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A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation. Three types of mutants were isolated. Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3. The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR. mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations. The mutator phenotype is recessive. Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans. No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain. The mutator's activity was little affected by the type of medium in which the strain was grown. Deoxyribonucleoside triphosphate pools were normal in mutR34. Intergenic recombination frequencies were the same in mutR and mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr times Fminus crosses when the recipeint was mutR34 as compared with mutR+. This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.

We developed a general procedure for the induction and identification of mutations in chromosomal essential genes that are located in a diploid region of Escherichia coli K-12. The partial diploidy is conferred by an episome that is temperature sensitive for replication so that a mutant strain will form microcolonies at 42 C on complete media if an essential chromosomal gene in the diploid region is defective. Mutations identified by this procedure can be classified into cistrons by a complementation method devised for the purpose. To verify that the procedure works in practice, we fused an episome covering the rif region with an Ftslac+ and used the resulting temperature-sensitive episome to identify chromosomal mutations in essential functions near rif. As expected, a certain proportion of the mutations were in the rif gene, an essential gene that codes for the beta subunit of ribonucleic acid polymerase.

The effects of pyrimidine limitation on chromosome replication and the control of ribosomal and transfer ribonucleic acid syntheses were investigated. Chromosome replication was studied by autoradiography of (3)H-thymine pulse-labeled cells. Pyrimidine limitation did not affect the fraction of cells incorporating radioactive thymine during a short pulse, indicating that when growth is limited by the supply of pyrimidine, the time required for chromosome duplication increases in proportion to the time required for cell duplication. Control of ribosomal RNA and transfer RNA syntheses was examined by chromatographing cell extracts on methylated albumin kieselguhr columns. When growth was controlled by carbon-nitrogen limitation, the ratio of tRNA to total RNA remained roughly constant at growth rates above 0.5 doublings per hour. During pyrimidine limitation, however, the control of rRNA synthesis was apparently dissociated from the control of tRNA synthesis: the ratio of tRNA to total RNA increased as the growth rate decreased.

Segregants from merodiploids heterozygous at two or more sites in the lac region were selected on the basis of containing a recombinant F-merogenote. Such recombinants frequently contained a recombinant chromosome as well. When the merodiploid was heterozygous at two sites, the frequency at which reciprocally recombinant chromosomes were present in the selected population was lower when the two marked sites were in the same gene and close together than when the sites were more widely separated. When the merodiploid was heterozygous at four sites and selection was made for an intragenic recombinational event, the recombinant chromosome was the reciprocal type about half the time and about half the time was not. Among the latter genotypes, most were nonrecombinant for the intragenic pair of markers. The data are consistent with a model in which recombination leads to the formation of two recombinant products, each containing a region of hybrid deoxyribonucleic acid.

The growth rate of a pyrimidine-requiring strain was controlled by limiting the concentration of exogenous orotic acid. As the steady state, pyrimidine-limited growth rate was decreased, the intracellular pyrimidine pools and the total nucleic acid per unit mass of culture also decreased. The ratio of deoxyribonucleic acid to protein remained constant, whereas the ratio of ribonucleic acid to protein decreased 30% over a threefold variation in growth rate (50- to 150-min doubling times). The intracellular uridine triphosphate and cytosine triphosphate pools also decreased (although not coordinately), and the pyrimidine biosynthetic enzymes were derepressed. Cell size was unaffected by pyrimidine-mediated variation of the growth rate.

Herman, Robert K. (Lawrence University, Appleton, Wis.). Reciprocal recombination of chromosome and F-merogenote in Escherichia coli. J. Bacteriol. 90:1664-1668. 1965.-Mitotic recombination of an F-merogenote with the bacterial chromosome was observed under conditions where both recombinant episome and reciprocally recombinant chromosome, if formed and if passed on to the same progeny, could be detected. Recombinant strains selected on the basis of having a recombinant F-merogenote were found frequently to contain a recombinant chromosome of the reciprocal type.

ICR-191, an acridine half-mustard known to cause frameshift mutations in bacteria, was used to induce Lac(-) mutations revertible by ICR-191. The reversion rates of several of these mutations were stimulated approximately twofold by the presence of lac inducer. The stimulatory effect of inducer was attributable to gene induction rather than some other effect of inducer, since inducer did not stimulate reversion in a regulator constitutive strain. The stimulatory effect was not observed unless the gene to be reverted was induced during the period of exposure to ICR-191. The presence of a strong polar (nonsense) mutation on the operator side of a frameshift mutation abolished the stimulatory effect of inducer on reversion of the frameshift mutation by ICR-191. (As expected, a nonpolar mutation on the operator side of the frameshift mutation did not affect inducer-stimulated reversion.) It was concluded that some aspect of transcription or translation, or both, in the neighborhood of the ICR-191-induced mutation stimulated reversion by ICR-191. A recA mutation had no effect on reversion by ICR-191 in the presence or absence of inducer. In one mutant, gene induction depressed reversion by ICR-191 about sevenfold. The difference between this exceptional strain and other mutants was not attributable to different genetic backgrounds but seemed to be an inherent difference in the original Lac(-) mutations.