Sensitivity of meiotic yeast cells to ultraviolet light

Sporulating cells of Saccharomyces cerevisiae show an increasing sensitivity to ultraviolet irradiation. Maximum sensitivity is reached at a time comparable to meiotic prophase. Sensitivity is expressed as reduced sporulation after the irradiation. The uv effect can be efficiently reversed by photoreactivating light. Viability is also more severely affected during premeiotic DNA synthesis and during meiosis than in earlier stages in sporulation. Cells left in sporulation medium after the irradiation show a reduced viability compared with the cells plated immediately after the irradiation. Non-sporulating diploids do not acquire sensitivity when exposed to sporulation medium, hence the sensitivity is related to the sporulation process. That meiosis itself is affected, rather than spore formation alone, is evident from experiments in which the uv irradiation interferes with the uncovering of a recessive marker and with commitment to meiosis. It is proposed that during meiotic prophase, the DNA repair system is different from that found in vegetative cells.     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE

Effects of the rad52 mutation in Saccharomyces cerevisiae on meiotic, γ-ray-induced, UV-induced and spontaneous mitotic recombination were studied. The rad52/rad52 diploids undergo premeiotic DNA synthesis; sporulation occurs but inviable spores are produced. Both intra and intergenic recombination during meiosis were examined in cells transferred from sporulation medium to vegetative medium at different time intervals. No intragenic recombination was observed at the his1–1/his1–315 and trp5–2/trp5–48 heteroalleles. Gene-centromere recombination also was not observed in rad52/rad52 diploids. No γ-ray- or UV-induced intragenic mitotic recombination is seen in rad52/rad52 diploids. The rate of spontaneous mitotic recombination is lowered five-fold at the his1–1/his1–315 and leu1–c/leu1–12 heteroalleles. Spontaneous reversion rates of both his1–1 and his1–315 were elevated 10 to 20 fold in rad52/rad52 diploids.—The RAD52 gene function is required for spontaneous mitotic recombination, UV- and γ-ray-induced mitotic recombination and meiotic recombination.     

The Genetic Control of Cell Growth and Development in Yeast Saccharomyces cerevisiae: Disturbed Sporulation in Diploids with a Decreased Activity of the Ras/cAMP Signal Transduction Pathway

Seven haploid strains (four with the MAT mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with changed activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34°C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

The genetic control of cell growth and development in yeast Saccharomyces cerevisiae. Disturbed sporulation in diploids with decreased activity of the Ras/cAMP signal transduction pathway

Seven haploid strains (four with the MAT alpha mating type and three with the MATa mating type) were selected from the Peterhof genetic collection of yeast. Previous phenotypic analysis assigned six of these strains to a physiological group of strains with a lower activity of the Ras/cAMP signal transduction pathway. The haploids were crossed, and the resulting 12 diploids showed higher glycogen accumulation, tolerance to heat shock and nitrogen starvation, and sporulation in complete media. Ten of the diploids expressed the hypersporulation phenotype (higher sporulation efficiency). The phenotypic characters of these ten diploids suggested a reduced activity of the Ras/cAMP pathway. All 12 diploids were tested for sporulation and production of two groups of asci (those with one or two spores and those with three or four spores) as dependent on culture conditions (21, 30, or 34 degrees C; standard sporulation medium or a complete medium containing potassium acetate or glycerol in place of glucose). Sporulation proved to depend on temperature and medium composition. The results are collated with the data on yeast phenotypes associated with a lower activity of the Ras/cAMP signal transduction pathway.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Initiation of meiosis in cell cycle initiation mutants of Saccharomyces cerevisiae

Control of the initiation of meiosis in yeast was examined in diploids homozygous for one of four different temperature-sensitive mutations that affect “start” of the mitotic cell cycle. Two of the mutations, cdc28 and tra3, bring about deficiencies in the initiation of meiosis, while cdc25 and cdc35 do not prevent initiation of normal meiosis at both permissive and restrictive temperatures. Moreover, diploids homozygous for the latter two mutations are capable of initiating meiosis in rich growth media upon transfer to the high, non-permissive temperature. This unique feature contrasts with the behavior of other yeast strains which require a starvation sporulation medium for initiation of meiosis. It is suggested that the initiation of meiosis includes functions that are shared with “start” of the mitotic cell cycle, as well as functions related to the choice between the two processes. Meiosis in vegetative media at the restrictive temperature (in cdc25 or cdc35 homozygotes) may be important for the study of chemical and physiological phenomena resulting from the meiotic process and not from adaptation to the sporulation medium.     

Two-dimensional protein patterns during growth and sporulation in Saccharomyces cerevisiae.

Proteins synthesized by Saccharomyces cerevisiae in presporulation and sporulation media were compared by using sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast strains. Total cellular proteins were labeled with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms and/or fluorograms showed some 700 spots per gel. Nine proteins were synthesized by a/alpha cells which were specific to vegetative, log-phase conditions. During incubation in sporulation medium, sporulating (a/alpha) cells synthesized 11 proteins not present in vegetatively growing cell. These same 11 proteins, however, were synthesized by nonsporulating (a/a and alpha/alpha) cells on sporulation medium as well. Nonsporulating diploids (a/a and alpha/alpha) were also examined with the electron microscope at various times during their incubation in sporulation medium. Certain cellular responses found to be unique to meiotic yeast cells in previous studies were exhibited by the nonsporulating controls. The degree to which all cell types (a/alpha, a/a, and alpha/alpha) were committed to sporulation was also determined by shifting cells from sporulation medium to vegetative medium. Some commitment to the meiotic pathway was observed in both the a/alpha and the a/a, alpha/alpha cells.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

The Effect of Ochre Suppression on Meiosis and Ascospore Formation in Saccharomyces

The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.     

Sporulation of Clostridium acetobutylicum P262 in a Defined Medium

A defined minimal sporulation medium for Clostridium acetobutylicum P262, which produces high levels of solvents, is described. The overall sporulation sequence was similar to that of other endospore-forming bacteria. However, we observed a presporulation stage, during which swollen phase-bright cells which contained large amounts of granulose formed. During sporulation, the initiation of spore coat formation occurred before the onset of cortex formation. Other Clostridium strains tested showed marked variations in ability to grow and sporulate in various minimal media.     

Historical and Contemporary NaCl Concentrations Affect the Duration and Distribution of Lag Times from Individual Spores of Nonproteolytic Clostridium botulinum

In this study we determined the effect of NaCl concentration during sporulation (0 or 3.0% [wt/vol] added NaCl) and subsequent growth (0 or 2.0% [wt/vol] added NaCl) on the distributions of times associated with various stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B. The effects of NaCl on the probability of germination and the probability of subsequent growth were also determined. Spore populations exhibited considerable heterogeneity at all stages of lag phase for each condition tested. Germination time did not correlate strongly with the times for later stages in the lag phase, such as outgrowth and doubling time. Addition of NaCl to either the sporulation or growth media increased the mean times for, and variability of, all the measured stages of the lag phase (germination, emergence, time to one mature cell, and time to first doubling). There was a synergistic interaction between the inhibitory effects of NaCl in the sporulation medium and the inhibitory effects of NaCl in the subsequent growth medium on the total lag time and each of its stages. Addition of NaCl to either the sporulation medium or the growth medium reduced both the probability of germination and the probability of a germinated spore developing into a mature cell, but the interaction was not synergistic. Spores formed in medium with added NaCl were not better adapted to subsequent growth in suboptimal osmotic conditions than spores formed in medium with no added NaCl were. Knowledge of the distribution of lag times for individual spores and quantification of the biovariability within lag time distributions may provide insight into the underlying mechanisms and can be used to improve predictions of growth in food and to refine risk assessments.     

Sporulation of Bacillus stearothermophilus

A broth medium containing tryptone and manganese sulfate supported heavy sporulation of Bacillus stearothermophilus ATCC 7953 (NCA 1518) and four isolates identified as B. stearothermophilus. Maximal spore yields were obtained by use of inocula grown anaerobically in a medium containing glucose with aeration of sporulation medium via bubbling. After an extended stationary period, sporulation occurred concurrently with vegetative growth between 6 and 8 hr of incubation at 60 C. Omission of glucose from the inoculum or use of a “young” (2 hr) inoculum abolished the stationary period, but decreased spore yields. A requirement of oxygen for rapid vegetative growth and sporulation was demonstrated. Manganese (15 to 30 ppm) stimulated sporulation but did not enhance cell growth.     

Sporulation of protoplasts

Vegetative cells ofBacillus megaterium formed protoplasts in a sucrose-stabilized medium under the influence of lysozyme. The protoplasts sporulated during subsequent incubation. The morphological properties, germination, resistance to u. v. irradiation and thermo-resistance of protoplast spores were the same as with normal cells. It thus appears that in the later sporulation stages the spore formation occurs, without participation of the sporangium cell wall.     

Effect of pH on Adenine and Amino Acid Uptake During Sporulation in Saccharomyces cerevisiae

During the early stages of sporulation in Saccharomyces cerevisiae, the pH of the acetate sporulation medium rises to values of 8.0 or higher. Associated with this rise in pH is a reduced cell permeability to certain precursors of ribonucleic acid (RNA), deoxyribonucleic acid or protein. Uptake of adenine, alanine, and leucine was optimal at pH 5.6 to 6.0, but sporulation was inhibited when the sporulation medium was buffered below pH 7.0. Cellular impermeability can be largely overcome by adjusting the acetate sporulation medium to pH 6.0 for optimal uptake of ¹⁴ C-adenine during short pulses without any apparent effect on sporulation. Sporulating cells pulse-labeled 20 min at pH 6.0 incorporated 40 times more ¹⁴C-adenine into RNA than sporulating cells pulse-labeled at pH 8.0. This increased incorporation can be attributed to a 100-fold increase in labeled adenosine triphosphate in cells pulse-labeled at pH 6.0 where maximum uptake occurs.     

Nuclear and mitochondr1al DNA synthesis during yeast sporulation

Nuclear and mitochondrial DNA synthesis during sporulation of Saccharomyces cerevisiae has been studied in a wild-type (aα) strain and 3 sporulation deficient strains. We find that in a strain carrying a dominant mutation which prevents sporulation, nuclear DNA synthesis is initiated but not completed; mitochondrial DNA synthesis, on the other hand, does take place. In aa and αα diploids no initiation of nuclear DNA synthesis is seen to occur, and only a very low level of mitochondrial DNA synthesis is observed. We conclude that mitochondrial DNA synthesis in sporulation medium is uncoupled from nuclear DNA synthesis. In addition, the steps at which the sporulation process is arrested in aa and αα cells and in the dominant mutant can be ordered in time as being before and after the initiation of nuclear DNA synthesis.     

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.     

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 (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα 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 αα 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°, but not at 23°. 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.     

Enhanced Gene Conversion and Postmeiotic Segregation in Pachytene-Arrested SACCHAROMYCES CEREVISIAE

Previous study has demonstrated that incubation of yeast cells of strain AP-1 in sporulation medium at 36° permits them to begin meiosis but that they become arrested at pachytene and undergo enhanced intragenic recombination between ade2 heteroalleles. Tetrad analysis was undertaken to characterize the altered program of meiotic recombination more widely. In one set of experiments, pachytene-arrested cells were permitted to resume sporulation upon transfer to the permissive temperature. In the resulting asci, both postmeiotic segregation and gene conversion were increased several-fold at a number of loci relative to unarrested controls, whereas reciprocal recombination increased two- to threefold. Another set of experiments analyzed the genetic consequences of inducing the pachytene-arrested cells to revert directly to mitotic growth without completion of meiosis. The appearance of homozygous sectors from heterozygous markers revealed that these cells had become committed to appreciable recombination but that reciprocal exchange was less frequent than in normal asci. Taken together, the data indicate that pachytene arrest rendered the cells committed to enhanced recombination upon resumption of sporulation but that most of the crossing over did not occur until release from the arrest. —The genetic basis of pachytene arrest by AP-1 was investigated by mating each of its parents with progeny of strain Y55, which is able to sporulate at 36°. Both of these diploids sporulated at 36°, and asci from the one studied further exhibited 2:2 segregation of the sporulation defect, indicating that pachytene arrest is dependent on a recessive, temperature-sensitive allele at a chromosomal locus.