Yeast hexokinase mutants.

Using yeast mutants, it is shown that growth on glucose occurs even in the absence of both hexokinase A and hexokinase B; fructose growth requires at least one of these two enzymes. Expression of hexokinase A and of glucokinase seem to be regulated.     

The Biological Role of Glycerol in Yeast Cells. Yeast as Glycerol Producers

Applied Biochemistry and Microbiology - This review contains information on the physiological role of glycerol as an osmoprotective and cryoprotective factor in the vital activity of yeast cells....     

Nobel Lecture. Yeast and cancer

The discovery of genes that control cell division in yeast, and their relation to cancer, is reviewed.     

Yeast lacking superoxide dismutase. Isolation of genetic suppressors.

Null mutants of superoxide dismutase (SOD) in Saccharomyces cerevisiae are associated with a number of biochemical defects. In addition to being hypersensitive to oxygen toxicity, strains containing deletions in both the SOD1 (encoding Cu/Zn-SOD) and SOD2 (encoding Mn-SOD) genes are defective in sporulation, are associated with a high mutation rate, and are unable to biosynthesize lysine and methionine. The sod-linked defect in lysine metabolism was explored in detail and was found to occur at an early step in lysine biosynthesis, evidently at the level of the alpha-amino adipate transaminase. To better understand the role of SOD in cell metabolism, our laboratory has isolated yeast suppressors that have bypassed the SOD defect ("bsd" strains), that is, S. cerevisiae cells lacking SOD, yet resistant to oxygen toxicity. Two nuclear bsd complementation groups have been identified, and both suppress a variety of biological defects associated with sod1 and sod2 null mutants. These results demonstrate that a single gene mutation can alleviate the requirement for SOD in cell growth. Both bsd complementation groups are unable to utilize many non-fermentable carbon sources, suggesting a possible suppressor-linked defect in electron transport.     

Ends-in Vs. Ends-Out Recombination in Yeast

Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for ``omega' insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks.     

Mechanisms of Fatty Acid Toxicity for Yeast.

Neal, A. L. (Rutgers, The State University, New Brunswick, N.J.), Joan O. Weinstock, and J. Oliver Lampen. Mechanisms of fatty acid toxicity for yeast. J. Bacteriol. 90:126-131. 1965.-The internal pH of stationary- and log-phase yeast cells dropped quite rapidly when the cells were exposed to acetate buffers at pH 4 and 3, whereas no, or much less, acidification occurred with pyruvate or phosphate. Although inhibition of respiration and glycolysis was almost instantaneous when the cells were exposed to 0.2 m acetate at pH 4, the effect was not permanent and could be reversed by washing them with water or phosphate buffer. Irreversible inhibition did occur, however, at 0.5 m acetate under the same conditions; there was a marked decrease in several glycolytic enzyme systems, which undoubtedly contributed to the irreversible nature of the inhibition. In cell-free homogenates, various low-molecular-weight monocarboxylic acids exhibited about the same inhibitory effect on glycolysis; structural differences such as branching or unsaturation did not cause a marked change in their inhibitory effect. Also, glycolysis was much more sensitive to dicarboxylic acids such as succinate and phthalate than to acetate; phthalate was more inhibitory than succinate. This is in contrast with the noninhibitory nature of succinate and phthalate to whole cells, even at pH 4. Pyruvic acid decarboxylation was inhibited by phthalate but not by succinate. The greater toxic effect of phthalic acid may be due to the fixed steric configuration of its carboxyl groups, as compared with those of succinic acid.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Yeast metallothionein. Sequence and metal-binding properties

The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein.