High efficiency transformation of Kluyveromyces marxianus by a replicative plasmid

Kluyveromyces marxianus can be transformed with an efficiency of 10⁵ transformants/g of DNA by a replicative plasmid using electroporation. In order to obtain this efficiency, we isolated ura^- mutants cells which can be complemented by the URA3 gene from Saccharomyces cerevisiae. The URA3 gene and KARS2, a replicative origin from Kluyveromyces lactis which functions in K. marxianus, were ligated together in a plasmid which can be used as a vector to transform this strain.     

Specific gene probes as tools in yeast taxonomy

Summary The genePDC1 ofSaccharomyces cerevisiae coding for pyruvate decarboxylase (E.C. 4.1.1.1.) was used as a hybridization probe to detect gene sequence homologies in different strains ofSaccharomyces cerevisiae and in other yeast species. Additionally six other genes coding for glycolytic enzymes as well as the genesURA3 of the pyrimidine synthetic andTRP1 of the tryptophan synthetic pathways were used. A restriction polymorphism for the BamH1 fragments carrying thePDC1 gene was evident within the speciesSaccharomyces cerevisiae. All strains definitely declared asSaccharomyces cerevisiae showed the same restriction patterns. Hybridizations of different intensities were observed only with species in the familySaccharomycetaceae. Hybridizations with different genes showed different degrees of conservation for certain DNA sequences.     

As in Saccharomyces cerevisiae,aspartate transcarbamoylase is assembled on a multifunctional protein including a dihydroorotase-like cryptic domain in Schizosaccharomyces pombe

The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Cloning and sequencing of URA10, a second gene encoding orotate phosphoribosyl transferase in Saccharomyces cerevisiae

Summary Orotate phosphoribosyl transferase (OPRTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.     

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP

Summary Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5 monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup ^-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.     

A genetic analysis of an alpha-amylase super-secretor in yeast; implications for the regulatory pathway

Summary Extracellular glucoamylase activity was increased by a gene, which is present in super-secretor, but absent in low-secretor, strains of the yeast Saccharomyces cerevisiae. Genetic data indicated that this super-secretor gene is linked to the STA3 structural gene for glucoamylase. This gene appears to act specifically since it increased the secretion of glucoamylase but not of other secreted enzymes like acid phosphatase and invertase.     

Two new multi-purpose multicopy Schizosaccharomyces pombe shuttle vectors,pSP1 and pSP2

Plasmids pSP1 and pSP2 are two new Schizosaccharomyces pombe ars1 multicopy vectors with the Saccharomyces cerevisiae LEU2 and URA3 genes as selectable markers. They are derivatives of S. cerevisiae integrative plasmids. These plasmids allow classical molecular genetic techniques, such as mutagenesis, nested deletions and sequencing, to be performed directly.     

Reciprocal mitotic recombination is the predominant mechanism for the loss of a heterozygous gene in saccharomyces cerevisiae

The loss of a functional copy of a heterozygous tumor suppressor gene represents an important step during neoplastic transformation. In order to learn more about the genetic events that lead to spontaneous and drug-induced loss of heterozygosity, a diploid Saccharomyces cerevisiae strain was constructed that allows the detection of the loss of a heterozygous gene by means of direct selection. The strain contains a single functional URA3 gene copy inserted at the ADE2 locus located on the right arm of chromosome 15. In addition, the chromosome contains two other phenotypic marker genes, HIS3 which is located distal from URA3, and PHO80 which is closely linked to the centromere. The homologous chromosome lacks all three marker genes. Loss of the heterozygous copy of URA3 can easily be detected by 5-fluoro-orotic acid resistance of the resulting clones. Simple phenotypic tests of the resistant clones further allows one to distinguish whether the loss of the URA3 gene copy occurred by crossing over, chromosomal loss, or point mutation and gene conversion. Loss of heterozygosity was found to be induced in a dose-dependent fashion by UV radiation and by several chemical agents. All the tested mutagens induced loss of heterozygosity predominantly by crossing over. © 1994 Wiley-Liss, Inc.     

Induction of heterothallic strains and their genetic and physiological characterization in a homothallic strain of the yeast Saccharomyces exiguus

Summary We isolated heterothallic strains from a homothallic strain of S. exiguus by mutagenization with UV or ethylmethanesulfonate (EMS). A gene, not linked to the mating-type locus, was found to control homothallism in the yeast, as in S. cerevisiae. Pheromone of S. exiguus (^se pheromone) induced formation of large pear-shaped cells (shmooing) in a strains of S. exiguus, S. cerevisiae, and S. kluyveri, and sexual agglutinability of an inducible a strain of S. cerevisiae. ^se Pheromone is a peptidyl substance a little different from pheromone of S. cerevisiae. a Pheromone of S. exiguus acts only on a cells of S. exiguus. Contrary to the above results, neither sexual agglutination nor zygote formation occurred among these three Saccharomyces yeasts.     

Isolation of a Chlamydomonas reinhardtii telomer by functional complementation in yeast

We attempted to determine whether Chlamydomonas reinhardtii telomeres, which do not form G-quartet structures readily in vitro, are able to nucleate telomere addition in Saccharomyces cerevisiae. Restricted C. reinhardtii genomic DNA was ligated to a linear S. cerevisiae vector lacking a telomere. A C. reinhardtii telomere ligated to this unprotected end allowed vector replication as a linear DNA molecule in S. cerevisiae. DNA sequencing revealed common [T₄AG₃]_n and variant T₆AG₃ and T₅AG₃ C. reinhardtii telomere repeats capped by S. cerevisiae telomere repeat units. The recognition of a C. reinhardtii telomere by the telomere maintenance machinery of S. cerevisiae is consistent with a common theme for telomere structure in organisms with divergent telomere repeats.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

Use of sulfite resistance in Saccharomyces cerevisiae as a dominant selectable marker

Two S. cerevisiae genes were found to exhibit dominant phenotypes useful for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZF1-4, which confers sulfite resistance, was originally isolated and identified as RSU1-4, but the two genes are shown here to be allelic. Cysteine 57 in wild-type Fzf1p was found to be replaced by tyrosine in Fzf1-4p. Multicopy SSU1, which also confers sulfite resistance, was found to be somewhat less efficient. In both cases, a period of outgrowth in non-selective medium following transformation was found to be necessary. The number of transformants obtained was found to be strain-dependent, and also to depend on the sulfite concentration used during selection. Undesirable background growth of non-transformants was not observed at cell densities as high as 2.5 × 10⁷/plate. In two ura3 laboratory strains where selection for URA3 was applied independently of that for sulfite, the transformation efficiency for sulfite resistance was about 50% that for uracil prototrophy. Received: 1 July / 9 August 1999     

Localization of yeast glucoamylase genes by PFGE and OFAGE

Summary Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.     

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.     

Chromosomal polymorphism of the yeast Yarrowia lipolytica and related species: electrophoretic karyotyping and hybridization with cloned genes

Significant differences in electrophoretic karyotyping patterns were found among 27 strains of Y. lipolytica. Twenty-one of these strains were classified into four groups of similar karyotypes while six strains showed unique karyotypes. Chromosomal DNAs of different strains were hybridized with cloned genes of Y. lipolytica (URA3, LEU2, ARS18 and ARS68), which revealed four different bands in most strains. We conclude that the haploid chromosome number of Y. lipolytica is at least four, and possibly five or six. Electrophoretic karyotyping and hybridization with cloned genes of Y. lipolytica provided evidence of a large divergence between Y. lipolytica and related species of Saccharomycopsis, Endomycopsella and Endomyces.     

A genetic analysis of glucoamylase activity in the diastatic yeast Saccharomyces cerevisiae NCYC 625

Summary The wild diastatic yeast Saccharomyces cerevisiae NCYC 625 has been shown to be homozygous for the glucoamylase-specifying gene STA2. spoII-1-mapping has positioned STA2 on chromosome II. Expression of STA2 is suppressed in some but not all diploids capable of sporulation, and is also inhibited by unlinked nuclear suppressor genes (SGL) found in some S. cerevisiae tester strains. EMS-induced glucoamylase-negative mutants often contain STA2-suppressor mutations. Depending on the allelic status of GEP1, a nuclear gene which also appears able to antagonise SGL-mediated suppression, STA2 expression can be blocked in petite mutants.     

Virus-like particles from the yeast Yarrowia lipolytica

Summary Four out of the 24 strains of the yeast Yarrowia lipolytica we have checked for the presence of virus-like particles (VLPs) proved to contain encapsidated double-stranded RNA (dsRNA) molecules, 4.9 kb long. A major VLP polypeptide of MW 80,000 was observed in all 4 cases, and a second one of MW 77,000 in three cases. dsRNA from the VLPs harboring only the larger polypeptide showed little homology with the 3 others. We have found no homology between VLP dsRNAs and host DNA or dsRNAs from Saccharomyces cerevisiae, and no relationship between the presence of VLPs and a possible killer phenomenon in Y. lipolytica.     

Conversion of homothallic yeast to heterothallism through HO gene disruption

A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho::neo disrupted allele was constructed by cloning a dominant selectable marker, the bacterial neo gene, within the HO gene. Transformation of a homothallic diploid S. cerevisiae strain with plasmid DNA containing the ho::neo allele yielded G418-resistant yeast transformants in which one of the HO alleles was replaced by the disrupted ho::neo allele. Meiotic tetrad analysis of four-spored asci from these G418-resistant transformants gave rise to haploid heterothallic and diploid homothallic tetrad progeny. The presence of the ho::neo and HO alleles in the heterothallic and homothallic progeny was confirmed by Souther-blot analysis.