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  • Saccharomyces cerevisiae  (34)
  • Springer  (34)
  • Cell Press
  • Elsevier
  • 1985-1989  (34)
  • 1980-1984
  • 1986  (34)
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  • 1985-1989  (34)
  • 1980-1984
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  • 1
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    Springer
    Current genetics 10 (1986), S. 943-945 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; α-factor ; Protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary When Mat a cells are treated with α-factor prior to being protoplasted and fused, the frequency of karyogamy is higher than in unarrested controls.
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  • 2
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    Current genetics 10 (1986), S. 665-670 
    ISSN: 1432-0983
    Keywords: Multiple drug resistance ; Genetic mapping ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In the yeast Saccharomyces cerevisiae, two nuclear pleiotropic drug resistance mutations pdr3-1 (former designation muc PR) and pdr3-2 (former designation DRI9/T7) have been selected as resistant to mucidin and as resistant to chloramphenicol plus cycloheximide, respectively. The pdr3 mutations were found not to affect the plasma membrane ATPase activity measured in a crude membrane fraction. Meiotic mapping using strains with standard genetic markers revealed that mutation pdr3-1 is centromere linked on the left arm of chromosome II at a distance of 5.9 ± 3.3 cM from its centromere and 11.6 ± 3.1 cM from the marker pet9. The centromere linked pdr3-2 mutation exhibited also genetic linkage to pet9 with a map distance of 9.8 ± 3.2 cM. These results indicate that pdr3-1 and pdr3-2 are alleles of the same pleiotropic drug resistance locus PDR3 which is involved in the control of the plasma membrane permeability in yeast.
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  • 3
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    Current genetics 10 (1986), S. 657-664 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Amino acid biosynthesis ; General control ; GCD-genes ; GCN-genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the “general control derepressed” (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: “constitutive derepression” and “slow growth”. Epistatis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the “constitutive derepression” phenotype, and not to “slow growth”; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.
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  • 4
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    Current genetics 10 (1986), S. 491-494 
    ISSN: 1432-0983
    Keywords: Protoplast fusion ; Saccharomyces cerevisiae ; Schwanniomyces castellii ; Starch fermentability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Prototrophic hybrids have been obtained by the fusion of various auxotrophic haploid strains of Saccharomyces cerepisiae and Schwanniomyces castellii. The fusion hybrids showed starch fermentability which derived from one of the fusion parents, S. castellii. Surprisingly, these fusion hybrids were found to exhibit excellent sporulation and spore germination. The progenies of these fusion hybrids showed a few aberrant segregations, but mostly normal segregation for auxotrophic genetic markers. They also showed many tetrads with an apparently digenic segregation (2:2, 3:1 and 4:0) for starch fermentation. On the other hand, mating types of segregants of the fusion hybrids were determined by the prototrophic recovery method. Consequently, tetrad types for mating type were mostly 2a:1α:1 non-mater and several asci showed tetrad types of 2a:2 non-mater and 2a:2α. The 60 prototrophic fusion hybrids and its segregants did not secrete α-amylase on the starch agar plate. However, all of the data suggested that fusion hybrid could carry two dominant genes (STAB and STAC) to ferment starch, and that the two genes STAB and STA2 may be identical or allelic as may be the genes STAC and STA3.
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  • 5
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    Current genetics 11 (1986), S. 93-96 
    ISSN: 1432-0983
    Keywords: Uracil permease gene ; Saccharomyces cerevisiae ; Chromosomal mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The gene FUR4, coding for the uracil permease in Saccharomyces cerevisiae, was mapped on chromosome II, at a distance of 7.8 cM from the centromere on the right arm of the chromosome. In a first step, we used the chromosome loss mapping method developed by Falco and Botstein (1983) to determine on which chromosome the gene mapped. After the observation that FUR4 was closely linked to GAL10, one of the three genes forming the gal cluster (Bassel and Mortimer 1971), we could determine precisely the position of the gene on chromosome II.
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  • 6
    ISSN: 1432-0983
    Keywords: Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; DNA ligase ; DNA damage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary UV-irradiation of stationary phase cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a 9-fold and 90-fold increase in transcript levels from the respective DNA ligase genes CDC9 and CDC17, whereas exponential cells show only 3-fold and 2-fold increases. Induction of CDC9 after MMS treatment and γ-irradiation was also observed by using a CDC9-lacZ translational fusion and assaying for β-galactosidase. Surprisingly, irradiation of S. cerevisiae induces only a 50% increase in DNA ligase itself, probably reflecting the extremely high in vivo stability of the enzyme. The UV-induction of ligase may be part of a “fail-safe” mechanism which, together with the enzyme stability, ensures adequate supplies of this essential enzyme.
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  • 7
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    Current genetics 11 (1986), S. 193-200 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Ty elements ; Transposable elements ; Retroviruses ; tRNA genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have isolated and characterized a Ty element from a yeast cosmid library which exhibits several unsual features: it is flanked by non-homologous delta elements and directly associated with a singular delta element. A tRNA(Glu3) gene and tRNA(Cys) gene are found in conjunction with this element, located in opposite orientation on either end of it. The sequence information now available for several Ty elements has been used in a detailed comparative analysis to determine conserved features among the Ty elements, preferably between class I elements and a class II element. Highly conserved sequence motifs appear to be located at the borders of particular segments that correspond to the putative protein domains of the Tys. Furthermore, we include a comparison of the best-conserved amino acid homologies for these putative proteins of Ty elements, transposable elements from other organisms and several retroviral proviruses to confirm their close structural resemblance.
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  • 8
    ISSN: 1432-0983
    Keywords: Rho°-petites ; Lycorine ; Mitochondrial DNA replication ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This paper describes the isolation and characterization of mutants affected in the maintenance of the mitochondrial (mt) genome. The rationale of the screening procedure is based on the observation that the alkaloid lycorine inhibits growth of rho −-mutants, whereas rho°-mutants, devoid of mt DNA, are resistant to this drug (Del Giudice et al. 1984). Fourteen temperature sensitive mutants have been isolated that display the following phenotype: -Growth on fermentable medium at 23°C and 35°C (exclusion of general temperature-sensitive mutants). -no growth at 23°C and growth at 35°C on fermentable medium containing lycorine (selection for mutants producing rho°-petites). -growth at 23°C and no growth an 35°C on non-fermentable medium (selection for temperature-dependent loss of respiratory competence). These mutants were termed tmm (for temperature sensitive maintenance of mt genome). Mutant tmm1-1 was analyzed genetically and biochemically. It carries a recessive nuclear mutation which gives rise to 90–95% cytoplasmic petites at the non-permissive temperature. The population of petites consists of more than 95% rho°-petites as shown by their resistance to lycorine, by staining with 4′,6-diamidino-2-phenylindole (DAPI), and by Southern hybridization with mt DNA probes. Wild-type control cultures produced approximately 1% petites with less than 10% rho°-mutants.
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  • 9
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    Current genetics 11 (1986), S. 211-215 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Hyperresistance ; DNA damaging agents ; Genotoxic effects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinohne-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.
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  • 10
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    Current genetics 11 (1986), S. 217-225 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Gene cloning ; Invertase genes ; Multicopy vector
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Six unlinked loci for invertase structural genes are known in the yeast Saccharomyces cerevisiae: SUC1-SUC5 and SUC7. These genes are similar in structure and expression but not identical. Different yeast strains possess none, one or several of these genes. We have isolated the genes SUC1-SUC5, subcloned them into the multicopy vector YEp24 and compared the expression of the five SUC genes in one recipient strain. SUC2 was isolated by transformation of a suc0 strain with a gene pool and complementation to sucrose fermentation. SUC4 was cloned from a minipool of chromosomal fragments which were shown to contain SUC4 by Southern hybridization. SUC1, SUC3 and SUC5 were isolated using the method of plasmid eviction. A plasmid containing regions flanking SUC4 was integrated next to these SUC genes. The plasmid together with the SUC genes were then cut out of the chromosome using an appropriate restriction endonuclease. The length of chromosomal DNA fragments containing the different SUC genes were 4.8 kb for SUC1, 5.2 kb for SUC2, 4.8 kb for SUC3, 12.8 kb for SUC4 and 17.2 kb for SUC5. Fragments containing the complete SUC genes and the sequences controlling their expression were subcloned into YEp24 and transformed into a strain without any active invertase gene. Invertase activity of transformants was measured after growth repressing (8% glucose) and derepressing (2% raffinose) conditions. As expected from results with strains carrying the individual SUC genes in a chromosomal location, the SUC genes were expressed to a different extent.
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  • 11
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nucleo-mitochondrial interactions ; Mitochondrial status ; Dominant lycorine resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutants resistant to 200 µg/ml of the alkaloid lycorine (LYC R) in non-fermentable substrate were isolated after nitrosoguanidine mutagenesis. Tetrad analysis and growth of heterozygous (LYC R/lyc s) diploids from two different mutants revealed that a single nuclear and dominant mutation is responsible for the resistant phenotype. In the wild type total protein synthesis is only slightly inhibited, whereas DNA and RNA synthesis is lowered to about 30% of the control. In the lycorine resistant mutants all macromolecular syntheses are unaffected by the drug.
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  • 12
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Cytochrome c ; Regulatory gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The CYP1 gene has previously been identified as coding for a positive trans active factor that activates the expression of CYC1 and CYP3, which are the structural genes for isol- and iso2-cytochrome c. Two phenotypically distinct classes of CYP1 mutations can be obtained indicating that CYC1 and CYP3 are differentially regulated by the product of CYP1. The HAP1 gene codes for a product which has previously been proved to be necessary for the expression of the heme dependent CYC1-UAS1 cis regulatory sequence. In this article, we show by complementation and recombination that CYP1 and HAP1 are the same gene, moreover we identify hap1-1 as an iso2-cytochrome c underproducer mutation of the CYP1 gene.
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  • 13
    ISSN: 1432-0983
    Keywords: agα1 Mutant ; Agglutination ; Gene dose effect ; Mapping ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A recessive agα1 mutation leads to specific defect in sexual agglutinability specifically in α cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cryR 1, closely linked to the mating type locus, was used to select α/α strains which emerged from α/α strains by mitotic nonreciprocal recombination, to genetically analyse agα1, since agα1 is expressed only in α mating type. The agα1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of α cells was shown to be dependent on the dose of the AGα1 gene, using α/α isogenic strains carrying AGα1/AGα1, AGα1/agα1 or agα1/agα1. The sst2-1 mutation did not suppress the agα1 mutation. Based on these results, function of the AGα1 gene is discussed.
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  • 14
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    Archives of microbiology 145 (1986), S. 27-31 
    ISSN: 1432-072X
    Keywords: Nitrite ; Sulfite ; Saccharomyces cerevisiae ; ATP ; Energy metabolism ; Inorganic phosphate ; Glyceraldehyde-3-phosphate dehydrogenase ; Glucose-6-phosphate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F1ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.
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  • 15
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    Archives of microbiology 145 (1986), S. 104-106 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; α Factor ; Trifluoperazine ; Chlorpromazine ; Calmodulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Saccharomyces cerevisiae α factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with α factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by α factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of α factor on MAT a cells.
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  • 16
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    Archives of microbiology 146 (1986), S. 214-220 
    ISSN: 1432-072X
    Keywords: Yeasts ; Cell wall ; Mannoproteins ; Aculeacin A ; Glucan ; Protoplasts ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33000 mannoprotein into the wall network. This was concomitant with the excretion of the latter molecule into the growth medium. Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin. The protoplast walls did lack this component and contained only minor amounts of the 33000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of the 33000 species were immunodetected in the supernatants from treated and untreated protoplasts. These results point to the existence of specific interactions between the glucan network of the yeast cell surface and some of the wall mannoproteins. On the other hand, the presence of a population of SDS-solubilizable mannoproteins in the wall was independent of glucan levels.
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  • 17
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    Archives of microbiology 146 (1986), S. 221-226 
    ISSN: 1432-072X
    Keywords: Exoglucanase ; Saccharomyces cerevisiae ; Secretory mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (〈65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.
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  • 18
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    Archives of microbiology 145 (1986), S. 32-38 
    ISSN: 1432-072X
    Keywords: Sulphate assimilation ; Adenylylsulphate 3′-phosphotransferase EC 2.7.1.25 ; Escherichia coli ; Saccharomyces cerevisiae ; Enzyme purification ; Enzyme regulation ; Thiredoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3′-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B (“blue” or “red”) and chromatofocusing on polybuffer PBE 94tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps. The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it desintegrated into assumingly identical smaller subunits (E. coli rom Mr 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). Both forms, dimer and monomer were found catalytically active. Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.
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  • 19
    ISSN: 1432-072X
    Keywords: Bacillus megaterium ; Saccharomyces cerevisiae ; Ethionine ; Protein degradation ; Abnormal protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Degradation of abnormal proteins in Bacillus megaterium and Saccharomyces cerevisiae in vivo was compared with that in cell-free extracts. Protein degradation in vivo, when the cells were labelled with 14C-leucine during growth in the presence of ethionine, was affected by the concentration of the analogue used. Proteins synthesized in the presence of 0.2–1 mM ethionine were degraded most rapidly in both organisms. The proteolytic enzyme system of yeast degraded the analogue-containing proteins in vitro faster than the normal proteins. This holds also for proteins synthesized in the presence of 5 mM ethionine, whose degradation in vivo was impaired. The proteolytic system of B. megaterium, on the other hand, was unable in vitro to differentiate between normal and abnormal proteins. Denatured proteins underwent preferential degradation over normal and ethionine-containing proteins.
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  • 20
    ISSN: 1573-4935
    Keywords: Candida utilis ; cellulase ; DNAse ; β-glucuronidase ; Hansenula jadinii ; protoplast ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; spheroplast ; yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.
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  • 21
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    Molecular genetics and genomics 203 (1986), S. 73-78 
    ISSN: 1617-4623
    Keywords: Catalase T ; CTT1 ; Saccharomyces cerevisiae ; Yeast ; Heme control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The 5′-flanking region of the Saccharomyces cerevisiae catalase T gene (CTT1) and the part of the gene coding for the N-terminus of catalase T were sequenced. 5′-Ends of transcripts of the region were located by S1 nuclease mapping and primer extension. To analyse control elements in the upstream region, a CTT1-lacZ gene fusion was constructed. Deletion analysis was carried out within a part of the 5′-flanking region showing homology to the upstream region of the yeast CYC1 gene. Like the CTT1 gene, this gene is controlled by heme, oxygen and glucose. The results obtained show that the CTT1 gene is positively controlled by heme. Tentative evidence has been obtained for the involvement of upstream sequences homologous to USA1 and USA2 of the CYC1 gene in heme control. Further, a negative site has been located between the upstream activator sites and the transcription start. Within this negative region a ten base-pair sequence was detected that shows high homology to a sequence located within a negative control region of the CYC1 gene and some homology to the negative control elements of the S. cerevisiae CAR1 and CAR2 genes.
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  • 22
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Gene expression ; Cell cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae. Consistent with the results reported previously by Storms et al. (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication. Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respecitively, exhibited relatively constant levels of transcription throughout the cell cycle. These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation. Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote.
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  • 23
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    Molecular genetics and genomics 205 (1986), S. 422-427 
    ISSN: 1617-4623
    Keywords: SUC2 gene ; Active chromatin ; DNase I hypersensitive sites ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DNase I sensitivity of chromatin of the yeast SUC2 gene, which encodes two forms of invertase, has been studied both in the genome and in a multicopy plasmid carrying the gene and its flanking sequences. Whereas little if any difference in the DNase I sensitivity of the flanking regions was found between the repressed and the derepressed states, derepression of the gene was accompanied by a large increase in the sensitivity of the transcribed region. A well-defined DNase I hypersensitive site was found centered at ∼ 120 bp downstream from the end of the coding region. This site seems to be flanked in the 3′ non-coding region by strictly positioned nucleosomes, and the structure of this region changes upon derepression. In the 5′ non-conding region two DNase I hypersensitive sites have been found flanking the TATA box and a set of three closely spaced hypersensitive sites occurs in an upstream regulatory sequence. The structure of these latter sites depends on the on-off state of transcription.
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  • 24
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; ARO3 gene ; DAHP synthase ; Regulation ; Gene cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Regulation of the two isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase; EC 4.1.2.15) encoded by the genes ARO3 and ARO4 of Saccharomyces cerevisiae was studied. Both genes were shown to respond equally well to the general control of amino acid biosynthesis. Strains with mutations in these two genes were obtained by selecting first for a single aro3 mutation and afterwards for a double aro3 aro4 mutation. Gene ARO3, coding for the phenylanine-dependent isozyme of DAHP synthase was cloned on the 2 μm multicopy vector pJDB207 by complementation of mutation aro3-1 in yeast. The ARO3 gene, carried originally on a 9.6 kb BamHI fragment (plasmid pME541A), was subcloned on a 1.9 kb HindIII-XbaI fragment (plasmid pME543). A transcript of about 1.5 kb was shown to proceed from the HindIII towards the XbaI site. Expression from the 9.6 kb as well as from the 1.9 kb fragment was normal on a multicopy vector, since in both cases DAHP synthase levels of about 50-fold the wild-type level were observed.
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  • 25
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Arginase ; Regulation of gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We localized the chromosomal targets of several of the regulatory controls of expression of theCAR1 gene. Fusion tolacZ of several fragments of the 5′ non-coding region showed that induction ofCAR1 by arginine is positively regulated by the products of theARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affectsCYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory allelesCARGRII andCARGRIII is not situated in the 5′ non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5′—nitrogen catabolite repression—activation by arginine—CARGRI-mediated repression—CAR1.
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  • 26
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    Molecular genetics and genomics 202 (1986), S. 42-47 
    ISSN: 1617-4623
    Keywords: Minicells ; Histidinol phosphatase ; Recombinant plasmids ; Salmonella typhimurium ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysis.
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  • 27
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    Molecular genetics and genomics 202 (1986), S. 120-124 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Ribosomal protein ; Edeine resistant mutant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The r-proteins of an edeine-resistant mutant of Saccharomyces cerevisiae were compared to those of the wild-type strain by using two different two-dimensional electrophoretic techniques: (1) the Kaltschmidt-Wittmann method and, (2) the Kaltschmidt-Wittmann system, in the first dimension and the Na Dodecyl-SO4 system in the second. With the first technique, the results indicate that the patterns of basic ribosomal proteins are similar in the two strains. However, the pattern of acidic ribosomal proteins of the mutant revealed an additional protein band with respect to the normal one. Using the other technique, the patterns of basic and acidic ribosomal proteins of the mutant demonstrated a similarity to the corresponding pattern of the wild-type strain. The data disclose that an acidic ribosomal protein of the mutant may have two forms with different electrophoretic mobilities and similar molecular weights.
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  • 28
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    Molecular genetics and genomics 202 (1986), S. 421-428 
    ISSN: 1617-4623
    Keywords: Recombination intermediates ; Mitochondrial DNA ; Electron microscopy ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an ∼4,600 pb monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand “Holliday” type recombination intermediates. Digestion of both non-crosslinked and crosslinked and mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.
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  • 29
    ISSN: 1617-4623
    Keywords: Excision repair ; denV gene ; UV radiation ; Saccharomyces cerevisiae ; Phage T4
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The denV gene of bacteriophage T4 was reconstituted from two overlapping DNA fragments cloned in M13 vectors. The coding region of the intact gene was tailored into a series of plasmid vectors containing different promoters suitable for expression of the gene in E. coli and in yeast. Induction of the TAC promoter with IPTG resulted in overexpression of the gene, which was lethal to E. coli. Expression of the TACdenV gene in the absence of IPTG, or the use of the yeast GAL1 or ADH promoters resulted in partial complementation of the UV sensitivity of uvrA, uvrB, uvrC and recA mutants of E. coli and rad1, rad2, rad3, rad4 and rad10 mutants of S. cerevisiae. The extent of denV-mediated reactivation of excision-defective mutants was approximately equal to that of photoreactivation of such strains. Excision proficient E. coli cells transformed with a plasmid containing the denV gene were slightly more resistant to ultraviolet (UV) radiation than control cells without the denV gene. On the other hand, excision proficient yeast cells were slightly more sensitive to killing by UV radiation following transformation with a plasmid containing the denV gene. This effect was more pronounced in yeast mutants of the RAD52 epistasis group.
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  • 30
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    Molecular genetics and genomics 204 (1986), S. 98-102 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Minichromosomes ; Centromere ; Copy number ; Mitotic stability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-Δ4 allele (as well as the URA3 and TRP1 markers) yielded His+ transformants at 0.1%–50% the frequency of Ura+ Trp+ transformants. Additional His+ derivatives arose on continuous growth of transformants originally scored as His- Ura+ Trp+. In all cases, the His+ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His+ phenotype was lost more readily than the Ura+ Trp+ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura+ Trp+ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.
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  • 31
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    Molecular genetics and genomics 204 (1986), S. 496-504 
    ISSN: 1617-4623
    Keywords: Heterothallic mating type switching ; Saccharomyces cerevisiae ; RAD52 ; RAD3
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mating type interconversion of the yeast Saccharomyces cerevisiae is mediated by intrachromosomal gene conversion. Whereas homothallic switching is initiated by an endonuclease that produces a DNA double-strand cut within MAT, heterothallic strains lack this activity. In order to identify functions essential for initiation and realisation of heterothallic switching, repair-deficient strains carrying the rad52 or the rad3 mutation were constructed and tested for spontaneous and induced heterothallic switching frequencies. The wild type RAD52 function is essential for spontaneous and induced switching as well as for the intrachromosomal crossing over which produces a deleted ring chromosome III. The rad3 mutation had almost no influence on spontaneous or X-ray induced switching, but it does reduce induction by ultraviolet radiation. The data are interpreted to indicate that heterothallic switching is accomplished via recombinogenic repair, perhaps of a double-strand break. The conversion event as well as the crossing over event leading to a change in mating type are equally affected by the rad52 mutation and therefore perhaps associated.
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  • 32
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    Molecular genetics and genomics 204 (1986), S. 310-316 
    ISSN: 1617-4623
    Keywords: Gene replacement ; PGI1 deletion ; Glucose-6-P ; Glucose dependence ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The structural gene PG11 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PG11 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PG11 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1Δ haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1Δ strains did not grow in glucose as sole carbon source. On the other hand pgi1Δ/pgi1Δ diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1Δ mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that (a) the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, (b) glucose-6-P is essential in yeasts, and (c) the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.
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  • 33
    ISSN: 1617-4623
    Keywords: Centromere ; In vitro mutagenesis ; Mitosis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Centromeres most likely consist of DNA (CEN DNA) interacting with specific proteins. In Saccharomyces cerevisiae a clear picture has emerged of a 120 bp sequence that is characteristic of CEN DNA. We have investigated the 25 bp centromere DNA element (CDEIII) that represents the right part of a CEN DNA. We showed using a series of mutants generated in vitro that the right most triple A of the consensus sequence TGT.T.TG.. TTCCGAA.....AAA participates in the assembly of a functional centromere and that no further sequences to the right are needed. Distance changes between the centre dyad TTCCGAA and the triple A have two effects: Addition of one base pair leads to a reduction, and addition of two or four base pairs to a loss of centromere function implying a participation of the centre dyad and the triple A region in protein binding. Indeed, a synthetic ologonucleotide of 39 bp containing CDEIII shows specific protein binding.
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  • 34
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    Journal of industrial microbiology and biotechnology 1 (1986), S. 187-193 
    ISSN: 1476-5535
    Keywords: Saccharomyces cerevisiae ; Cytochromec ; Superoxide dismutase ; Catalase ; Oxyradicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Two strains ofSaccharomyces cerevisiae were used to study the synthesis of superoxide dismutase. One strain (cytochromec-deficient) contained 5–10% of the normal amounts of total cytochromec, while the other strain was a wild type. The cytochromec-deficient mutant had lower specific growth rate, growth yield, and oxygen uptake than the wild type. The superoxide dismutase and catalase activities, in both strains, were significantly lower under anaerobic than under aerobic conditions. Furthermore, under aerobic conditions the mutant contained higher levels of superoxide dismutase than the wild type which may be attributed to the higher intracellular flux of superoxide radicals caused by the cytochromec deficiency. The mutant also showed a lower level of catalase which was due to glucose repression.
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