RECOMBINATION DURING TRANSFORMATION AS A SOURCE OF CHIMERIC MAMMALIAN ARTIFICIAL CHROMOSOMES IN YEAST (YACS)
RECOMBINATION DURING TRANSFORMATION AS A SOURCE OF CHIMERIC MAMMALIAN ARTIFICIAL CHROMOSOMES IN YEAST (YACS)
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DOI：
10.1093/nar/22.20.4154
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发表时间：
1994-10-11
影响因子：
14.9
通讯作者：
RESNICK, MA
中科院分区：
文献类型：
--
作者：
LARIONOV, V;KOUPRINA, N;RESNICK, MA
在酵母中以人工染色体形式克隆的哺乳动物DNA(YAC)通常是在非连续DNA之间形成的嵌合体。使用人和小鼠的YAC对，我们研究了转化过程中的重组或随后的有丝分裂生长对嵌合YAC形成的贡献。从含有同源或异源YAC的酵母菌株对的DNA在典型的YAC文库开发条件下转化为第三个菌株。选择一个YAC，然后确定第二个的存在。大分子的共渗透率是50%，这是通过识别不同YAC的标记的共转化而得出的。在大约一半接受两个同源YAC的细胞中，YAC发生了重组。共转化依赖于重组，因为当YAC是异源时，共转化减少了近10倍。虽然同源YAC之间的有丝分裂重组比酵母染色体高出近100倍，但仍远低于转化过程中观察到的水平。为了研究常见的Alu重复序列在嵌合体形成中的作用，用不同的人YAC和一段未选择的DNA片段转化球体体，DNA片段的一端含有Alu，另一端含有端粒。当使用未断裂的YAC时，1%至6%的所选YAC可以整合片段，而当YAC被破坏时，这一比例为49%。我们认为Alu‘s或其他常见的重复序列可能是嵌合YAC的一个重要来源。由于当RAD52突变体为受体时，YAC或YAC与含有Alu的片段之间形成的嵌合体的频率降低，并且由于YAC内的缺失减少，RAD52和可能的其他重组缺陷突变体有望对YAC文库的开发有用。
Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) frequently are chimeras formed between noncontiguous DNAs. Using pairs of human and mouse YACs we examined the contribution of recombination during transformation or subsequent mitotic growth to chimeric YAC formation. The DNA from pairs of yeast strains containing homologous or heterologous YACs was transformed into a third strain under conditions typical for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large molecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving two homologous YACs, the YACs had undergone recombination. Co-transformation depends on recombination since it was reduced nearly 10-fold when the YACs were heterologous. While mitotic recombination between homologous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To investigate the role of commonly occurring Alu repeats in chimera formation, spheroplasts were transformed with various human YACs and an unselected DNA fragment containing an Alu at one end and a telomere at the other. When unbroken YACs were used, between 1 and 6% of the selected YACs could incorporate the fragment as compared to 49% when the YACs were broken. We propose that Alu's or other commonly occurring repeats could be an important source of chimeric YACs. Since the frequency of chimeras formed between YACs or a YAC and an Alu-containing fragment was reduced when a rad52 mutant was the recipient and since intra-YAC deletions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development.