A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1.
A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1.
复制标题
噬菌体 f1 基因 V 蛋白温度敏感变体的遗传选择。
DOI：
10.1093/nar/16.18.9027
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发表时间：
1988
影响因子：
14.9
通讯作者：
Zabin,HB
中科院分区：
文献类型：
--
作者：
Terwilliger,TC;Fulford,WD;Zabin,HB
基于一种质粒编码的噬菌体F1基因的活性，发展了互补的阴性和阳性遗传选择。负选择是基于基因Vine蛋白的活性。结肠细胞可显著减少F1相关病毒对这些细胞的感染。为了对表达活性基因V蛋白的细胞进行筛选，用噬菌体R386感染细胞，R386是F1的衍生物，对氯霉素具有抗性，并在抗生素存在的情况下进行平板培养。那些含有基因V蛋白的细胞很少感染病毒，在氯霉素存在的情况下无法生长；那些不包含基因V蛋白的细胞很容易感染，在抗生素存在的情况下可以生长。正向遗传选择包括从质粒中切除基因V序列，并用它们取代含有琥珀突变的噬菌体F1衍生体的基因V。只有那些编码活性基因V蛋白的基因才能支持噬菌体的生长和产生斑块。这两种基因选择可以组合在一起，以产生编码温度敏感基因V蛋白的大量丰富基因。
Complementary negative and positive genetic selections based on the activity of a plasmid-encoded bacteriophage f1 geneVare developed. The negative selection is based on an activity of thegeneVprotein inE. colicells which markedly reduces the infection of those cells by f1-related viruses. In order to select against cells expressing active geneVprotein, the cells are infected with the phage R386, a derivative of f1 which confers resistance to chloramphenicol, and are plated in the presence of the antibiotic. Those cells which contain geneVprotein are infrequently infected with the virus and are unable to grow in the presence of chloramphenicol; those which do not contain the geneVprotein are readily infected and can grow in the presence of the antibiotic. The positive genetic selection consists of excising the geneVsequences from the plasmids and using them to replace the geneVof a bacteriophage f1 derivative containing an amber mutation in geneV. Only those genes which encode an active geneVprotein can support phage growth and yield plaques. The two genetic selections can be combined in order to yield a substantial enrichment for genes encoding temperature-sensitive geneVproteins.