Molecular cloning, DNA structure, and RNA analysis of the arginase gene in Saccharomyces cerevisiae. A study of cis-dominant regulatory mutations.

The Saccharomyces cerevisiae gene cargA + or CAR1 , encoding arginase has been cloned by recovering function in transformed yeast cells. It was used to analyse RNA and chromosomal DNA from six strains bearing cis-dominant regulatory mutations leading to constitutive arginase synthesis. The DNA from the four cargA + O- strains in which constitutive arginase synthesis was independent of the mating-type functions showed no detectable differences with the wild- typye . The cargA + O- mutations were, therefore, small alterations, possibly single base substitutions. On the other hand, the cargA + Oh-1 and cargA + Oh-2 mutations, leading to a constitutive and mating-type dependent arginase synthesis, were identified as insertions. Their size and restriction pattern strongly suggested that they were induced by the Ty1 yeast transposable element. This was confirmed by cloning and analysis of the cargA + Oh-1 mutant gene. The concentration of arginase RNA was significantly increased in the mutants, indicating that the regulation of arginase synthesis was exerted, at least in part, at the level of RNA synthesis or stability. In the cargA + Oh-2 strain the Ty1 element was located at a distance of approximately 600 base pairs from the insertion present in the cargA + Oh-1 strain. This result suggests either a surprisingly large arginase regulatory region or an indirect influence of the Ty1 element on gene expression over long distances.     

The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance.

To precisely define the functional sequence of the CHO1 gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the original 4.5-kilobase (kb) CHO1 clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of cho1 mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression. Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHO1 gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opi1, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When cho1 mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in cho1 cells.     

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.     

Isolation and characterization of mutants that produce the allantoin-degrading enzymes constitutively in Saccharomyces cerevisiae.

Degradation of allantoin, allantoate, or urea by Saccharomyces cerevisiae requires the participation of four enzymes and four transport systems. Production of the four enzymes and one of the active transport systems is inducible; allophanate, the last intermediate of the pathway, functions as the inducer. The involvement of allophanate in the expression of five distinct genes suggested that they might be regulated by a common element. This suggestion is now supported by the isolation of a new class of mutants (dal80). Strains possessing lesions in the DAL80 locus produce the five inducible activities at high, constitutive levels. Comparable constitutive levels of activity were also observed in doubly mutant strains (durl dal80) which are unable to synthesize allophanate. This, with the observation that arginase activity remained at its uninduced, basal level in strains mutated at the DAL80 locus, eliminates internal induction as the basis for constitutive enzyme synthesis. Mutations in dal80 are recessive to wild-type alleles. The DAL80 locus has been located and is not linked to any of the structural genes of the allantoin pathway. Synthesis of the five enzymes produced constitutively in dal80-1-containing mutants remains normally sensitive to nitrogen repression even though the dal80-1 mutation is present. From these observations we conclude that production of the allantoin-degrading enzymes is regulated by the DAL80 gene product and that induction and repression of enzyme synthesis can be cleanly separated mutationally.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site.

URS1 is known to be a repressor binding site in Saccharomyces cerevisiae that negatively regulates expression of many genes including CAR1 (arginase), several required for sporulation, mating type switching, inositol metabolism, and oxidative carbon metabolism. In addition to the proteins previously shown to directly bind to the URS1 site, we show here that the UME6 gene product is required for URS1 to mediate repression of gene expression in the absence of inducer. We also show that mutations in the CAR80 (CARGRI) gene are allelic to those in UME6.     

Expression of the ROAM mutations in Saccharomyces cerevisiae: involvement of trans-acting regulatory elements and relation with the Ty1 transcription

The regulatory mutations in Saccharomyces cerevisiae designated cargA + Oh, cargB + Oh, and durOh are alterations in the control regions of the respective structural genes. The alteration causing the cargA + Oh mutation has been shown to be an insertion of a Ty1 element in the 5' noncoding region of the CAR1 ( cargA ) locus. All three mutations cause overproduction of their corresponding gene products and belong to the ROAM family of mutations (Regulated Overproducing Allele responding to Mating signals) in yeast. The amount of overproduction in ROAM mutants is determined, at least in part, by signals that control mating functions in yeast. We report the identification of two genetic loci that regulate Oh mutant gene expression but that do not affect mating ability. These loci are defined by the recessive roc mutations ( ROAM mutation control) that reduce the amount of overproduction caused by the cargA + Oh, cargB + Oh, and durOh mutations. RNAs homologous to CAR1 ( cargA ), DUR1 ,2 and Ty1 DNA probes were analyzed by the Northern hybridization technique. In comparison with wild-type strains, cargA + Oh and durOh mutant strains grown on ammonia medium contain increased amounts of CAR1 and DUR1 ,2 RNA. This RNA overproduction is diminished in MATa/MAT alpha diploid strains as well as in haploid strains that also carry the ste7 mutation which prevents mating or that carry either of the roc1 or roc2 mutant alleles. The amount of RNA homologous to Ty1 DNA is also reduced in ste7 , roc1 , and roc2 mutant strains. This reduction is not observed in a strain with the ste5 mutation, which prevents mating but has no effect on overproduction of ROAM mutant gene products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of tryptophanase in Escherichia coli: Isolation and characterization of a structural-gene mutant and two regulatory mutants

Summary A mutant of E. coli has been isolated that is temperature-sensitive in respect of tryptophanase. When incubated at 60°C, cell-free extracts of the mutant suffer inactivation of enzyme activity much more rapidly than similar extracts of the wild type. After lysogeny with a specialized transducing phage carrying the wild-type tryptophanase gene, the mutant is able to synthesize tryptophanase that is wild-type in its response to treatment at 60°C. It is concluded that the mutation lies in the structural gene for the enzyme.Two further mutants have been isolated that synthesize tryptophanase constitutively. One mutation renders synthesis of the enzyme indifferent to the presence of inducer; the other mutation allows synthesis of the enzyme in the absence of inducer at about 35% of the fully induced wild-type rate. Neither mutation alleviates catabolite repression. Genetic mapping shows that the constitutive mutations lie very close to the structural-gene mutation, on the side of the structural gene distant from bglR.     

The regulation of hexokinase and phosphoglucomutase activity in Aspergillus nidulans.

Summary The levels of glucose-6-phosphate and 6-phosphogluconate dehydrogenase in wildtype cells of Aspergillus nidulans varied with the carbon and nitrogen source. In general, hexokinase activity did not vary with carbon or nitrogen source. The ammonium derepressed mutant amrA1 had only 50% of the wildtype level of hexokinase. Phosphoglucomutase activity was low in wildtype cells grown with nitrate, but high in cells grown with ammonium when glucose was the carbon source. A non-inducible mutant, nirA ^-1, in the regulatory gene for nitrate reductase, had high phosphoglucomutase activity when grown with nitrate or ammonium. A constitutive mutant nirA ^c1, in the regulatory gene for nitrate reductase had low phosphoglucomutase activity when grown with nitrate or ammonium. The mutants nir ^-1 and nirA ^c1 are recessive and semi-dominant respectively for abnormal phosphoglucomutase activity.