Screening for protein-protein interactions in the yeast two-hybrid system.
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Protein Binding↗
Biomedical subjects
R A Woods
Publications and source records attributed to R A Woods.
High-efficiency transformation of plasmid DNA into yeast.
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Culture Media↗
Genetic transformation of yeast.
Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi. This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae. The application of protocols developed for S. cerevisiae to other important yeast species is discussed. The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed.
Gene Transfer Techniques↗
Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system.
The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.
Cloning, Molecular↗
Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.
An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.
Acetates↗
Interaction of the yeast RAD7 and SIR3 proteins: implications for DNA repair and chromatin structure.
We have used the two-hybrid system to identify proteins that interact with the product of RAD7, a gene involved in DNA repair. A screen of a yeast genomic DNA-GAL4 activation domain (GAD) fusion gene library allowed the isolation of plasmids containing sequences corresponding to the 3' end of the SIR3 gene. This gene is known to be involved in the production of transcriptionally silent DNA at the cryptic mating-type cassettes and at telomeres. The cloned sequences coded for amino acids 307-979 of the Sir3 protein. A sir3 deletion allele, constructed in an isogenic rad7-deletion strain, rescued approximately one-quarter of the UV sensitivity associated with the rad7 deletion, indicating that the two genes interact genetically. Radiolabeled fusion proteins, made with the glutathione S-transferase (GST) gene in the vector pGEX-2T, were purified from Escherichia coli and shown to interact in vitro. This evidence suggests that the Sir3 protein interacts with the Rad7 protein to allow the nucleotide excision repair complex access to transcriptionally inactive chromatin. The proportions of 5-FOA-resistant cells in cultures from isogenic RAD+ and rad7-delta strains containing a telomeric URA3 gene were similar, suggesting that the RAD7 gene is not involved in the production or structure of transcriptionally silent chromatin at the telomeres. RAD7-dependent DNA repair of transcriptionally silent chromatin was shown not to induce expression of a telomeric copy of the URA3 gene, suggesting that repair of transcriptionally silent chromatin differs from transcriptionally active chromatin. Expression of a telomeric copy of the URA3 gene was stimulated in a rad7-delta mutant, suggesting that repair of lesions in the absence of Rad7 can result in the activation of transcriptionally silenced genes.
Base Sequence↗
OSHA breaks a rule.
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Legislation, Medical↗
Improved method for high efficiency transformation of intact yeast cells.
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Genetic Techniques↗
The chromosome 19 linkage group LDLR, C3, LW, APOC2, LU, SE in man.
The data establish linkage in both sexes for LDLR:LW (zeta = 8.43 at theta = 0.00) and in the male for LDLR:LU (zeta = 3.31 at theta = 0.00) and for LW:APOC2 (zeta = 3.90 at theta = 0.00). They confirm LDLR:C3 and APOC2:LU linkage in both sexes, and LW:LU linkage in the male. The loci constitute two tightly linked gene clusters, LDLR, C3, LW and APOC2, LU, SE, distinguished by measurable linkage in female meioses within but not between clusters. Argument is supported for a 19p13.2-cen position for LW and a long arm position for LU and SE.
Apolipoprotein C-II↗
The LW:C3 recombination fraction in female meioses.
No recombinants between LW and C3, using a C3 DNA probe, were observed in 16 female meioses: z 4.216 at theta = 0.00. Combined with the data of Sistonen (1984) the recombination fraction between LW and C3 is estimated to be 0.09 (z 3.773) in females.
Antigens↗
A hot aldehyde-peroxide fixation method for electron microscopy of the free-living nematode Caenorhabditis elegans.
An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.
Animals↗
The effects of amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on Caenorhabditis elegans.
The paralyzing effects of the anthelmintic drugs amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on whole and cut C. elegans were investigated. The minimum effective concentrations with whole worms were 350 and 180 microM, respectively, compared to only 4 microM for another anthelmintic drug, levamisole. After rendering the worms permeable by cutting them at their approximate midsections, the minimum effective concentrations were: amidantel 0.30 microM, deacylated amidantel 0.07 microM and levamisole 0.15 microM. Comparison of the effects produced by amidantel and deacylated amidantel with those produced by levamisole, a known cholinergic agonist, suggested a common mode of action for all three drugs. The drugs were moderately potent inhibitors of both E. electricus and C. elegans acetylcholinesterase but at concentrations too high to account for their abilities to contract cut worms. Their primary mode of action appears to be as agonists at the level of the acetylcholine receptor, a view supported by the observation that their effects may be blocked by the nicotinic antagonists d-tubocurarine and gallamine.
Acetylcholine↗
Adenine phosphoribosyltransferase mutants in Saccharomyces cerevisiae.
Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2,4,2,7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase (EC 2,4,2,14). The mutants were recessive and defined a single gene, apt1. They did not excrete purine when combined with ade4+. The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 apt1 responded to both adenine and hypoxanthine. Mutants deficient in adenine aminohydrolase (EC 3,5,4,2) activity, aah1, and hypoxanthine:guanine phosphoribosyltransferase (EC 2,4,2,8) activity, hpt1, were used to synthesize the genotypes apt1 hpt1 aah+ and apt1 hpt+ aah1. The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of apt1 mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.
Adenine Phosphoribosyltransferase↗
Glomerular & vascular changes in malignant hypertension.
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Animals↗
Hypoxanthine: guanine phosphoribosyltransferase mutants in Saccharomyces cerevisiae.
Yeast mutants lacking activity of the enzyme hypoxanthine:guanine phosphoribosyltransferase (H:G-PRT) have been isolated by selecting for resistance to 8-azaguanine in a strain carrying the wild type allele, ade4%, of the gene coding for amidophosphoribosyltransferase (PRPPAT), the first enzyme of de novo purine synthesis. The mutants excrete purines and are cross-resistant to 8-azaadenine. They are recessive and represent a single complementation group, designated hpt1. Ade4-su, a prototrophic allele of ade4 with reduced activity of PRPPAT, is epistatic to hpt1, suppressing purine excretion and resistance to azaadenine but not resistance to azaguanine. The genotype ade2hpt1 does not respond to hypoxanthine. Hpt1 complements and is not closely linked to the purine excreting mutants pur1 to pur5. Hpt1 and pur6, a regultory mutant of PRPPAT, are also unlinked but do not complement, suggesting a protein-protein interaction between H:G-PRT and PRPPAT. Mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis, inhibits the growth of hpt1 and hpt1+. Xanthine allows both genotypes to grow in the presence of MPA whereas guanine only allows growth of hpt1+. Activity of A-PRT, X-PRT and H:G-PRT is present in hpt+. Hpt1 lacks activity of H:G-PRT but has normal A-PRT and X-PRT.
Alleles↗
Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae.
Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.
Amidophosphoribosyltransferase↗
Identification of ergosta-8,24(28)-dien-3 beta,6 alpha-diol in A delta 8 goes to delta 7 sterol isomerase-blocked yeast mutant.
In addition to the monohydroxysterols found in the delta 8 goes to delta 7 isomerase-blocked Saccharomyces cerevisiae mutant erg 2, a noval dihydroxysterol, ergosta-8,24(38)-dien-3 beta,6 alpha-diol, was isolated. This sterol accumulated to the extent of 2.1% of the total sterol fraction when this mutant was treated with 23-azacholesterol, a known inhibitor of the 24-methylene-sterol-24(28)-reductase.
Chromatography, Gas↗
The nephrotoxicity of p-aminophenol. II. The effect of metabolic inhibitors and inducers.
Inducers and inhibitors of the microsomal mixed function oxidase system have no consistent effect upon the nephrotoxicity of p-aminophenol, or on binding of the compound in vivo to cell protein. p-[ring-3H]Aminophenol was bound in vitro to kidney microsomal protein and to a lesser extent to liver. The binding was enhanced by preincubation of the p-aminophenol in air and inhibited by ascorbate, GSH, N2 and NADPH. These findings indicate that in contrast to paracetamol hepatoxicity which is dependent upon the mixed function oxidase system, that nephrotoxicity of p-aminophenol is dependent upon oxidation to a toxic metabolite by some other pathway. A similar metabolite may be responsible for the nephrotoxic action of phenacetin.
Aminophenols↗