Local environment in yeast-based impedance biodosimeters strongly influences the measurable dose.

This work investigates the feasibility of yeast-based impedance measurements for retrospective dosimetry applications. The local environment around yeast cells in a previously developed film-badge was modeled using Geant4. A greater dose response was observed when yeast cells were surrounded by an aluminum-polymer structure, which acted as a conversion layer. Bench-top experiments were conducted using a jar-based dosimeter design that directly combined a finely-ground aluminum conversion medium with yeast powder. It was shown when irradiated in the presence of aluminum grains, yeast cells yielded a higher impedance signal, thereby indicating greater radiation-induced damage. Finally, in separate irradiation experiments, lead and aluminum sheets were placed behind yeast samples and the dosimeters were irradiated to 1 Gy. A 2-fold increase in the impedance signal was shown when samples were positioned in close contact with the lead sheet compared to the aluminum sheet. In all experiments, it was shown that the local environment significantly influences radiative energy deposition in yeast cells.

Two recessive suppressors of Saccharomyces cerevisiae cho1 that are unlinked but fall in the same complementation group.

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.

SACCHAROMYCES CEREVISIAE Recessive Suppressor That Circumvents Phosphatidylserine Deficiency.

Phenotypic reversion of six independent Saccharomyces cerevisiae cho1 mutants was shown to be due predominantly to mutation of an unlinked gene, eam1. The eam1 gene was located very close to ino1 on chromosome X by meiotic tetrad analysis. Recessive eam1 mutations did not correct the primary cho1 defect in phosphatidylserine synthesis but made endogenous ethanolamine available for sustained nitrogenous phospholipid synthesis. A novel biochemical contribution to nitrogenous lipid synthesis is indicated by the eam1 mutants.

Secretion can proceed uncoupled from net plasma membrane expansion in inositol-starved Saccharomyces cerevisiae.

Secretion of acid phosphatase and invertase was examined in an inositol-requiring ino1 mutant of the yeast Saccharomyces cerevisiae. Inositol starvation is known to block plasma membrane expansion, presumably due to restricted membrane phospholipid synthesis. If membrane expansion and extracellular protein secretion are accomplished by the same intracellular transport process, one would expect secretion to fail coordinately with cessation of plasma membrane growth in inositol-starved cells. In glucose-grown, inositol-starved cells, plasma membrane expansion and acid phosphatase secretion stopped coordinately, and intracellular acid phosphatase accumulated. In sucrose-grown, inositol-starved cells, plasma membrane growth halted, but secretion of both acid phosphatase and invertase continued until the onset of inositol-less death. Although glucose-grown and sucrose-grown cells differ in their ability to secrete when deprived of inositol, they exhibited the same disturbances in phospholipid synthesis. Phosphatidylinositol synthesis failed, and its precursors phosphatidic acid and CDP-diglyceride accumulated equally in both cultures. Sucrose-grown yeast cells appear to accomplish normal levels of extracellular protein secretion by an inositol-independent mechanism. In glucose-grown yeasts, both plasma membrane expansion and secretion are inositol dependent.

Plasma membrane expansion terminates in Saccharomyces cerevisiae secretion-defective mutants while phospholipid synthesis continues.

Phospholipid synthesis activity and plasma membrane growth have been studied in the Saccharomyces cerevisiae temperature-sensitive, secretion-defective mutants isolated by Novick and Schekman (Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979; Novick et al., Cell 21:205-215, 1980). The mutants, sec1 through sec23, do not grow at 37 degrees C and exhibit lower rates of phospholipid synthesis than does the wild-type strain X2180. None of the mutants exhibits a decline in lipid synthesis rapid enough to explain secretion failure. Plasma membrane growth was assessed indirectly by examining the osmotic sensitivity of spheroplasts derived from cultures transferred from 24 to 37 degrees C. Spheroplasts from the normal-growing strain X2180 exhibited a small rapid increase in osmotic sensitivity and stabilized at a more sensitive state. Spheroplasts from the sec mutants exposed to the same temperature shift exhibited progressively increasing osmotic sensitivity. Cycloheximide treatment prevented progressive increases in osmotic fragility. These data are compatible with the hypothesis that plasma membrane expansion is restricted in the sec mutants. During incubation at 37 degrees C, the accumulation of intracellular materials within the no-longer expanding plasma membrane exerts osmotic stress on the membrane, increasing with time. The gene products defective in Novick and Schekman's sec mutants appear to be required for both extracellular protein secretion and plasma membrane growth in yeast cells.

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium.

When radiolabeled serine is incubated with a particulate fraction from Saccharomyces cerevisiae, radioactivity is incorporated initially into phosphatidylserine and gradually appears in phosphatidylethanolamine. Because decarboxylation of phosphatidylserine is blocked by hydroxylamine, phosphatidylserine synthase can be assayed separately. The yeast phosphatidylserine synthase activity 1) exhibits a divalent cation requirement; 2) is stimulated by exogenous CDP-diolein (apparent Km = 0.17 mM); 3) has an apparent Km = 4 mM for L-serine; 4) has a neutral pH optimum; 5) is inhibited by p-hydroxymercuribenzoate; and 6) is reversible in the presence of 5'-CMP, but not 2'-CMP, 3'-CMP, or 5'-AMP. The phospholipid-synthesizing activity is solubilized with Triton X-100 and the enzymatic parameters have been compared with the particulate form of the enzyme. Detergent extracts catalyze the conversion of exogenous purified [31P]CDP-diglyceride to [32P]phosphatidylserine in the presence of Mn2+ and L-serine. Enzyme preparations from cells grown in the presence of choline, that have reduced phospholipid methylation activity (Waechter, C. J., Steiner, M. R., and Lester, R. L. (1969) J. Biol. Chem. 244, 3419-3422), also have substantially less phosphatidylserine synthase activity compared to identical preparations grown in the absence of choline. When choline, phosphocholine, CDP-choline, and phosphatidylcholine are present in vitro, there is no direct inhibitory effect on phosphatidylserine synthase activity. While the inclusion of choline in the growth medium caused a significant reduction in phosphatidylserine synthase activity, it did not appreciably effect the apparent Km values for L-serine and CDP-diglyceride. These results are consistent with choline-grown cells containing less phosphatidylserine synthase activity because of lower amounts of enzyme present or perhaps less active enzyme due to covalent modification.

Yeast mutants auxotrophic for choline or ethanolamine.

Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures.

Isolation and characterization of an actinomycin D-sensitive mutant of Saccharomyces cerevisiae.

A single mutation in Saccharomyces cerevisiae conferred sensitivity to low concentrations of actinomycin D. Treatment with actinomycin D preferentially inhibited synthesis of rRNA's. Residual rRNA synthesized was processed normally. Total protein synthesis and inducibility of the enzyme maltase were relatively unaffected at concentrations of actinomycin D which severely inhibited rRNA synthesis.

Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae.

Physiological states associated with inositol starvation of spheroplasts of Saccharomyces cerevisiae were investigated and compared with conditions preceding death of starved whole cells. In the absence of synthesis of inositol-containing lipids, cell surface expansion terminated after one doubling of whole cells. In spheroplasts, cessation of membrane expansion was apparently followed by rapid development of an osmotic imbalance, causing lysis. Continued synthesis and accumulation of cytoplasmic constituents within the limited cell volume were implicated as a cause of the osmotic imbalance. In whole cells, an increase in internal osmotic pressure also follows termination of membrane and cell wall expansion. The cell wall prevents lysis, allowing a state of increasing cytoplasmic osmotic pressure to persist in the period preceding onset of inositol-less death.

Growth and metabolism of inositol-starved Saccharomyces cerevisiae.

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death". The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in a imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death.