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.

Aminopterin, an analogue of folic acid, binds very strongly to the enzyme dihydrofolate reductase, preventing the generation of tetrahydrofolate, the cofactor of a number of Ci transfers (Huennekens, 1963). The synthesis of thymine, the purines, serine and methionine is thereby prevented and the inhibition of bacterial growth by aminopterin can only be overcome by supplying these metabolites. Ryan, Yanagisawa & Okada (1961) made the surprising observation with Escherichia coli that among the survivors of a prolonged incubation in the presence of high concentrations (400,ug./ml.) of

K 12 bacteria, carrying an autonomous F factor or an F-prime factor, enhances the fertility of the population by increasing the number of cells which can transfer the bacterial chromosome. In contrast, under similar conditions the fertility of irradiated Hfr populations falls in proportion to the survivors. Following irradiation, the effect begins to develop after about 30 min. incubation in broth at 37", reaches a peak at about 90 min., and thereafter slowly declines. The effect develops with similar kinetics during post-irradiation incubation in minimal medium as in broth, provided the bacteria have been minimal-grown; in the case of broth-grown cells, appearance of the effect in minimal medium is greatly delayed. A comparison of the kinetics of the effect with the growth of the population as a whole shows that the u.v.-induced donor state is not inheritable. Mitomycin C, which resembles U.V. radiation in producing DNA damage repairable by a mechanism involving excision of single-stranded fragments, also induces new donor bacteria. Other agents such as X-rays and methyl methansulphonate (MMS) do not stimulate the production of new donors but may enhance the recombination frequency since cells killed by them may continue to act as chromosome donors. The effect is not shown either by uvr mutants (unable to excise thymine dimers) or by rec mutants (unable to mediate recombination) which carry an F-prime factor. A possible mechanism is suggested whereby the excision of single-stranded fragments of the bacterial chromosome, during the repair of U.V. damage, facilitates pairing with homologous regions of the complementary sex factor strand. A recombination event, mediated by breakage and covalent bonding, then joins a free end of the excised DNA strand to the paired sex factor strand. In this way, recombination connects sex factor and chromosome by only a single strand instead of by the two strands which normally leads to insertion and the formation of an Hfr chromosome. It is postulated that such a structure can be transferred at conjugation, but is incapable of more than one cycle of replication.

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