Viable variants in VA-RNA, gene of an Ad2-Ad5 recombinant.
Viable variants in VA-RNA, gene of an Ad2-Ad5 recombinant.
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VA-RNA（Ad2-Ad5 重组体的基因）中的可行变体。
DOI：
10.1016/0042-6822(81)90302-0
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发表时间：
1981
期刊：
影响因子：
3.7
通讯作者：
Chinnadurai,G
中科院分区：
文献类型：
--
作者：
Rajagopalan,S;Chinnadurai,G
We have developed a simple method for selecting viral mutants whose DNA lack one of the sites of a restriction endonuclease that has more than one site on the viral genome. We have used this method for isolating viable variants of an Ad2-Ad5 recombinant that lack aBamHI cleavage site located at mp 29 spanning the VA-RNAIgene. The viral DNA-protein complex was prepared from a stock of the Ad2-Ad5 recombinant passed under high m.o.i., and cleaved withBamHI. We would expectBamHI to cleave the parental Ad2-Ad5 recombinant DNA at two sites, 29 and 59.5, but to cleave naturally arising mutant DNAs at one or the other site. With mutation at mp 29 site,BamHI cleavage would produce a fusion fragment from mp 0–59.5. Our objective, then, was to recombine the mutant DNA fragmentin vivowith an overlapping fragment containing sequences from the right half of the genome, e.g.,SalA (mp 45.9–100) to produce an infectious mutant DNA with a singleBamHI site at mp 59.5. DNA protein complex was digested withBamHI and cotransfected with the DNA-protein complex cleaved withSalI, and the resulting plaques were screened for DNAs lacking theBamHI site at mp 29. Two such mutants were isolated, one of which is probably a deletion mutant (203) and the other an insertion mutant (204). Mutant 203 made VA-RNAIof slightly higher molecular weight and mutant 204 made VA-RNAIof slightly higher molecular weight. These mutants grew to levels comparable to that of the parental virus in KB cells indicating that these mutations did not alter the growth of the mutants. The method described here should also be valuable in isolating mutants elsewhere in the genome.
