14-3-3 (Bmh) proteins inhibit transcription activation by Adr1 through direct binding to its regulatory domain.

14-3-3 proteins, known as Bmh in yeast, are ubiquitous, highly conserved proteins that function as adaptors in signal transduction pathways by binding to phosphorylated proteins to activate, inactivate, or sequester their substrates. Bmh proteins have an important role in glucose repression by binding to Reg1, the regulatory subunit of Glc7, a protein phosphatase that inactivates the AMP-activated protein kinase Snf1. We describe here another role for Bmh in glucose repression. We show that Bmh binds to the Snf1-dependent transcription factor Adr1 and inhibits its transcriptional activity. Bmh binds within the regulatory domain of Adr1 between amino acids 215 and 260, the location of mutant ADR1(c) alleles that deregulate Adr1 activity. This provides the first explanation for the phenotype resulting from these mutations. Bmh inhibits Gal4-Adr1 fusion protein activity by binding to the Ser230 region and blocking the function of a nearby cryptic activating region. ADR1(c) alleles, or the inactivation of Bmh, relieve the inhibition and Snf1 dependence of this activating region, indicating that the phosphorylation of Ser230 and Bmh are important for the inactivation of Gal4-Adr1. The Bmh binding domain is conserved in orthologs of Adr1, suggesting that it acquired an important biological function before the whole-genome duplication of the ancestor of S. cerevisiae.

Evidence that human epididymal protein ARP plays a role in gamete fusion through complementary sites on the surface of the human egg.

Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca(2+) ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.

Evidence for a Graves' disease susceptibility locus at chromosome Xp11 in a United Kingdom population.

Graves' disease (GD), which has a strong female preponderance (female/male ratio, >5:1), is inherited as a complex genetic trait. Loci for GD have started to be defined using genome-wide approaches for genetic linkage. To date, 3 loci have been confirmed in at least 2 cohorts of GD patients, the strongest effect being at the cytotoxic T lymphocyte antigen-4 (CTLA-4) locus on chromosome 2q33 in our population. Two other loci for GD have recently been proposed, but not confirmed, on chromosomes Xq21 (GD3) and 14q31 (GD1). We studied a cohort of 75 sibling pairs with GD from the United Kingdom for linkage to 12 markers over a 83-cM region of the X chromosome and for 8 markers over a 36-cM region of 14q31-q33. A peak multipoint nonparametric linkage score of 2.21 (P = 0.014) was found at marker DXS8083 on Xp11, which increased to a nonparametric linkage score of 3.18 (P = 0.001) in data that had been conditioned for allele sharing at the CTLA-4 locus under an epistatic model. There was no evidence to support linkage of GD to Xq21.33-q22 (GD3) or at the 14q31-q33 (GD1) region in our population. A locus with a moderate contribution to GD susceptibility (lambda(s) = 1.4) is likely to exist in the Xp11 region, but we are unable to confirm that the GD1 or the GD3 regions contain major susceptibility loci in our United Kingdom GD population.

Trinucleotide repeats are clustered in regulatory genes in Saccharomyces cerevisiae.

The genome of Saccharomyces cerevisiae contains numerous unstable microsatellite sequences. Mononucleotide and dinucleotide repeats are rarely found in ORFs, and when present in an ORF are frequently located in an intron or at the C terminus of the protein, suggesting that their instability is deleterious to gene function. DNA trinucleotide repeats (TNRs) are found at a higher-than-expected frequency within ORFs, and the amino acids encoded by the TNRs represent a biased set. TNRs are rarely conserved between genes with related sequences, suggesting high instability or a recent origin. The genes in which TNRs are most frequently found are related to cellular regulation. The protein structural database is notably lacking in proteins containing amino acid tracts, suggesting that they are not located in structured regions of a protein but are rather located between domains. This conclusion is consistent with the location of amino acid tracts in two protein families. The preferred location of TNRs within the ORFs of genes related to cellular regulation together with their instability suggest that TNRs could have an important role in speciation. Specifically, TNRs could serve as hot spots for recombination leading to domain swapping, or mutation of TNRs could allow rapid evolution of new domains of protein structure.

Evidence for a new Graves disease susceptibility locus at chromosome 18q21.

Graves disease (GD) is a common autoimmune thyroid disorder that is inherited as a complex multigenic trait. By using a single microsatellite marker at each locus, we screened the type 1 diabetes loci IDDM4, IDDM5, IDDM6, IDDM8, and IDDM10 and the fucosyltransferase-2 locus for linkage in sib pairs with GD. This showed a two-point nonparametric linkage (NPL) score of 1.57 (P=.06) at the IDDM6 marker D18S41, but NPL scores were <1.0 at the other five loci. Thus, the investigation of the IDDM6 locus was extended by genotyping 11 microsatellite markers spanning 48 cM across chromosome 18q12-q22 in 81 sib pairs affected with autoimmune thyroid disease (AITD). Multipoint analysis, designating all AITD sib pairs as affected, showed a peak NPL score of 3.46 (P=.0003), at the marker D18S487. Designation of only GD cases as affected (74 sib pairs) showed a peak NPL score of 3.09 (P=.001). Linkage to this region has been demonstrated in type 1 diabetes (IDDM6), rheumatoid arthritis, and systemic lupus erythematosus, which suggests that this locus may have a role in several forms of autoimmunity.

Evolution of a glucose-regulated ADH gene in the genus Saccharomyces.

To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues. Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade. We cloned and characterized ADH promoters from S. bayanus, S. douglasii, and S. kluyveri. The ADH promoters from S. bayanus and S. douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract. The expression of a reporter gene driven by the S. bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S. cerevisiae. One S. kluyveri promoter was also glucose-repressed and ADR1-dependent in S. cerevisiae. The other S. kluyveri ADH promoter was expressed constitutively and was ADR1-independent. Although showing little sequence conservation with the S. cerevisiae ADH2 promoter, the glucose-repressed S. kluyveri promoter contains numerous potential binding sites for Adr1. The glucose-repressed ADH from S. kluyveri is a mitochondrial isozyme most closely related to S. cerevisiae ADHIII. ADR1 homologues from S. douglasii and S. paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S. cerevisiae and S. bayanus. No ADR1 homologue could be detected in S. kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved.

Association analysis of the cytotoxic T lymphocyte antigen-4 (CTLA-4) and autoimmune regulator-1 (AIRE-1) genes in sporadic autoimmune Addison's disease.

Although autoimmune Addison's disease (AAD) may occur as a component of the monogenic autoimmune polyendocrinopathy type 1 syndrome (APS1), it is most commonly found as an isolated disorder or associated with the autoimmune polyendocrinopathy type 2 syndrome (APS2). It is likely that sporadic (non-APS1) AAD is inherited as a complex trait; however, apart from the major histocompatibility complex, the susceptibility genes remain unknown. We have examined polymorphisms at two non-major histocompatibility complex candidate susceptibility loci in sporadic (non-APS1) AAD: the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene and the autoimmune regulator (AIRE-1) gene. DNA samples from AAD subjects (n = 90) and local controls (n = 144 for CTLA-4; n = 576 for AIRE-1) were analyzed for the CTLA-4A/G polymorphism in exon 1 of the CTLA-4 gene and for the common mutant AIRE-1 allele (964de113) in United Kingdom subjects with APS1, by using the restriction enzymes Bst7II and BsrBI, respectively. There was an association of the G allele at CTLA-4A/G in AAD subjects (P = 0.008 vs. controls), which was stronger in subjects with AAD as a component of APS2 than in subjects with isolated AAD. In contrast, the mutant AIRE-1 964del13 allele was carried in one each of the 576 (0.2%) control subjects and the 90 (1.1%) AAD subjects as a heterozygote (P = 0.254, not significant), suggesting that this common AIRE-1 gene abnormality does not have a major role in sporadic (non-APS1) AAD.

An accessory DNA binding motif in the zinc finger protein Adr1 assists stable binding to DNA and can be replaced by a third finger.

The DNA binding domain of Adr1, the protein derived from alcohol dehydrogenase regulatory gene 1, is unusual for zinc finger proteins in that it consists of two classical Cys2His2 zinc fingers and an amino-terminal proximal accessory region termed PAR. PAR is unstructured in the free protein and becomes structured in the DNA-bound form. We investigated the role of PAR in DNA binding using molecular and biochemical approaches, and its importance for activation in vivo, using Adr1-dependent reporter genes. PAR was unimportant for DNA binding when a third finger was added to Adr1, and its importance was diminished but not eliminated by mutations in finger two that increased DNA binding affinity. The kinetic rate constants for three Adr1 proteins containing or lacking PAR were determined by surface plasmon resonance. PAR increased the on rate and decreased the off rate for specific DNA sites for Adr1 containing wild-type fingers one and two. Surprisingly, PAR had no significant effect on the kinetic rate constants when a third finger was present, or when single-stranded DNA was used as the substrate for DNA binding. A mutant form of Adr1-F1F2 in which finger 2 makes three base-specific contacts with DNA had a higher affinity for DNA than Adr1 containing three fingers, yet the mutant protein still depended on PAR for optimal binding affinity. The ability to activate transcription in vivo was correlated with a low dissociation rate, suggesting that stabilizing an activator at the promoter might be rate-limiting for transcription in vivo. PAR may have evolved to lend additional stability to DNA-Adr1 complexes encompassing short binding sites. In addition, PAR may have a role in transcription at a step after DNA binding since deletion of PAR from Adr1 with three fingers decreased activation in vivo but had no effect on DNA binding kinetics.

Post-translational regulation of Adr1 activity is mediated by its DNA binding domain.

ADR1 encodes a transcriptional activator that regulates genes involved in carbon source utilization in Saccharomyces cerevisiae. ADR1 is itself repressed by glucose, but the significance of this repression for regulating target genes is not known. To test if the reduction in Adr1 levels contributes to glucose repression of ADH2 expression, we generated yeast strains in which the level of Adr1 produced during growth in glucose-containing medium is similar to that present in wild-type cells grown in the absence of glucose. In these Adr1-overproducing strains, ADH2 expression remained tightly repressed, and UAS1, the element in the ADH2 promoter that binds Adr1, was sufficient to maintain glucose repression. Post-translational modification of Adr1 activity is implicated in repression, since ADH2 derepression occurred in the absence of de novo protein synthesis. The N-terminal 172 amino acids of Adr1, containing the DNA binding and nuclear localization domains, fused to the Herpesvirus VP16-encoded transcription activation domain, conferred regulated expression at UAS1. Nuclear localization of an Adr1-GFP fusion protein was not glucose-regulated, suggesting that the DNA binding domain of Adr1 is sufficient to confer regulated expression on target genes. A Gal4-Adr1 fusion protein was unable to confer glucose repression at GAL4-dependent promoters, suggesting that regulation mediated by ADR1 is specific to UAS1.

Functional analysis of the yeast Glc7-binding protein Reg1 identifies a protein phosphatase type 1-binding motif as essential for repression of ADH2 expression.

In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

The cytotoxic T lymphocyte antigen-4 is a major Graves' disease locus.

Graves' disease (GD) is an autoimmune thyroid disorder that is inherited as a complex trait. We have genotyped 77 affected sib-pairs with autoimmune thyroid disease for eight polymorphic markers spanning the cytotoxic T lymphocyte antigen-4 ( CTLA-4 ) region of chromosome 2q31-q33, and for five markers spanning the major histocompatibility complex ( MHC ) region of chromosome 6p21. Non-parametric analysis showed linkage of GD to the CTLA-4 region with a peak non-parametric linkage (NPL) score of 3.43 ( P = 0.0004) at the marker D2S117. The proportion of affected full-sibs sharing zero alleles (z0) reached a minimum of 0.113 close to D2S117, giving a locus-specific lambdas for this region of 2.2. Families with brother-sister sib-pairs showed a peak NPL of 3.46 ( P = 0.0003, lambdas > 10) at D2S117, compared with 2.00 ( P = 0.02, lambdas = 1.9) in the families with only affected females, suggesting a stronger influence in families with affected males. Association between GD and the G allele of the Thr17Ala polymorphism within the CTLA-4 gene ( CTLA4A/G ) was observed using unaffected sib controls ( P = 0.005). Lesser evidence for linkage was found at the MHC locus, with a peak NPL score of 1.95 ( P = 0.026), between the markers D6S273 and TNFalpha. We demonstrate that the CTLA-4 locus (lambdas = 2.2) and the MHC locus (lambdas = 1.6) together confer approximately 50% of the inherited susceptibility to GD disease in our population.

Characterization of a p53-related activation domain in Adr1p that is sufficient for ADR1-dependent gene expression.

The yeast transcriptional activator Adr1p controls expression of the glucose-repressible alcohol dehydrogenase gene (ADH2), genes involved in glycerol metabolism, and genes required for peroxisome biogenesis and function. Previous data suggested that promoter-specific activation domains might contribute to expression of the different types of ADR1-dependent genes. By using gene fusions encoding the Gal4p DNA binding domain and portions of Adr1p, we identified a single, strong acidic activation domain spanning amino acids 420-462 of Adr1p. Both acidic and hydrophobic amino acids within this activation domain were important for its function. The critical hydrophobic residues are in a motif previously identified in p53 and related acidic activators. A mini-Adr1 protein consisting of the DNA binding domain of Adr1p fused to this 42-residue activation domain carried out all of the known functions of wild-type ADR1. It conferred stringent glucose repression on the ADH2 locus and on UAS1-containing reporter genes. The putative inhibitory region of Adr1p encompassing the protein kinase A phosphorylation site at Ser-230 is thus not essential for glucose repression mediated by ADR1. Mini-ADR1 allowed efficient derepression of gene expression. In addition it complemented an ADR1-null allele for growth on glycerol and oleate media, indicating efficient activation of genes required for glycerol metabolism and peroxisome biogenesis. Thus, a single activation domain can activate all ADR1-dependent promoters.

Meiotic inheritance of functional Gal80S gene product in Saccharomyces cerevisiae.

Transcription factors inherited during meiosis play a crucial role directing subsequent gene activity. Factors of maternal origin have been shown to influence the pattern of early zygotic transcription during Drosophila and Xenopus embryogenesis. Nevertheless, little is known regarding the meiotic inheritance of the vast majority of transcription factors. In the case of yeast meiosis, for example, it is not yet known whether any of the multitude of transcription factors expressed in the diploid are transmitted to haploid spores in functional form. Here we use a GAL1-STE4 reporter whose activity is detectable in single living cells to identify a transcription factor inherited during sporulation in Saccharomyces cerevisiae. We show that functional Gal80S repressor is meiotically inherited at levels reflecting its expression in the diploid parent.

Cyclic AMP-dependent protein kinase inhibits ADH2 expression in part by decreasing expression of the transcription factor gene ADR1.

In Saccharomyces cerevisiae, the unregulated cyclic AMP-dependent protein kinase (cAPK) activity of bcy1 mutant cells inhibits expression of the glucose-repressible ADH2 gene. The transcription factor Adr1p is thought to be the primary target of cAPK. Here we demonstrate that the decreased abundance of Adr1p in bcy1 mutant cells contributes to the inhibition of ADH2 expression. Activation of ADH2 transcription was blocked in bcy1 mutant cells, and UAS1, the Adr1p binding site in the ADH2 promoter, was sufficient to mediate this effect. Concurrent with this loss of transcriptional activation was an up to 30-fold reduction in the level of Adr1p. Mutating the strong cAPK phosphorylation site at serine 230 did not suppress this effect. Analysis of ADR1 mRNA levels and ADR1-lacZ expression suggested that decreased ADR1 transcription was responsible for the reduced protein level. In contrast to the ADH2 promoter, however, deletion analysis suggested that cAPK does not act through a discrete DNA element in the ADR1 promoter. The amount of Adr1p found in bcy1 mutant cells should have been sufficient to support 23% of the wild-type level of ADH2 expression. Since no ADH2 expression was detectable in bcy1 mutant cells, cAPK must also act by other mechanisms. Overexpression of Adr1p only partially restored ADH2 expression, indicating that some of these mechanisms may impinge upon events at or subsequent to the ADR1-dependent step in ADH2 transcriptional activation.

The incidence of diabetes mellitus in an English community: a 20-year follow-up of the Whickham Survey.

The original Whickham Survey documented the prevalence of diabetes and lipid disorders in a sample of 2779 adults aged 18 years and over, which matched the British population structure. The aim of the 20-year follow-up study was to determine the incidence and natural history of diabetes. Outcomes in terms of morbidity and mortality at follow-up were determined in over 97% of the original population. Ninety-four subjects had been identified and treated for diabetes since the first survey, including 17 subjects identified as having a fasting plasma glucose > or = 7.8 mmol l-1 at follow-up. The incidence of diabetes for the total population was 2.2 1000-1 year-1 (95% confidence interval 1.8, 2.6). The risk factors identified at first survey were corrected for age, cut-off at the 95 centile and entered into a log linear model. Those which strongly predicted development of diabetes in the total population were fasting blood glucose (odds ratio (OR) (with 95% confidence intervals) = 2.3 (1.5, 3.5)) and body mass index (OR = 2.2 (1.5, 3.3)) in men, and fasting blood glucose (OR = 2.6 (1.7, 4.1)) and fasting serum triglyceride (OR = 2.8 (1.8, 4.4)) in women. A logit model has enabled the calculation of the probability of developing diabetes 20 years later. It was the characteristics of becoming older such as obesity, hypertriglyceridaemia, and raised fasting blood glucose, rather than age itself, which were associated with the development of diabetes.

Isolation and identification of genes activating UAS2-dependent ADH2 expression in Saccharomyces cerevisiae.

Two cis-acting elements have been identified that act synergistically to regulate expression of the glucose-repressed alcohol dehydrogenase 2 (ADH2) gene. UAS1 is bound by the trans-activator Adr1p. UAS2 is thought to be the binding site for an unidentified regulatory protein. A genetic selection based on a UAS2-dependent ADH2 reporter was devised to isolate genes capable of activating UAS2-dependent transcription. One set of UAS2-dependent genes contained SPT6/CRE2/SSN20. Multicopy SPT6 caused improper expression of chromosomal ADH2. A second set of UAS2-dependent clones contained a previously uncharacterized open reading frame designated MEU1 (Multicopy Enhancer of UAS2). A frame shift mutation in MEU1 abolished its ability to activate UAS2-dependent gene expression. Multicopy MEU1 expression suppressed the constitutive ADH2 expression caused by cre2-1. Disruption of MEU1 reduced endogenous ADH2 expression about twofold but had no effect on cell viability or growth. No homologues of MEU1 were identified by low-stringency Southern hybridization of yeast genomic DNA, and no significant homologues were found in the sequence data bases. A MEU1/beta-gal fusion protein was not localized to a particular region of the cell. MEU1 is linked to PPR1 on chromosome XII.

The development of ischemic heart disease in relation to autoimmune thyroid disease in a 20-year follow-up study of an English community.

The original Whickham Survey documented risk factors for cardiovascular disease and the prevalence of thyroid disorders in a sample of 2779 adults that closely matched the British population. A 20-year follow-up study has determined outcomes in terms of morbidity and mortality from ischemic heart disease in over 97% of the original survey population. Analysis of deaths from all causes and from ischemic heart disease showed no association with antithyroid antibody status identified at first survey. A multiple logistic regression using the development of ischemic heart disease in the total population at follow-up as the dependent variable found that the significant predictor variables for men were age, cholesterol, mean arterial blood pressure, smoking history, and skinfold thickness index. For women only age, cholesterol, and mean arterial blood pressure were significant. The presence of autoimmune thyroid disease, as defined by either hypothyroidism, positive antithyroid antibodies, or raised serum thyrotropin at first survey, was not significant. A retrospective cohort study of a subsample of women identified at first survey with positive antithyroid antibodies or raised serum thyrotropin and closely matched controls found no significant association with mortality or development of ischemic heart disease. There is no evidence from this study to suggest that evidence of autoimmune thyroid disease identified 20 years ago is associated with an increased risk of ischemic heart disease.

A single amino acid substitution in zinc finger 2 of Adr1p changes its binding specificity at two positions in UAS1.

The two zinc fingers of the yeast transcription factor Adr1p recognize the 6 bp sequence TTG GAG site in which the first finger, a His-X3-His finger, recognizes the G-rich triplet (GAG) and the second zinc finger, a His-X4-His finger, recognizes the T-rich sequence (TTG). Mutations were introduced into the alpha-helical region of the second finger and the resultant mutant proteins were analyzed for DNA binding affinity and specificity in vitro and in vivo. Substituting His for Leu in the third position (+3) of the helix created a new binding specificity at two positions in the binding site. The mutant with His replacing Leu146(L146H) bound with high affinity to GGG GGG and with low affinity to TTG GGG. The single substitution at position +3 in the helix had the same effect on DNA binding specificity as substitution of the whole helix of the second finger with the helix of finger one. Changing Asp145 to Ala in the presence of His146 changed the apparent binding site of finger 2 to GT/CG. The L146H mutant zinc finger protein had the same binding specificity in vivo as in vitro. Changing the spacing between the His residues that ligand zinc in the second finger from four to three, the spacing found in the first finger of Adr1p and Zif268, did not alter the specificity or affinity of the wild-type or mutant protein.

Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.