HOMOLOGOUS RECOMBINATION IN HUMAN CELL EXTRACTS

通过同源重组修饰活细胞中的基因的能力 基因置换治疗的意义及部位特异性基因 修改。在过去的几年里，已经证明了 哺乳动物的体细胞具有所有必要的酶来调节 外源导入的质粒之间的同源重组。这个 这一过程的机制和酶的实际性质介导 人们的反应尚不清楚。作为迈向详细基因研究的第一步， 哺乳动物尊贵基因的生化和分子生物学研究 重组，我们已经开发出一种无细胞系统，能够 催化这一反应。该检测包括共同孵化两个非 PSV2neo，a的回复、非重叠缺失突变体 真核-原核穿梭载体与细胞提取物和使用 产生的DNA用于直接分析或转化重组 缺陷型(recA-)大肠杆菌。目前的提案涉及一项详细的 细胞萃取物的分析。PSV2neo质粒将被修改为 这两个质粒会有不同的限制性内切酶。这些 将使用限制性内切酶酶切位点多态来检测产品 重组和演绎基因转化和互换的作用 重组。将对细胞提取液进行检测，以确定是否存在 启动和完成所需的酶活性 重组反应。生物和生化分析都将是 使用。一种参与DNA重组酶蛋白的分离 用分级法和抗体亲和力进行链交换反应 还提出了立柱的建议。一种高度特异的抗体也将用于 从噬菌体表达载体中筛选人cDNA文库进行分离 重组酶基因的表达。将该基因插入哺乳动物表达系统中 载体及其促进同源重组的能力将是 学习。与该基因相对应的基因组克隆的分离及其表达 并提出了表征方法。

The ability to modify genes in living cells by homologous recombination has significance to gene replacement therapy as well as site specific gene modification. During the past few years it has been demonstrated that mammalian somatic cells have all the enzymes necessary to mediate homologous recombination between exogenously introduced plasmids. The mechanism of this process and the actual nature of enzymes mediating the reactions are not known. As a first step towards a detailed genetic, biochemical and molecular biological understanding of mammalian honologous recombination, we have developed a cell-free system which is capable of catalyzing this reaction. The assay involves co-incubation of two non reverting, non-overlapping deletion mutants of PSV2neo, a eukaryotic-prokaryotic shuttle vector with the cell extracts and using the resulting DNA for direct analysis or transformation of recombination deficient (recA-) E-coli. The current proposal involves a detailed analysis of the cell extracts. The PSV2neo plasmids will be modified so that the two plasmids will have different restriction enzyme sites. THese restriction enzyme site polymorphisms will be used to examine the products of recombination and deduce the role of gene conversion and reciprocal recombination. The cell extracts will be tested for the presence of enzymatic activities that are required for the initiation and completion of the recombination reaction. Both biological and biochemical assays will be used. Isolation of a recombinase protein which would be involved in a DNA strand exchange reaction by fractionation methods and by antibody affinity columns is also proposed. A highly specific antibody will also be used to screen a human cDNA library in a bacteriophage expression vector to isolate the recombinase cDNA. The cDNA will be inserted into mammalian expression vectors and its ability to enhance homologous recombination will be studies. Isolation of a genomic clone corresponding to the cDNA and its characterization are also proposed.