Increased dihydrofolate reductase synthesis in Diplococcus pneumoniae following translatable alteration of the structural gene. 3. Further evidence on the extent of genic involvement.

Abstract

VIDENCE for the occurrence of mutation in the dihydrofolate reductase E gene of Diplococcus pneumoniae, which alters both the amount and structure of the corresponding enzyme, has been presented in two previous reports from this laboratory. (SIROTNAK and HACHTEL 1969; SIROTNAK, WILLIAMS and HACHTEL 1969). This form of dual biochemical effect is similar in some respects to that observed in the L-arabinose operon of Escherichia coli (ENGLESBERG 1961; LEE and ENGLESBERG 1963) and appears to involve an increase in the rate of dihydrofolate reductase synthesis as a result of a “missense” alteration of the structural gene. Other genetic and enzymological findings lending further support to this idea are presented here. In addition to their effect on antifolate binding by the enzyme, a number of these mutations was also found to have a marked effect on stability. Of special significance, also, are results indicating that increased levels of dihydrofolate reductase are not due to genetic alteration of only a specific region of the gene, but occur as a result of mutation at any one of a large number of sites encompassing a major portion, if not all, of the gene. Data relating to a number of extreme dual-effect mutations were presented in a preliminary report ( SIROTNAK 1969). Recent evidence was also given by BERBERICH and LEVINTHAL (1969) for the occurrence of a similar type of mutation in the dihydrofolate reductase gene of Salmonella typhimurium. MATERIALS AND METHODS Many of the procedures used during the present study have already been described in detail (SIROTNAK and HACHTEL 1969; SIROTNAK, WILLIAMS and HACHTEL 1969). Media used for the isolation of mutant strains, for genetic experiments and for culture production have also been given (SIROTNAK, LUNT and HUTCHISON 1964).