Studies on Mechanisms of DNA Rearrangements in Mammalian Cells.

(1)分裂酵母Schizosaccharomices pombe同源重组和细胞生长基因的分子分析。我们分离到了与出芽酵母酿酒酵母DST2基因同源的S. pombe基因，其产物具有DNA链交换活性。用S. cereviceae DST2的探针筛选pombe基因组文库，确定阳性克隆的核苷酸序列。在该克隆中发现了编码991个氨基酸的约3 kb的开放前导框，称为dhp1 （S. p-ombe的DST2同源物）。该产物具有DNA链交换活性，是有丝分裂细胞生长所必需的。此外，我们还分离出DST2的另一个同源物，称为dhp2。与dhpl相反，dhp2不是有丝分裂细胞生长所必需的。利用DST2同源物分析pombe的同源重组机制。(2)哺乳动物细胞同源重组基因的克隆与分析。利用对dhpl基因氨基酸序列的分析结果，分离出人CDNA进行同源重组，取得了成功。我们正在分析该人类CDNA及其产物的结构和功能。我们也在尝试分离小鼠基因进行同源重组和构建同源重组基因有缺陷的突变小鼠。(3) sv40衍生穿梭载体dna在哺乳动物细胞中同源重组的体内外研究。在有SV40大T抗原存在的情况下，将在galK^+标记两侧含有直接重复序列的SV40衍生穿梭载体与HeLa细胞质提取物一起孵育。用E. coligal^-检测载体dna是否形成galK缺失。使用该检测系统，galk缺失的形成以高频率被检测到，并且依赖于DNA复制的SV40起源和SV40大T抗原的存在。该体外系统将成为阐明哺乳动物细胞同源重组的生化方面的有力工具。(4) DNA拓扑异构酶在非法囚犯重组中的作用。我们发现小牛胸腺DNA拓扑异构酶II在体外系统中介导两个噬菌体λ DNA分子之间的重组。重组分子含有重复或缺失，大多数交叉发生在非同源的lambda DNA序列之间，通过重组连接的序列来判断。因此，犊牛胸腺DNA拓扑异构酶II介导的重组是一种非法重组，类似于我们之前指出的大肠杆菌DNA旋切酶或噬菌体T4 DNA拓扑异构酶介导的重组。此外，我们还分析了拓扑异构酶抑制剂在哺乳动物培养细胞中的作用。DNA拓扑异构酶II抑制剂VM26刺激mon]Cey细胞中sv40来源DNA的缺失形成，暗示拓扑异构酶II参与哺乳动物细胞中缺失的形成。10. 关键词：少

(1) Molecular analysis of genes for homologous recombination and cell growth from fission yeast Schizosaccharomices pombe. We have isolated S. pombe genes for homologous to budding yeast S. cereviseae DST2 gene, whose product have the activity of DNA strand exchange. S. pombe genomic library was screened with a probe from S. cereviceae DST2 and nucleotide sequences of positive clones were determined. Approximately 3 kb open leading frame which encodes 991 amino acids was found in the resulted clones and is called dhp1 (DST2 homologue of S. p-ombe). The product had the activity of DNA strand exchange and was essential for the mitotic cell growth. Furthermore we also isolated another homologue of the DST2, which is called dhp2. In contrast to the dhpl, the dhp2 is not essential for mitotic cell growth. The DST2 homologues will be used to analyze the mechanism of homologous recombination in S. pombe.(2) Cloning and analysis of gene (s) for homologous recombination in mammalian cells. The … More result of amino acid sequence of the dhpl gene was used to isolate human CDNA for homologous recombination and we succeeded it. We are analyzing the structure and function of this human CDNA and its product. We are also trying to isolate mouse genes for homologous recombination and to construct mutant mice which are defective in the gene for homologous recombination.(3) In vivo and vitro study of homologous recombination of SV40-derived shuttle vector DNAs in mammalian cells. SV40-derived shuttle vectors which contain direct repeats in the both sides of the galK^+ marker were incubated with an extract from HeLa cytosol in the presence of SV40 large T antigen. The vector DNAs were assayed for galK deletion formation using E. coligal^-. Using this assay system, the formation of galk deletion was detected at a high frequency and is dependent on the presence of both SV40 origin of DNA replication and SV40 large T antigen. This in vitro system will become a powerful tool for elucidating biochemical aspects of homologous recombination in mammalian cells.(4) Role of DNA topoisomerases on illeg, inmate recombination. We found that calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoiscimerase, which we have previously indicated. Furthermore, we have also analyzed the effects of topoisomerase inhibitors in mammalian cultured cells. The DNA topoisomerase II inhibitor VM26 stimulated deletion formation in SV40-derived DNA in mon]Cey cells, implicating that topoisomerase II participates in deletion formation in mammalian cells. 10. KEY WORDS Less