Fujimura R K, Tavtigian S V, Choy T L, Roop B C
J Virol. 1985 Feb;53(2):495-500. doi: 10.1128/JVI.53.2.495-500.1985.
Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.
分离出了含有噬菌体T5 DNA聚合酶基因的DNA片段。通过用限制性内切酶消化产生的纯化DNA片段转化大肠杆菌来确定该基因的物理位置，并且用转化后的细胞来拯救T5的琥珀突变体，这些突变体在DNA聚合酶基因上发生了突变。该方法适用于任何其他具有低回复频率突变的基因。我们研究了T5 DNA聚合酶基因的以下突变，按照标准惯例从左到右读取（D. J. McCorquodale，《微生物学评论》4：101 - 159，1975）：D7、D8、aml、ts5E - ts53、am6和D9。发现这些位点位于总长为3600个碱基对的三条DNA片段内。由于T5 DNA聚合酶的结构基因估计长2600个碱基对，所以整个结构基因可能位于这些片段中。它们位于距DNA左端距离的58.3%至61.3%处。根据转化试验的结果，含有聚合酶基因的DNA左端片段（1100个碱基对）有位点D7和D8，右端片段（1600个碱基对）有位点D9。这些结果与遗传图谱一致。
