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  • 1
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The occurrence of “up promoter” mutations appears to be a general feature of eukaryotic gene regulation. Such mutations have been described in organisms as different as Yeast, Aspergillus, and Drosophila (Douglas and Hawthorne, 1966; Arst and Scazzochio, 1975; Hynes, 1975; Ciriacy, 1976; Chovnick et al., 1976), yet little is known about the underlying regulatory mechanisms. Some years ago, Clavilier et al. (Clavilier et al., 1969) discovered in S. cerevisiae regulatory mutations enhancing the synthesis of iso-2 cytochrome c. One of these mutations was found to entail a secondary phenotype prohibiting growth on a glucose medium containing high levels of chloramphenicol (Péré-Aubert, 1970). In this paper evidence is presented that: 1. This regulatory mutation, CYP3-15, is linked to the structural gene of iso-2 cytochrome c, is cis dominant, and therefore qualifies as an “up promoter” mutation of the iso-2 cytochrome c producing unit. 2. The secondary phenotype entailed by CYP3-15 never occurs in the absence of the main phenotype of iso-2 cytochrome c stimulation, and is a consequence of the extension into a nearby function of the genetic alteration corresponding to CYP3-15. This secondary phenotype is manifest whatever the genotype at the CYC1 locus, including in the presence of the wild type allele. 3. Despite being cis dominant, CYP3-15 is still receptive to the effect of three other independent regulatory mutations. One of these, which reduces both the iso-1 and iso-2 cytochrome c level, is a null allele of the CYP1 locus. This later locus was also originally identified by mutations which enhance, to various degrees, the amount of iso-2 cytochrome c produced by the cell (Clavilier et al., 1976).
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary CYP1 is a trans acting regulatory locus modulating both iso 1- and iso 2-cytochrome c synthesis. Genetical analysis of various mutated alleles has allowed us to identify the gene product as a positive regulatory element. The region of the target sequence of the CYP1 product on the iso 2-cytochrome c structural gene was located by molecular and genetic analysis of two cis acting mutations located at the CYP3 locus: CYP3-36 and CYP3-4, which have been shown to arise from the integration of TY1 elements near the promoter site. Determination of the amount of iso 2-cytochrome c synthesized by strains bearing various genetic constructions, in which the cis acting mutations were associated with different alleles of the CYP1 trans acting locus, showed that TY1 inserted into CYP3-36 extinguishes the activation function due to a mutated overproducer allele CYP1-18, while CYP3-4 amplifies this function. This result identifies at least a part of the target sequence of the CYP1 product within the region separating the two TY1 insertions. To clone the CYP1 gene, we took advantage of the iso 2-cytochrome c overproducer phenotype of the mutated allele CYP1-18, which confers a Lactate+ phenotype on an iso 1-cytochrome c-deficient strain. Such a phenotype allowed the isolation of a recombinant plasmid YEpJFM1 carrying the mutated allele, able to complement on lactate medium a lactate - recipient strain. The identity of the YEpJFM1 sequence with the chromosomal gene was confirmed by homologous recombination at the CYP1 locus.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 228 (1991), S. 300-306 
    ISSN: 1617-4623
    Keywords: Positive and negative regulation ; Cytochrome P450 ; Coproporphyrinogen oxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1 + and cyp1° deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme-sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; CYP1(HAP1) protein ; Electron transport ; Oxygen and heme regulation ; Trans regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under hemedeficient conditions is dependent upon DNA binding.
    Type of Medium: Electronic Resource
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