Three aldo–keto reductases of the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an industrially important yeast, which is also used extensively as a model eukaryote. The S. cerevisiae genome has been sequenced in its entirety and therefore represents an ideal organism in which to carry out functional analysis of genes. We have identified several open reading frames in the S. cerevisiae genome which show significant similarity to members of the aldo–keto reductase superfamily. The physiological roles of these gene products have not been previously determined, but their similarity to other enzymes suggests they may perform roles in carbohydrate metabolism and detoxification pathways. Cloning and expression of three of these enzymes has allowed their substrate specificities to be determined. Expression profiling and gene disruption analysis will allow potential roles for these enzymes within the cell to be examined.     

Inactivation of exogenous β-lactamase in transformed yeast Saccharomyces cerevisiae

Summary The stability of bacterial -lactamase in transformedSacharomyces cerevisiae grown on glucose was studied. A culture of a prototrophic strain showed marked inactivation shortly before the stationary phase. This was also observed in cells starved of nitrogen. The level of reserve carbohydrate was lower both in the stationary-phase culture of the auxotroph and in the glucose-starved culture of the prototroph, where less inactivation was observed. Such a close correlation suggests that inactivation may be triggered mainly in response to nitrogen-limitation which regulates reserve carbohydrate metabolism.     

Temperature dependency of induction of sexual agglutinability by α pheromone in the yeast Saccharomyces cerevisiae

When pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.Non-common abbreviations PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethylsulfonyl fluoride     

Expression of an endoglucanase from Tribolium castaneum （TcEG 1 ） in Saccharomyces cerevisiae

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation （saccharification） and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum （TcEG 1）, and evaluated the activity of the transgenic product （rTcEG 1）. Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional β-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.     

Activities of elements of the yeast α-factor precursor leader at different stages of somatropin secretion by Saccharomyces cerevisiae

An analytical system for a quantitative assessment of contributions of pre- and pro-components of the yeast α-factor leader to key stages of somatropin secretion in Saccharomyces cerevisiae has been designed. Using this system, it was shown that: 1—the α-factor pro-peptide has contributed almost equally to the secretion at the Erv29p-dependent stage of vesicular transport and at the translocation stage; 2—of the two above stages, the Erv29p-dependent stage could only be optimized by using of two copies of the α-factor pro-peptide; 3—when the α-factor pre-peptide and the artificial pro-peptide with significantly greater hydrophobicity were compared it was found that somatropin secretion apparently depended on the translocation mode as higher secretion was achieved with hydrophobic artificial pre-peptide associated with the co-translational translocation.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically and investigated for its inhibition with a series of sulfonamides and one sulfamate. The enzyme showed high CO₂ hydrase activity, with a k_cat of 9.4 × 10⁵ s^?1, and k_cat/K_M of 9.8 × 10⁷ M^?1 s^?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K_Is in the range of 0.976–18.45 μM. Better inhibition (K_Is in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K_Is in the range of 82.6–133 nM. The best inhibitor (K_I of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.     

Vacuoles: The sole compartments of digestive enzymes in yeast (Saccharomyces cerevisiae)?

Almost all the vacuoles (about 95%) remained intact after polybase-induced lysis of the yeast protoplasts. These vacuoles could be sedimentated together with other cell organelles which were equally well preserved, leaving as a supernatant a cytosol fraction which was essentially uncontaminated by the contents of disrupted vacuoles. After density gradient centrifugation more than half of the vacuoles were recovered in a fraction which was highly purified as judged from the measurement of several marker enzymes and from light and electron microscopic observations. Polyphosphate, which has been shown to be located exclusively in the vacuolar sap of protoplasts, was used as a vacuolar marker to determine the yields of vacuoles in the different fractions obtained from the density gradients. It was also used to assess the overall distribution of lytic enzymes in the cytosol and in the vacuome.The results indicate that the following enzyme activities are mostly, if not exclusively (>90%), located in the vacuome, probably all in the typical large vacuoles present in the protoplasts: exo-and endopolyphosphatase, proteases A and B, carboxypeptidase Y, an aminopeptidase, RNase, -mannosidase, and phosphatases which hydrolyze a number of different substrates. The polyphosphatases are thus in the same compartment as the polyphosphate. The activities of some other hydrolases, notably of a Mg²⁺ dependent, Oligomycin and NaN₃ insensitive ATPase and alkaline phosphatase, were partially associated with the vacuoles. The activities of pyrophosphatase, tripolyphosphatase, -glucosidase, and aminopeptidase active in the presence of EDTA, were located almost exclusively in the soluble, cytosolic fraction.Non-Standard Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - BSA bovine serum albumin - BTNA N-benzoyl-L-tyrosine-p-nitroanilide - LeuNA leucine-p-nitroanilide - LysNA lysine-p-nitroanilide - PIPES pinerazine-N,N-bis-2-ethanesulfonic acid - PP polyphosphate - Tri-PP tripolyphosphate     

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B

Background

A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat.

Results

We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91 % of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87 % of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B.

Conclusions

We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1803-y) contains supplementary material, which is available to authorized users.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .     

DNA-length polymorphisms of chromosome III in the yeast Saccharomyces cerevisiae

The chromosome-sized DNAs of sporulation-deficient mutants, which had been isolated by mutagenizing spores of a homothallic diploid strain (MT98a-3D) of Saccharomyces cerevisiae, were analyzed by pulsed-field gel electrophoresis. While the size of chromosome III DNA of the parent strain was 450 kb, some mutants had one or more chromosome III DNAs of 350 kb, 450 kb, 530 kb and 630 kb. No size variation was observed for other chromosomes. Chromosome III DNAs of laboratory-stock strains, except MT98a-3D, were in the neighborhood of 350 kb. Size variation of chromosome III was observed at a high frequency in spore clones derived from MT98a-3D strain. The results suggest that DNA-length polymorphisms of chromosome III are generated by the loss or addition of a specific DNA unit of approximately 100 kb.     

Strategies for the Genetic Manipulation of Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries. In this article the plethora of methods available for manipulating the genome of S. cerevisiae are reviewed. This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods. Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.     

Common physical map of four Brucella bacteriophage genomes

Abstract DNAs isolated from four strains of Brucella bacteriophages were studied by restriction endonuclease mapping and Southern blot analysis. In all strains the genome was composed of a 38 kb (25.1 × 10⁶ dalton) double-stranded circular DNA. The physical map was the same for the four genomes and Southern blot hybridization of restriction endonuclease fragments with the Tbilissi strain DNA as a probe showed complete homology between the four DNAs. Thus, the four phage strains appear to be identical, the specific host range of each originating from minor changes in phage or Brucella receptors or both.     

Interaction between ORC and Cdt1p of Saccharomyces cerevisiae

Origin recognition complex (ORC), a six-protein complex, is the most likely initiator of chromosomal DNA replication in eukaryotes. Throughout the cell cycle, ORC binds to chromatin at origins of DNA replication and functions as a 'landing pad' for the binding of other proteins, including Cdt1p, to form a prereplicative complex. In this study, we used yeast two-hybrid analysis to examine the interaction between Cdt1p and every ORC subunit. We observed potent interaction with Orc6p, and weaker interaction with Orc2p and Orc5p. Coimmunoprecipitation assay confirmed that Cdt1p interacted with Orc6p, as well as with Orc1p and Orc2p. We mapped the C-terminal region, and a middle region of Orc6p (amino acids residues 394-435, and 121-175, respectively), as important for interaction with Cdt1p. Cdt1p was purified to examine its direct interaction with ORC, and its effect on the activity of ORC. Glutathione-S-transferase pull-down analysis revealed that Cdt1p binds directly to ORC. Cdt1p neither bound to origin DNA and ATP nor affected ORC-binding to origin DNA and ATP. These results suggest that interaction of Cdt1p with ORC is involved in the formation of the prereplicative complex, rather than in regulation of the activity of ORC.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.