Caffeine resistance of Saccharomyces cerevisiae

Four caffeine-resistant haploid isolates, two resistant to 50 mM caffeine and two resistant to 100 mM caffeine, were genetically analyzed. Complementation and tetrad analysis indicated that all four mutations are alleles of the same locus. All four isolates demonstrated incomplete dominance when hybridized to the wild-type strain and dominance of high to low resistance when hybridized to one another. Differences in caffeine resistance were found between wild-type grande cells and its petite derivative.     

成熟天花粉蛋白基因在酵母中的表达

本文利用DNA重组技术,将酵母α因子的启动子和信号序列与成熟天花粉蛋白基因融合,从而构建了天花粉蛋白的酵母表达载体。将该载体转入酵母细胞,转化子在选择培养基中培养24小时后,获得了高效表达。表达的天花粉蛋白位于细胞内。     

Expression of chicken beta-actin in Saccharomyces cerevisiae

Actin interacts with a number of so-called actin-binding proteins which participate at various stages of the cell motility process such as regulation of filament formation, assembly and disassembly of filaments, force generation and depolymerization. Gene technology makes a precise mapping of the interacting surfaces on the actin molecules possible by studying specifically designed actin mutants expressed in a suitable organism. In addition, the production of engineered actin will become increasingly important when the three-dimensional structure of actin is determined. Chicken beta-actin can be produced in large quantities in Escherichia coli but such actin shows only a limited biological activity and thus seems to be of minor interest in future studies of structure-function relationships of this molecule. To circumvent the problem of a denatured bacterial protein, the yeast Saccharomyces cerevisiae was chosen as an alternative organism to express actin. This paper describes the expression, isolation and characterization of the yeast-produced chicken beta-actin. From a 12-liter culture of yeast cells, 500 micrograms of polymerizable beta-actin was isolated.     

Expression of Ty-lacZ fusions in Saccharomyces cerevisiae

We have determined the nucleotide sequence of about 520 bp spanning the 5' delta regions (Figure 1) of two Tyl elements. There is an open reading frame running out of the deltas for at least 180 nucleotides into the internal region of each element. The functional significance of these open reading frames has been tested by fusing them to a defective E.coli lacZ gene. Expression of B-galactosidase in yeast transformants containing these fusions shows that Tyl elements contain functional translation signals.     

Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity.     

Spontaneous and induced non-specific drug resistance in Saccharomyces cerevisiae.

Nitrous acid, diepoxybutane and methyl methane sulfonate induce effectively non-mitochondrial chloramphenicol-resistant mutants cross-resistant to other drugs. HNO2 induces also unstable erythromycin resistant mutants. The ability of the mutants to grow on antibiotic media can be modified by detergents, guanidine hydrochloride or increased osmotic pressure of the medium. This suggests that the resistance is due to changes in cell membrane permeability similar to those described by Rank, Robertson and Philips (1975b). Multiple drug-resistant mutants selected for chloramphenicol resistance show an increased sensitivity to ethidium bromide in glucose medium. Therefore the mutations involved increase probably nuclear envelope permeability to the latter drug. Results of genetic analyses of non-mitochondrial capr and eryr mutants suggest strongly that in most, if not all, cases the resistance is determined by interaction between nuclear and extranuclear factors.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Expression of a proteolipid gene from a high-copy-number plasmid confers trifluoperazine resistance to Saccharomyces cerevisiae.

A wild-type haploid yeast strain was transformed with a library of wild-type yeast DNA fragments ligated into a high-copy-number plasmid vector (YEp24). The pooled URA+ transformants were plated on rich medium containing a lethal concentration of trifluoperazine (TFP). Plasmids rescued into Escherichia coli from TFP-resistant yeast colonies contained overlapping DNA fragments from a unique region of yeast chromosome XVI. Deletion and disruption experiments, mini-Tn10 LUK hop analysis, and DNA sequencing defined a novel gene with significant amino acid identity to bovine and yeast vacuoletype proteolipid subunits. This is the second locus identified that can be altered to confer TFP resistance to Saccharomyces cerevisiae and that has significant amino acid identity to a vacuolar ATPase subunit. This suggests that a target for TFP in S. cerevisiae is the electrogenic membranes of the vacuolar network and that alteration of expression or activity of vacuolar proton ATPase subunits is a general mechanism for TFP resistance in this yeast.     

Nuclear inheritance of resistance to antimycin A in Saccharomyces cerevisiae.

Summary A group of 30 independent mutants of Saccharomyces cerevisiae, resistant to the respiratory inhibitor antimycin A, was investigated from a genetical and biochemical point of view. All the mutants can be grouped into two nuclear loci: AMY1 maps on the VII chromosome, between leu 1 and trp 5; AMY2 is close to its centromere on either chromosome XVIII or XIX. Both genes do not affect mitochondrial structures or functions.     

Extra-chromosomal inheritance of rhodamine 6G resistance in Saccharomyces cerevisiae.

Summary Rhodamine 6G was found to be a specific inhibitor of aerobic growth of yeast, having no effect on fermentative growth. A single step spontaneous mutant of S. cerevisiae resistant to rhodamine 6G was isolated, which showed cross-resistance to the ATPase inhibitors venturicidin and triethyltin, to the uncoupler 1799, to bongkrekic acid and to cycloheximide, but not to oligomycin or to the inhibitors of mitochondrial protein synthesis, chloramphenicol and erythromycin. The genetic analysis of this mutant showed that both nuclear and cytoplasmic (but apparently not mitochondrial) factors may be involved in the determination of the mutation. The behaviour is discussed as a possible function for 2 micron circular (omicron) DNA.     

Allelism of pleiotropic drug resistance in Saccharomyces cerevisiae

Allelism of pleiotropic drug resistant (pdr) mutants was evaluated by complementation tests, linkage to chromosome-VII centromere markers and response to a partial suppressor (sur). Complementation tests were confounded by incomplete dominance and somatic segregation. Phenotypic suppression by sur was observed for all mutant and wild type alleles and thus could not be used to distinguish alleles. Five different alleles were tentatively identified by their close linkage to leul; 88 tetrads from three factor crosses produced the following linkages--leul (4.7) pdrl (17.0) trp5. Resistance of DRI 9/T7, a [cir o] strain of French origin, was not inherited as an allele of pdr but was controlled by a different pleiotropic centromere linked gene. An evaluation of published data suggest that antl, AMYl, till, cyh3, BOR2, and axe1 may be alleles of pdr. Thus pdr appears to be an allele that influences permeability to many inhibitors.     

Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli.

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.     

Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae

There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155:995-1000, 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted.     

Expression of human placental aromatase in Saccharomyces cerevisiae

A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.     

Expression of human asparagine synthetase in Saccharomyces cerevisiae

Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.     

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also found proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wild-type cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RASsc1 mRNA and immunoprecipitation of the translation products that the cloned RASsc1 gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.