Kouprina N, Eldarov M, Moyzis R, Resnick M, Larionov V
Laboratory of Molecular Genetics, NIEHS, Research Triangle Park, North Carolina 27709.
Genomics. 1994 May 1;21(1):7-17. doi: 10.1006/geno.1994.1218.
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.
含有哺乳动物DNA的酵母人工染色体（YACs）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之间的相互作用所致。为了研究参与此类重排的因素，我们开发了一种能够标记物理变化的遗传系统。一个Alu-HIS3-Alu盒已被定位到一个有丝分裂稳定的YAC上，该YAC含有一条360kb的人类2号染色体DNA插入片段。检测了五个在不同位置整合了该盒的YAC在转化为酵母及后续生长过程中内部HIS3标记的丢失情况。在有丝分裂生长的细胞中，内部标记丢失的平均频率约为1.0×10⁻⁴。对保留两个端粒标记的His⁻ YAC进行物理分析表明，标记的丢失是由于缺失（20 - 90kb）。这些结果与转化后YACs所得到的结果形成对比。近33%的重新转化的YACs缺乏内部HIS3标记。与转化相关的丢失也是由于80至260kb不等的缺失。用亲本人类YAC和另一个含有390kb小鼠DNA插入片段的有丝分裂稳定YAC进行重新转化后，也得到了类似的结果。当宿主是缺失RAD52基因的重组缺陷菌株时，人类YACs中与转化相关的高缺失水平降低了10倍以上。与同基因的Rad⁺菌株相比，rad52突变菌株中有丝分裂生长期间人类YAC内部的不稳定性水平也显著降低。然而，用含有小鼠DNA的YAC对rad52突变体进行重新转化，产生的改变水平与野生型菌株所观察到的相当。因此，在YACs中与转化相关的缺失肯定还有其他遗传因素参与。我们认为这些YACs和菌株可作为研究YAC完整性的有用工具。在这些研究过程中，在有丝分裂繁殖的YACs中发现了一类独特的缺失，它是由端粒区域和HIS3盒中相同序列之间的重组导致的。除了已知的YAC“断裂”方法外，这可能提供一种产生内部缺失的手段以及一种替代的定位方法。
