Susan A. Henry

Selection of spontaneous mutants by inositol starvation in yeast

SummaryA new method for the routine isolation of mutations of spontaneous origin in the yeast, Saccharomyces cerevisiae, is reported. The technique is based on the observation that inositol auxotrophs die when deprived of inositol. However, if marromolecular synthesis is inhibited, most of the cells survive. Appropriate manipulation of inositol requiring mutants can threfore result in the selective survival of cells possesing mutations which affect macromolecular synthesis. Since reversion to inositol prototrophy can be a major source of interference in efficient selection, a haploid double mutant strain has been constructed which reverts to inositol prototrophy with a frequency estimated to be several orders of magnitude lower than the expected frequency of single, spontaneous mutational events. Using this strain, enrichment in excess of 10000 fold has been obtained for various classes of auxotrophic mutants. Spontaneous temperature sensitive mutants have also been obtained.

Characterization of a yeast regulatory mutant constitutive for synthesis of inositol-1-phosphate synthase

SummaryThe enzyme inositol-1-phosphate synthase is repressed at least 50-fold in wild type yeast grown in inositol-supplemented media. Mutants which synthesize this enzyme constitutively have been isolated using a selection procedure based on excretion of inositol into the growth medium by putative mutants. Biochemical analysis of one of the mutants (opi1-1) confirmed that the nature of the mutations is regulatory, and not in the structural gene for the enzyme. Immunoprecipitation of crude extracts with antibody directed against purified inositol-1-phosphate synthase showed that a protein which reacts with the antibody is present in the mutant grown under both repressing and derepressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed. Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and drepressing conditions. Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD. These analyses indicated that purified mutant synthase was similar to the wild type enzyme.

The mechanism of interallelic complementation at the INO1 locus in yeast: Immunological analysis of mutants

SummaryThe ino1 locus of yeast has been demonstrated to be the structural gene for the repressible enzyme, L-myo-inositol-1-phosphate synthase (Donahue and Henry 1981 a). We have screened a large number of allelic representatives of the ino1 locus for the presence of protein which cross reacts with antibody produced in response to purified wild type inositol-1-phosphate synthase. Approximately 50% of all ino1 representatives screened by immunoprecipitation produce a protein of 62,000 molecular weight, identical in size to the wild type enzyme subunit. These mutants (termed crm+) were tested for expression of the 62,000 MW protein under conditions which are repressing for the wild type enzyme (greater than 25 μM exogenous inositol). The protein produced by the crm+ mutants, like the active enzyme in wild type yeast, is repressed in the presence of high levels of exogenous inositol. In addition, we have reassessed the interallelic complementation pattern observed among mutants at the ino1 locus. The entire pattern of interallelic complementation is temperature sensitive.

Synthesis of specific membrane proteins is a function of DNA replication and phospholipid synthesis in Caulobacter crescentus

Abstract Analysis of the effects on membrane function and protein composition of altering phospholipid synthesis in Caulobacter crescentus showed that, like other bacteria, C. crescentus continues to induce a lactose transport system and to synthesize most membrane proteins. However, we show that the incorporation of a set of outer membrane proteins primarily synthesized in stalked cells is dependent on DNA replication which, in turn, is dependent on membrane phospholipid synthesis. Furthermore, the incorporation of another set of membrane proteins, two of which are synthesized primarily in the swarmer cell, appears to be independent of the replication of the chromosome but to be directly dependent on phospholipid synthesis. We have also found that when phospholipid synthesis is blocked, the synthesis of the flagellar proteins is inhibited and that this effect may be mediated by the primary inhibition of DNA replication. Newton has presented evidence that the synthesis of flagellar proteins is dependent on specific execution points in DNA replication and that this connection serves as a temporal regulator of differential protein synthesis (Osley et al. , 1977; Sheffery & Newton, 1981). We suggest here that a direct link between the replicating chromosome and the growing membrane might serve, in turn, to dictate the site of membrane assembly of newly synthesized gene products.

The effect of termination of membrane phospholipid synthesis on cell-dependent events in caulobacter☆

Abstract Membrane phospholipid synthesis in Caulobacter crescentus has been shown to be related to the expression of specific cell cycle events. DNA synthesis was immediately inhibited if phospholipid synthesis was terminated either by glycerol starvation of a glycerol auxotroph or by treatment of mutant and wildtype cultures with cerulenin. Termination of phospholipid synthesis, by either method, resulted in the inhibition of stalk elongation, flagellum biogenesis and cell division. The inability to form a stalk appears to be directly due to the cessation of phospholipid synthesis, whereas the inhibition of flagella formation and cell division is likely a result of the secondary effect on DNA replication. Two cell cycle events, the ejection of the flagellum and stalk initiation, were shown to be independent of phospholipid synthesis and DNA replication.

INOSITOL-1-PHOSPHATE SYNTHASE MUTANTS OF THE YEAST, SACCHAROMYCES CEREVISIAE

SUMMARY: More than 100 independent inositol-requiring mutants of Saccharomyces cerevisiae have been obtained by ethyl methane sulfonate mutagenesis. Representative mutants have been shown to lack the enzyme inositol-1-phosphate synthase. The mutants fall into 15 major complementation groups. Several of the complementation groups are represented by mutants which are simultaneously respiratory deficient (petite). The petite phenotype cosegregates with the inositol requirement. Progress in the genetic analysis of the mutants is reported.