Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases

Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition. Received: 25 January / 15 March 1999     

Heterologous expression of human 5-aminolevulinate dehydratase in Saccharomyces cerevisiae

Summary A cDNA coding for human 5-aminolevulinate dehydratase was placed in a yeast expression vector under the control of the GAL10 promoter. The resulting multicopy plasmid was then used to transform a yeast mutant which contains a defective hem2 gene coding for 5-aminolevulinate dehydratase. Expression of the human cDNA was shown in four ways: (1) restoration of normal growth on glycerol/galactose as primary carbon source, (2) decrease in intracellular 5-aminolevulinic acid concentration, (3) restoration of cytochrome biosynthesis and (4) direct, in situ assay of 5-aminolevulinic acid dehydratase. Curing transformed cells of plasmid restored the hem2 mutant phenotype. This heterologous system could be used to produce large quantities of human 5-aminolevulinic acid dehydratase for physical and biochemical studies.     

Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae

Using a gene bank of C. albicans, the lysine-permease deficiency in a strain of S. cerevisiae was complemented, and the restriction map of the corresponding C. albicans DNA fragment was constructed. Its expression in S. cerevisiae showed that the permease of C. albicans actively transports arginine (K_T=18 mol/l, J_max=26 nmol/min per mg dry weight), lysine (K_T=12 mol/l, J_max=18 nmol/min per mg dry weight), histidine (K_T=37 mol/l, J_max=9.7 nmol/min per mg dry weight), as well as their toxic analogues canavanine and thialysine, with high affinity. The intracellular concentration of basic amino acids transported into S. cerevisiae by the C. albicans permease reaches more than a thousand-times-higher value compared to the external concentration in the medium. Accumulated amino acids do not leave the cells. The uptake is strongly reduced by the protonophores and inhibitors of plasma membrane H⁺-ATPase.     

The REV2 gene of Saccharomyces cerevisiae: cloning and DNA sequence

Summary The REV2 gene of Saccharomyces cerevisiae was cloned and sequenced; it contains an open reading frame of 1985 bp with a coding potential of 662 amino acids. Interruption of the chromosomal REV2 gene by integrating the URA3 gene coupled with partial deletion of the 3 terminal region produced viable haploid rev2 mutants. This indicates that the REV2 gene is non-essential for growth. The rev2 mutant is slightly more UV-sensitive than strains carrying various rev2 alleles (rev2-1, rev2x, rad5-1, rad5-8). The putative Rev2 protein is probably a globular protein containing a highly conserved nucleotide-binding site and two zinc-finger domains.     

Interspecific protoplast fusion between the yeasts Saccharomyces cerevisiae and Saccharomyces fermentati

Summary Protoplasts of Saccharomyces cerevisiae his1 trp2 resistant to acriflavine and able to ferment galactose and of Saccharomyces fennentati arg resistant to DL-p-fluorophenylalanine and able to ferment lactose were fused. As a result of fusion two types of prototrophic hybrids were obtained. Type 1 hybrids were able to grow on medium with galactose or lactose as sole carbon source and were sensitive to acriflavine and resistant to DL-p-pfluorophenylalanine. Type 2 hybrids were able to grow on medium with galactose as sole carbon source and were resistant to acriflavine and sensitive to DL-p-fluorophenylalanine.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.     

Multiple allelic states of the cyh2 gene cause low- and high-level cycloheximide resistance in Saccharomyces cerevisiae

Summary At least four different mutations at the cyh2 locus (rp1X; gene product: YL24) of Saccharomyces cerevisiae confer cycloheximide resistance. The mutant YL24 proteins are either more basic (high-level resistant phenotype), more acidic (low-level resistant phenotype), or unchanged in their electrophoretic mobility (both low-and high-level resistant phenotypes). None of the mutations at other loci seem to induce high-level resistance to cycloheximide.     

Time-dependent mitotic recombination in Saccharomyces cerevisiae

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.     

Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in Saccharomyces cerevisiae

A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-β-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1 _S ). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae. Received: 1 March 1996 / 9 April 1996     

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene

Summary Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc⁺ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc⁺ colonies by UV irradiation. It is postulated that these UV-induced cdc⁺ colonies arise as the result infidelity in DNA replication.     

Identification and functional analysis of a Kluyveromyces lactis homologue of the SPT4 gene of Saccharomyces cerevisiae

Sequence analysis of a DNA fragment containing the KlCOX18 gene originating from chromosome II of the yeast Kluyveromyces lactis revealed the presence of an adjacent open reading frame (ORF) for a protein exhibiting 78.4% identity with the Saccharomyces cerevisiae Spt4p. Based on the identical length (102 aa) and the conservation of the zinc-finger motif found in Spt4p we named this ORF KlSPT4. When expressed in S. cerevisiae the KlSPT4 gene complemented all spt4 mutant phenotypes. It is proposed that KlSpt4p, like its S. cerevisiae counterpart is a protein involved in the establishment or maintenance of the chromatin structure that influences the expression of many yeast genes. Received: 15 June / 31 August 1998     

Molecular characterisation of GTP1, a Saccharomyces cerevisiae gene encoding a small GTP-binding protein

DNA sequence analysis upstream of the yeast DNA repair gene SNM1 revealed gene GTP1 with an ORF of 573 bp on chromosome XIII. The putative amino-acid sequence of the encoded protein shows homology to proteins of the ARF-class of small GTP-binding proteins. Homology within GTP-binding motifs is highly conserved. Gene disruption showed that GTP1 is not an essential gene and that it has no influence on the expression of the DNA repair gene SNM1 with which it shares a 191-bp promoter region.     

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

Summary We have developed a system for assaying pyrimidine dimers in the 2 m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m^–2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.     

Newly synthesised DNA of high molecular weight in the yeast Saccharomyces cerevisiae

Summary Two species of newly synthesised DNA larger than average replicons have been found in yeast. Their molecular weights are 60 million and 90 million daltons respectively. The exact nature of these molecules is not certain. They may represent entirely novel species of cellular DNA or they could be concatameric replication intermediates of some particular fraction of DNA, such as mitochondrial DNA or rDNA. Alternatively they could result from the fusion of adjacent completed replicons in a small cluster.     

Mitotic hyperploidy for chromosomes VIII and III in Saccharomyces cerevisiae

Summary The arg4–8 and cup1 ^s markers comprise a copy-number-dependent signal device in the yeast Saccharomyces cerevisiae. These alleles permit reliable discrimination between euploid and disomic haploids as well as between euploid and trisomic diploids. To investigate and compare inherent inter-chromosomal differences as regards propensity for hyperploidy, we transplaced arg4–8 and cup1 ^s by deleting them from chromosome VIII and then re-introducing them at the leu2 locus on chromosome III. The rate of chromosome gain was significantly greater for the chromosome III construct compared to the native chromosome VIII, in both diploid and haploid strains. In addition, more coincident aneuploidy for other chromosomes was found among chromosome VIII hyperploids compared to chromosome III hyperploids.     

Instability of Saccharomyces cerevisiae heterokaryons

Summary We have constructed heterokaryons of Saccharomyces cerevisiae by crossing kar1 — mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from kar1 — crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable.This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability.