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PubMed1993

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences.

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 microns origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of 'ZAP'-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.

Amino Acid Sequence↗
PubMed2002

A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution.

We surveyed the sequenced Saccharomyces cerevisiae genome (strain S288C) comprehensively for open reading frames (ORFs) that could encode full-length proteins but contain obvious mid-sequence disablements (frameshifts or premature stop codons). These pseudogenic features are termed disabled ORFs (dORFs). Using homology to annotated yeast ORFs and non-yeast proteins plus a simple region extension procedure, we have found 183 dORFs. Combined with the 38 existing annotations for potential dORFs, we have a total pool of up to 221 dORFs, corresponding to less than approximately 3% of the proteome. Additionally, we found 20 pairs of annotated ORFs for yeast that could be merged into a single ORF (termed a mORF) by read-through of the intervening stop codon, and may comprise a complete ORF in other yeast strains. Focussing on a core pool of 98 dORFs with a verifying protein homology, we find that most dORFs are substantially decayed, with approximately 90% having two or more disablements, and approximately 60% having four or more. dORFs are much more yeast-proteome specific than live yeast genes (having about half the chance that they are related to a non-yeast protein). They show a dramatically increased density at the telomeres of chromosomes, relative to genes. A microarray study shows that some dORFs are expressed even though they carry multiple disablements, and thus may be more resistant to nonsense-mediated decay. Many of the dORFs may be involved in responding to environmental stresses, as the largest functional groups include growth inhibition, flocculation, and the SRP/TIP1 family. Our results have important implications for proteome evolution. The characteristics of the dORF population suggest the sorts of genes that are likely to fall in and out of usage (and vary in copy number) in a strain-specific way and highlight the role of subtelomeric regions in engendering this diversity. Our results also have important implications for the effects of the [PSI+] prion. The dORFs disabled by only a single stop and the mORFs (together totalling 35) provide an estimate for the extent of the sequence population that can be resurrected readily through the demonstrated ability of the [PSI+] prion to cause nonsense-codon read-through. Also, the dORFs and mORFs that we find have properties (e.g. growth inhibition, flocculation, vanadate resistance, stress response) that are potentially related to the ability of [PSI+] to engender substantial phenotypic variation in yeast strains under different environmental conditions. (See genecensus.org/pseudogene for further information.)

Chromosomes, Fungal↗
PubMed1997

Using GCN4 as a reporter of eIF2 alpha phosphorylation and translational regulation in yeast.

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the alpha subunit of the translation initiation factor elF2, which causes an inhibition of general translation. In contrast, in the yeast Saccharomyces cerevisiae phosphorylation of elF2 alpha on serine-51 by the GCN2 protein kinase mediates the translational induction of GCN4 expression. The unique structure of the GCN4 mRNA makes GCN4 expression especially sensitive to elF2 alpha phosphorylation, and the simple microbiological tests developed in yeast to analyze GCN4 expression serve as good reporters of elF2 alpha phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian elF2 alpha kinases will functionally substitute for GCN2. Structure-function analyses of translation factors or translational regulators can also be performed by assaying for effects on general and GCN4-specific translation. Three tests can be used to study elF2 alpha phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, while GCN4 expression can be analyzed using sensitive replicaplating tests. Second, GCN4 translation can be quantitated by measuring expression from GCN4-lacZ reporter constructs. Finally, isoelectric focusing gels can be used to directly monitor in vivo phosphorylation of elF2 alpha in yeast.

DNA-Binding Proteins↗
PubMed1993

Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast.

The aim of this research was to determine whether the structural homology between the O2 gene, a maize transcriptional activator, and the GCN4 gene, a yeast transcriptional factor, is reflected at the level of function. The O2 cDNA was cloned in the yeast expression vector pEMBLyex4 under the control of a hybrid inducible promoter, and used to transform the yeast Saccharomyces cerevisiae. Transformed yeast cells produced O2 mRNA and a polypeptide immunoreactive with anti-O2 antibodies during growth in galactose. The heterologous protein was correctly translocated into the yeast nuclei, as demonstrated by immunofluorescence, indicating that the nuclear targeting sequences of maize are recognized by yeast cells. Further experiments demonstrated the ability of O2 to rescue a gcn4 mutant grown in the presence of aminotriazole, an inhibitor of the HIS3 gene product, suggesting that O2 activates the HIS3 gene, gene normally under control of GCN4. It was shown that the O2 protein is able to trans-activate the HIS4 promoter in yeast cells and binds to it in vitro. The sequence protected by O2, TGACTC, is also the binding site for GCN4. Finally, the expression of O2 protein in yeast did not produce alterations during batch growth at 30 degrees C, while transformants expressing O2 protein showed a conditionally lethal phenotype when grown in galactose at 36 degrees C; this phenotype mimics the behaviour of gcd mutants. The results support the idea that basic mechanisms of transcription control have been highly conserved in eukaryotes.

Cloning, Molecular↗
PubMed1986

Medium for susceptibility testing and yeast phase conversion of Blastomyces dermatitidis.

Cottonseed protein agar and a modified Tween-albumin casein hydrolysate (TAC) medium were compared for the yeast phase conversion of Blastomyces dermatitidis strains including fresh isolates as well as strains maintained in long-term storage. It was found that both media converted all the B. dermatitidis (mycelial phase) strains studied to yeast phase in three days. The TAC medium has the added advantage that it is clear and the growth can be recognized earlier than in the opaque cottonseed agar medium. The conversion in most cases was more than 95% and the morphology of the yeast cells was uniformly typical with broad base budding. There was a striking difference between the sensitivity of the yeast and mycelial phases of B. dermatitidis strains. The yeast phase was usually more sensitive to Amphotericin B than the mycelial phase of B. dermatitidis. Similarly, the yeast phases of four out of six strains were more sensitive to ketoconazole than their respective mycelial phases, while two strains showed identical sensitivity in cottonseed agar. The yeast phase organism was more susceptible to Amphotericin B when cottonseed medium was used whereas the yeast phase showed more susceptibility to ketoconazole in TAC medium. Since the sensitivity among the various strains differed, it is necessary to determine the antifungal susceptibility of the pathogenic phase of the organism for initiating proper therapy and monitoring effectiveness.

Amphotericin B↗
PubMed1982

Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex.

Two yeast DNA pools inserted in a hybrid Escherichia coli-yeast vector pFL1 were used to transform E. coli and yeast aspartate-transcarbamylase-less strains to prototrophy. From the first pool--a BamHI yeast DNA digest--a 6.4 kb BamHI fragment was recovered that gave good complementation of the E. coli auxotrophy but poor complementation of the yeast auxotrophy. From the second pool--a partial Sau3A yeast DNA digest--five independent plasmids complementing either E. coli, yeast, or both were recovered. Each of the five plasmids possessed sequences in common with the 6.4 kb BamHI fragment. One of these plasmids, which complemented the two URA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the full URA2 gene. A restriction map of the URA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization with URA2 messenger RNA, allowing us to estimate the limits of the gene.

Aspartate Carbamoyltransferase↗
PubMedDate not supplied

Glucose tolerance factor potentiation of insulin action in adipocytes from rats raised on a torula yeast diet cannot be attributed to a deficiency of chromium or glucose tolerance factor activity in the diet.

The nature of the dietary component responsible for adipocytes having the ability to respond to Glucose Tolerance Factor (GTF) was investigated. Rats were raised on either a control diet or one of three diets differing only in the protein source (torula yeast, brewer's yeast, or casein). Only in adipocytes from rats fed the torula yeast diet did a GTF fraction prepared from brewer's yeast potentiate the action of suboptimal concentrations of insulin in the incorporation of label from D-[1-14C]-glucose and D-[U-14C]-glucose into CO2 and fatty acids. It was concluded that this potentiation was not the result of a deficiency of GTF activity in torula yeast, because a GTF fraction prepared from torula yeast had similar insulin potentiating activity. Differences in response among diets were not owing to differences in levels of amino acids or owing to concentrations of 22 (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mo, Na, Ni, P, Pb S, Se, Si, Sn, Sr, Zn) of the 23 trace elements investigated. The level of Mn was low in all diets, but particularly low in the torula yeast diet. Mn deficiencies have previously been implicated in perturbations of glucose metabolism, so that it is possible that this deficiency may be responsible for the effects attributed to the torula yeast diet.

Adipose Tissue↗
PubMed1997

Enzymic activity of whole cells entrapped in reversed micelles. Studies on alpha-amylase and invertase in the entrapped yeast cells.

Studies have been conducted on the enzymic activity of Baker's yeast and also of Brewer's yeast entrapped into the reversed micelles formed by cetyl pyridinium chloride (CPC1) in n-hexane. The activities of alpha-amylase and invertase enzymes in the entrapped cells have been estimated and compared with those in the control experiments where there was no entrapment. The following significant observations have been made: 1. except for invertase, enzymes in Brewer's yeast, the entrapped yeast cells showed enhanced enzymic activities; 2. when the yeast cells were entrapped inside the reversed micelles along with substrates of the two enzymes, alpha-amylase, and invertase, the activity of each of these enzymes showed a further enhancement in comparison to that showed in the experiments in which substrates of the individual enzymes alone were entrapped-the phenomenon of synergism; 3. when the yeast cells and the respective substrates were entrapped inside separate reversed micelles and the solutions containing entrapped cells and entrapped substrates were mixed, the activities of the individual enzymes, alpha-amylase and invertase, showed further enhancement in comparison to the case in which the cells and the substrates were entrapped inside the same reversed micelle (in this case also the phenomenon of synergism was observed); and (4) In the case of experiments in which there was no entrapment, it was observed that the presence of substrates induced more release of enzymes from the yeast cells. These observations on yeast cells, which to the best of our knowledge have not been reported before, should be biotechnologically relevant.

Culture Media↗
PubMed2004

Coupling gravitational and flow field-flow fractionation, and size-distribution analysis of whole yeast cells.

This work continues the project on field-flow fractionation characterisation of whole wine-making yeast cells reported in previous papers. When yeast cells are fractionated by gravitational field-flow fractionation and cell sizing of the collected fractions is achieved by the electrosensing zone technique (Coulter counter), it is shown that yeast cell retention depends on differences between physical indexes of yeast cells other than size. Scanning electron microscopy on collected fractions actually shows co-elution of yeast cells of different size and shape. Otherwise, the observed agreement between the particle size distribution analysis obtained by means of the Coulter counter and by flow field-flow fractionation, which employs a second mobile phase flow as applied field instead of Earth's gravity, indicates that yeast cell density can play a major role in the gravitational field-flow fractionation retention mechanism of yeast cells, in which flow field-flow fractionation retention is independent of particle density. Flow field-flow fractionation is then coupled off-line to gravitational field-flow fractionation for more accurate characterisation of the doubly-fractionated cells. Coupling gravitational and flow field-flow fractionation eventually furnishes more information on the multipolydispersity indexes of yeast cells, in particular on their shape and density polydispersity.

Cell Fractionation↗
PubMed2006

Evaluation of image processing programs for accurate measurement of budding and fission yeast morphology.

To study the cellular functions of gene products, various yeast morphological mutants have been investigated. To describe yeast morphology objectively, we have developed image processing programs for budding and fission yeast. The programs, named CalMorph for budding yeast and F-CalMorph for fission yeast, directly process microscopic images and generate quantitative data about yeast cell shape, nuclear shape and location, and actin distribution. Using CalMorph, we can easily and quickly obtain various quantitative data reproducibly. To study the utility and reliability of CalMorph, we evaluated its data in three ways: (1) The programs extracted three-dimensional bud information from two-dimensional digital images with a low error rate (<1%). (2) The absolute values of the diameters of manufactured fluorescent beads calculated with CalMorph were very close to those given in the manufacturer's data sheet. (3) The programs generated reproducible data consistent with that obtained by hand. Based on these results, we determined that CalMorph could monitor yeast morphological changes accompanied by the progression of the cell cycle. We discuss the potential of the CalMorph series as a novel tool for the analysis of yeast cell morphology.

Cell Division↗
PubMed2005

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs.

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H(+)-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Antifungal Agents↗
PubMed1998

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica.

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20 degrees C, and the other psychrotrophic, capable of growth at temperatures above 20 degrees C. In addition to species--specific heat shock prote in (hsp) profiles, a heat shock (15 degrees C-25 degrees C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15 degrees C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25 degrees C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles.

Antarctic Regions↗
PubMed2004

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash.

Two separate 4 (bacterial concentrations)x6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1x10(6), 1x10(7) and 1x10(8) cells/ml mash. Yeast concentrations were 0, 1x10(6), 1x10(7), 2x10(7), 3x10(7), and 4x10(7) cells/ml mash. An increased yeast inoculation rate of 3x10(7) cells/ml resulted in a greater than 80% decrease (P < 0.001) and a greater than 55% decrease (P < 0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1x10(8) lactobacilli/ml. No differences (P > 0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1x10(7) or 1x10(8) lactobacilli/ml, a reduction of 0.7-0.9% v/v (P < 0.005) and a reduction of 0.4-0.6% v/v (P < 0.005) in the final ethanol produced was observed in mashes inoculated with 1x10(6) and 1x10(7) yeast cells/ml, respectively. At higher yeast inoculation rates of 3x10(7) or 4x10(7) cells/ml, no differences (P > 0.35) were observed in the final ethanol produced even when the mash was infected with 1x10(8) lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3x10(7) yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.

Equipment Contamination↗
PubMed1992

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.

Amino Acid Sequence↗
PubMed1992

Ca(2+)-dependent ubiquitination of calmodulin in yeast.

Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.

Animals↗
PubMed1990

Identification of peptides from autolysates of Saccharomyces cerevisiae that exhibit glucose tolerance factor activity in a yeast assay.

1. Cationic fractions were isolated from a low chromium (less than 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast systems as an assay for GTF.

Amino Acids↗
PubMed1993

Recombinant yeast in drug metabolism.

The usefulness of cDNA-directed expression of human hepatic P450s in yeast for the in vitro study of drug metabolism is emphasized. The major advantages of yeast expression are: (i) relatively high yields of heterologous P450 (approximately 5-10 nmol/l of culture medium) can be obtained; (ii) the expressed P450s are directly active in yeast microsomes, allowing the determination of specific catalytic activities of individual isoforms, which is a prerequisite for the prediction of metabolic pathways for new drug candidates; (iii) transformed yeast microsomes can also be used to study the specific affinity of individual P450s for various substrates and the formation of P450-metabolite complexes by difference visible spectroscopy; such studies can help to predict drug interactions. The advantages of expression in yeast with respect to biochemical studies of drug metabolism are illustrated with data about P450 NF25 (P450 3A4), the major form of human liver. Expressed P450 NF25 is obtained in a functionally active state, and some specific catalytic activities observed in liver microsomes could be reproduced directly with transformed yeast microsomes. The use of genomically modified yeast strains coexpressing human cytochrome b5 and/or overexpressing yeast P450-reductase allowed us to optimize these catalytic activities. In particular, this coexpression system was useful in the study of the in vitro formation of a P450 NF25 Fe(II)-RNO complex. Such inhibitory complexes have been implied in numerous drug interactions involving P450 3A4.

Animals↗
PubMed1990

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast.

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.

Alcohol Dehydrogenase↗