Graduate Thesis Or Dissertation
 

Effects of physiological state upon nitrosoguanidine-mediated allelic recombination in Saccharomyces cerevisiae

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  • Saccharomyces cerevisiae strain XS-380, a. heteroallelic diploid strain, carries six genetically analyzed loci on the left arm of chromosome VII. Nitrosoguanidine (NMG) at low concentrations was observed to cause allelic recombination (either mitotic recombination or gene conversion) of the heteroallelic loci with low lethality. The NMG-inducible heteroallelic system was used in studies of the relationship of mutagenic action to DNA replication in both randomly and synchronously growing yeast cultures. Studies of asynchronous cultures in balanced logarithmic growth, in the early stationary phase, and in the early phases of amino acid starvation indicated that each locus had a characteristic recombinant response. Two of these conditions, amino acid starvation and early stationary phase, produced a drastic reduction in numbers of induced recombinants. Moreover, after onset of amino acid starvation, a timed series of samples could be individually induced by pulse mutagenesis to show that the mutagen response decreased by the time that DNA synthesis stopped. Similar results were also obtained by substituting cycloheximide treatment for the amino acid starvation conditions in the time course experiment. However, a definite relationship of mutagenic response to DNA replication could not be demonstrated by amino acid starvation or cycloheximide inhibition, because coordinate protein synthesis may be necessary for the maintenance of DNA synthesis. Synchronous cultures also produced ambiguous responses to NMG-mediated pulse mutagenesis. The frequency of inducible mitotic gene conversion increased rapidly before initiation of a discrete synchronous doubling of total DNA. In contrast, high levels of inducible mitotic recombination could also be obtained during the period of DNA replication with decreasing levels toward the end of the first round of DNA doubling. Most of the markers on the chromosome arm displayed similar cyclic variations in recombination frequency. It was suggested that the cyclic activity of DNA repair enzymes may be responsible for the variations of inducible prototroph frequencies during synchronous growth. No definite direction of recombination or polarity could be observed on the chromosome.
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