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

利用NSING基因打靶技术建立了11号染色体P53基因座带有neo-tk盒的小鼠胚胎干细胞系。这些ES细胞系首次提供了一个研究染色体同源重组现象的系统。已经证明，该方法包括两个步骤的基因打靶，普遍能够用任何DNA序列替换基因。在该方法的发展过程中，基因转化被观察到是完全意想不到的伪同源重组体的克隆。大约25%的p53基因靶向克隆是假同源重组体。此外，野生型(W)和靶向型(T)等位基因在W/T杂合子中也被识别为重组修复。这种重组修复一直发生在两个方向上。这些成果已经发表(Gondo等人，BioChem.BiPhys.Res.Comm.，1994)Three Possib…对于重组修复的分子机制，已经提出了更多的LE模型。其一是p53基因及其邻近区域的切除-修复型基因转化。第二个可行的假设是，在细胞周期的S时期之后，两个同系物的姐妹染色单体之间进行有丝分裂重组，然后将相同的等位基因分离到相同的子细胞。最后一个模型假定同源的两个姐妹染色单体在有丝分裂时不分离，导致整个染色体获得纯合性，就像在单亲二体中发现的那样。为了检验这三个工作假说，有必要建立一个高度多态的ES细胞系，该细胞系具有分子标记，使我们能够区分这些多态。因此，从熊本大学的S.Aizawa博士那里获得了一个这样的多态ES细胞系TT2。用CBA和C57BL/6两个近交系的杂合子建立了TT2 ES细胞系。再次进行基因打靶，新建立了17个携带neotk盒的TT2 ES细胞克隆。此外，还对P53基因所在的11号染色体上的分子标记进行了研究，迄今已发现4个SSLP标记在CBA和C57BL/6之间具有多态性。利用该系统，可以研究哺乳动物基因组中特定染色体区域的同源重组的分子机制。11号染色体上更多的多态标记目前正在研究中。较少

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