Regulation of phospholipid metabolism in yeast.

Regulation of phospholipid metabolism in the model eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe was examined in order to further understand how cells maintain specific phospholipid compositions. Cardiolipin (CL) biosynthesis in S. cerevisiae was studied by trying to identify genes involved in the expression of phosphatidylglycerophosphate (PGP) synthase, which catalyzes the committed step in CL formation. Complementing an E. coli PGP synthase mutation with yeast genomic DNA, screening for PGP synthase expression in S. cerevisiae cytochrome c oxidase mutants, and screening for PGP synthase overproduction in wild type S. cerevisiae cells transformed with a high copy yeast genomic library did not lead to the identification of genes encoding this enzyme. S. cerevisiae petite mutants (bearing nuclear mutations affecting mitochondrial function) were also assayed for PGP synthase activity, since PGP synthase is presumed to be a nuclear encoded mitochondrial enzyme. While a mutant with low PGP synthase activity was not identified, petite strain C92 had a 4-fold increase in PGP synthase activity. However, high enzyme activity did not co-segregate with the petite phenotype. In another approach, mutagenized yeast cells were screened for defective incorporation of the PGP synthase substrate glycerol-3-phosphate into lipid products by replica printing colony autoradiography. Although no PGP synthase mutants were isolated in this screen, mutants with deficient incorporation of glycerol-3-phosphate into lyso-phosphatidic acid (lyso-PA), phosphatidic acid (PA), and diacylglycerol were identified and characterized. Regulation of S. cerevisiae glycerophosphate acytransferase (GPAT) and dihydroxyacetonephosphate acyltransferase (DHAPAT), enzymes involved in the synthesis of lyso-PA and PA, was subsequently analyzed. GPAT and DHAPAT activities were both derepressed in wild type cells grown on a nonfermentable carbon source but were not affected by inositol, a key regulator of expression of many yeast phospholipid biosynthetic enzymes in both S. cerevisiae and S. pombe. In addition, inositol-less death was shown to occur in S. pombe, although it was strain- and cell concentration-dependent. This loss in viability as S. pombe cells were starved for inositol was used as a powerful enrichment procedure for mutant isolation in this organism.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103456/1/9319588.pdfDescription of 9319588.pdf : Restricted to UM users only

Stratégies d'apprentissage et réussite au secondaire : un passeport pour les élèves en difficulté

"Matériel reproductible inclus"--Couv.Bibliogr.: p. [307]-312.Titre original: Academic success strategies for adolescents with learning disabilities and ADHD

Mutant enrichment of Schizosaccharomyces pombe by inositol-less death.

Enrichment procedures, such as those utilizing inositol-less death, have proven to be extremely powerful for increasing the efficiency of identification of spontaneous mutants in a variety of procaryotic and eucaryotic organisms. We characterized inositol-less death in several widely used strains of the inositol-requiring yeast Schizosaccharomyces pombe and determined conditions under which this phenomenon can be used to enrich for mutants. Conflicting reports in the literature on the effects of inositol starvation upon viability of S. pombe had cast doubt on the suitability of using inositol-less death in a mutant enrichment procedure for this organism. We determined that inositol-less death was strain dependent, with differences in viability of up to 5 orders of magnitude observed between the most-sensitive strain, 972, and the least-sensitive strain, SP837. Inositol-less death was also dependent upon the cell concentration at the time of initiation of starvation. While inositol-less death occurred at all four temperatures tested, the kinetics of death was slower at 16 degrees C than at 23, 30, or 37 degrees C. Inositol-less death was observed during growth in fermentable and nonfermentable carbon sources, although loss of viability in glycerol-ethanol was significantly slower than that in glucose, sucrose, or raffinose. The feasibility of exploiting inositol-less death to enrich for spontaneous mutants was demonstrated by the identification of amino acid auxotrophs, nucleotide auxotrophs, carbon source utilization mutants, and temperature-sensitive mutants. By varying starvation conditions, some mutants were recovered at frequencies as high as 5.7 x 10(-2), orders of magnitude higher than the spontaneous mutation rate