Biomedical subjects

S G Oliver

Publications and source records attributed to S G Oliver.

At least 19 recordsLinked to original sources

PubMed2001

Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation.

Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short-flanking homology (SFH) or long-flanking homology (LFH) gene replacement. Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs. Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S-methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur-containing amino-acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response. The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10-fold under sulphate starvation. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Amino Acid Transport Systems↗

PubMed2001

Towards a truly integrative biology through the functional genomics of yeast.

A complete library of mutant Saccharomyces cerevisiae strains, each deleted for a single representative of yeast's 6000 protein-encoding genes, has been constructed. This represents a major biological resource for the study of eukaryotic functional genomics. However, yeast is also being used as a test-bed for the development of functional genomic technologies at all levels of analysis, including the transcriptome, proteome and metabolome.

Fungal Proteins↗

PubMed2001

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

Adenine Nucleotides↗

PubMed2000

Exploring redundancy in the yeast genome: an improved strategy for use of the cre-loxP system.

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers (kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Blotting, Southern↗

PubMed2000

Chromosomal evolution in Saccharomyces.

The chromosomal speciation model invokes chromosomal rearrangements as the primary cause of reproductive isolation. In a heterozygous carrier, chromosomes bearing reciprocal translocations mis-segregate at meiosis, resulting in reduced fertility or complete sterility. Thus, chromosomal rearrangements act as a post-zygotic isolating mechanism. Reproductive isolation in yeast is due to post-zygotic barriers, as many species mate successfully but the hybrids are sterile. Reciprocal translocations are thought to be the main form of large-scale rearrangement since the hypothesized duplication of the whole yeast genome 10(8) years ago. To test the chromosomal speciation model in yeast, we have characterized chromosomal translocations among the genomes of six closely related species in the Saccharomyces 'sensu stricto' complex. Here we show that rearrangements have occurred between closely related species, whereas more distant ones have colinear genomes. Thus, chromosomal rearrangements are not a prerequisite for speciation in yeast and the rate of formation of translocations is not constant. These rearrangements appear to result from ectopic recombination between Ty elements or other repeated sequences.

Chromosomes, Fungal↗

PubMed2000

Functional analysis of six novel ORFs on the left arm of chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control.

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. Complementation of the yll035w deletion with its cognate clone, and a promoter-substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonstrated that YLL035w is under cell-cycle regulation. Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non-essential ORFs. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Cell Cycle↗

PubMed2000

Functional analysis of eight open reading frames on chromosomes XII and XIV of Saccharomyces cerevisiae.

Deletion, together with basic functional and bioinformatic analyses has been carried out on eight novel ORFs discovered during the sequencing of the Saccharomyces cerevisiae genome. Six ORFs (YLL049w, YLL051c, YLL052c, YLL053c, YLL054c and YLL055w) located on the left arm, and one (YLR130c) on the right arm, of chromosome XII, and an eighth ORF (YNL331c) on the left arm of the chromosome XIV, have been investigated. ORFs were deleted by the SFH-PCR gene-replacement strategy. Basic functional analysis revealed no obvious phenotype for any of the eight ORFs. Bioinformatic analysis, however, revealed possible functions for seven of the ORFs on the basis of the amino acid sequence similarity of their predicted protein products to those of proteins with known functions. ORF YLL051c (FRE6) shows similarity to iron transport proteins, such as ferric reductase. YLL052c and YLL053c appear to be aquaporins. The product of YLL054c (Yll054p) is highly similar to the oleate-specific transcriptional activator protein (Pip2p), which is involved in the peroxisomal induction pathway (pip). ORF YLL055w is similar to Dal5p, allantoate permease, and may play role in allantoin transport. YLR130c (ZRT2) is a low-affinity zinc transporter protein. YNL331c is also named AAD14, which is induced by chemicals that induce oxidative stress by depleting the cell of glutathione.

Chromosomes, Fungal↗

PubMed2000

Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides.

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.

Alcohol Dehydrogenase↗

PubMed2000

Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species.

The genes encoding GDP-mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3-SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine-mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP-mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP-mannose in the fungal pathogen C. albicans.

Candida albicans↗

PubMed2000

Conceptual modelling of genomic information.

MOTIVATION: Genome sequencing projects are making available complete records of the genetic make-up of organisms. These core data sets are themselves complex, and present challenges to those who seek to store, analyse and present the information. However, in addition to the sequence data, high throughput experiments are making available distinctive new data sets on protein interactions, the phenotypic consequences of gene deletions, and on the transcriptome, proteome, and metabolome. The effective description and management of such data is of considerable importance to bioinformatics in the post-genomic era. The provision of clear and intuitive models of complex information is surprisingly challenging, and this paper presents conceptual models for a range of important emerging information resources in bioinformatics. It is hoped that these can be of benefit to bioinformaticians as they attempt to integrate genetic and phenotypic data with that from genomic sequences, in order to both assign gene functions and elucidate the different pathways of gene action and interaction. RESULTS: This paper presents a collection of conceptual (i.e. implementation-independent) data models for genomic data. These conceptual models are amenable to (more or less direct) implementation on different computing platforms.

Computational Biology↗

PubMed1999

Analysis of a continuous-culture technique for the selection of mutants tolerant to extreme environmental stress.

An analysis is presented of a continuous-culture technique named "Brown and Oliver Interactive-Continuous Selection" (BOICS; Brown and Oliver, 1982), that was devised for the selection of microbial mutants tolerant to extreme environmental stress. The case in which the stress is due to a growth inhibitor is considered. The possible steady outcomes of competition between mutants in BOICS are analyzed under the assumption that the specific growth rates of the competing mutants depend only on the inhibitor concentration. The analysis indicates that BOICS selects mutants that sustain a given specific growth rate (equal to the dilution rate in the selection experiment) at an increased concentration of the inhibitor. Simulation results support the steady-state analysis. Further simulation results show that BOICS may fail if the specific growth rate of a mutant has to be considered to depend on a factor beside the inhibitor concentration. The choice of experimental conditions that promote the success of BOICS is discussed. An interpretation of BOICS emerges from the analysis. Namely, that BOICS implements (at least approximately) a strategy for the selection of tolerant mutants in which (1) all factors in the growth environment, beside the stress of interest (inhibitor concentration) are maintained constant, and (2) the stress is adjusted as the culture evolves so that it is always maximized subject to a specified minimum value of the mean specific-growth rate being maintained. This interpretation suggests new (possibly improved) ways of implementing essentially the same selection technique. It is noted that a chemostat implements the same strategy in the case that the stress is due to the lack of a growth-rate-limiting nutrient. The outcome of selection for inhibitor tolerant mutants in chemostats, turbidostats, and in BOICS is compared. Only BOICS selects specifically for mutants tolerant to extreme concentrations of the inhibitor.

Adaptation, Physiological↗

PubMed1999

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae.

Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Alleles↗

PubMed1999

Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae.

The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast cells in order to release their intracellular contents. To develop such a method, a tightly regulated promoter, pMET3, has been used to control three genes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of these regulation cassettes, pMET3-SRB1/PSA1 and pMET3-PKC1, have been integrated at the chromosomal loci of the respective genes in order to overcome problems of plasmid instability. Although repression of PDE2 did not cause cell lysis, cells depleted of Srb1p/Psa1p gradually lost their viability and integrity, releasing about 10% of total protein into the medium. Repression of PKC1 led to extensive cell lysis, accompanied by the release of 45% of cellular protein into the medium. A double mutant, carrying both pMET3-SRB1/PSA1 and pMET3-PKC1 cassettes in place of SRB1/PSA1 and PKC1, was constructed and found to permit the efficient release of both homologous and heterologous proteins. &copy 1999 John Wiley & Sons, Inc.,

Base Sequence↗

PubMed1999

Transcript analysis of 250 novel yeast genes from chromosome XIV.

The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi-steady-state conditions (exponential growth on rich fermentative, rich non-fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo-stress, heat-shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACT1 mRNA. The levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift; five were repressed by glucose; six were induced in response to nitrogen starvation; three were induced in stationary phase; five were induced by osmo-stress; four were induced by heat-shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome-wide basis.

Blotting, Northern↗

PubMed1999

Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

Alcohol Dehydrogenase↗

PubMed1999

Analysis of the seven-member AAD gene set demonstrates that genetic redundancy in yeast may be more apparent than real.

Saccharomyces cerevisiae has seven genes encoding proteins with a high degree (>85%) of amino-acid sequence identity to the aryl-alcohol dehydrogenase of the lignin-degrading, filamentous fungus, Phanerochaete chrysosporium. All but one member of this gene set are telomere associated. Moreover, all contain a sequence similar to the DNA-binding site of the Yap1p transcriptional activator either upstream of or within their coding sequences. The expression of the AAD genes was found to be induced by chemicals, such as diamide and diethyl maleic acid ester (DEME), that cause an oxidative shock by inactivating the glutathione (GSH) reservoir of the cells. In contrast, the oxidizing agent hydrogen peroxide has no effect on the expression of these genes. We found that the response to anti-GSH agents was Yap1p dependent. The very high level of nucleotide sequence similarity between the AAD genes makes it difficult to determine if they are all involved in the oxidative-stress response. The use of single and multiple aad deletants demonstrated that only AAD4 (YDL243c) and AAD6 (YFL056/57c) respond to the oxidative stress. Of these two genes, only AAD4 is likely to be functional since the YFL056/57c open reading frame is interrupted by a stop codon. Thus, in terms of the function in response to oxidative stress, the sevenfold redundancy of the AAD gene set is more apparent than real.

Alcohol Oxidoreductases↗

PubMed1998

Characterization of recombinant Saccharomyces cerevisiae manganese-containing superoxide dismutase and its H30A and K170R mutants expressed in Escherichia coli.

All known Mn-containing superoxide dismutases (MnSODs) have a highly conserved histidine (His-30 in Escherichia coli FeSOD) in the active-site channel, and nearly all have an active-site arginine (Arg-170) that has been proposed to play a combined structural and functional role [Chan et al., Arch. Biochem. Biophys. 279, 195-201 (1990)]. In Saccharomyces cerevisiae MnSOD, the active-site arginine is replaced by a lysine. The S. cerevisiae MnSOD gene has been cloned and expressed in E. coli, and H30A and K170R site-specific mutants have been prepared. The purified recombinant native (RN) and mutant enzymes were compared to one another and to the native enzyme purified from S. cerevisiae (SC) in terms of activity, temperature stability, and sensitivity to 2,4,6-trinitrobenzenesulfonate (TNBS) and phenylglyoxal (PG). All enzymes had high specific activities (SC = 5000, RN = 5600, H30A = 4500, K170R = 4600) (U/mg, using the pyrogallol assay). SC, RN, and H30A were very stable at 75 degreesC (pH 8.0), with half-lives of 4.7, 2.8, and 2.7 h, respectively, while K170R had a much greater temperature lability, with a half-life of 0.36 h under these conditions. TNBS (0.5 mM, pH 9.0, 25 degreesC) rapidly inactivated SC, RN, and H30A, with half-lives of 3. 5, 5.1, and 5.5 min, respectively, but only slowly inactivated K170R, with a half-life of 101 min. PG (20 mM, pH 9.0, 25 degreesC) caused very slow inactivation of SC, RN, and H30A by biphasic kinetics, and each enzyme retained >/=25% activity after 3 h of modification. K170R, on the other hand, was completely inactivated by PG under these conditions by first-order kinetics, with a half-life of 7.0 min. The data suggest that His-30, a residue highly conserved in the active-site channel of MnSODs and FeSODs, does not play a crucial role in catalysis or stability. In addition, Lys-170, a residue that is almost always arginine in the numerous other MnSODs and FeSODs sequenced to date, can be replaced by arginine with no loss of catalytic activity, but K170R is less stable and Arg-170 in this mutant is more exposed than the corresponding arginine in other SODs. RN and SC showed some surprising differences. Thus, while the specific activities of RN and SC are very similar, SC is more stable to inactivation at 75 degreesC, and less susceptible to inactivation by phenylglyoxal, than RN. These data suggest that there may be slight differences in the tertiary structures of SC, the native enzyme expressed in S. cerevisiae, and RN, the recombinant native enzyme expressed in E. coli.

Alanine↗

PubMed1998

Quantitative analysis of yeast gene function using competition experiments in continuous culture.

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments is continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype.

Culture Media↗