Simultaneous production of buds on mother and daughter cells of Saccharomyces cerevisiae in the presence of hydroxyurea

Individual budding yeast cells, Saccharomyces cerevisiae, enclosedin small culture chambers were observed through two buddingcycles to examine their behavior during growth and division.In the nutrient medium (YHG medium), the duration of the buddingcycles was 77 min for mother cells and 90 min for daughter cells;a 13-min time lag between the two durations. Continuous exposureof cells to 16 or 32 mM hydroxyurea extended the duration ofthe cycles and increased the volume of the cells, resultingin the formation of abnormally large and equal-sized mother-daughterpairs. Each cell of these pairs subsequently produced buds simultaneously.Stained cell nuclei showed simultaneous nuclear division. Thissynchronous budding on mother-daughter pairs was repeated inthe next budding cycle. The coordination of growth with divisionis discussed in relation to these results. (Received August 11, 1979; )     

Growth cycle of daughter and mother cells ofSaccharomyces cerevisiae

Maximum values of specific rate of RNA synthesis, specific growth rate and a critical cell size determined by the surface to volume ratioS/V =1.0 are the factors which control the onset of budding in daughter cells. The increased rate of RNA synthesis is due not only to daughter cells but also to all buds formed on mother cells.     

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.     

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.     

Interaction of ATP17 gene with SUP45 and SUP35 genes in Saccharomyces cerevisiae yeast

Genes SUP35 and SUP45 have been identified in the saccharomycete yeast as genes controlling termination of translation in cytoplasmic ribosomes. However, many facts indicate that the control of translation termination is not the only function of these genes. This work is devoted to studying one of the pleiotropic effects of sup35 and sup45 mutations, a respiratory deficiency. The compensation for this deficiency in mutants for either gene can occur due to a mutation in the ATP17 gene encoding the f-subunit of mitochondrial F1F0 ATP synthase. It is assumed that the observed interaction can be related to the system of co-translational protein import into mitochondria.     

Mitochondrial genome integrity mutations uncouple the yeast Saccharomyces cerevisiae ATP synthase

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the alpha-, beta-, and gamma-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the gamma-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk.     

Micromolar HgCl2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells

Low concentrations of HgCl2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 microM HgCl2, the increase in ATP lasted for about 30 min, while at 10 microM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.     

Severing all ties between mother and daughter: cell separation in budding yeast

At the end of nuclear division in the budding yeast, acto-myosin ring contraction and cytokinesis occur between mother and daughter cells. This is followed by cell separation, after which mother and daughter cells go their separate ways. While cell separation may be the last event that takes place between the two cells, it is nonetheless under tight regulation which ensures that both cells are viable upon separation. It is becoming increasingly clear that the components of the cell separation machinery are controlled at various levels, including the temporal and spatial regulation of the genes encoding for the components and the specific localization of the components to the neck. In addition, these regulatory controls are co-ordinated with exit from mitosis, thereby placing a mechanistic link between the end of mitosis and cell separation. More importantly, the success of the cell separation event is contingent upon the presence of a trilaminar septum, whose assembly is dependent on a host of proteins which localize to the neck over the span of one cell division cycle.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

A putative second adenylate kinase-encoding gene from the yeast Saccharomyces cerevisiae.

Sequencing of the region upstream from the yeast RAD3 gene has revealed an open reading frame (ORF) of 225 amino acids (aa) that could encode a 25.3-kDa polypeptide. The predicted aa sequence of this ORF is homologous with that of several eukaryotic adenylate kinase (Adk)-encoding genes, including the yeast gene, ADK1. These findings suggest that the yeast Saccharomyces cerevisiae has a second Adk-encoding gene, tentatively designated as ADK2.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

RNA asymmetric distribution and daughter/mother differentiation in yeast

Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching. ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have been identified. It is composed of four localization elements (zipcodes), within the ASH1 mRNA, and at least three proteins, She1p/Myo4p, She2p and She3p. Whereas the movement of the RNA can be attributed to direct interaction with myosin, the regulation of the RNA expression is less well understood. Recent insights have revealed a role for translation that might have a key function in the regulation of Ash1 protein sorting.     

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.     

A dominant interfering mutation (CYR3) of the Saccharomyces cerevisiae RAS2 gene.

The dominant cyclic AMP-requiring mutation CYR3 had been previously reported as a mutation in the regulatory subunit of cyclic AMP-dependent protein kinase. However, recharacterization revealed that the CYR3 mutation was a nonconditional dominant lethal mutation and was a missense allele of RAS2 which results from the substitution of aspartic acid for glycine at amino acid 22.