GENE CONVERSION ASSOCIATED WITH HOMOLOGOUS RECOMBINATION AND DNA REPAIR OF THE MOUSE.

利用nsing基因靶向技术，在小鼠胚胎干细胞（ES） 11号染色体p53位点上建立了携带neo-tk盒的细胞系。这些胚胎干细胞系首次为研究染色体同源重组现象提供了一个系统。已经证明，该方法包括两个步骤的基因靶向普遍能够取代基因与任何DNA序列。在该方法的开发过程中，基因转化被观察到为完全意想不到的伪同源重组的克隆。大约25%的p53基因靶向克隆是伪同源重组。此外，在W/T杂合子中，野生型(W)和靶型(T)等位基因之间也存在重组修复。这种重组修复在任何方向都一致地发生。这些成果已经发表（Gondo, et al., biochemistry . biophysics . res . common）。对于重组修复的分子机制，已经提出了三种可能的模型。一种是在p53基因的有限区域及其附近发生切除修复型的基因转换。第二个可行的假设是，在细胞周期的S期后，两个同源物的姐妹染色单体之间发生有丝分裂重组，随后相同的等位基因分离到相同的子细胞中。最后一个模型假定在有丝分裂时同源的两个姐妹染色单体没有分离，导致整个染色体的纯合性获得，就像在单亲二体中发现的那样。为了检验这三个工作假设，有必要有一个高度多态性的胚胎干细胞系与分子标记，使我们能够区分这些多态性。因此，从熊本大学的s . aizawa博士那里获得了这种多态性ES细胞系之一TT2。以CBA和C57BL/6两种自交系的杂合子建立TT2 ES细胞系。再次进行基因靶向，新建立了17个携带网络盒的TT2 ES细胞克隆。对p53基因所在的11号染色体上的分子标记也进行了研究，目前发现4个SSLP标记在CBA和C57BL/6之间存在多态性。利用该系统，研究哺乳动物基因组中特定区域染色体同源重组的分子机制成为可能。目前正在研究11号染色体上更多的多态标记。少

Mouse embryonic stem (ES) cell lines that carried a neo-tk cassette at the p53 locus on the chromosome 11 were established by nsing gene targeting. These ES cell lines for the first time provided a system to investigate the phenomena with respect to the homologous recombination of chromosomes. It has been demonstrated that the method encompassing two steps of gene targeting universally enabled to replace a gene with any DNA sequences. During the process of the development of this method, gene conversions were observed as clones of totally unexpected types of pseudo-homologous recombinants. Approximately 25% of the p53 gene-targeted clones were pseudo-homologous recombinants. In addition, the recombination repair has been also recognized between the wild-type (W) and targeted-type (T) alleles in theW/T heterozygote. This recombination repair occurred to either direction consistently. These achievements were already published (Gondo, et al., Biochem.Biophys.Res.Commun., 1994)Three possib … More le models have been proposed for the molecular mechanism of the recombination repair. One is the gene conversion of the excision-repair type at the limited region of the p53 gene and its vicinities. The second feasible hypothesis is the mitotic recombination between the sister chromatids of the two homologues after S period of the cell cycle and subsequent segregation of the same alleles to the same daughter cells. The last model postulates no segregation of two sister chromatids of a homologue at mitosis resulting a gain of homozygosity of the entire chromosome as found in uniparental disomy. In order the examine the three working hypotheses, it is necessary to have a highly polymorphic ES cell line with molecular markers which allow us to distinguish those polymorphisms. Thus, one of such polymorphic ES cell line, TT2, was obtained from Dr.S.Aizawa (Kumamoto University). The TT2 ES cell line was established from a heterozygote of two inbred strains, CBA and C57BL/6. The gene targeting was performed again and 17 of the TT2 ES cell clones carrying the neotk cassette were newly established. The molecular markers on the chromosome 11 on which the p53 gene is were also investigated and 4 SSLP markers have been found so far to be polymorphic between CBA and C57BL/6.By using this system, it became plausible to study the molecular mechanisms of the homologous recombination at a defined region of the chromosomes in the mammalian genome. More polymorphic markers on the chromosome 11 have been currently under investigation. Less