Scearce L M, Masker W
Temple University School of Medicine, Department of Biochemistry and Molecular Biology, Philadelphia, PA 19140.
Mutat Res. 1993 Aug;288(2):301-10. doi: 10.1016/0027-5107(93)90098-z.
Deletion mutagenesis in bacteriophage T7 was studied with an insertion-reversion assay involving phage containing inserts of foreign DNA that form 10-bp direct repeats. The precise deletion of the insert restores the function of the non-essential gene and is easily assayed by growth on selection strains of E. coli. Similar inserts with unique direct repeats were placed in either gene 1.2 (dGTPase Inhibitor) or gene 1.3 (DNA ligase). The deletion rates of the inserts were quantified with Luria and Delbrück fluctuation tests. Deletion rates were similar for inserts in both genes indicating that the rates of deletion were not unique to either specific site, or the sequence of the direct repeats. Deletion was independent of functional T7 ligase at 37 degrees C, while an increase in the rate of deletion was noted in some ligase-deficient phage at 43 degrees C. The effect of E. coli DNA Polymerase I on deletion rate was tested and found to decrease deletion rate 60% with the polA1 mutation and 90% with the polA546ex mutation.
利用一种插入-回复分析方法对噬菌体T7中的缺失诱变进行了研究,该分析涉及含有形成10个碱基对直接重复序列的外源DNA插入片段的噬菌体。插入片段的精确缺失恢复了非必需基因的功能,并且通过在大肠杆菌选择菌株上生长很容易进行检测。具有独特直接重复序列的类似插入片段被置于基因1.2(dGTP酶抑制剂)或基因1.3(DNA连接酶)中。用卢里亚和德尔布吕克波动试验对插入片段的缺失率进行了定量。两个基因中插入片段的缺失率相似,这表明缺失率并非特定于任何一个特定位点或直接重复序列的序列。在37℃时,缺失与功能性T7连接酶无关,而在43℃时,在一些连接酶缺陷型噬菌体中观察到缺失率增加。测试了大肠杆菌DNA聚合酶I对缺失率的影响,发现polA1突变使缺失率降低60%,polA546ex突变使缺失率降低90%。