Increased demand for NAD+ relative to ATP drives aerobic glycolysis

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Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion

Abstract Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40°C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.     

Nonrecombinant Meiosis I Nondisjunction in Saccharomyces Cerevisiae Induced by Trna Ochre Suppressors

The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.     

Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation.

In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion. We showed that single-stranded regions were formed on both sides of the double-strand break prior to the formation of the product. The kinetics of the single-stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain. In rad50 mutants, single-stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate. Product formation was largely blocked in the rad52 mutant. In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked. rad50 appears to act before or at the same stage as rad52. We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other. Deletions formed preterentially between the homologous regions closest to the double-strand break. By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp. In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner.     

Crl Mutants of Saccharomyces Cerevisiae Resemble Both Mutants Affecting General Control of Amino Acid Biosynthesis and Omnipotent Translational Suppressor Mutants

Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.     

Cycloheximide-Resistant Temperature-Sensitive Lethal Mutations of Saccharomyces Cerevisiae

We describe the isolation and preliminary characterization of a set of pleiotropic mutations resistant to the minimum inhibitory concentration of cycloheximide and screened for ts (temperature-sensitive) growth. These mutations fall into 22 complementation groups of cycloheximide resistant ts lethal mutations (crl). None of the crl mutations appears to be allelic with previously isolated mutations. Fifteen of the CRL loci have been mapped. At the nonpermissive temperature (37°), these mutants arrest late in the cell cycle after several cell divisions. Half of these mutants are also unable to grow at very low temperatures (5°). Although mutants from all of the 22 complementation groups exhibit similar temperature-sensitive phenotypes, an extragenic suppressor of the ts lethality of crl3 does not relieve the ts lethality of most other crl mutants. A second suppressor mutation allows crl10, crl12, and crl14 to grow at 37° but does not suppress the ts lethality of the remaining crl mutants. We also describe two new methods for the enrichment of auxotrophic mutations from a wild-type yeast strain.