The Influence of DNA Sequences on Recombination in Mammalian Cells

哺乳动物细胞有能力进行同源 重组 染色体上的重组频率 然而，在基因转移实验中， 检测通常被高水平的非- 同源重组 各种质粒系统具有 现在已经开发出来，可以进行非常详细的研究， 在培养物中生长的细胞中的同源重组。 的系统 已经进一步开发了用于重组活性的测定 从这些细胞的细胞核制备的提取物中进行。我们 已经开始纯化一种酶活性， 同源DNA分子之间的交换。 据信 重组的过程基本上是相似的 有机体 从对简单生物体的研究中， 重组的特征已被确定为： 启动事件，可能涉及对特定DNA的偏好 序列;同源分子间的链交换;错配 校正所得异源双链体;最后拆分 突触分子的结构。 我们建议研究酶学 和DNA序列的特异性 过程、起始和链交换。 两种DNA 序列，Z DNA和高变小卫星， 与促进真核生物重组有关。 我们 设计了一对质粒/噬菌体载体， 用于测定neo基因中的重组， 在每一个中的同源位点插入特定序列， 以确定它们对复合的影响。 Z DNA 小卫星核心重复序列将被插入这些 载体及其对重组频率的影响 通过转染到人EJ细胞中测定。 是 认为这些序列的存在可能会刺激 重组过程中的起始和/或配对事件。 的 这些序列对重组的影响也将被 在浸提物催化的体外试验中检查 由EJ核制备。 哺乳动物同源重组的研究进展 细胞是非常感兴趣的遗传学家谁希望， 了解基本的遗传机制， 会给。 此外，在他们的基因中替换基因的能力 在哺乳动物基因组中的正确位置 重要性和商业价值。

Mammalian cells have the ability to carry out homologous recombination. The frequency of recombination in chromosomal genes, however,is low and in gene transfer experiments its detection is usually obscured by high levels of non- homologous recombination. A variety of plasmid systems have now been developed that allow quite detailed studies of homologous recombination in cells grown in culture. The systems have been further developed assays for recombinational activity in extracts prepared from nuclei of such cells can be done. We have begun to purify an enzyme activity capable of strand exchange between homologous DNA molecules. It is believed that the process of recombination is essentially similar in all organisms. From studies on simpler organisms the general features of recombination have been determined to be: an initiation event, possibly involving preference for specific DNA sequences; strand exchange between homologous molecules; mismatch correction ofthe resulting heteroduplex; and finally resolution of the synapsed molecules. We propose to study the enzymology and DNA sequence specificity of the first two stages of this process, initiation and strand exchange. Two types of DNA sequences, Z DNA and the hypervariable minisatellites, have been implicated in promoting recombination in eukaryotes. We have designed a pair of plasmid/phage vectors that can be used to assay recombination in the neo gene and allow specific sequences to be inserted at homologous sites in each substrate to determine their effect on recombination. Z DNA and mini-satellite core repeats will be inserted into these vectors and their effects on recombination frequency determined by transfection into human EJ cells. It is thought that the presence of such sequences may stimulate the initiation and/or pairing events during recombination. The effects of these sequences on recombination will also be examined in the in vitro assay catalyzed by extracts prepared from EJ nuclei. The achievement of homologous recombination in mammalian cells is of significant interest to geneticists who wish to understand basic genetic mechanisms for what insights it would give. Also, the ability to replace genes in their correct position in the mammalian genome would have both fundamental and commercial importance.