The Influence of DNA Sequences on Recombination in Mammalian Cells

哺乳动物细胞具有进行同源重组的能力。然而，染色体基因重组的频率很低，在基因转移实验中，其检测通常被高水平的非同源重组所掩盖。现在已经开发了多种质粒系统，可以对培养细胞中的同源重组进行相当详细的研究。该系统已得到进一步发展，可以对从这种细胞的细胞核制备的提取物中的重组活性进行测定。我们已经开始纯化一种能够在同源DNA分子之间进行链交换的酶活性。人们认为，所有生物体的重组过程本质上是相似的。从对较简单生物体的研究来看，重组的一般特征已被确定为：起始事件，可能涉及对特定DNA序列的偏好；同源分子间的链交换；所产生的杂双工错配校正；最后是突触分子的分解。我们建议研究这一过程的前两个阶段，起始和链交换的酶学和DNA序列特异性。两种类型的DNA序列，Z DNA和高变小卫星，已经涉及到促进真核生物的重组。我们设计了一对质粒/噬菌体载体，可用于检测neo基因的重组，并允许在每个底物的同源位点插入特定序列，以确定其对重组的影响。将Z DNA和迷你卫星核心重复序列插入这些载体中，并通过转染人EJ细胞来确定其对重组频率的影响。据认为，这些序列的存在可能刺激重组过程中的起始和/或配对事件。这些序列对重组的影响也将在体外实验中由EJ核提取物催化进行检验。在哺乳动物细胞中实现同源重组对于希望了解基本遗传机制的遗传学家来说是非常有意义的。此外，替换哺乳动物基因组中正确位置的基因的能力将具有基础和商业重要性。

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.