Robert K. Mortimer

A genetic study of X-ray sensitive mutants in yeast

Abstract A set of 64 mutants of Saccharomyces cerevisiae that confer sensitivity to X-ray inactivation were analyzed genetically to determine the number of genetic loci involved. The mode of interaction of various combinations of mutants was also determined. A minimum of 17 genes, when mutant, increase X-ray sensitivity of yeast, primarily by eliminating the resistance of budding haploid cells and by removing the shoulder on the survival curves of diploid cells. Eight mutant loci affect principally X-ray sensitivity while the remaining genes also control sensitivity to ultraviolet. Some of the genes when homozygous block sporulation or result in partial or complete sterility. Examination of the survival responses of multiple-mutant strains indicated a minimum of two pathways in the repair of X-ray damage. A number of the mutants have been mapped and these were found to be dispersed over the genome.

On the origins of wine yeast

There is still a lack of agreement concerning the relative contribution of wine yeast that may originate in the vineyard compared to that which may originate in the cellar. Part of this controversy is due to the extreme difficulty of finding Saccharomyces cerevisiae on the grapes. We estimate that only about one in one-thousand grape berries carries wine yeast. However, we have found that grape berries that are damaged (i.e. the skin is broken) are very rich depositories of microorganisms including S. cerevisiae, and that one in four such berries is S. cerevisiae-positive. These positive berries have between 100,000 and 1,000,000 wine yeast cells on them, and there is evidence that these yeasts are clonal. We believe that the yeasts are brought to the berries by insects such as bees, wasps, and Drosophila and that they multiply in the rich medium of the grape interior. Even though there are many cells of S. cerevisiae on the damaged berries, they are in a definite minority. All the other organisms that are found in wine fermentations are also present on these berries, and their total numbers are in the range of 10 million to 100 million cells per berry.

Evidence for S. cerevisiae Fermentation in Ancient Wine

Saccharomyces cerevisiae is the principal yeast used in modern fermentation processes, including winemaking, breadmaking, and brewing. From residue present inside one of the earliest known wine jars from Egypt, we have extracted, amplified, and sequenced ribosomal DNA from S.cerevisiae. These results indicate that this organism was probably responsible for wine fermentation by at least 3150 B.C. This inference has major implications for the evolution of bread and beer yeasts, since it suggests that S. cerevisiae yeast, which occurs naturally on the surface bloom of grapes, was also used as an inoculum to ferment cereal products.

Nucleotide sequence of the RAD57 gene of Saccharomyces cerevisiae

We have determined the nucleotide (nt) sequence of the RAD57 gene of Saccharomyces cerevisiae. RAD57 contains an open reading frame of 1380 bp. The deduced amino acid sequence of 460 residues contains a potential nt-binding sequence and shows significant similarity to the preliminary sequence of RAD51.

Allelic Mapping in Yeast by X-ray-Induced Mitotic Reversion

A new method for determining the sequence of mutational sites is based on the linear dose-effect relation for x-ray induction of allelic recombination in Saccharomyces cerevisiae. Mutations at two loci have been mapped by this method. The use of x-ray simplifies allelic mapping and greatly increases its sensitivity.

Mechanisms of Meiotic Gene Conversion, or “Wanderings on a Foreign Strand”

INTRODUCTION Over the last decade and a half, studies in organisms amenable to tetrad (or octad) analysis have provided a reasonably complete descriptive inventory of intragenic recombination (Fogel et al. 1979; Nicolas 1979; Rossignol et al. 1979; Sang and Whitehouse 1979). This paper aims to provide an overview of the relevant data. At the same time it attempts a synthesis of the new information in the expectation that a scaffold will emerge from which testable molecular hypotheses concerning generalized and intragenic recombination can be constructed. A comprehensive review of the literature concerning meiotic recombination will not be attempted. Much of the data have been cogently summarized in texts by Esser and Kunnen (1967), Whitehouse (1969), Grell (1974), Kushev (1974), Catcheside (1977), Fincham et al. (1979), Stahl (1979b) and in several critical reviews by Emerson (1969), Mortimer and Hawthorne (1969), Fogel and Mortimer (1971), Radding (1973), Hotchkiss (1974), Hastings (1975), Esposito and Esposito (1977), Pukkila (1977), Resnick (1979), Stahl (1979a), and Petes (1980b). Here, we approach the problem of intragenic meiotic recombination or recombinagenic events within short DNA segments by considering four questions. What is the phenomenon of gene conversion? What are the methods that can be employed to study this phenomenon? What are the salient features of the conversion process? Can methodologies of recombinant DNA technology be adapted to advance the analysis of intragenic recombination from the genetic level to the molecular level? We may begin by asking, What is meiotic gene conversion? In organisms subject...

Sequence of RAD54, a Saccharomyces cerevisiae gene involved in recombination and repair

The complete nucleotide sequence of the RAD54 gene of the yeast Saccharomyces cerevisiae has been determined. The sequenced region contains an open reading frame of 2694 bp, and the predicted RAD54 protein has a potential nucleotide-binding site and possible nuclear targeting sequences. Northern analysis reveals a transcript of approx. 3.0 kb which is induced following x-ray irradiation.

STUDIES OF THE GENETIC MECHANISM OF RADIATION-INDUCED MITOTIC SEGREGATION IN YEAST.

SummarySectoring was induced with x-rays or ultraviolet in a diploid yeast strain heterozygous for seven genes located on one chromosome arm. The frequencies of sectoring of different genes were approximately linearly related to their distance from the centromere. If two or more adjacent genes sectored, the event could be explained by mitotic crossing over. Sectoring of single genes, however, was mostly nonreciprocal and resembled a conversion-type event. Approximately 80% of the sectored colonies could be explained single mitotic crossovers in one of the intergenic regions.

Genotypic characterization of strains of commercial wine yeasts by tetrad analysis

The objective of this work was to use tetrad analysis to define the genotypes of a number of commercially available wine yeasts for a range of characteristics related to wine making. The levels of sporulation and spore viability of 13 wine yeasts were determined. Sporulation was very low in one strain and varied from low to high in the other 12 strains. Spore viability of these 12 strains varied from 0-95% and this range was comparable to a large sample of naturally-occurring wine strains. Colonies from viable spores, predominantly from 4-spored asci, from 11 strains were characterized for the ten traits: homothallism/heterothallism, fermentation of sucrose, galactose, maltose; growth on glycerol (nonfermentable); slow growth on glucose and glycerol; level of sulfide production; copper resistance; putative presence of a recessive lethal mutation (inviability of at least two spores/tetrad); yellow pigment (in colonies) on sugar media. The number of heterozygosities for these ten characteristics varied from zero to seven in 11 strains, and eight strains were genetically distinct. Another three strains, distinct from these eight strains, were identical for the ten characteristics and also equivalent for the levels of sporulation and spore viability. Although these three strains are marketed under different designations, there is a strong probability that they were derived from a common ancestral strain. The genotypic characterization of these 11 strains constitutes an important foundation for their identification and their use in breeding programs.

Influence of linear energy transfer and oxygen tension on the effectiveness of ionizing radiations for induction of mutations and lethality in Saccharomyces cerevisiae.

The radiations available from heavy-ion linear accelerators (HILAC) permit an extension of radiobiological studies to regions of linear energy transfer (LET) not previously available. The variation of the relative biological effectiveness (RBE) as a function of LET for the different particle beams has been determined for a variety of effects in different biological systems (1-5). For inactivation of protein molecules or viruses (3), the efficiency decreases continuously with increasing LET. For induction of lethality in yeast (1, 2) or in mammalian cells (5), the RBE rises to a maximum and then decreases as the LET increases. It was of interest to determine if the relation between RBE and LET for the induction of mutations paralleled those for effects on molecules or viruses, or was similar to the relation observed for induction of lethality in cells. Stapleton et al. (6), using Aspergillus, found that a-particles were more effective for killing but less effective for mutation than Xrays. Deering (4) has reported that oxygen ions are less effective for both killing and mutation than are less densely ionizing radiations, although mutability at high LET was decreased relatively more. Hrishi and James (7) have found that thermal neutrons are more effective than X-rays for inducing reversions in histidinerequiring diploid yeast cells. For a given degree of inactivation, however, neutrons were less effective for inducing mutation. The present study is concerned with the determination of the relative effectiveness of a number of particle beams in inducing specific genetic effects and lethality in yeast cells.