Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe

Summary Mutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, K_T, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher K_T for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.I.B.M.C., C.N.R.S., 15, rue René Descartes, F-67000 Strasbourg, France     

Cloning,sequencing and analysis of the yeastS. uvarum ERG10 gene encoding acetoacetyl CoA thiolase

Summary TheERG10 gene specific toS. uvarum, a brewing yeast, has been cloned by complementation of anS. cerevisiae erg10 mutant.S. uvarum contains two differentERG10 genes. One of these is similar to theS. cerevisiae ERG10 gene; they are structurally different, but functionally homologous. The clonedERG10 gene has been located on chromosome XVI, and we have shown that it is allelic to the previously isolatedtsm0115 mutants. Northern blot and sequence analysis indicate that theERG10 gene is highly expressed, and biochemical and genetic evidence show that it encodes the cytoplasmic acetoacetyl CoA thiolase.     

Cloning of the thymidylate synthetase gene (thyPIG 3) from theBacillus subtilis temperate phage IG 3

Summary ThethyPIG 3 gene fromBacillus subtilis bacteriophage IG 3 was cloned in the plasmid pHV 33. Two recombinant plasmids, pISL 61 and pISL 62 carrying that gene are effective in transforming to thymine prototrophy bothEscherichia coli (by complementation) andB. subtilis (by complementation and recombination). The comparison of cloned fragment containing thethyPIG 3 gene and thethyP 3 gene from phage 3 T, by restriction analysis and DNA hybridization, suggests a strong homology between the two. ThethyPIG 3 gene was mapped in this study in the central region of the IG 3 genome.     

The ogd1 and kgd1 mutants lacking 2-oxoglutarate dehydrogenase activity in yeast are allelic and can be differentiated by the cloned amber suppressor

The activity of mitochondrial 2-oxoglutarate dehydrogenase in S. cerevisiae can be impaired either by the ogd1 or the kgd1 mutation. The OGD1 gene and two suppressor genes were isolated by complementation of the ogd1 mutant. The complementation of the kdg1 mutant by the OGD1 gene, an allelism test, and meiotic mapping, revealed that the ogd1 and kgd1 mutations are allelic. The two mutations were differentiated by the cloned suppressor gene which was able to partially complement ogd1, but not kgd1. The molecular analysis of the suppressor gene revealed its identity with the natural tRNA _CAG ^Gln gene found in the upstream region of URA10.     

Basidiomycetousras cDNA functionally replaces its homolog genes in yeast

It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomyceteLentinus edodes (namedLeras cDNA) can functionally replace its homolog genes (ScRAS1 andScRAS2) in the yeastSaccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of bothRAS genes. The strain replaced by aLeras–cDNA-carrying plasmid, however, grew slower than the strains replaced by aScRAS1– or aScRAS2–carrying plasmid. The intracellular level of cAMP in the strain harboring theLeras–cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying theS. cerevisiae cAMP-dependent protein kinase catalytic subunit C₁ gene,TPK1, but was lower than that in a strain harboring anScRAS2–carrying plasmid. These results suggest that theLeras cDNA can complement theras1 ^– ras2^– mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing theScRAS2 gene.     

Identification of a lys2 mutant of Candida maltosa by means of transformation

Summary We have isolated five mutants of Candida maltos, which lack the 2-aminoadipate reductase activity, an enzyme involved in the lysine biosynthesis. By means of complementation analysis using protoplast fusion, the isolated mutants were divided into two complementation groups. Thereof the C. maltosa strain G457 could be transformed by the plasmids pDP12 and pDP13, which contain the L YS2-coding gene of Saccharomyces cerevisiae. On the basis of our presented results obtained by studies on hybridization, stability, and recovery of plasmids from C. maltosa transformants, we suggest that transformation does proceed integratively.Abbreviations AA 2-aminoadipate - PRA phosphoribosylanthranilate     

Cloning and molecular analysis of theIsi1(rfaF) gene ofNeisseria meningitidiswhich encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells

Neisseria meningitidis, but notHaemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. TheIsi1gene, which had been shown to be involved in LPS biosynthesis ofNeisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology withIsi1ofN. gonorrhoeaeand therfaFgene ofSalmonella typhimuriumwhich encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the clonedIsi1gene, as a functionalrfaFhomologue, was confirmed by the complementation of aS. typhimurium rfaFmutant using a P22 phage sensitivity test. AnIsi1mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type,Isi1, andgalEstrains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galEmutant) or the majority (Isimutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.     

Differences in the regulation of aldehyde dehydrogenase genes inAspergillus niger andAspergillus nidulans

Summary In order to study mechanisms of gene regulation inA. niger, and to compare these to similar systems inA. nidulans, a gene encoding an aldehyde dehydrogenase enzyme has been cloned. In wild-type strains ofA. niger the gene shows expression which is regulated by induction and repression. Levels of induction by various compounds and the extent of repression under various growth conditions differs from that seen for theA. nidulans aldA gene. Unlike the A.nidulans aldA gene, theA. niger gene has both carbon catabolite repressible and nonrepressible induction mechanisms. Studies of heterologous expression of theA. niger gene inA. nidulans have shown that its expression is regulated by thealcR gene product ofA. nidulans.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase

Summary The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.     

Chromium sensitive mutants of the yeast Saccharomyces cerevisiae

Summary Ten mutants of Saccharomyces cerevisiae sensitive to CrO₃ were obtained and genetically analyzed. All of them were recessive. Segregation of the sensitivity after crossing them with a wild type strain indicated that the sensitivity of some mutants was determined by single mutations and that of others was caused by multiple mutations. Among 6 mutations found in mutants having single mutations responsible for the sensitivity, 6 loci were distinguished by complementation. One of them was identified as LYS7, the gene previously known as the structure gene for homocitric dehydrase, in terms of co-segregation and co-reversion of chromium sensitivity and lysine dependency which did not complement an authentic lys7 mutation.     

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.     

Molecular cloning of the PEL1 gene of Saccharomyces cerevisiae that is essential for the viability of petite mutants

The PEL1 gene of Saccharomyces cerevisiae is essential for the cell viability of mitochondrial petite mutants, for the ability to utilize glycerol and ethanol on synthetic medium, and for cell growth at higher temperatures. By tetrad analysis the gene was assigned to chromosome III, centromere proximal of LEU2. The PEL1 gene has been isolated and cloned by the complementation of a pel1 mutation. The molecular analysis of the chromosomal insert carrying PEL1 revealed that this gene corresponds to the YCL4W open reading frame on the complete DNA sequence of chromosome III. The putative Pel1 protein is characterized by a low molecular weight of approximately 17 kDa, a low codon adaptation index, and a high leucine content.     

Molecular cloning of the ARG7 gene of Schizosaccharomyces pombe encoding argininosuccinate lyase

Summary A gene bank of Sau3A partially restricted Schizosaccharomyces pombe DNA in YEp13 was used to transform an arg4 mutant of Saccharomyces cerevisiae. One colony was recovered which contained the YEp13 plasmid bearing a large insert complementing the argininosuccinate lyase (ASL) mutation. As shown by restriction mapping and subcloning experiments, the DNA sequence required for complementation is localized on a 2 kb BamHI-BamHI fragment. The plasmid complemented several S. cerevisiae arg4 mutants of independent origin and a S. pombe arg7 mutant lacking ASL. Low but significant ASL activities were detected in crude extracts of these transformants. No complementation of the E. coli argH mutant was observed. Southern blot hybridizations showed that the insert originates from the S. pombe genome. No cross-hybridization was found between this sequence and S. cerevisiae DNA. It can be concluded that the cloned DNA fragment bears the S. pombe ARG7 gene coding for ASL.Abbreviations ASL argininosuccinate lyase - bp base pair - kb kilobase pair     

Evidence for the control of a mutation in lysine catabolism by the mating type in Yarrowia lipolytica

Summary Reversions of a mutation lye 1.5 blocking the first step of the lysine catabolic pathway were isolated. These reversions mapped inside the LYC1 locus. They exhibited an alteration of the regulation of the lysine pool after nitrogen and phosphorus starvation. This phenomenon did not appear in sexual diploids homozygous for the reversion. Diploids homozygous for the mating type were constructed by protoplast fusion. They displayed a pattern similar to that of the haploids. We conclude that the expression of this mutation is under the control of the physiological state of the mating type.Preliminary accounts were presented at the We International Congress of Genetics (New Delhi, December 1983)     

The primary structure of the leul + gene of Schizosaccharomyces pombe

Summary A DNA fragment which carries the leul gene encoding beta-isopropylmalate dehydrogenase in Schizosaccharomyces pombe has been isolated by complementation of an E. coli leuB mutation. This 1.5 kb DNA fragment complements not only the S. pombe leul mutation, but also the S. cerevisiae leu2 mutation. The nucleotide sequence of the essential part of the leul gene and its flanking regions was determined. This sequence contains an open reading frame of 371 codons, from which a protein having a Mr = 39,732 can be predicted. The deduced amino acid sequence and its codon usage were compared with those of the S. cerevisiae LEU2 protein. The cloned DNA will be a useful marker when transforming S pombe.     

Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, -aminoadipate reductase. The LYS5 gene of S. cerevisiae was cloned by functional complementation of a lys5 mutant, X4004-3A, using a YEp24 plasmid library. The cloned LYS5 gene was contained within a 7.5 kb DNA insert of the recombinant plasmid pSC5. Cloning of LYS5 gene was confirmed by second cycle transformation of a lys5 mutant with the pSC5 plasmid, growth response studies, and plasmid loss experiments with Lys5 ⁺ transformants. Analysis of restriction digests of the pSC5 plasmid revealed 3 EcoRI, 5 PvuII, 1 PstI, 1 BglII and 2 HpaI sites in the 7.5 kb insert. A 3.9 kb internal pSC5 fragment hybridized only to the plasmid pSC5, but no homology was observed with LYS2 DNA or the YEp24 vector. The pSC5 transformed Lys5 ⁺ cells and the wild-type strain exhibited same level of -aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2 ⁺ transformants consistently had five times greater -aminoadipate reductase activity when compared with the wildtype and the Lys5 ⁺ transformant. The -aminoadipate reductase activity was repressed in lysine-grown wildtype and Lys5 ⁺ transformed cells but not in Lys2 ⁺ transformed cells. A Lys2 ⁺ and Lys5 ⁺ double transformant exhibited higher a-aminoadipate reductase activity than lys2 ⁺ or lys5 ⁺ transformant.