Genetic Analysis of Petite Mutants of SACCHAROMYCES CEREVISIAE : Transmissional Types

We have studied a number of petite [rho^- ] mutants of Saccharomyces cerevisiae induced in a wild-type strain of mitochondrial genotype [ome^- CHL ^R ERY^S OLI^S_1,2,3 PAR^S] by Berenil and ethidium bromide, all of which have retained two mitochondrial genetic markers, [CHL^R] and [ERY^S], but have lost all other known markers. Though stable in their ability to retain these markers in their genome, these mutants vary widely among themselves in suppressiveness and in the extent to which the markers are transmitted on crossing to a common wild-type tested strain. In appropriate crosses all of the strains examined in this study demonstrate mitochondrial polarity, and thus have also retained the [ome^-] locus in a functional form; however, five different transmissional types were obtained, several of them quite unusual, particularly among the strains originally induced by Berenil. One of the most interesting types is the one that appears to reverse the parental genotypes with [CHL^R ERY^S] predominating over [CHL^S ERY^R] in the diploid [rho+] progeny, rather than the reverse, which is characteristic of analogous crosses with [rho+] or other petites. Mutants in this class also exhibited low or no suppressiveness. Since all of the petites reported here are derived from the same wild-type parent, and so have the same nuclear background, we have interpreted the transmissional differences as being due to different intramolecular arrangements of largely common retained sequences.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

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.     

Gene Duplication in SACCHAROMYCES CEREVISIAE

Five indepdendent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found.     

Genetic Analysis of Mutants of SACCHAROMYCES CEREVISIAE Resistant to the Herbicide Sulfometuron Methyl

Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media. Sixty-six spontaneous mutants resistant to SM were isolated. All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive. The mutations occur in three linkage groups, designated SMR1, smr2 and smr3. Several lines of evidence demonstrate that the SMR1 mutations (47 dominant and four semidominant) are alleles of ILV2 which encodes acetolactate synthase (ALS), the target of SM. First, SMR1 mutations result in the production of ALS enzyme activity with increased resistance to SM. Second, molecular cloning of the ILV2 gene permitted the isolation of mutations in the cloned gene which result in the production of SM-resistant ALS. Finally, SMR1 mutations map at the ILV2 locus. The smr2 mutations (four recessive, two dominant and one semidominant) map at the pdr 1 (pleiotropic drug resistance) locus and show cross-resistance to other inhibitors, typical of mutations at this locus. The smr3 mutations (six recessive and two dominant) define a new gene which maps approximately midway between ADE2 and HIS3 on the right arm of chromosome XV.     

Gene Duplication as a Mechanism of Genetic Adaptation in SACCHAROMYCES CEREVISIAE

It has been shown that specific mutations of the gene that codes for the general acid monophophatase (Aphtase) of S. cerevisiae can increase the affinity of this enzyme for beta-glycerophosphate (BGP) and thereby provide this organism with the capacity to exploit extremely low concentrations of this organic phosphate (Francis and Hansche 1973). In this report two additional avenues are demonstrated to be available to this organism for increasing its capacity to exploit low concentrations of organic phosphates. One avenue is through mutations that increase the amount of Aphtase that associates with the cell wall, where it catalizes the hydrolysis of exogenous organic phosphates. The other avenue is through duplication of the gene that codes for Aphtase, doubling the amount of Aphtase synthesized.--The spontaneous duplication of the structural gene of Aphtase and the incorporation of the duplicate into this experimental population as a means of exploiting low concentrations of exogenous organic phosphates provides direct support for the first step of the mechanism through which new metabolic functions are postulated to evolve.     

Mitochondrial Genetics. Xii. an Oligomycin-Resistant Mutant Localized at a New Mitochondrial Locus in SACCHAROMYCES CEREVISIAE

Three antibiotic-resistance mutations were isolated from strain FL496–2B: two are independent Mendelian genes, one conferring both oligomycin and venturicidin resistance (oli^R₄₉₆) and the other conferring cycloheximide resistance (cyh^R₄₉₆). The third is a mitochondrial mutation, O^R₉, and confers a low level of oligomycin resistance to cells (in vivo) but not to the extracted mitochondrial ATPase (in vitro). This mutation is located on the mitochondrial DNA at a new locus [OLI4] linked to [OLI2] and independent from [OLI1] and [OLI3] and from the other mitochondrial loci.

All three mutations (O ^R₉, oli^R₄₉₆, cyh^R₄₉₆ ) were found without any selection, in the same prototrophic haploid strain, which contained unknown resistances to antibiotics.

Some physiological, genetical and biochemical properties of the mitochondrial mutation are described.

     

Rearrangement of the Genetic Map of Chromosome VII of SACCHAROMYCES CEREVISIAE

The genetic map of the right arm of chromosome VII of Saccharomyces cerevisiae includes markers on a distal segment for which meiotic linkage to the centromere-proximal marker cly8 has not previously been demonstrated. According to the currently accepted map, SUF4 is the most distal marker on the right arm. We have shown by tetrad analysis that SUF4 is linked to cly8 and ade6. The genetic distance between SUF4 and cly8 is 29 cM. These data indicate that the genetic map of the right arm of chromosome VII should be revised by inverting the orientation of the distal segment so that SUF4 is located near cly8, and SUC1 and MAL1 are the most distal markers. With this revision, all of the polymeric fermentation markers that have been mapped are located at the ends of chromosomes.     

Genetic Analysis of Multiple Drug Cross Resistance in SACCHAROMYCES CEREVISIAE: a Nuclear-Mitochondrial Gene Interaction

A mutant of the yeast Saccharomyces cerevisiae, cross resistant to several antibiotics, was isolated in our laboratory and subjected to genetic analysis. Tetrad analysis of diploids obtained from crosses between the resistant mutant and a sensitive wild-type strain suggest that the multiple resistance to the five agents, oligomycin (OLI), rhodamine 6G (RHG), tetracycline (TCN), chloramphenicol (CAP) and cycloheximide (CHX) is determined by a single nuclear gene, ant1, and requires several cytoplasmic genes for expression of resistance to oligomycin, rhodamine 6G and tetracycline. --Vegetatively growing diploid clones derived from the cross ant1 [RHO+] X +[RHO+] show mitotic segregation of two phenotypic classes for the drugs OLI, RHG TCN. Diploids derived from the two reciprocal crosses, ant1 [RHO+] X +[RHO-] and ant1 [RHO-] X +[RHO+], fail to exhibit mitotic segregation. These results are consistent with our hypothesis concerning the involvement of cytoplasmic loci. They suggest, in addition, that these loci are associated with mitochondrial DNA (mtDNA). --Evidence for this association is provided by the demonstration of genetic linkage between the cytoplasmic loci involved in the interaction, RHG-1, TCN-1 and OLI-5, and two well-characterized mitochondrial loci, ERY and CAP. --We have mapped the nuclear ant1 locus 3.3 cM from the centromere-linked gene, leu1, on the same side of the centromere of chromosome VII as leu1. --In the light of these findings, we discuss the claims made by several authors of the episomal nature of mutations similar to the one described here, as well as of the possible involvement of yeast 2 mu DNA in such mutations.     

Genetic Properties of Mutations at the PEP4 Locus in SACCHAROMYCES CEREVISIAE

Yeast cells that inherit mutations at the PEP4 locus exhibit a pronounced phenotypic lag in the expression of the mutant phenotype imparted by these mutations. This lag appears to extend to all of the enzymes that are affected by the pep4-3 mutation. For at least two of the enzymatic activities, phenotypic lag shows mitotic cosegregation. Phenotypic lag is found for meiotic progeny and for mitotic segregants from heterokaryons. The phenotypic lag in the expression of the carboxypeptidase Y deficiency is abolished by nonsense mutations in either PRC1, the structural gene for carboxypeptidase Y, or PRB1, the structural gene for proteinase B. Models to explain these observations are proposed.     

Mating-Type Differentiation by Transposition of Controlling Elements in SACCHAROMYCES CEREVISIAE

The nonfunctional mutation of the homothallic gene HML alpha, designated hml alpha, produced two mutant alleles, hml alpha-1 and hml alpha-2. Both mutant clones were mixed cultures consisting of a mating-type cells and nonmating haploid cells. The frequencies of the two cell types were different, and a few diploid cells able to sporulate were found in the hml alpha-2 mutant. Conversions of an a mating-type cell to nonmater, and vice versa, were observed in both mutants. The conversion of an a mating phenotype to nonmating is postulated to occur by alteration of the a mating type to the sterile mating-type allele in the hml alpha-1 mutant. In tetrad dissection of prototrophic diploids that were obtained by rare mating of hml alpha-1 mutants with a heterothallic strain having the MATa ho HMRa HMLa genotype, many mating-deficient haploid segregants were found, while alpha mating-type segregants were observed in a similar diploid using an hml alpha-2 mutant. The mating-type-deficient haploid segregants were supposed to have the sterile alpha mating-type allele because the nonmating genetic trait always segregated with the mating-type locus. Sporogenous diploid cells obtained in the hml alpha-2 mutant clone had the MATa/MAT alpha HO/HO HMRa/HMRa hml alpha-2/hml alpha-2 genotype. These observations suggested that the hml alpha-1 allele produces a transposable element that gives rise to the sterile alpha mating type by transposition into the mating-type locus, and that the hml alpha-2 allele produces an element that provides alpha mating-type information, but is defective in the structure for transposition.     

Mapping Chromosomal Genes of SACCHAROMYCES CEREVISIAE Using an Improved Genetic Mapping Method

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + approximately n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and alpha strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.