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E T Young

Publications and source records attributed to E T Young.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Alcohol Dehydrogenase↗
PubMed1994

Intrapulmonary shunting causing hypoxaemia in a case of carcinoid syndrome.

A case of metastatic carcinoid syndrome with hypoxaemia is described. The hypoxaemia appeared to be due to intrapulmonary shunting. There was improvement in resting and exercise arterial oxygen saturations following chemotherapy with streptozotocin and 5-fluorouracil. This was followed by a decrease in the tumour bulk which may have led to a reduction in the secretion of the vasodilator products responsible for shunting. Some features of this case suggest that the hypoxaemia might have been due to dilatation of precapillary and capillary vessels of the pulmonary micro-circulation. These features are part of the so-called 'hepatopulmonary syndrome', of which this case appears to represent a less severe form.

Adult↗
PubMed1994

Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.

Alcohol Dehydrogenase↗
PubMed1993

ADH2 expression is repressed by REG1 independently of mutations that alter the phosphorylation of the yeast transcription factor ADR1.

In Saccharomyces cerevisiae, expression of the ADH2 gene is undetectable during growth on glucose. The transcription factor ADR1 is required to fully activate expression when glucose becomes depleted. Partial activation during growth on glucose occurred in cells carrying a constitutive allele of ADR1 in which the phosphorylatable serine of a cyclic AMP (cAMP)-dependent protein kinase phosphorylation site had been changed to alanine. When glucose was removed from the growth medium, a substantial increase in the level of this constitutive expression was observed for both the ADH2 gene and a reporter construct containing the ADR1 binding site. This suggests that glucose can block ADR1-mediated activation independently of cAMP-dependent phosphorylation at serine 230. REG1/HEX2/SRN1 was identified as a potential serine 230-independent repressor of ADH2 expression. Yeast strains carrying a deletion of the REG1 gene, reg1-1966, showed a large increase in ADR1-dependent expression of ADH2 during growth on glucose. A smaller increase in ADR1-independent expression was also observed. Additionally, an increase in the level of ADR1 expression and posttranslational modification of the ADR1 protein were observed. When the reg1-1966 allele was combined with various ADR1 constitutive alleles, the level of ADH2 expression was synergistically elevated. This indicates that REG1 can act independently of phosphorylation at serine 230. Our results suggest that glucose repression in the presence of ADR1 constitutive alleles occurs primarily through a REG1-dependent pathway which appears to affect ADH2 transcription at multiple levels.

Alcohol Dehydrogenase↗
PubMed1992

Identification and characterization of three genes that affect expression of ADH2 in Saccharomyces cerevisiae.

Using a new selection protocol we have identified and preliminarily characterized three new loci (ADR7, ADR8 and ADR9) which affect ADH2 (alcohol dehydrogenase isozyme II) expression. Mutants were selected which activate ADH2 expression in the presence of an over-expressed, normally inactive ADR1 allele. The mutants had very similar phenotypes with the exception that one was temperature sensitive for growth. In the absence of any ADR1 allele, the mutants allowed ADH2 to partially escape glucose repression. However, unlike wildtype strains deleted for ADR1, the mutants were able to efficiently derepress ADH2. The mutations allowed a small escape from glucose repression for secreted invertase, but had no effect on the glucose repression of isocitrate lyase or malate dehydrogenase. The mutations were shown to be nonallelic to a wide variety of previously characterized mutations, including mutations that affect other glucose-repressed enzymes.

Alcohol Dehydrogenase↗
PubMed1992

A mutation outside the two zinc fingers of ADR1 can suppress defects in either finger.

A second-site mutation that restored DNA binding to ADR1 mutants altered at different positions in the two zinc fingers was identified. This mutation (called IS1) was a conservative change of arginine 91 to lysine in a region amino terminal to the two zinc fingers and known from previous experiments to be necessary for DNA binding. IS1 increased binding to the UAS1 sequence two- to sevenfold for various ADR1 mutants and twofold for wild-type ADR1. The change of arginine 91 to glycine decreased binding twofold, suggesting that this arginine is involved in DNA binding in the wild-type protein. The increase in binding by IS1 did not involve protein-protein interactions between the two ADR1 monomers, nor did it require the presence of the sequences flanking UAS1. However, the effect of IS1 was influenced by the sequence of the first finger, suggesting that interactions between the region amino terminal to the fingers and the fingers themselves could exist. A model for the role of the amino-terminal region based on these results and sequence homologies with other DNA-binding motifs is proposed.

Alleles↗
PubMed1992

Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.

Amino Acid Sequence↗
PubMed1991

Alanine scanning site-directed mutagenesis of the zinc fingers of transcription factor ADR1: residues that contact DNA and that transactivate.

To identify functionally important amino acids in the two zinc fingers of transcription factor ADR1 [alcohol dehydrogenase (ADH) II synthesis regulator], oligonucleotide-directed mutagenesis was used to substitute alanine for the original amino acid at each position in both fingers. The effects of these mutations on DNA binding and thermal stability of ADR1 in vitro and on activation of ADH2 expression in vivo were measured. The DNA binding activity was remarkably heatstable. Amino acids that are candidates for DNA contact sites were identified in the finger-tip and alpha-helical region of each finger, three in the first finger and two in the second. Unexpectedly, an acidic residue in the first finger was essential for transactivation, but its replacement by alanine had no effect on DNA binding. Substitution at several highly conserved positions did not affect ADR1 functions. The ADR1 zinc fingers appear to make relatively few energetically significant contacts to DNA, perhaps as few as three in the first finger and one in the second.

Alanine↗
PubMed1991

Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

Alcohol Dehydrogenase↗
PubMed1990

Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure.

The "zinc finger" model [Miller, J., McLachlan, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614; Brown, R. S., Sander, C. & Argos, P. (1985) FEBS Lett. 186, 271-274] makes both specific structural and specific functional predictions about zinc finger consensus sequences that can be tested with a combination of genetic, molecular biological, and biophysical techniques. The yeast transcription factor ADR1 contains two adjacent zinc finger domains; genetic and deletion analyses showed that amino acid substitutions and deletions in the zinc finger domains resulted in the loss of protein activity. To test the structural and folding predictions of the zinc finger model, peptides encompassing each of the ADR1 fingers were synthesized (ADR1a and ADR1b) as well as a mutant finger peptide (del138) deleted for a single amino acid residue. The folding and metal-binding characteristics of these were assessed by 1H nuclear magnetic resonance (NMR) and visible spectroscopy. While a single unique conformational species was detected for the two wild-type peptides upon tetrahedral binding of zinc, the deletion peptide did not bind zinc with tetrahedral geometry, nor did it fold into a zinc finger domain. The metal-binding and folding results found with the mutant peptide were similar to those obtained when thiol alkylation or imidazole protonation of the wild-type peptides was performed. These data indicate that ligand spacing and both thiol and imidazole participation in zinc binding are specific and necessary requirements for zinc finger folding, which provides direct support for the initial predictions of the model.

Alkylation↗
PubMed1990

cAMP-dependent phosphorylation and inactivation of yeast transcription factor ADR1 does not affect DNA binding.

Transcription factor ADR1 increases the level of ADH2 gene expression 200-fold by binding to a palindromic upstream activation sequence (UAS1) in the glucose-repressible ADH2 promoter in Saccharomyces cerevisiae. cAMP-dependent protein kinase (cAPK) phosphorylates ADR1 in vitro and a yeast strain with elevated cAPK activity inhibits the ability of ADR1 to activate ADH2 transcription in vivo [Cherry, J. R., Johnson, T. R., Dollard, C., Schuster, J. R. & Denis, C. L. (1988) Cell 56, 409-419]. Intact ADR1 protein was detected at comparable levels in extracts made from repressed or derepressed yeast cells, indicating that glucose repression is not due to absence of ADR1. ADR1 in extracts made from glucose-repressed and -derepressed cells bound UAS1 DNA with similar affinities despite having greatly different abilities to activate ADH2 gene expression in vivo. A mutant form of ADR1 encoded by ADR1-5c, which has an altered consensus sequence for phosphorylation by cAPK conferred constitutive expression on ADH2 but bound DNA to the same extent as wild-type ADR1 protein. Similarly, normal DNA binding was seen for ADR1 produced in mutants with altered levels of cAPK activity. Because inactivation of ADR1 by phosphorylation has no detectable effect on either DNA binding or ADR1 levels, ADR1 probably binds to UAS1 constitutively and phosphorylation prevents it from promoting transcription.

Alcohol Dehydrogenase↗
PubMed1990

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

Alcohol Dehydrogenase↗
PubMed1989

Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae.

A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADR1-dependent activation upon a heterologous Saccharomyces cerevisiae promoter. UAS1 was nonfunctional when placed within an intron 3' to the transcription start site. The 11-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1. Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation. This result suggests that a specific orientation of sequences within UAS1 is necessary for ADR1-dependent activation. We determined that an ADR1-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional 11-bp half palindrome. However, the 11 bp did not confer UAS activity, suggesting that ADR1 binding is not sufficient for activation in vivo. ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences. We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYC1-lacZ 20-fold when combined with UAS1. UAS2 permitted ADR1-independent, glucose-regulated expression of the hybrid gene. Consistent with this observation, ADR1 did not form a detectable complex with UAS2. Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.

Alcohol Dehydrogenase↗
PubMed1989

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.

Acetylglucosaminidase↗
PubMed1989

Localization of a minimal binding domain and activation regions in yeast regulatory protein ADR1.

ADR1 is a transcription factor required for activation of the glucose-repressible alcohol dehydrogenase 2 (ADH2) gene in Saccharomyces cerevisiae. ADR1 has two zinc finger domains between amino acids 102 and 159, and it binds to an upstream activation sequence (UAS1) in the ADH2 promoter. A functional dissection of ADR1 was performed by using a series of amino- and carboxy-terminal deletion mutants of ADR1, most of which were fused to the Escherichia coli beta-galactosidase. These deletion mutants were assayed for binding to UAS1 in vitro, for the ability to activate ADH2 transcription in vivo, and for level of expression. Deletion of ADR1 amino acids 150 to 172 and 76 to 98 eliminated DNA binding in vitro, which accounted for the loss of transcriptional activation in vivo. Results with the former deletion mutant indicated that both of the ADR1 zinc fingers are necessary for sequence-specific DNA binding. Results with the latter deletion mutant suggested that at least part of the sequence between amino acids 76 to 98, in addition to the two finger domains, is required for high-affinity DNA binding. The smallest fusion protein able to activate ADH2 transcription, containing ADR1 amino acids 76 to 172, was much less active in vivo than was the longest fusion protein containing amino acids 1 to 642 of ADR1. In addition, multiple regions of the ADR1 polypeptide (including amino acids 40 to 76, 260 to 302, and 302 to 505), which are required for full activation of ADH2, were identified. An ADR1-beta-galactosidase fusion protein containing only the amino-terminal 16 amino acids of ADR1 was present at a much higher level than were larger fusion proteins, which suggested that the sequences within ADR1 influence the expression of the gene fusion.

Amino Acid Sequence↗
PubMed1989

Serial studies on the affinity and heterogeneity of human autoantibodies to thyroglobulin.

The functional affinity and heterogeneity of autoantibodies to thyroglobulin (Tg) were measured by an IgG subclass-specific solid-phase competition ELISA in patients with autoimmune thyroid disease. High-affinity IgG1 and IgG4 antibodies formed the major anti-Tg response. Both titre and affinity of IgG3 and IgG2 anti-Tg were generally low but in some Hashimoto's disease patients high-affinity IgG2 anti-Tg were found and IgG2 anti-Tg, unlike those of other subclasses, showed very restricted heterogeneity. The affinity of IgG4 anti-Tg was similar in patients with thyroid disease and their clinically euthyroid (normal) relatives. In contrast, a progressive increase in IgG1 anti-Tg affinity was seen in clinically euthyroid individuals compared with their relatives with thyroid disease and high titred Hashimoto's disease patients, suggesting that either rising titres of high affinity IgG1 anti-Tg or affinity maturation of IgG1 anti-Tg may be indicative of impending hypothyroidism.

Antibody Affinity↗
PubMed1988

The yeast ADR6 gene encodes homopolymeric amino acid sequences and a potential metal-binding domain.

The ADR6 gene of Saccharomyces cerevisiae has an open reading frame which could encode a polypeptide of 1314 amino acids. The predicted mRNA encodes a protein with homopolymeric stretches of asparagine and threonine, particularly near its amino terminus and contains additional sequences consisting of polyglutamine repeats. The predicted protein also contains a potential metal binding (Cys)4-type finger near its carboxy-terminus. An ADR6/beta-galactosidase fusion protein was predominantly nuclear in location, consistent with its role as an activator of ADH2 transcription.

Alcohol Dehydrogenase↗