Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

Procedures used in the induction of mitotic recombination and mutation in the yeast Saccharomyces cerevisiae.

Techniques are described for the use of various yeast strains to detect the induction of (1) mitotic crossing-over, (2) mitotic gene conversion, (3) forward mutation and (4) reverse mutation. The technique for the detection of mitotic crossing over is based on a diploid that carries two different alleles of the gene locus ade2. These alleles differ in their extent of colony pigmentation engendered on low-adenine media, and they complement each other to the effect that the diploid is white. Mitotic crossing over results in the formation of twin-sectored colonies with a red and a pink sector. The technique for the detection of mitotic gene conversion is based on the use of a heteroallelic diploid carrying two non-complementing alleles that cause a nutritional requirement. Mitotic gene conversion leads to the restoration of intact and dominant wild-type alleles that alleviate the nutritional requirement so that convertant cells can be selected on a minimal medium. The forward mutation technique is based on the use of a haploid strain with a defect in the ade2-gene locus which causes the formation of red colonies. Induction of forward mutation in a number of other loci prevents the accumulation of this red pigment so that induction of mutation can be detected by the formation of pink and white colonies. The reverse mutation technique is based on the restoration or compensation of a mutational defect causing a growth requirement. Mutants can be selected for on a minimal medium.     

Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae

Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes differ significantly (3.80x10(-10) at URA3 and 6.44x10(-10) at CAN1) and we propose a definition for the effective target size of genes (the probability that a mutation inactivates the gene) that acknowledges that the mutation rate is nonuniform across the genome.     

Respiratory mutation and galactose metabolism in yeast Saccharomyces cerevisiae.

Restriction in growth on galactose as unique source of energy due to respiratory deficiency resulting from mutation in a gene gal probably different from gal 3 is described.     

Iron requirement for GAL gene induction in the yeast Saccharomyces cerevisiae

Iron is an essential nutrient. Its deficiency hinders the synthesis of ATP and DNA. We report that galactose metabolism is defective when iron availability is restricted. Our data support this connection because 1) galactose-mediated induction of GAL promoter-dependent gene expression was diminished by iron limitation, and 2) iron-deficient mutants grew slowly on galactose-containing medium. These two defects were immediately corrected by iron replacement. Inherited defects in human galactose metabolism are characteristic of the disease called galactosemia. Our findings suggest that iron-deficient galactosemic individuals might be more severely compromised than iron-replete individuals. This work shows that iron homeostasis and galactose metabolism are linked with one another.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Isoprenoide pathway and cell proliferation in the yeast Saccharomyces cerevisiae]

Yeast mutants blocked in farnesyl diphosphate (FPP) synthetase have been isolated. Their specific phenotype is likely linked to a lowering in the FPP pool required for protein prenylation. The structural gene of FPP synthetase has been isolated. Complete inactivation of FPP synthetase by gene disruption is letal for the yeast cells.     

Some aspects of catalase induction in baker's yeast (Saccharomyces cerevisiae)

Yeasts grown in anaerobic liquid media produced catalase in response to the presence of H₂O₂ in the growth medium. The fact that some of the induced enzyme was active at the cell surface, bound either to the cell wall or cell-surface membrane, eliminated the need to crush cells in order to release the enzyme complement. Instead, catalase production was monitored by using H₂O₂-reagent strips to detect changes in the level of H₂O₂, in the growth medium. In addition, catalase induction in yeasts was found to be temperature-sensitive. It is suggested that biology teachers in schools might find the following experiments useful for demonstrating essential features of substrate-induced enzyme synthesis, based on the Jacob-Monod model, and for showing that the activity of certain genes can be modified by environmental temperature.     

Effect of stress on the life span of the yeast Saccharomyces cerevisiae

A correlation is known to exist in yeast and other organisms between the cellular resistance to stress and the life span. The aim of this study was to examine whether stress treatment does affect the generative life span of yeast cells. Both heat shock (38 degrees C, 30 min) and osmotic stress (0.3 M NaCl, 1 h) applied cyclically were found to increase the mean and maximum life span of Saccharomyces cerevisiae. Both effects were more pronounced in superoxide dismutase-deficient yeast strains (up to 50% prolongation of mean life span and up to 30% prolongation of maximum life span) than in their wild-type counterparts. These data point to the importance of the antioxidant barrier in the stress-induced prolongation of yeast life span.     

Adaptive mutation in Saccharomyces cerevisiae

Adaptive mutation is a generic term for processes that allow individual cells of nonproliferating cell populations to acquire advantageous mutations and thereby to overcome the strong selective pressure of proliferation-limiting environmental conditions. Prerequisites for an occurrence of adaptive mutation are that the selective conditions are nonlethal and that a restart of proliferation may be accomplished by some genetic change in principle. The importance of adaptive mutation is derived from the assumption that it may, on the one hand, result in an accelerated evolution of microorganisms and, on the other, in multicellular organisms may contribute to a breakout of somatic cells from negative growth regulation, i.e., to cancerogenesis. Most information on adaptive mutation in eukaryotes has been gained with the budding yeast Saccharomyces cerevisiae. This review focuses comprehensively on adaptive mutation in this organism and summarizes our current understanding of this issue.     

The effect of the cdc9 mutation on premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

A diploid homozygous for cdc9, a conditional mutation defective in DNA ligase [2], has been used to investigate the role of this enzyme in premeiotic DNA synthesis. The cdc9 ligase has the same effect on premeiotic as on mitotic DNA synthesis and at the restrictive temperature the newly synthesized DNA is recovered in small fragments. A difference has been observed, however, between meiotic and mitotic cells, namely in their ability to join together these fragments on return to the permissive temperature. In mitotic cells this can be readly demonstrated within 50 min, whereas in contrast little joining was detected in meiotic cells, even after 2 h at the permissive temperature.     

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.     

Genetic analysis of spontaneous suppressors of the pho85 mutation in the yeast Saccharomyces cerevisiae

Chaperones are known to play an important role in complexation of cyclin-dependent kinases with cyclins. In yeast cells growing in the presence of phosphate, cyclin-dependent kinase Pho85p and cyclin Pho80p form a complex and phosphorylate activator Pho4p. As a result, Pho4p is exported from the nucleus, and the PHO5 gene is not transcribed. The mutations suppressing the pho85 mutation were analyzed in order to identify genes which code for chaperones involved in the formation of the Pho80p-Pho85p complex in the presence of environmental phosphate. Dominant mutations DSP1, DSP2, and DSP4-6 were found. It is shown that the DSP1 gene is 2.1 cM away from the PHO85 gene on chromosome XVI and probably coincides with the EGD1 gene coding for a chaperone.     

Effect of Bacillus intermedius ribonuclease on properties of the yeast Saccharomyces cerevisiae

Effects of Bacillus intermedius ribonuclease on physiological, biochemical, and consumer properties of baker's yeast Saccharomyces cerevisiae were studied. This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors. The antistress effect of RNase correlated with an earlier start of the stationary growth phase and increased trehalose pool.     

Damage-induced recombination in the yeast Saccharomyces cerevisiae

Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome. In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans. Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans.