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  • 1
    ISSN: 1432-0983
    Keywords: Homoeologous recombination ; Ras ; SDC25 ; CDC25
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity. A chimera between the 3′ regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal CDC25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the CDC25 and the SDC25 gene products are structurally and functionally similar. The CDC25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.
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  • 2
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; Bud site selection ; Guanine exchange factor ; Ras
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.
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  • 3
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5′-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5′-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5′-phosphate carboxy-lyase and orotate phosphoribosyl transferase.
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  • 4
    ISSN: 1617-4623
    Keywords: Dictyostelium discoideum ; Gene disruption ; 5-Fluoroorotic acid ; UMP synthase ; Uracil auxotrophy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5′-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase− mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura − phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have analysed the contribution of the Msn2/4 transcription factors and the Ras-cAMP-proteine kinase A (PKA) pathway to the control of the yeast H2O2 response. Strains deleted for MSN2 and MSN4 are hypersensitive to H2O2, although they can still adapt to this oxidant. They are also unable to induce 27 proteins of the H2O2 stimulon as shown by quantitative two-dimensional gel analysis. This peculiar H2O2 tolerance defect, the nature of the proteins of the Msn2/4 regulon, and the partial overlap of this regulon with the Yap1 H2O2-response regulon, suggest an independent and distinctive role of these two H2O2 stress response pathways. A strain lacking PDE2, and therefore carrying high intracellular cAMP levels, is also hypersensitive to H2O2. In the presence of exogenous cAMP, this strain does not induce the entire H2O2 Msn2/4 regulon and some other proteins. This, and the normal H2O2 induction of a gene reporter under control of the Yap1 regulator when intracellular cAMP level are high, demonstrate that the Ras-cAMP pathway negatively affects the H2O2 stress response through Msn2/4. However, the high H2O2 sensitivity of a strain lacking the PKA-negative regulatory subunit Bcy1, is not only the consequence of the inhibition of Msn2/4 but also of Yap1 through a yet undefined mechanism.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [35S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25°C to 38°C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.
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  • 7
    ISSN: 0749-503X
    Keywords: Cyclic AMP ; nitrogen limitation ; resting state ; cell cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have identified a mutation called rcal (for rescue by cAMP) which allows adenylate cyclase-deficient mutants to divide in the presence of cAMP. We took advantage of this rcal mutation to study the effect of externally added cAMP on the onset of the resting state when cells are starved for ammonium. We measured the resistance of the cells to zymolyase treatment as a parameter of the resting state. We observed that the onset of the resting state is reversibly blocked by cAMP. This inhibitory effect of cAMP is discussed together with the cAMP control of the start. This leads us to propose a model in which the cAMP level, controlled by the availability of nutrients, should trigger the choice between the entry of the cell into the resting state and the initiation of a new division cycle.
    Additional Material: 3 Ill.
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