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.

Evolution and variation of the yeast (Saccharomyces) genome.

In this review we describe the role of the yeast Saccharomyces in the development of human societies including the use of this organism in the making of wine, bread, beer, and distilled beverages. We also discuss the tremendous diversity of yeast found in natural (i.e., noninoculated) wine fermentations and the scientific uses of yeast over the past 60 years. In conclusion, we present ideas on the model of "genome renewal" and the use of this model to explain the mode by which yeast has evolved and how diversity can be generated.

Genetic and physical maps of Saccharomyces cerevisiae.

Genetic and physical maps for the 16 chromosomes of Saccharomyces cerevisiae are presented. The genetic map is the result of 40 years of genetic analysis. The physical map was produced from the results of an international systematic sequencing effort. The data for the maps are accessible electronically from the Saccharomyces Genome Database (SGD: http://genome-www.stanford. edu/Saccharomyces/).

Genome renewal: a new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts.

We have analyzed by genetic means 43 strains of Saccharomyces that had been isolated from fermenting grape musts in Italy. Twenty eight of these strains were isolated from 28 cellars in the Region of Emilia Romagna. The other 15 strains came from 5 fermentations at four cellars near the city of Arpino, which is located south and east of Rome. We found that 20 of the 28 strains from Emilia Romagna were heterozygous at from one to seven loci. The balance were, within the limits of our detection, completely homozygous. All these strains appeared to be diploid and most were homozygous for the homothallism gene (HO/HO). Spore viability varied greatly between the different strains and showed an inverse relation with the degree of heterozygosity. Several of the strains, and in particular those from Arpino, yielded asci that came from genetically different cells. These different cells could be interpreted to have arisen from a heterozygote that had sporulated and, because of the HO gene, yielded homozygous diploid spore clones. We propose that natural wine yeast strains can undergo such changes and thereby change a multiple heterozygote into completely homozygous diploids, some of which may replace the original heterozygous diploid. We call this process 'genome renewal'.

Synergistic interactions between RAD5, RAD16 and RAD54, three partially homologous yeast DNA repair genes each in a different repair pathway.

Considerable homology has recently been noted between the proteins encoded by the RAD5, RAD16 and RAD54 genes of Saccharomyces cerevisiae. These genes are members of the RAD6, RAD3 and RAD50 epistasis groups, respectively, which correspond to the three major DNA repair pathways in yeast. These proteins also share homology with other eucaryotic proteins, including those encoded by SNF2 and MOT1 of yeast, brahma and lodestar of Drosophila and the human ERCC6 gene. The homology shares features with known helicases, suggesting a newly identified helicase subfamily. We have constructed a series of congenic single-, double- and triple-deletion mutants involving RAD5, RAD16 and RAD54 to examine the interactions between these genes. Each deletion mutation alone has only a moderate effect on survival after exposure to UV radiation. Each pairwise-double mutant exhibits marked synergism. The triple-deletion mutant displays further synergism. These results confirm the assignment of the RAD54 gene to the RAD50 epistasis group and suggest that the RAD16 gene play a larger role in DNA repair after exposure to UV radiation than has been suggested previously. Additionally, the proteins encoded by RAD5, RAD16 and RAD54 may compete for the same substrate after damage induced by UV radiation, possibly at an early step in their respective pathways.

Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51.

The RAD51 gene of Saccharomyces cerevisiae is required both for recombination and for the repair of DNA damage caused by X rays. Here we report the sequence and transcriptional regulation of this gene. The RAD51 protein shares significant homology (approximately 50%) over a 70-amino-acid with the RAD57 protein (J.A. Kans and R.K. Mortimer, Gene 105:139-140, 1991), the product of another yeast recombinational repair gene, and also moderate (approximately 27%), but potentially significant, homology with the bacterial RecA protein. The homologies cover a region that encodes a putative nucleotide binding site of the RAD51 protein. Sequences upstream of the coding region for RAD51 protein share homology with the damage response sequence element of RAD54, an upstream activating sequence required for damage regulation of the RAD54 transcript, and also contain two sites for restriction enzyme MluI; the presence of MluI restriction sites has been associated with cell cycle regulation. A 1.6-kb transcript corresponding to RAD51 was observed, and levels of this transcript increased rapidly after exposure to relatively low doses of X-rays. Additionally, RAD51 transcript levels were found to that of a group of genes involved primarily in DNA synthesis and replication which are thought to be coordinately cell cycle regulated. Cells arrested in early G1 were still capable of increasing levels of RAD51 transcript after irradiation, indicating that increased RAD51 transcript levels after X-ray exposure are not solely due to an X-ray-induced cessation of the cell cycle at a period when the level of RAD51 expression is normally high.

Identification of RAD16, a yeast excision repair gene homologous to the recombinational repair gene RAD54 and to the SNF2 gene involved in transcriptional activation.

The RAD54 gene of Saccharomyces cerevisiae is involved in the recombinational repair of DNA damage. The predicted amino acid sequence of the RAD54 protein shows significant homologies with the yeast SNF2 protein, which is required for the transcriptional activation of a number of diversely regulated genes. These proteins are 31% identical in a 492-amino acid region that includes presumed nucleotide and Mg2+ binding sites. We noted previously that the SNF2 protein also shares homology with a partial open reading frame (ORF) that was reported with the sequence of an adjacent gene. This ORF also shares homology with the RAD54 protein. To test whether this ORF is involved in transcriptional activation or DNA repair, yeast strains deleted for part of it have been isolated. These strains do not show a Snf-like phenotype, but they are UV sensitive. This gene has been identified as RAD16, a gene involved in the excision repair of DNA damage. Analysis of the rad16 deletion mutations indicates that RAD16 encodes a non-essential function and is not absolutely required for excision repair. Outside the region of homology to RAD54 and SNF2, the predicted RAD16 protein contains a novel cysteine-rich motif that may bind zinc and that has been found recently in eleven other proteins, including the yeast RAD18 protein. The homologies between RAD16, RAD54 and SNF2 are also shared by several additional, recently isolated yeast and Drosophila genes.

A mathematical model of interference for use in constructing linkage maps from tetrad data.

In determining genetic map distances it is necessary to infer crossover frequencies from the ratios of recombinant and parental progeny. To do this accurately, in intervals where multiple crossovers may occur, a mathematical model of chiasma interference must be assumed when mapping in organisms displaying such interference. In Saccharomyces cerevisiae the model most frequently used is that of R.W. Barratt. An alternative to this model is presented. This new model is implemented using a microcomputer and standard numerical methods. It is demonstrated to fit ranked tetrad data from Saccharomyces more closely than the Barratt model and thus generates more accurate estimates of map distances when used with two-point data. A computer program implementing the model has been developed for use in calculating map distances from tetrad data in Saccharomyces.

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.

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.

A quantitative model of DNA fragments generated by ionizing radiation, and possible experimental applications.

We derive an equation for observed frequencies of DNA fragments as a function of size. In this derivation, we consider an experimental system where fragments are generated by random, independent double-strand breaks on chromosomes (or other large DNA molecules) and then separated by size on agarose gels. When visualizing these fragments using Southern hybridization techniques (employing a site-specific probe), we predict an intensity distribution that has unusual properties. In particular, peaks in the fragment size distribution depend not only on standard breakage parameters, but also on the location of the hybridization site. Our model is consistent with experimental and theoretical results reported elsewhere, where measurements of peaks are used for the physical mapping of genes. Further, we propose that similar experiments might be suitable for precise measurements of the parameters of double-strand breakage (as an alternative to neutral filter elutions and neutral sucrose gradients) and for testing the assumption of random, independent breakage for different types of radiation.

A polymerization model of chiasma interference and corresponding computer simulation.

A model of chiasma interference is proposed and simulated on a computer. The model uses random events and a polymerization reaction to regulate meiotic recombination between and along chromosomes. A computer simulation of the model generates distributions of crossovers per chromosome arm, position of events along the chromosome arm, distance between crossovers in two-event tetrads, and coincidence as a function of distance. Outputs from the simulation are compared to data from Saccharomyces cerevisiae and the X chromosome of Drosophila melanogaster. The simulation demonstrates that the proposed model can produce the regulation of recombination observed in both genetic and cytological experiments. While the model was quantitatively compared to data from only Drosophila and Saccharomyces, the regulation observed in these species is qualitatively similar to the regulation of recombination observed in other organisms.

Random-breakage mapping, a rapid method for physically locating an internal sequence with respect to the ends of a DNA molecule.

We describe a method for determining the position of a cloned internal sequence with respect to the ends of a DNA molecule. The molecules are randomly broken at low frequency and the fragments are subjected to electrophoresis. Southern hybridization using the cloned DNA as a probe identifies only those fragments containing the sequence. The size distribution of these fragments is such that two threshold changes in intensity of signal are seen in the smear pattern below the unbroken molecules. The positions of the changes represent the distances from the sequence to each molecular end. The intensity changes arise because the natural ends of the molecules influence the fragment distribution obtained. From once-broken molecules, no fragments can arise that contain a given sequence and are shorter than the distance between that sequence and the nearest molecular end. We tested the method by using x-rays to induce breakage in yeast DNA. Genes of independently known position were mapped within whole chromosomes or Not I restriction fragments using Southern blots from gels of irradiated molecules. We present equations to predict fragment distribution as a function of break-frequency and position of the probed sequence.

Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast.

We describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome III (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. We demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints, we present data on the timing of commitment to meiotic recombination scored genetically. We have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-sized circles originating in part from sister-chromatid exchange, which we find to be frequent during meiosis.

ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.

Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes.

The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis.

The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/alpha cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of beta-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.