Studies on Mechanisms of DNA Rearrangements in Mammalian Cells.

(1)裂殖酵母裂殖酵母同源重组和细胞生长基因的分子分析。分离出S。pombe基因与芽殖酵母S.酿酒酵母DST 2基因，其产物具有DNA链交换活性。S.用来自S.并对阳性克隆进行了核苷酸序列测定。结果表明，该基因编码991个氨基酸，约3 kb的开放前导框被命名为dhp 1（S.dsp2同源基因）。p-ombe）。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，我们还分离了DST 2的另一个同源物，称为dhp 2。与dhpl相反，dhp 2对于有丝分裂细胞生长不是必需的。DST 2同源物将用于分析S.粟酒(2)哺乳动物细胞中同源重组基因的克隆和分析。的 ...更多信息 利用dhpl基因的氨基酸序列分析结果，成功地分离出了人cDNA，并对其进行了同源重组，目前正在分析该cDNA及其产物的结构和功能。我们也在尝试分离小鼠的同源重组基因，并构建同源重组基因缺陷的突变小鼠。(3)SV 40衍生穿梭载体DNA在哺乳动物细胞中同源重组的体内和体外研究。在SV 40大T抗原存在的情况下，将在galK^+标记的两侧含有直接重复序列的SV 40衍生穿梭载体与HeLa细胞质提取物一起孵育。用E.大肠杆菌。使用该测定系统，以高频率检测galk缺失的形成，并且其依赖于SV 40 DNA复制起点和SV 40大T抗原两者的存在。这种体外系统将成为阐明哺乳动物细胞中同源重组的生物化学方面的有力工具。(4)DNA拓扑异构酶在非法重组中的作用。我们发现，小牛胸腺DNA拓扑异构酶II介导的重组两个噬菌体λ DNA分子在体外系统。重组分子含有重复或缺失，并且大多数交叉发生在λ DNA的非同源序列之间，如通过重组连接点的序列所判断的。因此，由小牛胸腺DNA拓扑异构酶II介导的重组是一种非法重组，类似于我们先前指出的由大肠杆菌DNA促旋酶或噬菌体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VM 26刺激单核细胞中SV 40衍生DNA的缺失形成，暗示拓扑异构酶II参与哺乳动物细胞中的缺失形成。10.关键词减

(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