The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.     

The DAN1 gene of S. cerevisiae is regulated in parallel with the hypoxic genes,but by a different mechanism

The DAN1 gene is expressed under anaerobic conditions in yeast and completely repressed during aerobic growth. The function of the gene is unknown, and genetic disruption had no effect on fitness which could be detected, even upon prolonged anaerobic growth. Expression of DAN1 was constitutive in a heme-deficient strain, indicating that heme participates in repression. Expression was blocked by heme in anaerobic medium, suggesting that heme acts as a negative co-effector rather than through its metabolic functions, i.e., in the production of a co-effector. Expression of DAN1 was regulated in parallel with the hypoxic gene ANB1, showing identical kinetics of induction and dose response to heme. However, unlike ANB1, DAN1 is not regulated by the repressor of the hypoxic regulon, ROX1, as shown by observation of normal aerobic repression of DAN1 in a strain carrying a deletion of ROX1. These results indicate the existence of a parallel regulatory system which produces an identical response to oxygen by a different mechanism than that controlling the hypoxic regulon.     

Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae

The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.     

Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

Effects of null mutations in the hexokinase genes of Saccharomyces cerevisiae on catabolite repression.

Saccharomyces cerevisiae has two homologous hexokinases, I and II; they are 78% identical at the amino acid level. Either enzyme allows yeast cells to ferment fructose. Mutant strains without any hexokinase can still grow on glucose by using a third enzyme, glucokinase. Hexokinase II has been implicated in the control of catabolite repression in yeasts. We constructed null mutations in both hexokinase genes, HXK1 and HXK2, and studied their effect on the fermentation of fructose and on catabolite repression of three different genes in yeasts: SUC2, CYC1, and GAL10. The results indicate that hxk1 or hxk2 single null mutants can ferment fructose but that hxk1 hxk2 double mutants cannot. The hxk2 single mutant, as well as the double mutant, failed to show catabolite repression in all three systems, while the hxk1 null mutation had little or no effect on catabolite repression.     

Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene. Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present. The promoter element is located 5' to the URS in the ANB1 gene. Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity. The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function. A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.     

Multiple Elements and Auto-Repression Regulate Rox1, a Repressor of Hypoxic Genes in Saccharomyces Cerevisiae

The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.     

Identification of Reo1, a Gene Involved in Negative Regulation of Cox5b and Anb1 in Aerobically Grown Saccharomyces Cerevisiae

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.     

The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.

The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus.     

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.     

Regulation of nuclear genes encoding mitochondrial proteins in Saccharomyces cerevisiae.

Selection for mutants which release glucose repression of the CYB2 gene was used to identify genes which regulate repression of mitochondrial biogenesis. We have identified two of these as the previously described GRR1/CAT80 and ROX3 genes. Mutations in these genes not only release glucose repression of CYB2 but also generally release respiration of the mutants from glucose repression. In addition, both mutants are partially defective in CYB2 expression when grown on nonfermentable carbon sources, indicating a positive regulatory role as well. ROX3 was cloned by complementation of a glucose-inducible flocculating phenotype of an amber mutant and has been mapped as a new leftmost marker on chromosome 2. The ROX3 mutant has only a modest defect in glucose repression of GAL1 but is substantially compromised in galactose induction of GAL1 expression. This mutant also has increased SUC2 expression on nonrepressing carbon sources. We have also characterized the regulation of CYB2 in strains carrying null mutation in two other glucose repression genes, HXK2 and SSN6, and show that HXK2 is a negative regulator of CYB2, whereas SSN6 appears to be a positive effector of CYB2 expression.     

Isolation and characterization of respiratory-deficient mutants of Escherichia coli K-12.

Several mutants of Escherichia coli K-12 defective in aerobic metabolism were isolated. One such mutant was found to be deficient in cytochromes, heme, and catalase. Aerobically grown cells did not consume oxygen and could grow only on fermentable carbon sources. Supplementation of the growth medium with delta-aminolevulonic acid, protoporphyrin IX, or hemin did not restore aerobic metabolism. The lack of heme and catalase in mutant cells grown on glucose was not due to catabolite repression, since the addition of exogenous cyclic AMP did not restore the normal phenotype. When grown aerobically on complex medium containing glucose, the mutant produced lactic acid as the principal fermentation product. This pleotropic mutation was attributed to an inability of the cells to synthesize heme, and preliminary data mapped the mutation to between 8 and 13 min on the E. coli genome.     

Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.