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  • Yeast  (53)
  • Springer  (53)
  • Annual Reviews
  • Elsevier
  • International Union of Crystallography (IUCr)
  • 2005-2009
  • 1990-1994
  • 1985-1989  (42)
  • 1980-1984  (11)
  • 1989  (42)
  • 1981  (11)
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Publisher
  • Springer  (53)
  • Annual Reviews
  • Elsevier
  • International Union of Crystallography (IUCr)
  • Wiley-Blackwell  (8)
Years
  • 2005-2009
  • 1990-1994
  • 1985-1989  (42)
  • 1980-1984  (11)
Year
  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 4 (1989), S. 315-323 
    ISSN: 1476-5535
    Keywords: Sugar uptake ; Yeast ; Brewer's wort
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.
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  • 2
    Electronic Resource
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    Springer
    Journal of industrial microbiology and biotechnology 4 (1989), S. 49-53 
    ISSN: 1476-5535
    Keywords: l-Phenylacetyl carbinol ; Saccharomyces cerevisiae ; Yeast ; Benzaldehyde ; Biotransformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The rate of production ofl-phenylacetyl carbinol bySaccharomyces cerevisiae in reaction mixtures containing benzaldehyde with sucrose or pyruvate as cosubstrate was investigated in short 1 h incubations. The effect of yeast dose rate, sucrose and benzaldehyde concentration and pH on the rate of reaction was determined. Maximum biotransformation rates were obtained with concentrations of benzaldehyde, sucrose and yeast of 6 g, 40 g and 60 g/l, respectively. Negligible biotransformation rates were observed at a concentration of 8 g/l benzaldehyde. The reaction had a pH optimum of 4.0–4.5. Rates of bioconversion of benzaldehyde and selected substituted aromatic aldehydes using both sucrose and sodium pyruvate as cosubstrate were compared. The rate of aromatic alcohol production was much higher when sucrose was used rather than pyruvate.o-Tolualdehyde and 1-chlorobenzaldehyde were poor substrates for aromatic carbinol formation although the latter produced significant aromatic alcohol in sucrose-containing media. Yields of 2.74 and 3.80 g/l phenylacetyl carbinol were produced from sucrose and pyruvate, respectively, in a 1 h reaction period.
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  • 3
    ISSN: 1572-8773
    Keywords: Manganese ; Electron spin resonance ; Superoxide dismutase ; Saccharomyces cerevisiae ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Manganese accumulation was studied by room-temperature electron spin resonance (ESR) spectroscopy inSaccharomyces cerevisiae grown in the presence of increasing amounts of MnSO4. Mn2+ retention was nearly linear in intact cells for fractions related to both low-molecular-mass and macromolecular complexes (‘free’ and ‘bound’ Mn2+, respectively). A deviation from linearity was observed in cell extracts between the control value and 0.1 mM Mn2+, indicating more efficient accumulation at low Mn2+ concentrations. The difference in slopes between the two straight lines describing Mn2+ retention at concentrations lower and higher than 0.1 mM, respectively, was quite large for the free Mn2+ fraction. Furthermore it was unaffected by subsequent dialyses of the extracts, showing stable retention in the form of low-molecular-mass complexes. In contrast, the slope of the line describing retention of ‘bound’ Mn2+ at concentrations higher than 0.1 mM became less steep after subsequent dialyses of the cell extracts. This result indicates that the macromolecule-bound Mn2+ was essentially associated with particulate structures. In contrast to Cu2+, Mn2+ had no effect on the major enzyme activities involved in oxygen metabolism except for a slight increase of cyanide-resistant Mn-superoxide dismutase activity, due to dialyzable Mn2+ complexes.
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  • 4
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    Springer
    BioMetals 2 (1989), S. 50-54 
    ISSN: 1572-8773
    Keywords: Cu(I)8-thionein ; Yeast ; Extracellular ; Circular dichroism ; Fluorescence ; Electronic absorption
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The release of intact CU(I)8-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.
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  • 5
    ISSN: 1432-0983
    Keywords: Yeast ; Minichromosomes ; Impaired segregation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nondisjunction of artificial yeast minichromosomes (2:0 segregation events) during mitosis is accompanied by the appearance of cells containing more than one copy of the mini-chromosome. A mathematical simulation of this process has demonstrated that under certain conditions, a nondisjunction of the minichromosomes may result in their accumulation in a considerable portion of the cell population. An increase in the copy number of artificial minichromosomes as a result of impaired segregation has been used to develop a new experimental procedure for directly selecting yeast mutants showing an impaired segregation of artificial minichromosomes during mitosis. Four new genes, AMC1, AMC2, AMC3, and AMC4, which control the segregation of artificial minichromosomes in mitosis, have been identified (AMC-3 and AMC4 are mapped to chromosome IV and VII, respectively). Mutations in the genes AMC1–AMC4 also affect the mitotic transmission of natural chromosomes. We suggest that the genes AMC1, AMC2, AMC3, and AMC4 control the segregation of natural chromosomes in yeast.
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  • 6
    ISSN: 1432-0983
    Keywords: Yeast ; Diuron ; Respiration ; Nuclear genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In Saccharomyces cerevisiae, diuron blocks the respiration pathway at the level of the bc1 complex. Nuclear diuron-resistant mutations which confer in vitro resistance to mitochondrial NADH oxidase have been identified. Five mutations were found to be clustered at two distinct nuclear loci, DIU3 and DIU4. The distance between the two loci was estimated to be about 36.7 cM. These loci do not appear to be centromere-linked and did not show a linkage to any of the genes coding for bc1 complex subunits. DIU3 and DIU4 loci might, therefore, code for other components of the respiratory chain.
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  • 7
    ISSN: 1432-0983
    Keywords: Alcoholic fermentation ; Deletion mutant ; Pyruvate decarboxylase ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely neccessary for glucose fermentation in yeast.
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  • 8
    ISSN: 1432-0983
    Keywords: Yeast ; 2μm FRT duplication ; Intrachromosomal recombination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-Δ1 locus in a [cir°] background. The 1.5 kb BglII deletion of ilv2-Δ1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2μm site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir°] and [cir +] diploids formed by crossing the [cir°] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir°] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir +] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.
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  • 9
    ISSN: 1432-0983
    Keywords: Yeast ; Diuron ; Nuclear, mitochondrial mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In Saccharomyces cerevisiae, diuron blocks the respiratory pathway at the level of the bc1 complex. Two mitochondrially inherited loci, DIU1 and DIU2, located in the cytochrome b gene, and two nuclearly inherited loci, DIU3 and DIU4, have previously been identified. The present work genetically characterizes two double mutants. One mutant, Diu-217, carries two nuclearly inherited mutations, diu3-217a and diu-217b; the second mutant, Diu-783, carries the previously described nuclear mutation diu3-783 and a mitochondrial mutation diu2-783. Each mutation, independent of its location, exhibits a weak diuron resistance. The joint expression of two or three mutations leads to a cumulative or a cooperative enhanced diuron-resistant phenotype.
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  • 10
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondrial frameshift suppressor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A polypeptide chain-terminating mutation (M5631) previously has been shown to be a +1T insertion in the yeast mitochondrial gene oxi1, coding for subunit II of the cytochrome c oxidase. A spontaneously arisen frameshift suppressor (mfs-1) that is mitochondrially inherited suppresses this mutation to a considerable extent. The suppressor mutation was mapped by genetic and molecular analyses in the mitochondrial tRNASer-var1 region of the mitochondrial genome of the yeast S. cerevisiae. Genetic analyses show that the suppressor mfs-1 does not suppress other known mitochondrial frameshift mutations, or missense and nonsense mutations.
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  • 11
    ISSN: 1432-0983
    Keywords: Yeast ; Ribosomal protein gene ; Transcription activation ; Mutation ; Methylation interference
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Most ribosomal protein (rp-)genes in yeast are preceded by conserved sequence motifs that act as upstream transcription-activating sites (RPG box). These sequence elements have previously been shown to represent specific binding sites for a protein factor, TUF. Comparison of the various nucleotide elements identified so far indicates a remarkably high degree of variation in the respective sequences. On the other hand, a methylation interference study performed with one RPG box revealed close contact points with the TUF protein along the entire sequence. To investigate the sequence requirements of the RPG box, we inserted synthetic oligonucleotides that differed from the general consensus sequence ACACCCATACATTT at single positions into a deletion mutant of the L25 promoter that lacked its natural RPG elements. Transcription activity was estimated by Northern analyses of the cellular level of L25-galK hybrid transcripts. The results show that in the 3′ part of this sequence element single substitutions are allowed at all positions, in the 5′ part, however, the nucleotide requirements appear to be more stringent. In particular, the invariant C at position 5 of the consensus sequence is absolutely necessary for its enhancer function.
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  • 12
    ISSN: 1432-0983
    Keywords: Yeast ; oxi3 gene ; Petite genome ; Frameshift mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho −) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).
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  • 13
    ISSN: 1432-0983
    Keywords: Yeast ; Transcription ; RNA polymerase I ; Enhancer ; DNA-binding protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Using the gel retardation assay we have identified a protein that can specifically bind to a site within the enhancer of the 37S pre-ribosomal RNA operon in yeast, as well as to a site 210 by upstream of the site of transcription initiation of this operon. This protein (RBP1) has been partially purified by means of heparin-agarose chromatography and protects 20 by in the rDNA enhancer, and 25 by in the initiation region, against DNase I in an in vitro footprinting assay. In vivo footprinting studies using methylation of intact yeast cells with dimethylsulphate, indicate that the same binding sites are occupied in vivo as well. Deletions that abolish binding of RBP1 to the enhancer in vitro, as well as linker insertions into the RBP1 binding site in the initiation region that strongly diminish in vitro binding of RBP1, have no effect whatsoever on the enhancement of rDNA transcription in vivo. This was studied by deletion/mutation of the RBP1 binding site in vitro in an artificial ribosomal minigene and measuring the effect on the minigene transcription in vivo in yeast cells, transformed with the deleted/mutated minigenes. It can therefore be concluded that binding of RBP1 is not an important parameter in the functioning of the rDNA enhancer in yeast. Using the same minigene system we also show that RBP1 is not involved in termination of RNA polymerase I (PolI) transcription at the main terminator T2.
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  • 14
    ISSN: 1432-0983
    Keywords: Yeast ; Saccharomyces cerevisiae ; Nonsense suppression ; Omnipotent suppressors ; Gene mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Ten dominant omnipotent suppressors of Saccharomyces cerevisiae, which were previously shown to be different from SUP46, have been examined. Nine are mapped in a region between lys5 and cyh2 on the left arm of chromosome VII. These suppressors, like SUP46, manifest sensitivity to increased temperature and the antibiotics paromomycin and hygromycin B. In addition, they have an identical action spectrum. These results strongly suggest that they are allelic to each other and they are designated SUP138. The tenth is mapped to a position between his1 and arg6 on the right arm of chromosome V. This suppressor, named SUP139, does not manifest temperature sensitivity nor antibiotic sensitivity. SUP139 and SUP138, which are clearly distinguished by means of action spectrum, act on much fewer nonsense mutations than SUP46. It is now clear that dominant omnipotent suppressors arising at a single locus are homogeneous and that their efficiency is locus-dependent. The order of efficiency is SUP46〉SUP138〉SUP139.
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  • 15
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    Current genetics 16 (1989), S. 339-346 
    ISSN: 1432-0983
    Keywords: Yeast ; Transformation ; ss carrier DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 × 105 transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2μm origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
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  • 16
    ISSN: 1432-0983
    Keywords: Platinum compounds ; Yeast ; Repair mutants ; Interstrand cross-links ; DNA degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Four haploid yeast strains differing in proficiency for DNA repair were treated with cis- or transDDP. The wild type was least sensitive while the excision-deficient mutants rad1, rad2 and snm1exhibited higher sensitivities to either platinum compound. In all four strains tested cisDDP showed a two- to five-fold higher cytotoxicity than equimolar concentrations of transDDP. DNA interstrand cross-linking was caused by both agents in all strains. However, transDDP introduced more DNA cross-links at exposure times up to 6 h while cisDDP was the more active cross-linking agent at longer times. There was no clear-cut correlation of the number of DNA interstrand cross-links with survival. Formaldehyde-treated cells showed DNA with lower buoyant density due to proteinase K sensitive DNA-protein cross-linking; this effect was not observed after treatment with either platinum compound. Post-treatment incubation of wild-type cells exposed to cisDDP led to degradation of DNA by single and double-strand breaks, parallel with further increase of DNA interstrand cross-linking. DNA from transDDP-treated cells did not show extensive degradation although interstrand cross-links were lost during liquid holding.
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  • 17
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    Current genetics 16 (1989), S. 347-350 
    ISSN: 1432-0983
    Keywords: Yeast ; 7SL RNA ; Yarrowia lipolytica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have identified an abundant cytoplasmic 7S RNA in crude extracts of the yeast Yarrowia lipolytica. A cDNA probe was prepared from this RNA and used to screen a genomic library. The DNA sequence of a positive clone was determined and the end positions of the 7S RNA gene established by comparison with the sequence of the extremities of 7S RNA. This gene, designated SCR2, encodes a 270-nucleotide RNA that can be folded into a secondary structure similar to that of 7SL RNAs. This RNA is 94.4% homologous to a previously identified 7S RNA from this yeast, but is encoded by a separate gene with highly divergent flanking sequences.
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  • 18
    ISSN: 1432-0983
    Keywords: Yeast ; Repair ; Complementation ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; Gene cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.
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  • 19
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    Current genetics 15 (1989), S. 99-106 
    ISSN: 1432-0983
    Keywords: Yeast ; Isoleucyl-tRNA synthetase ; Isoleucine ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The isoleucyl-tRNA synthetase gene (ILS1) from the yeast Saccharomyces cerevisiae was cloned and sequenced. This gene was initially cloned because it cross-hybridizated to what is now presumed to be the isoleucyl-tRNA synthetase gene (cupC) from the protozoan Tetrahymena hhermophila. The ILS1 gene was determined to be 1,072 amino acids in length. A comparison with a recently published sequence of ILS1 1 from another laboratory (Englisch et al. 1987) was made and differences noted. Two promoter elements were detected, one for general amino acid control and one for constitutive transcription. A heat shock protein (hsp70) gene (probably SSA3) was found 237 by upstream from the ILS1 translation start site. The ILS1 amino acid sequence was compared to isoleucyl-tRNA synthetases from other organisms, as well as to valyl-, leucyl- and methionyl-tRNA synthetases. Regions of conservation between these enzymes were found.
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  • 20
    ISSN: 1432-0983
    Keywords: PDC3 ; Pyruvate decarboxylase ; Subunits ; Yeast ; Cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Biochemical evidence that pyruvate decarboxylase in S. cerevisiae might be constituted from two independently encoded subunits led us to question genetic evidence for a single structural gene. The main evidence for this was that three “structural” mutations appeared to be alleles of the same gene, PDC1 (Schmitt and Zimmermann 1982). We report that one of these mutations (pdcl-30) is not allelic either to other pdc1 alleles or to pdc2 mutations and therefore is has been renamed pdc3-30 thus identifying a new gene, PDC3. We have cloned the PDC3 gene, it represents a unique sequence in the genome and targeted integration shows tight linkage to the PDC3 locus. However, the size, abundance and regulation of the PDC3 transcript suggest that it does not encode a second structural gene. Possible functions for the PDC3 gene product are discussed.
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  • 21
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    Current genetics 16 (1989), S. 21-25 
    ISSN: 1432-0983
    Keywords: Yeast ; Vectors ; Stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 μm plasmid to completely eliminate the bacterial moiety upon introduction into yeast. A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth. These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression.
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  • 22
    ISSN: 1432-0983
    Keywords: Yeast ; Chromosome organization ; Acid phosphatase ; Telomere
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 17 kb region from near the right end of chromosome I of Saccharomyces cerevisiae was isolated on recombinant λ bacteriophages. This region contained the PH011 gene which was located only 3.4 kb from the right end of the chromosome. We found that this region also was repeated approximately 13 kb from the end of the chromosome VIII DNA molecule. The chromosome VIII sequence appears to be a previously unnamed acid phosphatase gene that we propose to call PH012. Thus, similar to the repeated SUC, MAL, X and Y' sequences, some members of the repeated acid phosphatase gene family also appear near the termini of yeast chromosomes.
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  • 23
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Intron splicing ; RNA maturase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the b14 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.
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  • 24
    ISSN: 1432-0983
    Keywords: Telomere Binding Activity (TBA) ; Yeast ; Telomeric binding sites ; RAP1 gene product
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Telomere Binding Activity (TBA), an abundant protein from Saccharomyces cerevisiae, was identified by its ability to bind to telomeric poly(C1–3A) sequence motifs. The substrate specificity of TBA has been analyzed in order to determine whether the activity binds to a unique structure assumed by the irregularly repeating telomeric sequences or whether the activity recognizes and binds to subset of specific sequences found within the telomere repeat tracts. Deletion analysis and DNase I protection assays demonstrate that TBA binds specifically to two poly(C1–3A) sequences that differ by one nucleotide. The methylation of four guanine residues, located at identical relative positions within these two binding sequences, interferes with TBA binding to the substrates. A synthetic olignucleotide containing a single TBA binding site can function as a TBA binding substrate. The TBA binding site shares homology with the binding sites reported for the Repressor/Activator Protein 1 (RAP1), Translation Upshift Factor (TUF) and General Regulatory Factor (GRFI) transcription factors, and TBA binds directly to RAP1/TUF/GRFI substrate sequences. Yeast TBA preparations and the RAP1 gene product expressed in E. coli cells are both similarly sensitive to in vitro protease digestion. Affinity-purified TBA extracts include a protein indistinguishable from RAP1 in binding specificity, size, and antigenicity. The binding affinity of TBA for the two telomeric poly(C1–3A) binding sites is higher than its affinity for any of the other binding substrates used for its identification. In extracts of yeast spheroplasts prepared by incubation of yeast cells with Zymolyase, an altered, proteolyzed form, of TBA (TBA-S) is present. TBA-S has a faster mobility in gel retardation assays and SDS-PAGE gels, yet it retains the DNA binding properties of standard TBA preparations: it binds to RAP1/TUF/GRFI substrates with the same relative binding affinity and protects poly(C1–3A) tracts from DNase I digestion with a “footprint” identical to that of standard TBA preparations.
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  • 25
    ISSN: 1432-0983
    Keywords: Yeast ; DNA methylation ; DNA methyltransferase ; rad mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary DNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD + and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD + strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m5C residues.
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  • 26
    ISSN: 1432-0983
    Keywords: Yeast ; Mating ; Sexual agglutination ; a-Specific mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Seven α-specific mutants specifically defective in sexual agglutinability were isolated. The other α mating functions exhibited by these mutants, designated sag mutants, such as the production of α pheromone and response to a mating pheromone, were normal. While the MATα sag1 cells did not agglutinate with wild-type a cells, the MATα sag1 cells did, indicating that the SAG1 gene is expressed only in α cells. The mutations were semi-dominant and fell into a single complementation group, SAG1, which was mapped near met3 on chromosome X. Complementation analysis showed that sag1 and aga1, the latter being a previously reported α-specific mutation, were mutations in the same gene.
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  • 27
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    Current genetics 15 (1989), S. 385-392 
    ISSN: 1432-0983
    Keywords: Yeast ; Meiosis ; Distributive disjunction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Distributive disjunction is defined by first meiotic division segregation of either two nonhomologous chromosomes that lack homologous pairing partners, or of two homologous chromosomes that have failed to undergo crossing-over. In the yeast Saccharomyces cerevisiae, plasmid minichromosomes, synthetic linear chromosomes and a fragment of a real chromosome have been observed to segregate from nonhomologous DNA species at the first meiotic divisions. Suggesting that this organism may have a distributive mechanism for chromosome segregation. However, it is not known whether intact chromosomes also participate in a distributive process. To determine whether intact, full length, S. cerevisiae chromosomes could segregate from nonhomologous chromosomal species, the meiotic behavior of an unpaired intact copy of chromosome I has been analyzed with respect to several centromere-containing circular plasmid minichromosomes. Strains monosomic or trisomic for chromosome I were transformed with centromere plasmids containing either homologous or nonhomologous inserts, sporulated, and analyzed genetically both for the presence of plasmid and for the number of copies of chromosome 1. Each plasmid segregated from an intact unpaired copy of chromosome I at the first meiotic division in a significant majority (63–93%) of the asci examined. These results suggest that intact chromosomes from S. cerevisiae are capable of distributive disjunction.
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  • 28
    ISSN: 1432-0983
    Keywords: Yeast ; Invertase ; Gene expression
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    Notes: Summary Gene SUC4 produced about four fold more invertase activity than did gene SUC5. However, these genes differ in only three positions located in the 5′ non-coding region. The difference in gene expression between SUC4 and SUC5 must be due to the G to A transition (position −497) and/or the C to T transition (position −460) in the upstream activator sequences. The sequence TACAAA present in SUC5 can play the same role than the TATAAA box of SUC4.
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  • 29
    ISSN: 1432-0983
    Keywords: Ribosomes ; Antisuppressor ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The yeast antisuppressor mutation, asu9-1 (Liebman and Cavenagh 1980) was found to cause an alteration in the 40S ribosomal subunit. Two-dimensional polyacrylamide gel electrophoresis patterns of the 40S ribosomal proteins from four different strains bearing the asu9-1 mutation all contained the same extra protein spot which was completely absent in five strains which did not carry the asu9 mutation.
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  • 30
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    Current genetics 3 (1981), S. 213-220 
    ISSN: 1432-0983
    Keywords: Mitochondrial genetics ; Yeast ; Suppressiveness ; Triploid analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A genetically defined highly suppressive petite yeast strain (ρ −cob+AsEoCoOoPo) was crossed with a grande strain carrying a multiply marked mitochondrial genome (ρ +ArErCrO rpr). Petite diploid progeny, isolated from individual zygotic clones consisting either of wholly petite or mixtures of grande and petite cells, were characterised genetically by crossing to grande haploids. The diploid petites were found to closely resemble the petite parent and in general not to carry mitochondrial markers from the grande parent. In the petites from the mixed clones recombination was detected, but only within the region of homology between the genomes. These observations are inconsistent with models of suppressiveness based on destructive recombination and suggest that the petite genome eliminates the grande genome from zygotic progeny through being preferentially replicated. The most plausible model to explain the observed pattern of zygotic clones postulates a limited number of mDNA replication sites in zygotes, competition for sites between input mDNA molecules and an advantage in this competition for suppressive ρ − mDNA.
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  • 31
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    Current genetics 3 (1981), S. 229-233 
    ISSN: 1432-0983
    Keywords: Yeast ; Nascent DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.
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  • 32
    ISSN: 1432-0983
    Keywords: Regulation ; Urea ; Catabolism ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Urea amidolyase and the high affinity urea uptake system are induced by allophanate. durM − and durL − recessive mutations, which are easily obtained, totally prevent this induction. They are not linked to each other nor to the concerned structural genes. Despite an intensive hunt, no mutation of repressor or classical operator type has been selected. We conclude that urea amidolyase and urea uptake induction involves at least two positive elements coded for by the durM and durL genes.
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  • 33
    ISSN: 1432-0983
    Keywords: Yeast ; Catalase ; Trehalose ; Glycogen
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Summary Mutations at the GLC1 locus in Saccharomyces cerevisiae result in a major deficiency in synthesis of catalase T, but do not affect catalase A. Three independent glc1 mutations were shown to have the same pleiotropic phenotype: catalase T deficiency, defective glycogen synthesis and defective trehalose accumulation. These three deficiencies appear to be determined by a single, nuclear gene. The possibility that glc1 mutations alter a protein kinase is considered.
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  • 34
    ISSN: 1432-0983
    Keywords: Arginine catabolism ; Regulation ; Ornithine transaminase ; Double induction ; Yeast
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Summary Yeast ornithine transaminase is known to be induced by arginine and ornithine, through the action of regulatory elements common to arginase induction. We show here that it is subject to a second induction circuit, that which is responsible for urea amidolyase and urea permease induction by allophanate and defined by the regulatory mutants durL − and durM −
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  • 35
    ISSN: 1432-072X
    Keywords: Pyrophosphate ; Polymerie acid-soluble poly-phosphates ; Budding process ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the cells of hybrid yeast strain Saccharomyces N.C.Y.C. 644 SU3 (Karlsberg collection), a large amount of pyrophosphate (30–300 μmol/g of dry weight) accumulates whatever the aeration conditions and the content of glucose in the medium. The content of pyrophosphate is 10–1000 times higher than that of ATP. At the early and mid-exponential growth phases two maxima of pyrophosphate accumulation are observable. The periods of maximal pyrophosphate accumulation in yeast coincide with those of the minimal content of polymeric acid-soluble polyphosphates and intense budding. In the light of the data obtained, the question is discussed as to the relationship between the metabolism of pyrophosphates and acid-soluble polyphosphates in yeast.
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  • 36
    ISSN: 1432-072X
    Keywords: Germination ; Glycogen ; Outgrowth ; Schizosaccharomyces pombe ; Spore ; Trehatase ; Trehalose ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Quantitative changes in various carbohydrates of Schizosaccharomyces, pombe spores during germination and outgrowth were studied. Trehalose decreased rapidly, shortly after onset of germination, while glycogen remained constant throughout germination and outgrowth. Alkali-insoluble carbohydrates decreased after the lag period of about 40 min. The content of alkali-soluble carbohydrates was constant during germination, but increased remarkably in parallel with germtube formation. The mechanism of rapid degradation of trehalose during germination was also studied. The activity of trehalase (EC 3.2.1.28) was detected only in the cell wall fraction of isolated spores. Trehalase activity in the cell wall fraction was not enhanced during germination. Trehalose was not found in the isolated spore walls, but in the soluble fraction. These facts strongly suggested that trehalose, and trehalase were spatially separated in dormant spores and that trehalase became accessible to trehalose upon germination.
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  • 37
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    Archives of microbiology 129 (1981), S. 47-48 
    ISSN: 1432-072X
    Keywords: Adenylate energy charge ; Phosphate ; Saccharomyces ; Sporulation ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.
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  • 38
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Yeast ; Phospholipase B ; Lysophospholipase ; Enzyme inhibition ; AMP ; Unesterified fatty acids
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    Topics: Biology
    Notes: Abstract Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 μM. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.
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  • 39
    ISSN: 1432-072X
    Keywords: Peroxisome ; Methanol ; Methylamine ; Yeast ; Hansenula polymorpha ; Alcohol oxidase ; Amino oxidase ; Catalase ; Catabolite inactivation ; Turnover ; Cytochemical localization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Growth of Hansenula polymorpha in shake flasks and chemostat cultures in the presence of methanol as the sole source of carbon and methylamine as the sole source of nitrogen was associated with the development of peroxisomes in the cells. The organelles were involved in the concurrent oxidation of these two compounds, since they contained both alcohol oxidase and amine oxidase, which are key enzymes in methanol and methylamine metabolism, respectively. In addition catalase was present. Peroxisomes with a completely crystalline substructure were observed in methanol-limited chemostat-grown cells. Amine oxidase probably formed an integral part of these crystalloids, whereas catalase was present in a freely diffusable form. Transfer of cells, grown in a methanol-limited chemostat in the presence of methylamine into glucose/ammonium sulphate media resulted in the loss of both alcohol oxidase and amine oxidase activity from the cells. This process was associated with degradation of the crystalline peroxisomes. However, when cells were transferred into glucose/methylamine media, amine oxidase activity only declined during 2 h after the transfer and thereafter increased again. This subsequent rise in amine oxidase activity was associated with the development of new peroxisomes in the cells in which degradation of the crystalline peroxisomes, originally present, continued. These newly formed organelles probably originated from peroxisomes which had not been affected by degradation. When in the methanollimited chemostat methylamine was replaced by ammonium sulphate, repression of the synthesis of amine oxidase was observed. However, inactivation of this enzyme or degradation of peroxisomes was not detected. The decrease of amine oxidase activity in the culture was accounted for by dilution of enzyme as a result of growth and washout.
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  • 40
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    Archives of microbiology 151 (1989), S. 198-202 
    ISSN: 1432-072X
    Keywords: Sexual agglutination ; Mating ; Saccharomyces cerevisiae ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.
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  • 41
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    Archives of microbiology 130 (1981), S. 87-89 
    ISSN: 1432-072X
    Keywords: Yeast ; Kluyveromyces ; 6-Deoxyglucose ; Glucose transport
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    Topics: Biology
    Notes: Abstract The inducible glucose transport system of the yeast, Kluyveromyces lactis, was studied using the nonmetabolizeable glucose analogue, 6-deoxyglucose. The free sugar analogue is transported into glucose-grown cells via a facilitated diffusion system as determined by the nonconcentrative uptake of the sugar analogue, by the failure of energy inhibitors to reduce the rate of transport and by exchange diffusion across the membrane. Free 6-deoxyglucose is also transported into succinate-grown cells passively. Induction experiments revealed that 6-deoxyglucose serves as a gratuitous inducer for the glucose transport system in this yeast.
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  • 42
    ISSN: 1432-072X
    Keywords: d-Xylose fermentation ; Aeration level ; Xylose reductase ; Xylitol dehydrogenase ; Yeast ; Candida shehatae ; Candida tenuis ; Pichia stipitis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The relationship between the degree of aerobiosis, xylitol production and the initial two key enzymes of d-xylose metabolism were investigated in the yeasts Pichia stipitis, Candida shehatae and C. tenuis. Anoxic conditions severely curtailed growth and retarded ethanol productivity. This, together with the inverse relationship between xylitol accumulation and aeration level, suggested a degree of redox imbalance. The ratios of NADH- to NADPH-linked xylose reductase were similar in all three yeasts and essentially independent of the degree of aerobiosis, and thus did not correlate with their differing capacities for ethanol production, xylitol accumulation or growth under the different conditions of aerobiosis. Under anoxic conditions the enzyme activity of Pichia stipitis decreased significantly, which possibly contributed to its weaker anoxic fermentation of xylose compared to C. shehatae.
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  • 43
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    Archives of microbiology 152 (1989), S. 564-566 
    ISSN: 1432-072X
    Keywords: l-Malate ; Schizosaccharomyces malidevorans ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast Schizosaccharomyces malidevorans utilizes l-malate when grown on glucose as the carbon source. A mutant of this yeast has been isolated which is dependent on the presence of both l-malate and glucose for growth. The mutant utilizes l-malate as rapidly as the wildtype and the utilization of glucose is greatly reduced. Other TCA cycle intermediates do not relieve the malate dependence.
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  • 44
    ISSN: 1617-4623
    Keywords: Autoregulation ; LacZ fusion protein ; Northern hybridization ; Regulatory circuit ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The mode of expression was investigated for two positive regulatory genes PHO2 and PHO4, whose products are indispensable for the transcriptional control of the structural genes of repressible acid phosphatase and the inorganic phosphate (Pi) transport system in Saccharomyces cerevisiae. Northern analysis of poly(A)+ RNA of the wild-type and the pho regulatory mutants with PHO4 DNA as hybridization probe and expressional analysis of a pho4′-'lacZ fused gene on a YEp plasmid revealed that PHO4 is expressed at a low level, constitutively, and independently of the PHO regulatory system and Pi in the medium. Similar analyses with PHO2 DNA indicated that PHO2 is expressed at an even lower level than PHO4, and is repressed by Pi and by the active PHO2 product, possibly at the translational level, while retaining a substantial level of basal activity.
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  • 45
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    Molecular genetics and genomics 216 (1989), S. 276-286 
    ISSN: 1617-4623
    Keywords: Methionine ; Initiator tRNA ; tRNA(met) ; Yeast ; Multigene family
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA I Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA I Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.
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  • 46
    ISSN: 1617-4623
    Keywords: Yeast ; Nuclear genes ; Mitochondrial translation ; Mitochondrial splicing ; Suppression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.
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  • 47
    ISSN: 1617-4623
    Keywords: Yeast ; Mitochondrial 15 S rRNA ; Ribosomal frameshifting ; Paromomycin resistance
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    Notes: Summary The leaky expression of the yeast mitochondrial geneoxi1, containing a frameshift mutation (+1), is caused by natural frameshift suppression, as shown previously (Fox and Weiss-Brummer 1980). A drastic decrease in the natural level of frameshifting is found in the presence of thepar r-454 mutation, localized at the 3′ end of the 15 S rRNA gene. This mutation causes resistance to the antibiotic paronomycin in the yeast strains D273-10B and KL14-4A (Li et al. 1982; Tabak et al. 1982). The results of this study imply that in the yeast strain 777-3A this mutation alone is sufficient for restriction of the level of natural frameshifting but is insufficient to confer resistance to paromomycin. A second mutation, arising spontaneously with a frequency of 10−4 leads, in combination with thepar r-454 mutation, to full paromomycin resistance in strain 777-3A.
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  • 48
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    Molecular genetics and genomics 215 (1989), S. 425-430 
    ISSN: 1617-4623
    Keywords: Yeast ; DNA repair ; RAD18 ; Chromosomal deletions ; Mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.
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  • 49
    ISSN: 1617-4623
    Keywords: Chromosome fragmentation ; Mapping ; PHO13 sequence ; Phosphatase ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The structural gene, PHO13, for the specific p-nitrophenyl phosphatase of Saccharomyces cerevisiae was cloned and its nucleotide sequence determined. The deduced PHO13 protein consists of 312 amino acids and its molecular weight is 34635. The disruption of the PHO13 gene produced no effect on cell growth, sporulation, or viability of ascospores. The PHO13 locus was mapped at 1.9 centimorgans from the HO locus on the left arm of chromosome IV. By chromosome fragmentation, the PHO13 locus was found to be located about 72 kb from the left-hand telomere of chromosome IV and distal to the HO locus.
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  • 50
    ISSN: 1617-4623
    Keywords: Centromere ; Chromatin ; Hypersensitive sites ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Isolated nuclei of Saccharomyces cerevisiae were incubated with five restriction nucleases. Out of the twenty-one recognition sequences for these nucleases in the centromere region of chromosome XIV, only five are accessible to cleavage. These sites map 11 by and 74 by to the left and 27 bp, 41 by and 290 by to the right, respectively, of the boundaries of the 118 by functional CEN14 DNA sequence. The distance between the sites accessible to cleavage and closest to CEN14 is 156 bp, suggesting this is the maximal size of DNA protected in CEN14 chromatin. The DNA in CEN14 chromatin protected against cleavage with DNase I and micrococcal nuclease overlaps almost completely with this region. Hypersensitive regions flanking both sides are approximately 60 by long. Analyses of other S. cerevisiae centromeres with footprinting techniques in intact cells or nucleolytic cleavages in isolated nuclei are discussed in relation to our results. We conclude that structural data of chromatin obtained with restriction nucleases are reliable and that the structure of CEN14 chromatin is representative for S. cerevisiae centromeres.
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  • 51
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    Molecular genetics and genomics 217 (1989), S. 40-46 
    ISSN: 1617-4623
    Keywords: Gene dosage ; Gene expression ; Regulatory circuit ; Signal transmission ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Expression of the repressible acid phosphatase (rAPase) gene, PHO5, of Saccharomyces cerevisiae is repressed by a certain level of inorganic phosphate (Pi) in the medium and is derepressed when the Pi concentration is lowered. The Pi signals are conveyed to PHO5 by a regulatory system consisting of proteins coded for by the PHO2, PHO4, PHO80 and PHO81 genes. We have found that the transcription of PHO81 is regulated by Pi through the PHO regulatory system. Increasing the dosage of PHO4 and PHO81 by ligating each gene to YEP13 gives rise to, respectively, considerable and weak synthesis of rAPase by cultivation of the transformants in high-Pi medium; but in low-Pi medium, increased dosage of PHO4 stimulates the rAPase synthesis significantly, whereas PHO81 has no effect. Increased dosage of PHO2 stimulates rAPase synthesis considerably in low-Pi but not in high-Pi. A coordinate increase of PHO80 cancels the dosage effect of PHO4, but not that of PHO81. Coordinate increases of PHO80 and PHO2 give rise to the same phenotype as an increased dosage of PHO80 alone. The level of the PHO4 protein was found to be the limiting factor of the rAPase synthesis and the copy number of the PHO5 gene not to be. These facts accord with the idea that the PHO80 protein transmits the Pi signals to the PHO5 gene via the PHO4 protein, whereas the PHO2 protein does not have a direct function in the signal transmission.
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  • 52
    ISSN: 1617-4623
    Keywords: Yeast ; MFα1 leader ; Gene fusion ; Secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The constuction of two fused genes is described. One involves the in-frame fusion of the yeast prepro-α-factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the α-factor promoter, expressed active β-galactosidase in α haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MFα1-lacZ fusion junction provided significantly higher levels of β-galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.
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  • 53
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    Journal of bioenergetics and biomembranes 21 (1989), S. 439-450 
    ISSN: 1573-6881
    Keywords: Yeast ; yeast mutant ; mitochondrial porin ; mitochondrial outer membrane ; lipid bilayer ; ion-channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than “normal” mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.
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