Current methods for Saccharomyces cerevisiae. I. Growth

Interest in the study of yeast biology has increased dramatically in the past few years. Since these organisms are eukaryotic, some phenomena observed in yeast may provide a useful model for similar phenomena in multicellular organisms. Yeast has several advantages as an experimental organism and many methods used for bacteria can be adapted to them. Yeast is simple to grow, cultures are easily maintained, and classical and molecular genetic techniques can be used. The ability to approach problems genetically and biochemically has lead to substantive progess with this group of organisms in areas such as cell biology (1) and gene expression (2). This review is intended to introduce investigators to practical techniques for the growth and radioactive labeling of yeast, primarily of Saccharomyces cerevisiae. For genetic techniques, readers are referred to a recent laboratory manual (3) and reviews (4,5).     

Acetate-Glyoxylate Medium for the Sporulation of Saccharomyces cerevisiae

S ummary : Studies on ascospore formation by Saccharomyces cerevisiae on sodium acetate agar with and without an addition of sodium glyoxylate showed that the rate of ascus formation was increased when glyoxylate was present. The final proportion of asci was not affected but, since cell multiplication (almost threefold) before ascus formation occurred only on the acetate-glyoxylate medium, on the basis of the number of asci/unit of inoculum the effect of glyoxylate was even more marked. If CO₂ was removed from the atmosphere around the cells, sporulation was strongly inhibited when the medium contained only acetate, but there was less inhibition when glyoxylate was present as well. Ascospores formed on the acetate-glyoxylate medium changed their staining properties after the third day. The majority then stained with safranin but not with malachite green.     

Lipid Synthesis During Sporulation of Saccharomyces cerevisiae

Lipid synthesis was studied in both sporulating (diploid) and nonsporulating (haploid) cells of Saccharomyces cerevisiae. Two phases of lipid synthesis occur in diploid cells transferred to sporulation medium. Phase I, which occurs during the first 12 h of exposure to sporulation medium, was also observed in the haploid strains. Phase II, occurring from the 20th to the 25th h, coincided with the appearance of mature asci and was observed only in the diploid cells. The majority of phospholipid synthesis took place during period I, whereas neutral lipid synthesis occurred during both periods. Phospholipid synthesis was virtually identical in both type and quantity in the sporulating and nonsporulating strains.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Cell Cycle Dependency of Sporulation in Saccharomyces cerevisiae

The study of sporulation in Saccharomyces cerevisiae is complicated by the fact that not all cells in the population complete sporulation and that the kinetics of development of those which do are not synchronous. By separating vegetative cells by zonal rotor centrifugation into fractions of increasing cell volume and hence progressive stages of the vegetative cell cycle, it was possible to observe sporulation of more homogeneous, synchronous populations. The capacity of S. cerevisiae to complete sporulation is low for small single cells at the beginning of the cell cycle and is greatest for large budded cells about to divide. The capacity of a cell to complete sporulation thus appears to be directly related to the stage in the vegetative cell cycle from which it was taken. The use of synchronously sporulating cultures made it possible to examine very early decision events leading to the commitment of a cell to sporulation. In addition, differences in the capacity of a mother and daughter cell produced by cell scission were examined.     

Sporulation of mitochondrial respiratory deficient mit- mutants of Saccharomyces cerevisiae.

Summary The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient mit ^- mutants. These mutants were isolated in an op1 strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1—cytochrome c oxidase subunit 2, OXI2 — subunit 3, OXI3 — subunit 1, and COB — cytochrome b.The ability of diploid mit ^- mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit ⁺ tetrads, both mit ⁺ and mit ^- tetrads, or only mit ^- tetrads. Mutants forming mit ^- tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some mit ^- mutants have retained the ability to perform meiosis and sporulation.     

Sporulation in single-spore isolates from amitrole-induced multispored asci of Saccharomyces cerevisiae.

Amitrole treatment causes multispored ascus production by cells of a yeast strain whose asci normally contain two diploid spores. Single spores were isolated from asci containing two to eight spores and their ability to germinate was determined. Cells in colonies grown from single spores sporulated in the same manner as the parent strain indicating that amitrole had not induced meiotic division in the developing asci.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Nature of Ribonucleic Acid Synthesis During Early Sporulation in Saccharomyces cerevisiae

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.     

Sporulation of Saccharomyces cerevisiae in the Absence of a Functional Mitochondrial Genome

The role of the mitochondrial system during sporulation of Saccharomyces cerevisiae was studied. Addition of ethidium bromide (EthBr) to cells growing in acetate medium resulted in the quantitative (>98%) conversion of the culture to the petite genotype in one generation. The cells were respiratory active (derepressed) but contained no mitochondrial deoxyribonucleic acid (mtDNA) as demonstrated by analytical ultracentrifugation in CsCl. When transferred to acetate sporulation medium, the culture sporulated. Ascus production was only slightly below that of the control culture. Synthesis of mtDNA occurred during sporulation in the control but not in the EthBr-treated culture. Mitochondrial protein synthesis was virtually eliminated in the EthBr-treated culture. Therefore, completely derepressed cells can sporulate without a functional mitochondrial genetic system. When partially repressed cells were treated with EthBr, no ascus formation was observed after transfer to sporulation medium. Control cultures underwent respiratory adaptation in sporulation medium and then sporulated. Extensive derepression of the respiratory system is thus required for sporulation, and this adaptation is dependent on a functional mitochondrial system. Our results suggest that once the cells are fully derepressed no mitochondrial genetic information has to be expressed during meiosis and ascus formation.     

Contribution à l'étude de la Sporulation chez Saccharomyces cerevisiae Hansen

The yeast strain employed designated as A 40 will sporulate if after growing on rich medium it is transferred to a neutral or alkaline pH medium which does not allow the vegetative division.A few carbon sources have been tested, and the acetate ion is the best one to induce the 3- or 4-spored asci. The cation sodium and potassium have a similar effect. A 14 to 16 hours' immersion in the sporulation medium induces the sporulation in an irreversible way. During this induction, the respiratory metabolism is altered: the oxydation speed of the exogenous substrate decreases while the endogenous reserve oxydation speed increases. Carbohydrate reserves accumulate, particularly trehalose and glycogen. The rate of proteins is not altered during the induction of sporulation.

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Zusammenfassung Eine Hefezelle des A 40 Stammes differenziert sich in einer Sporenmutterzelle wenn sie, nachdem sie sich auf einem reichen Medium entwickelt hatte, auf einem mit neutralen oder alkalinischen pH Medium übertragen wird, das die vegetative Vermehrung untersagt.Mehrere Kohlenstoffquellen wurden geprüft; das Acetat-Ion begünstigt die Bildung von Sporenmutterzellen mit 3 oder 4 Sporen am besten.Natrium- und Kaliumkationen weisen eine vergleichbare Wirkung auf.Ein 14 bis 16 Stunden Kontakt mit dem Sporenbildungsmedium induziert unwiderruflicher Weise die Sporenbildung. Im Laufe dieser Induktion wird der Atmungsstoffwechsel verändert: die Oxydationsgeschwindigkeit des exogenen Substrates nimmt ab, während die Oxydationsgeschwindigkeit der endogenen Vorräte zunimmt.Die Vorräte an Kohlenhydrate und besonders an Trehalose und an Glycogen werden aufgespeichert.Der Proteingehalt wird im Laufe der Induktion der Sporenbildung nicht verändert.

     

Appearance of a New Species of Ribonucleic Acid During Sporulation in Saccharomyces cerevisiae

In the course of study on ribonucleic acid (RNA) metabolism during sporulation in Saccharomyces cerevisiae, a new species of RNA (20S) was observed in sporulating cells by polyacrylamide gel electrophoresis. The relative content of this RNA to total RNA increased linearly early in sporulation. Labeled adenine was preferentially incorporated into 20S RNA during the early stages of sporulation. The correlation between the physiological and genetic control of sporulation and the synthesis of 20S RNA are discussed.     

Ergosterol determination in Saccharomyces cerevisiae. Comparison of different methods

Several methods for ergosterol determination in two different Saccharomyces cerevisiae strains were tested. The best results were obtained by a simple method including saponification with 8 M KOH, extraction of non-saponifiable lipids and ergosterol determination by reversed phase HPLC. The methods in which the samples were treated with 0.1 M HCl prior to hydrolysis with 2.5 or 4.5 M water-ethanolic KOH underestimated the ergosterol contents.     

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.     

Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae. II. Catalytic mechanism.

Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5' end of eukaryotic cytoplasmic tRNAHis species. A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT. In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate. The 5'-phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5')pp(5')N at the 5'-end of the tRNA. Finally, the 3'-hydroxyl of GTP adds to the activated 5' terminus of the tRNA with the release of AMP. This mechanism of tRNAHis guanylyltransferase is very similar to that of RNA ligases. dATP can substitute for ATP in this reaction. Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.     

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.