Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Adenosine kinase-deficient mutant of Saccharomyces cerevisiae

Abstract A cordycepin-resistant mutant strain of Saccharomyces cerevisiae (CD-R2) was found to be deficient in adenosine kinase. This mutant accumulated S-adenosylhomocysteine during growth in the presence of exogenous adenosine and it grew in a pseudohyphal manner in the presence of this nucleotide.     

NMR analysis of a cell division cycle mutant of Saccharomyces cerevisiae

cdc 19.1 is a temperature-sensitive lesion in the genome of Saccharomyces cerevisiae. The phenotype of this mutant is a cell cycle specific arrest in G1, which is expressed at 37 degrees C. In the present study, 31P- and 13C-NMR spectroscopy were used to analyze the metabolism of the mutant at the permissive and restrictive temperatures. Our results confirm previous findings which have indicated that cdc 19.1 contains temperature-sensitive pyruvate kinase activity. In contrast to previous findings, however, the present investigation demonstrates that restriction of pyruvate kinase activity in vivo takes as long as 24 h to be fully expressed. In addition, analysis by NMR has allowed us to assess the metabolic consequences of pyruvate kinase restriction which may contribute to the arrest of cell growth in the early G1 phase of the cell division cycle.     

Genetic and physiological analysis of UV-sensitive mutants of Saccharomyces cerevisiae

The use of tetrad analysis and complementation tests indicates that the groups of UV-sensitive mutants assigned the labels radI and rad3 are alleles of two single genes involved in the process of cellular repair of UV-induced damage in the yeast Saccharomyces cerevisiae.     

Genetic analysis of the mutant RD-50 in Saccharomyces cerevisiae. The role of nuclear and mitochondrial mutation

Inheritance of the mutant phenotype of respiratory deficience in RD-50 strain was studied. The deficience can be restored, giving respiratory sufficience, in crosses with rho0 testers. The "restorable" phenotype of the mutant, named RDc+, was shown to be determined by a nuclear pet-like mutation (pet50). The restorable RDc+ phenotype is stabilized in the presence of the pet50 allele, but can remain as such in the presence of the wild-type allele PET50. Restoration also takes place in cytoductants with the nucleus of kar partner. In order to explain the behaviour of the mitochondrial mutation mit50, we suppose it to be a microdeletion, capable of reversion, due to integration of a putative mt episome. Some features of the nuclear mutation pet50, particularly, its segregation in mitotic progeny of some revertants to respiratory competence point to its peculiarity. We suppose the mutation pet50 to be an insertion into the chromosomal PET50 gene. This insertion may be excised, remaining within the cell in the free state for some time, and then either eliminate or reintegrate into the chromosome.     

A potassium transport mutant of Saccharomyces cerevisiae

A mutant in Saccharomyces cerevisiae required one hundred times more K⁺ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K⁺ did not show significant differences in Rb⁺ transport. In the mutant, a rapid K⁺ loss induced by azide or incubation (4 h) in K⁺-free medium decreased the Rb⁺ transport K _m by one half; in the wild type, those treatments decreased the Rb⁺ K _m twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na⁺ transport and in the Na⁺ inhibition of Rb⁺ transport, either in normal-K⁺ cells or in K⁺-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K⁺ transport in S. cerevisiae.Abbreviations YPD yeast-peptone-dextrose medium     

Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene. Three mutant classes, which correspond to three different regions of the receptor protein, were observed. 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity. 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest. Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression. Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2. 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone. Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal. The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1. Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.     

A halotolerant mutant of Saccharomyces cerevisiae.

FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression.     

Properties of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae

We characterized a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae, TFL20, that has a mutation in the LEU4 gene. We monitored the concentration of extracellular i-AmOH and intracellular amino acids, and compared the ratios of gene expression in TFL20 with the wild-type strain, K30. We found that the LEU1, LEU2, and BAT1 genes were up-regulated in TFL20 for metabolism, and that TFL20 simultaneously produced as much i-AmOH and leucine as K30 does.     

Plasmid-mediated complementation of a delta-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant

Recombinant plasmids able to complement the Saccharomyces cerevisiae ole3 mutation were isolated. The nucleotide sequence responsible for complementation was localized to a 3.5-kb region. The level of delta-aminolevulinate (ALV) synthase activity in wild-type cells was six-fold lower than in plasmid-transformed ole3 mutant cells. Certain clones secreted a compound that supported growth of a lawn of adjacent ole3 mutant cells.     

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.