Using yeast genetics to generate a research environment.

Many of the same features of the yeast Saccharomyces cerevisiae that have made it so useful as a genetics and molecular biology research organism make it equally useful as a teaching organism. Furthermore, the fact that it is a modern research organism makes it all the more exciting to students and teachers. The unique characteristic of yeast as a unicellular, eukaryotic organism with a complete sexual life cycle is ideal for teaching. A simple monohybrid cross to explore dominance and recessiveness, a dihybrid cross to demonstrate independent assortment, pigmented adenine auxotrophs for investigating the fundamentals of gene action, and easily measured responses to ultraviolet radiation provide an array of appropriate laboratory tools that put real science in the hands of students and teachers. Direct collaborations between scientists and science teachers bring together complementing knowledge and experience, providing an effective and efficient way to adapt and simplify techniques and procedures to accommodate time and money constraints. Collaborations quickly identify technical and theoretical problems that must be solved for implementation in classrooms. They also provide a continuing stimulus to teachers and students to participate in the research process.

The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin.

Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of alpha factor, the peptide mating pheromone produced by mating-type alpha cells. We report here the DNA sequence of BAR1, the structural gene for barrier activity. The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases. Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site. Therefore, barrier protein is probably a protease that cleaves alpha factor. The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases. However, the BAR1 gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity.

Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the alpha mating pheromone, alpha-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BAR1, mat alpha 2 BAR1, and mat alpha 1 mat alpha 2 BAR1 strains, but not by MAT alpha BAR1, MATa/MAT alpha BAR1, mat alpha 1 BAR1, or MATa barl strains, demonstrating that it is under the control of both the MAT alpha 2 and the BAR1 genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to alpha-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for alpha-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The alpha-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for alpha-factor was found to be relatively insensitive to pH over a range of several units, the alpha-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BAR1 gene product in recovery of mating-type a cells from cell division arrest by alpha-factor.

Two temperature-sensitive mutants of Saccharomyces cerevisiae with altered expression of mating-type functions.

Two mutants of Saccharomyces cerevisiae have been isolated from normal haploid MAT alpha strains and characterized as having temperature-sensitive, pleiotropic phenotypes for functions associated with mating. At the permissive temperature, 23 degrees C, they were found to behave as normal MAT alpha haploids with respect to mating efficiency, sporulation in diploids formed with MAT a strains, secretion of alpha-factor, and failure to secrete the MATa-specific products, a-factor and Barrier. At higher temperatures they were found to decline in mating and sporulation efficiency and to express the a-specific functions. Genetic analysis established that one of these mutants, PE34, carries a temperature-sensitive allele of the MAT alpha 2 gene and that the other, PD7, carries a temperature-sensitive allele of the TUP1 gene.

Sensitivity of tryptophan, tyrosine and phenylalanine mutants of Saccharomyces cerevisiae to phenethyl alcohol.

Phenethyl alcohol inhibits the growth of many microorganisms. It is believed that the growth inhibition is mediated by its effect on the cell membrane. Differences between sensitive and resistant strains are suggested to be due to alterations in membrane structure. We report that, in some strains, an unexpected relationship exists between auxotrophy for tryptophan, tyrosine and phenylalanine and sensitivity to phenethyl alcohol.

The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates.

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.

Expression of radiation-induced mutations at the arginine permease (CAN1) locus in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the expression of resistance to the L-arginine analog, L-canavanine, after mutagenesis, is strongly dependent on the metabolic state of the cell. The frequency of mutations recovered after exposure to ultraviolet light or X rays was measured under a variety of culture conditions. The results indicate that the frequency of mutants recovered is determined by the following three factors: (1) The potential mutants still possess enough permease activity to take up some of the cell poison, and some are therefore killed before they can express the mutant genotype. The sensitivity is strongly influenced by the endogenous free arginine, which is in turn influenced by the growth medium. (2) The rapid decay of the permease molecules and the inability of the potential mutants to resynthesize this protein results in a rapidly increasing change of expression when selection is delayed. (3) During the time when the permease activity is decaying, repair of the mutagen-induced damage appears to occur.

Tetraploid Strains of SACCHAROMYCES CEREVISIAE That Are Trisomic for Chromosome III.

Meiotic segregation of several genes has been studied in tetraploid strains that are trisomic for chromosome III. The segregation data were compared to a computer simulation that assumes trivalent pairing of homologues involved in exchanges, followed by nonpreferential segregation. Trivalent pairing was characterized by higher frequencies of exchange as compared to bivalent pairing, and by the presence of spores resulting from at least double crossovers involving all three homologues. Trivalent segregation was characterized by a unique recombinant class. The strong interference normally exhibited in diploid meiotic recombination was not evident from the frequency of double crossovers in these strains.

Expression of cryptopleurine resistance in Saccharomyces cerevisiae.

An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.

Mutants of Saccharomyces cerevisiae resistant to the alpha mating-type factor.

Mutants that are resistant to alpha-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified alpha-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as non-mating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to alpha-factor by cells of mating-type a is essential for mating.

Suppression of two missense alleles of the TRP5 locus of Saccharomyces cerevisiae.

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cerevisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUP101. SUP101 does not suppress ochre alleles ade2-1, his5-2, arg4-17, lys1-1, amber alleles trp1-1, tyr7-1, or unclassified alleles at a number of other loci. These results indicate SUP101 is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozygous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the complete medium always segregated with the suppressor.

Genetic analysis of mutations affecting growth of Saccharomyces cerevisiae at low temperature.

A large number of genes control growth of the yeast Saccharomyces cerevisiae at low temperatures (< 10 degrees ). Approximately 47 percent of the mutants selected for inability to grow at 4-5 degrees C show increased sensitivity to cycloheximide. In 3 of 4 cases tested, supersensitivity to cycloheximide and inability to grow at the low temperature segregate together and thus appear to be effects of the same mutation. Since many cold-sensitive mutants of bacteria have been found to have altered ribosomes and since cycloheximide resistance in yeast can be caused by ribosomal changes, this suggests that the mutants having low-temperature-sensitive growth may be defective in ribosome-assembly processes at the low temperatures. Two of the lts loci, lts1 and lts3 have been located on chromosome VII and another two, lts4 and lts10 on chromosome IV. A mutation, cyh10, conferring cycloheximide resistance, but not cold sensitivity, has been located close to the centromere on chromosome II.

Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. I. Isolation and phenotypic characterization of nonmating mutants.

Nonmating mutants were also isolated from haploid strains of yeast of both mating types. The mutants were characterized with respect to their ability to produce and respond to specific yeast sex factors, their ability to mate at low frequencies, and the ability of the low-frequency diploids to sporulate. Loss of the ability to mate by either mating type was invariably accompanied by the loss of one or more, and in some cases, all, of the above capabilities. The results strongly indicate that the sex factors are functionally involved in the conjugation process.

Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. II. Genetic analysis of nonmating mutants.

Rare diploids formed by sterile mutants have been studied by tetrad analysis. Sixteen classes of mutants representing at least five distinct genetic loci have been defined. One group of mutations, isolated only in alpha, maps at the mating-type locus, while none of the others shows any linkage to mating type. Some of the mutations are nonspecific for mating type, while others act only on a or alpha. In addition, mutations were found that prevent sporulation when heterozygous in diploids. These appear to be mutations of the mating-type alleles.

Saccharomyces cerevisiae: a diffusible sex factor.

A hormone-like substance is secreted by alpha mating-type cells of heterothallic yeast strains. It induces in cells of the opposite mating type, a, a morphological change characteristic of the mating process. Secretion of this substance and mating ability have some common genetic determinants. In partially purified preparations, the substance has properties of an oligopeptide.

Physiological advantage of the mechanism of the tryptophan synthetase reaction.

Indole-accumulating and indole-utilizing tryptophan synthetase mutants of Saccharomyces cerevisiae were found to form complementing diploids with unusual growth properties. These strains grew at nearly the wild-type exponential rate, but only after an abnormally prolonged lag period of a duration that depended on the initial cell density. The exaggerated lag was reduced by adding low concentrations of indole in the medium. The growth properties were interpreted to be a consequence of the inability of these strains to synthesize tryptophan by the normal mechanism.

Genetic and biochemical studies of partially active tryptophan synthetase mutants of Saccharomyces cerevisiae.

Approximately 20% of the tryptophan synthetase mutants (tr(5)) of Saccharomyces cerevisiae retain activity in one of the half reactions catalyzed by this enzyme and have been identified as indole-accumulating or indole-utilizing tr(5) mutants by complementation tests. Ten indole-accumulating and six indole-utilizing mutants have been studied. For the half reactions they catalyze, these partially active mutants have from about one-half to twice the specific activities of the wild-type enzyme. Indole-accumulating mutant enzymes showed varying responses to pyridoxal phosphate and serine in the assay mixture. The partially active mutants were further characterized by their patterns of allelic complementation and their distribution on the fine-structure map of the locus. It was concluded that these mutants define two distinct functional regions of the tr(5) locus, corresponding to the two half reactions.