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PubMed1998

Triatoma infestans can be captured under natural climatic conditions using yeast-baited traps.

The effectiveness of a trap for triatomines baited with yeast cultures has been previously demonstrated in laboratory assays. We report results from assays testing yeast-traps for Triatoma infestans performed under natural climatic conditions. All assays were conducted at experimental chicken-coops colonised by bugs, situated at an endemic area for Chagas disease in Argentina. Two different models of traps were tested, for use either on the floor, or attached to walls. The results obtained clearly demonstrate that yeast-baited traps are effective for capturing triatomine bugs. Floor traps, when baited, captured significantly more bugs than their controls (t-test, P = 0.0008, k = 10). In addition, wall traps were also more effective, when compared to their controls (t-test, P = 0.011, k = 10). The maximum capture by a single yeast-trap was 39 bugs in one night, while for the same period the maximum capture by a control-trap was two bugs. Traps captured significantly more bugs at nights when maximum temperature was above 18 degrees C (t-test, P = 0.0002). Results indicate that yeast traps are capable of capturing significant numbers of free insects in one night. The potential use of yeast- traps for the detection of triatomine bugs is discussed.

Animals↗
PubMed1989

The effect of deletion of the genes encoding the 40 kDa subunit II or the 17 kDa subunit VI on the steady-state kinetics of yeast ubiquinol-cytochrome-c oxidoreductase.

Yeast ubiquinol-cytochrome c oxidoreductase is still active after inactivation of the genes encoding the 40 kDa Core II protein or the 17 kDa subunit VI (Oudshoorn et al. (1987) Eur. J. Biochem. 163, 97-103 and Schoppink et al. (1988) Eur. J. Biochem. 173, 115-122). The steady-state levels of several other subunits of Complex III are severely reduced in the 40 kDa0 mutant. The level of spectrally detectable Complex III cytochrome b in the mutant submitochondrial particles is about 5% of that of the wild type. However, when the steady-state activity of Complex III with respect to the cytochrome c reduction was examined, similar maximal turnover numbers and Km values were found for the mutated and the wild-type complexes, both when yeast cytochrome c and when horse-heart cytochrome c was used as electron acceptor. We therefore conclude that the Core II subunit of yeast Complex III plays no role in the binding of cytochrome c and that it has no major influence of the overall electron transport and on the binding of ubiquinol by the enzyme. Absence of the 17 kDa subunit VI of yeast Complex III, the homologous counterpart of the hinge protein of the bovine heart enzyme, resulted in a decrease in the rate of reduction of both horse-heart cytochrome c and yeast cytochrome c by Complex III under conditions of relatively high ionic strength. However, under conditions of optimal ionic strength, no difference could be seen in the maximal turnover numbers and Km values, neither with horse-heart cytochrome c nor with yeast cytochrome c between Complex III deficient in the 17 kDa protein and the wild-type complex. Binding of ATP to ferricytochrome c inhibits its reduction by Complex III under conditions of relatively high ionic strength. But when the 17 kDa protein is absent, this inhibition is also observed under optimal ionic-strength conditions. These results can be explained by assuming a stimulating role for the acidic 17 kDa protein in the association of basic cytochrome c with Complex III. This association is (part of) the rate-limiting step in the reduction of cytochrome c by Complex III under conditions of relatively high ionic strength or when this association is hindered, for instance, by binding of ATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed2003

Bright stable luminescent yeast using bacterial luciferase as a sensor.

Bright luminescent yeast cells with light intensities similar to bacteria containing luciferase (LuxAB) were generated by providing saturating nontoxic levels of the substrates for the bioluminescence reaction (FMNH(2)+O(2) and fatty aldehyde-->light). Z-9-Tetradecenal added to yeast (+luxAB) gave a luminescent signal close to that with decanal with the signal remaining strong for >24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min. Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal. Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the NADPH-specific FMN reductase (FRP) from Vibrio harveyi. The recognition of the nontoxicity and high luminescence generated by Z-9-tetradecenal and the generation of high levels of FMNH(2) in yeast by transformation with a flavin reductase provide evidence for the strong potential use of bacterial luciferase as the light-emitting sensor of choice in eukaryotic organisms.

Aldehydes↗
PubMed1999

Assessment of uncoupling activity of the human uncoupling protein 3 short form and three mutants of the uncoupling protein gene using a yeast heterologous expression system.

The human uncoupling protein 3 gene generates two mRNA transcripts, uncoupling protein 3L and uncoupling protein 3S, which are predicted to encode long and short forms of the uncoupling protein 3 protein, respectively. While uncoupling protein 3L is similar in length to the other known uncoupling proteins, uncoupling protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human uncoupling protein 3 gene, resulting in the exclusive expression of uncoupling protein 3S, and a number of point mutations in the uncoupling protein 3 gene have been described. This study compares the biochemical activity of uncoupling protein 3S as well as three mutants of the uncoupling protein 3 gene (V9M, V102I, R282C) with that of uncoupling protein 3L utilizing a yeast expression system. All proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the uncoupling function. Both uncoupling protein 3S and uncoupling protein 3L decreased the yeast growth rate by 35 and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, uncoupling protein 3S and uncoupling protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to uncoupling protein 1 (102% increase). A truncated version of uncoupling protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced uncoupling activity. The two naturally occurring uncoupling protein 3 mutants, V9M and V102I, were similar to uncoupling protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption. The R282C mutant had a reduced effect compared to uncoupling protein 3L. In summary, uncoupling protein 3S and the three mutants of uncoupling protein 3 appear to be functional proteins with biochemical activities similar to uncoupling protein 3L, although uncoupling protein 3S and the R282C mutant have a modestly reduced function.

Carrier Proteins↗
PubMed2000

Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential interference contrast microscopy.

In studies of phagocytosis there is a need to distinguish targets that are internalized by the cell from those that are bound to the cell surface. The present work describes a simple method by which internalized and surface-bound yeast particles can be identified by differential interference contrast microscopy, using trypan blue to stain surface-bound yeast particles. The method has the advantage that both internalized and surface-bound particles can be visualized without the need to switch the illumination source and/or filter sets, thus facilitating concurrent quantitation of binding and internalization. The method was evaluated with the phagocytosis-modulating agents horseradish peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as phagocytic cells. When macrophages are challenged with a particular type of target, they usually bind many more targets than they ingest. It was shown that yeast particles were arrested in the initial binding phase of phagocytosis depending on the region of macrophage plasma membrane where binding sites were formed. Failure of surface-bound yeast particles to trigger internalization was not due to modifications of the yeast particle surface. Nor was it due to binding to non-phagocytic receptors, or low-affinity receptor-ligand interactions. The glycoprotein HRP inhibited only the binding stage of phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization, inhibited both binding and internalization. However, when the yeast particles were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the internalization step. The assay described here may be useful in studies concerned with the function and expression of phagocytosis-mediating surface lectins.

Animals↗
PubMed1998

Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays.

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.

Animals↗
PubMed2000

Repair of UV damage in the fission yeast Schizosaccharomyces pombe.

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.

Caffeine↗
PubMed2001

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells.

We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.

Acetylation↗
PubMed2005

Yeast model systems for examining nitrogen oxide biochemistry/signaling.

The yeast Saccharomyces cerevisiae is an ideal model system for examining fundamental nitrogen oxide biochemistry. The utility of this model system lies in both the similarities and the differences between yeast and mammalian cells. The similarities between the two systems, with regards to many of the fundamental biochemical processes, allow studies in yeast to be extrapolated to mammalian systems. On the other hand, yeast has distinct differences that allow, for example, the facile examination of O2, pH, and genetic dependencies on a number of nitrogen oxide-mediated processes. Thus, the yeast system is amenable to experimentation that is otherwise problematic or impossible in mammalian systems. Herein, we present several examples of the utility of the yeast model system for studying the intimate details of basic nitrogen oxide biochemistry.

Antioxidants↗
PubMed2002

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.).

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.

Adjuvants, Immunologic↗
PubMed2002

Rapid detection of lytic antimicrobial activity against yeast and filamentous fungi.

A rapid method for assessing the lytic activity of antimicrobial agents against yeast and fungi has been developed. The assay is based on the release of the intracellular enzyme, maltase (alpha-glucosidase). The released maltase activity was measured colorimetrically by the production of p-nitrophenol from p-nitrophenyl-alpha-D-glucopyranoside (PNPG). The lytic activity of different antimicrobial compounds was measured against yeast cells or germinating spores of filamentous fungi. Lytic anti-yeast activity could be detected within 20 min incubation at 30 degrees C against Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans. Lytic anti-fungal activity appeared after 2 h of incubation at 30 degrees C against germinating spores of Aspergillus niger and Botrytis cinerea. Whole cells of either yeast or fungi did not hydrolyze sufficient PNPG within 3 h at 30 degrees C to yield a detectable color change. Lytic activity of enzymes (e.g., Lyticase), antibiotics (e.g., Amphotericin B), and an antibiotic-producing strain of bacteria were detected using the assay. The anti-yeast assay has been adapted to a 96-well microtiter format. Both assays provided a rapid, sensitive, and reproducible detection of lytic anti-yeast and anti-fungal activity.

Antifungal Agents↗
PubMed2003

Proteolytic yeasts isolated from raw, ripe tomatoes and metabiotic association of Geotrichum candidum with Salmonella.

Metabiotic associations between food-borne fungi and bacteria capable of causing human diseases are a public health concern. A survey of decayed and damaged, uncooked, ripe tomatoes was done to determine the presence and prevalence of yeasts capable of increasing the pH pulp tissue, thus creating a more favorable environment for survival and growth of enteric pathogens. Sixty-two of the 371 (16.7%) fungi isolated from 215 decayed or damaged tomatoes; 12 of the 62 (19.4%) yeasts showed proteolytic activity on gelatin agar (GA) and/or standard methods caseinate (SMC) agar. The pH of tomato pericarp (pulp) tissue from which 9 of the 12 yeasts were isolated ranged from 4.3 to 7.5 (mean=5.3) compared to 4.2-5.1 (mean=4.8) for sound pulp tissue in the same tomatoes. The 12 proteolytic yeasts consisted of four strains of Cryptococcus albidus, two strains each of Debaryomyces hansenii and Trichosporon pullulans, and one strain each of Cryptococcus humicolus, Cryptococcus laurentii, Geotrichum candidum, and Sporidiobolus pararoseus. Survival and growth characteristics of a five-serotype mixture of Salmonella co-inoculated with G. candidum into sound (not chill injured) and chill-injured tomatoes were studied. Storage of sound tomatoes at 15 degrees C for 10 days resulted in an increase in population of 7.6 log(10) cfu of Salmonella/g of a 2-g sample of co-infected pulp tissue. Increases were less in tissue inoculated with Salmonella only, Salmonella on day 0 followed by G. candidum on day 3, or G. candidum on day 0 followed by Salmonella on day 3. Trends were similar in sound inoculated tomatoes stored at 25 degrees C. Growth of Salmonella was enhanced in chill-injured tomatoes compared to sound tomatoes; a population of 10 log(10) cfu/g of chill-injured pulp tissue was reached within 10 days at 25 degrees C. Results clearly show that growth of a proteolytic, alkalinizing yeast such as G. candidum in raw tomatoes enhances conditions for growth of Salmonella. The removal of tomatoes infected with proteolytic yeasts and other fungi from lots intended for minimally processed tomato products is an essential step in reducing the risk of human diseases caused by pathogenic bacteria favored by increased pH of decayed pulp tissue.

Colony Count, Microbial↗
PubMed1997

Phosphorylation of Srp1p, the yeast nuclear localization signal receptor, in vitro and in vivo.

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.

Amino Acid Sequence↗
PubMed1980

Utilization of waste products of dehydrated onion industry for production of fodder yeast by Saccharomyces cerevisiae.

One strain of Saccharomyces cerevisiae was selected from different yeasts, isolated from black strap molasses. This microorganism was cultivated on seven fermentation media for the production of protein. Medium I exhibited the highest potentiality for formation of protein. Therefore strain 1 of S. cerevisiae and medium I were used for further studies in the formation of protein. Factors controlling production of protein were explored. The required incubation period for the fermentation process was 72 hrs, while the initial pH value of the medium was 6.0. Sucrose supported the microorganism for higher production of protein (40.96%), while the best concentration of sucrose was shown to be 10.0 g/l. The best inorganic and organic nitrogen sources for protein formation were (NH4)2HPO4, (NH4)3PO4 and yeast extract, respectively. The best concentrations of (NH4)2HPO4 and yeast extract, supporting protein formation, were 5.0 g/l and 10.0 g/l, respectively. Addition of MgSO4, ZnSO4, ferrous ammonium sulphate, copper sulphate, biotin, Ca-pantothenate, thiamine, pyridoxine, and inositol to the synthetic medium did not markedly influence high level of protein formation. Glutamic acid was the best amino acid, supporting protein formation by S. cerevisiae. Onion juice was found to be a good medium, after deletion of inhibitory volatile sulphur organic compounds, for the production of protein by S. cerevisiae. Addition of (NH4)2HPO4 to the best concentration of onion juice assisted the onion medium in production of fodder yeast, containing high level of protein. Addition of MgSO4 to onion juice and (NH4)2HPO4 did not increase the total nitrogen of the biomass. Fodder yeast, produced by onion juice medium, contained more valuable ingredients than fodder yeast, produced by synthetic medium.

Amino Acids↗
PubMed2000

Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.

The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.

Amino Acid Sequence↗
PubMed2002

Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range.

The ability to express heterologous genes in yeast has become indispensable for many biological research techniques. Expression systems that can be regulated are particularly useful because they allow an experimenter to control the timing and levels of gene expression. Despite their many advantages, however, surprisingly few conditional expression systems are available for yeast. Moreover, of those that have been described, many are not ideal either because they have high background expression levels, low induced levels, or because they require restrictive growth conditions. Here we describe a conditional expression system that takes advantage of the yeast MAL62 promoter (MAL62p), which can be controlled by adding maltose or glucose to the growth medium to induce or repress transcription, respectively. In addition, we use a mating scheme to dramatically increase the dynamic range of expression levels possible. We show that MAL62p background activity can be effectively eliminated by maintaining expression constructs in a mal(-) yeast strain. High-level expression can be induced in diploids formed by mating the mal(-) strain with a MAL(+) strain. A similar mating scheme may be useful for other conditional expression systems as well. Among other uses, this approach should aid high throughput yeast two-hybrid assays, which rely on maintaining large libraries of expression strains, which are eventually mated to conduct assays for protein interactions. We demonstrate a two-hybrid system in which MAL62p is used in conjunction with the yeast GAL1 promoter to independently regulate expression of both hybrid proteins, and to allow detection of interactions involving toxic proteins.

Amino Acid Sequence↗
PubMed2003

Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90beta, a property that can be exploited in screens for new Hsp90 chaperone inhibitors.

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90beta or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90beta in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90beta relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.

Anti-Bacterial Agents↗
PubMed2003

Using yeast to place human genes in functional categories.

The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines. One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast. We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively. Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain.

Cerebellum↗