The Influence of DNA Sequences on Recombination in Mammalian Cells

点击翻译按钮获取中文摘要

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.