影响因子：
4.4
通讯作者：
LARIONOV, V
中科院分区：
文献类型：
--
作者：
KOUPRINA, N;ELDAROV, M;LARIONOV, V
含有哺乳动物DNA的酵母人工染色体在转化和繁殖过程中可能会发生缺失，这可能是由于重复DNA之间的相互作用造成的。为了研究参与这种重排的因素，我们开发了一种可以发出物理变化信号的遗传系统。Alu-HIS3-Alu盒被定位于含有人2号染色体360kb DNA插入的有丝分裂稳定的YAC。研究了5个在不同位置整合了该盒的YAC在转化酵母和随后的生长过程中内部HIS3标记的丢失。在有丝分裂生长的细胞中，内部标记丢失的平均频率约为1.0x10(-4)。对保留两个端粒标记的His(-)YAC的物理分析表明，该标记的丢失是由于缺失(20-90kb)。这些结果与YAC变换后得到的结果形成了对比。近33%的重新转化的YAC缺乏内部HIS3标记。与转化相关的丢失也是由于80kb到260kb的缺失。用亲本人YAC和另一个含有390kb小鼠DNA插入片段的有丝分裂稳定的YAC进行再转化后，得到了类似的结果。当宿主是缺失RAD52基因的重组缺陷菌株时，人YAC中与转化相关的高水平缺失减少了10倍以上。与同基因Rad(+)株相比，rad52突变株在有丝分裂生长过程中的人YAC内部不稳定性水平也显著降低。然而，用含有YAC的小鼠DNA重新转化rad52突变体，产生的变化水平与观察到的野生型菌株相当。因此，YAC中与转化相关的缺失一定有额外的遗传因素参与。我们认为这些YAC和菌株可以作为研究YAC完整性的有用工具。在这些研究过程中，在有丝分裂传播的YAC中发现了一种独特的缺失类别，这种缺失是端粒区和HIS3盒中相同序列重组的结果。除了已知的YAC‘’碎片化‘’方法之外，这可以提供一种用于产生内部缺失的手段以及一种用于映射的替代方法。(C)1994年学术出版社。
Yeast artificial chromosomes (YACs) containing mammalian DNA potentially can undergo deletions during transformation and propagation, possibly due to interactions between repeat DNAs. To study factors involved in such rearrangements, we developed a genetic system that can signal physical changes. An Alu-HIS3-Alu cassette has been targeted to a mitotically stable YAC containing a 360-kb DNA insert of human chromosome 2. Five YACs with the cassette integrated at different positions were examined for loss of the internal HIS3 marker during transformation into yeast and subsequent growth. The average frequency of the internal marker loss in mitotically growing cells was approximately 1.0 x 10(-4). Physical analysis of His(-) YACs retaining both telomeric markers demonstrated that loss of the marker was due to deletions (20-90 kb). These results contrast with those obtained with YACs following transformation. Nearly 33% of the retransformed YACs lacked the internal HIS3 marker. The transformation-associated loss was also due to deletions varying from 80 to 260 kb. Similar results were obtained following retransformation with the parent human YAC and another mitotically stable YAC containing a 390-kb insert of mouse DNA The high level of transformation-associated deletions in the human YACs was reduced over 10-fold when the host was a recombination-deficient strain deleted for the RAD52 gene. The level of internal human YAC instability during mitotic growth was also significantly decreased in the rad52 mutant strain compared to that in the isogenic Rad(+) strain. However, retransformation of the rad52 mutant with a YAC-containing mouse DNA yielded comparable levels of alterations to those observed for the wildtype strain. Thus, there must be additional genetic factors involved in transformation-associated deletions in YACs. We propose that these YACs and strains can be useful tools for investigating YAC integrity. During the course of these studies a unique category of deletions was identified in mitotically propagated YACs that result from recombination between identical sequences in the telomeric region and the HIS3 cassette. In addition to the known YAC ''fragmentation'' method, this may provide a means for generating internal deletions as well as an alternative method for mapping. (C) 1994 Academic Press, Inc.