Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae

Summary The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated multifunctional since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively derepressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.     

Nitrogen metabolite repression in Aspergillus nidulans: A farewell to tamA?

Summary Previous work has established that nitrogen metabolite repression in Aspergillus nidulans is mediated by the positive acting regulatory gene areA. Pateman and Kinghorn (1977) proposed that the gene tamA plays an equally important regulatory role in nitrogen metabolite repression as the result of work with tamA^r-50, an allele leading to inability to utilise nitrogen sources other than ammonium, and tamA^d-1, an allele leading to nitrogen metabolite derepression. Both tamA^r-50 and tamA^d-1 were subsequently lost. We have therefore attempted to reconstruct Pateman and Kinghorn's work with tamA. We propose that tamA^r-50 was in fact a pyroB^– tamA^– double mutation. pyroB^– mutations lead to a block in vitamin B₆ biosynthesis which can be supplemented by extremely high concentrations of ammonium. tamA^– mutations, possibly as the result of a membrane alteration, reduce the concentration of ammonium required to supplement the pyroB^– auxotrophy. There is, however, no evidence that pyroB^– or tamA- mutations, alone or in combination, affect the regulation of the levels of a number of enzymes subject to nitrogen metabolite repression. Reversion of pyroB^– strains constitutes a powerful positive selection technique for obtaining a wide variety of mutations in glnA, the probable structural gene for glutamine synthetase. We suggest that the nitrogen metabolite derepressed phenotype attributed to tamA^d-1 might have resulted from an extremely leaky glnA^– mutation.     

Cloning,sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene

Summary The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5 and 3 gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragments has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The idenity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae ghd-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

Identification and characterization of four new GCD genes in Saccharomyces cerevisiae

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.     

High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.     

Bile acids and their amidates inhibit 11β-hydroxysteroid dehydrogenase obtained from rat kidney

Recently it has ben demonstrated that interaction of corticosteroids with extraadrenal target cells can effectively be modulated by metabolic transformation of the steroid hormone. As far as 11-hydroxylated glucocorticoids are concerned 11-hydroxysteroid dehydrogenase (11-HSD) is the most important enzyme charged with target cell metabolism. Inhibition of 11-HSD function either by genetically transmitted deficiency or by exogenous enzyme inhibitors causes severe pathophysiological derangements, which result in a syndrome of apparent mineralocorticoid excess. In the present paper we have tested whether or not endogenous inhibitors of this enzyme system might exist. The effects of the main naturally occurring mono-, di-, and trihydroxylated bile acids in man on 11-HSD have been studied in in vitro experiments. Using rat renal microsomes it could be demonstrated that unconjugated bile acids of all three classes as well as the corresponding glycine and taurine amidates effectively inhibit oxidative as well as reductive activity of 11-HSD, with lithocholic acid and chenodeoxycholic acid being the most potent compounds. It is concluded that bile acids are potent endogenous inhibitors of 11-HSD and, therefore, could participate in abnormalities of cortisol metabolism observed in liver cirrhosis and extrahepatic biliary obstruction and, possibly, after ingestion of bile acids.Abbreviations CA cholic acid - CDCA chenodeoxycholic acid - DCA deoxycholic acid - GCA glycocholic acid - GCDCA glycochenodeoxycholic acid - GLCA glycolithocholic acid - 11-HSD 11-hydroxysteroid dehydrogenase (EC 1.1.1.146) - IC₅₀ molar concentration of bile acid at 50% inhibition of enzyme activity - LCA lithocholic acid - TDCA taurodeoxycholic acid - TLCA taurolithocholic acid - TUDCA tauroursodeoxycholic acid - UDCA ursodeoxycholic acid Supported by the Deutsche Forschungsgemeinschaft Hi 97/16 1-4. Parts of this study have been presented at the 21st meeting of the Gesellschaft für Nephrologie [23]     

The recombinational hot spot mutation ade6-M26 of Schizosaccharomyces pombe stimulates recombination at sites in a nearby interval

Summary With the help of in vitro constructed intragenic double mutants, we investigated the influence of the recombinational hot spot mutation ade6-M26 on meiotic recombination between two additional ade6 mutations proximal to it. Recombination was stimulated four-fold when M26 was present in a heterozygous condition and ten-fold when homozygous. M26 itself remained unaffected in a substantial number of these events. This indicates that the stimulation can not only be due to a preferred conversion of M26 to wild-type with co-conversion of the second mutation in cis. A model is proposed in which M26 acts as an entry site for recombinational enzymes.     

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3 flanking regions, all genes present both the 3 box, a conserved sequence required for 3 processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis.     

Genetic mapping of two pairs of linked ribosomal protein genes in Saccharomyces cerevisiae

Summary We have used the 2 mapping method described by Falco and Botstein (1983) and tetrad analysis to map four ribosomal protein genes (two linked pairs) in S. cerevisiae. One pair (rp28–rp55 copy 1) is on chromosome XV, 14 cM proximal to ARG8. The other pair (rp55–rp28 copy 2) is 19 cM from the centromere on the left arm of chromosome XIV. To map copy 1 we used the E. coli -galactosidase gene rather than a yeast gene to mark the ribosomal protein chromosomal locus. This provided a more sensitive color screening assay for chromosome loss in the 2 method. It also removed the restriction that the mapping tester strains must be mutant for the plasmid marker.     

Starvation for His-tRNAHis in yeast causes translational arrest without a high level of misincorporation of glutamine at histidine codons

Summary The hts1.1 temperature-sensitive histidinyl-tRNA synthetase mutation enables Saccharomyces cerevisiae to be starved for His-tRNA^His by upshift to the non-permissive temperature of 38°C. If yeast behaves similarly to bacterial and mammalian cells, this lack of His-tRNA^His should greatly enhance misreading at histidine codons (CAU/CAC) by Gln-tRNA^Gln, resulting in substitution of the neutral amino acid glutamine in place of histidine, a basic amino acid. Such misreading causes the isoelectric point (pI) of proteins to shift to lower values, and is readily detectable as stuttering on two-dimensional (2D) protein gels. By gel analysis of pulse-labelled proteins of hts1.1 yeast cells that were overexpressing phosphoglycerate kinase (PGK), our study sought to detect this specific translational error in PGK protein. It was not detected by this relatively sensitive technique, indicating that missense errors due to glutamine insertion at histidine codons do not occur in yeast at the readily-detectable level found in bacterial and mammalian cells.     

Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1251 base pairs encoding approximately 47-kDA polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alkl encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis -galactosidase) was tested. Expression of P450alkl gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining -galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.