Improved Method for the Isolation of Thymine-Requiring Mutants of Escherichia coli
Improved Method for the Isolation of Thymine-Requiring Mutants of Escherichia coli
复制标题
大肠杆菌需要胸腺嘧啶突变体的改进方法
作者：
K. Stacey;E. Simson
在Okada， Yanagisawa和Ryan （Z. Vererbungslehre 92:403, 1961）观察到在高浓度氨基雀呤，胸腺嘧啶，嘌呤和丝氨酸存在下培养的细菌中含有数量惊人的无法合成自身胸腺嘧啶供应的细胞之前，很少发现需要胸腺嘧啶的大肠杆菌突变体。他们发现，这些胸腺嘧啶依赖性突变体在氨基蝶呤存在的情况下比亲本菌株生长得快得多，即使它们都提供了足够数量的代谢物，而这些代谢物的合成被氨基蝶呤阻断。这些观察结果为广泛使用的分离这类特殊突变体的方法提供了基础。Okada， Homma和Sonohara （J. Bacteriol. 84:602, 1962）报道了简化培养基的更高效率。我们还发现，如果使用完全最小的培养基，通常是M9，只含有胸腺嘧啶（50,g/ml）和氨基蝶呤（300,ug/ml）作为分离培养基，胸腺突变体的产量大大增加。其他代谢物的缺乏，而四氢叶酸的合成是必需的，似乎增加了选择压力，有利于胸腺嘧啶依赖性突变体。在一些实验中，在这种培养基中生长48小时后，不能自己制造胸腺嘧啶的菌落形成者的百分比接近100%。在这些实验中，从需要维生素B1（硫胺素）的菌株中分离胸腺突变体要困难得多。Pine （J. Bacteriol. 79: 827,1960）提供的证据表明，氨基蝶呤主要通过硫胺素渗透酶主动运输进入枯草芽孢杆菌细胞，并且添加少量的硫胺素或类似的嘧啶对氨基蝶呤引起的生长抑制有很大的保护作用（Pine， J. Bacteriol. 79: 835,1960）。培养基中添加的B1很可能同样保护了需要B1的大肠杆菌菌株。抑制大肠杆菌所需的高浓度氨基蝶呤是必要的，因为像B。
Thymine-requiring mutants of Escherichia coli were rarely found before the observation by Okada, Yanagisawa, and Ryan (Z. Vererbungslehre 92:403, 1961) that cultures of bacteria grown in the presence of high concentrations of aminopterin, thymine, purines, and serine contain a surprising number of cells unable to synthesize their own supply of thymine. They showed that these thymine-dependent mutants could grow much faster in the presence of aminopterin than the parent strains, even though both were supplied with adequate amounts of those metabolites whose syntheses are blocked by aminopterin. These observations provided the basis for a widely used method of isolation of this special class of mutants. The greater efficiency of a simplified medium was reported by Okada, Homma, and Sonohara (J. Bacteriol. 84:602, 1962). We also found that the yield of thymutants was greatly increased if a completely minimal medium, usually M9, containing only thymine (50 ,g/ml) in addition to aminopterin (300 ,ug/ml) was used as the isolation medium. The absence of the other metabolites, for whose synthesis tetrahydrofolate is required, appears to increase the selection pressure in favor of the thymine-dependent mutants. In some experiments, after 48 hr of growth in this medium, the percentage of colony-formers unable to make their own supply of thymine approached 100%. In these experiments, it was much more difficult to isolate thymutants from strains that require vitamin B1 (thiamine). Pine (J. Bacteriol. 79:827, 1960) presented evidence that aminopterin enters the cells of Bacillus subtilis mainly by active transport by the thiamine permease, and that the addition of small amounts of thiamine or a similar pyrimidine affords a great deal of protection against the growth inhibition caused by aminopterin (Pine, J. Bacteriol. 79:835, 1960). It is likely that the added B1 in the medium similarly protects the B1-requiring strains of E. coli. The high concentrations of aminopterin required to inhibit E. coli are necessary because, like B.