Inositol Starvation Research Articles

Inositol-less death in yeast results in a simultaneous increase in intracellular viscosity

Inositol auxotrophs of yeast developing on isositol-deficient medium continue protein synthesis for 4-6 h, lose viability rapidly after 6 h, and show an increase in cytoplasmic viscosity as measured by spin label rotational motion. Cycloheximide prevents the rapid loss of cell viability, stops protein synthesis, and simultaneously prevents an increase in cytoplasmic viscosity. From these observations, we infer that intracellular translational diffusion is upset as a consequence of inositol starvation. Cell death may be caused by a modified intracellular diffusion environment.

Ageing of Neurospora crassa. III. Induction of cellular death and clonal senescence of an inositol-less mutant by inositol starvation and the protective effect of dietary antioxidants

Clonal growth rate and cellular viability of an inositol-less mutant of Neurospora crassa decline rapidly during deprivation of dietary inositol. Dietary antioxidants, either nordihydroguaiaretic acid, vitamin E or 3,5-di- tert.-butyl-4-hydroxybenzyl alcohol, protected cells and clones of the mutant from death and senescence. Membrane deterioration with associated lipid autoxidation and consequent deleterious free radical reactions appear to be, in part at least, the causes of the mutant's death and senescence.

Inositol-requiring mutants of Saccharomyces cerevisiae.

Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular. - Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes. - Part of this work has been reported in preliminary form (CULBERTSON and HENRY 1974).

Mating type and sporulation in yeast. I. Mutations which alter mating-type control over sporulation.

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aalpha) can sporulate but cannot mate; homozygous aa and alpha-alpha diploids can mate, but cannot sporulate. From an alpha-alpha diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as alpha-alpha cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4alpha spores. The mutant gene which allows alpha-alpha cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant alpha-alpha and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32 degrees, but not at 23 degrees. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.

Selection of spontaneous mutants by inositol starvation in yeast.

A 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 macromolecular synthesis is inhibited, most of the cells survive. Appropriate manipulation of inositol requiring mutants can therefore result in the selective survival of cells possessing 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 10,000 fold has been obtained for various classes of auxotrophic mutants. Spontaneous temperature sensitive mutants have also been obtained.

The Isolation and Partial Characterization of Two Novel Sphingolipids from Neurospora crassa: Di(Inositolphosphoryl)ceramide and [(gal)3glu]ceramide

Neurospora crassa strains labeled uniformly with ^(32)P_i and [^3H]inositol exhibit at least six phospholipid components containing ^3H when separated by paper chromatography. One of the major components is phosphatidylinositol. Other components, which account for 40 to 60% of the lipid-extractable ^3H in various strains, are stable to mild alkaline methanolysis and appear to be sphingolipids with equivalent amounts of inositol and phosphorus. The major phosphosphingolipid was purified by means of differential solubility and by column chromatography on porous silica beads. This substance contains equivalent amounts of hydroxysphinganine and hydroxytetracosanoic acid and 2 eq each of myoinositol, phosphorus, and sodium. Alkaline degradation yielded 2 eq of inositol monophosphate and periodate degradation gave a C-15 fragment. The elemental composition of this compound also fits the formulation, (inositol-P)_2-ceramide. A [^3H]inositol pulse-chase experiment carried out with an inositol-requiring mutant in exponential growth shows labeled inositol accumulating in the sphingolipid accompanied by decreased labeling in phosphatidylinositol and the acid-soluble fraction. These changes also occur when the chase is carried out during inositol starvation suggesting that degradation of phosphatidylinositol and formation of sphingolipid occurs in the absence of growth. A neutral glycosphingolipid was also obtained as a by-product of the phospholipid purification. This substance is provisionally formulated as the ceramide tetrahexoside: [(gal)_3glu]-N-hydroxytetracosonyl-hydroxysphinganine.

INCREASED FREQUENCY OF AUXOTROPHIC MUTANTS OF USTILAGO HORDEI AFTER COMBINED UV IRRADIATION AND INOSITOL STARVATION

Auxotrophic mutants of Ustilago hordei were recovered after UV irradiation of a wild-type culture, inositol starvation of an inositol-requiring culture and a combination of the two methods. 1.9% of the survivors of UV irradiation followed by inositol starvation were found to carry a requirement additional to inositol. Inositol starvation by itself gave only 0.27% recovery while UV irradiation alone was even less efficient.