Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

Calcium uptake during the cell cycle of Saccharomyces cerevisiae

Synchronous culture of the budding yeast Saccharomyces cerevisiae was obtained by sucrose density gradient selection with 90–100% of yeast synchronized by using the cells in the bottom. In these adult cells bud emergence is coincident with an increase in calcium uptake at 100 min of the culture, followed by a return to basal values which are maintained until the end of the first cell cycle of study. The phenothiazine derivatives, trifluoperazine and chlorpromazine inhibit bud emergence and trifluoperazine also increases calcium uptake.     

Production of functional IgM Fab fragments by Saccharomyces cerevisiae

The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain cDNA was truncated at two different sites, yielding genes coding for the complete VH domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3′ terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a cDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments.     

Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol

The effects of d-limonene concentration, enzyme loading, and pH on ethanol production from simultaneous saccharification and fermentation (SSF) of citrus peel waste by Saccharomyces cerevisiae were studied at 37 °C. Prior to SSF, citrus peel waste underwent a steam explosion process to remove more than 90% of the initial d-limonene present in the peel waste. d-Limonene is known to inhibit yeast growth and experiments were performed where d-limonene was added back to peel to determine threshold inhibition amounts. Ethanol concentrations after 24 h were reduced in fermentations with initial d-limonene concentrations greater than or equal to 0.33% (v/v) and final (24 h) d-limonene concentrations greater than or equal to 0.14% (v/v). Ethanol production was reduced when enzyme loadings were (IU or FPU/g peel dry solids) less than 25, pectinase; 0.02, cellulase; and 13, beta-glucosidase. Ethanol production was greatest when the initial pH of the peel waste was adjusted to 6.0.     

NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae

The nucleolar protein Nop2p is an essential gene product that is required for pre-rRNA processing and ribosome biogenesis in Saccharomyces cerevisiae (Hong, B. et al., 1997, Mol. Cell. Biol., 17, 378–388). A search for proteins similar to Nop2p identified a novel yeast gene product that also shares significant homology with the human proliferation associated nucleolar protein p120. The gene encoding this 78 kDa protein was termed NCL1 (for nuclear protein 1; corresponding to YBL024w). Ncl1p and Nop2p contain an evolutionarily conserved motif that has been termed the ‘NOL1/NOP2/fmu family signature' (NOL1 encodes p120). Epitope tagged Ncl1p was found to be localized to the nucleus, including the nucleolus, and was concentrated at the nuclear periphery. NCL1 is not essential. Strains containing a disruption of NCL1, or strains overexpressing NCL1, grow essentially identically to wildtype NCL1 strains on a number of different media and at different temperatures. Disruption of NCL1 does not affect steady-state levels of large and small ribosome subunits, monoribosomes, and polyribosomes. However, disruption of NCL1 leads to increased sensitivity to the antibiotic paromomycin.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Pilot scale vinification process using immobilized Candida stellata cells and Saccharomyces cerevisiae

Sequential grape juice fermentation first with immobilized Candida stellata and then with an inoculum of Saccharomyces cerevisiae was carried out at pilot scale and under non-sterile conditions in order to evaluate the dynamics of yeast microflora and their influence on the analytical profile of wine. Non-Saccharomyces yeast were adequately controlled while S. cerevisiae wild strains were consistently present after 3 days of fermentation and could compete with the inoculated S. cerevisiae strain. However, the metabolism of immobilized C. stellata cells strongly influenced the analytical profile of wines with a consistent increase in glycerol (70%) and succinic acid content in comparison with values for a S. cerevisiae fermentation control.     

Effect of hot trub and particle addition on fermentation performance of Saccharomyces cerevisiae

In this investigation, the effect of hot trub (a precipitation product of the wort boiling process in beer manufacturing) addition on fermentation performance was observed under variation of yeast vitality, and origin and the amount of hot trub. Its addition improved suspended cell concentrations for all yeast vitalities tested, and the more trub was added, the greater the effect. Further, pilot-scale fermentations showed significantly lower pH values and an accelerated extract degradation, thus, advancing fermentation by roughly 1 day for hot trub addition versus the fermentation of extremely bright wort. Since the positive effect of trub has often been associated with its particulate characteristics, fermentations with fractionated model particles, such as poly(vinylpyrrolidones) and kieselguhr, of different particle sizes were carried out under variation of yeast vitality and particle amounts. The addition of both particle types also improved fermentation performance, however, the effect was not as great as that of hot trub. Particulate material may improve the development of CO₂ from the fermenting medium, thus reducing its concentration and inhibitory effect on yeast metabolism. The most effective fraction of kieselguhr had a 40 μm peak which also occurred in particle size distributions of all hot trubs investigated. This could be of particular interest when discussing particle effects.     

In vivo analysis of chromatin following nystatin-mediated import of active enzymes into Saccharomyces cerevisiae

In vivo DNA-protein interactions are usually studied at the molecular level using DNA-degrading agents of low molecular weight. In order to be useful, macromolecular probes of chromatin structure, such as enzymes must first cross the cell membrane. In this paper we describe the introduction and evaluation of macromolecules with enzymatic activity into yeast spheroplasts treated with the polyene antibiotic nystatin. We report the low resolution analysis of chromatin structure in the promoter region of the Saccharomyces cerevisiae gene encoding DNA topoisomerase I by this technique using micrococcal nuclease and restriction enzymes.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.     

Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cervisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MAT locus. The difference in levels of stimulation between MATa/MAT diploid and MAT haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m²), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MAT gene was introduced by DNA transformation into a MATa/mat::LEU2 ⁺ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATA gene was introduced by DNA transformation into a MAT haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.     

Effect of donor copy number on the rate of gene conversion in the yeast Saccharomyces cerevisiae

Summary Nonreciprocal recombination (gene conversion) between homologous sequences at nonhomologous locations in the genome occurs readily in the yeast Saccharomyces cerevisiae. In order to test whether the rate of gene conversion is dependent on the number of homologous copies available in the cell to act as donors of information, the level of conversion of a defined allele was measured in strains carrying plasmids containing homologous sequences. The level of recombination was elevated in a strain carrying multiple copies of the plasmid, compared with the same strain carrying a single copy of the homologous sequences either on a plasmid or integrated in the genome. Thus, the level of conversion is proportional to the number of copies of donor sequences present in the cell. We discuss these results within the framework of currently favoured models of recombination.     

Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae

In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast. For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene. A 400 by deletion in plasmid DNA had no effect on recombinational plasmid repair. However, a 400 by deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process. We have also studied the competition between recombination and ligation in the repair of linearized plasmids. Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA. This situation results in a decreased probability of non-recombinational (i.e. ligation) repair of linearized plasmid DNA.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.