Biomedical subjects

J S Sloan

Publications and source records attributed to J S Sloan.

7 recordsLinked to original sources

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

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.

Alcohol Dehydrogenase↗

PubMed1998

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.

Alcohol Dehydrogenase↗

PubMed1991

Characterization of genes encoding hydroxypyruvate reductase in cucumber.

Several clones corresponding to the gene encoding NADH-dependent hydroxypyruvate reductase have been isolated from a cucumber genomic library. Restriction mapping indicates the presence of two HPR genes, hpr-A and hpr-B, in the cucumber genome. Examination of the DNAs of individual plants suggests that hpr-A and hpr-B are most likely alleles at a single locus. The sequence of a 6.7 kb genomic fragment that includes the entire transcribed region, 2.2 kb of 5' flanking sequence, and about 0.8 kb of 3' flanking sequence reveals the presence of 12 introns in hpr-A. These introns are AT-rich relative to the exons. The donor sequence at the 5' end of the sixth intron contains an unusual dinucleotide, GC, rather than the nearly invariant GT. Primer extension analysis maps the transcription initiation site to 61 nucleotides upstream of the translation initiation codon. An AT-rich stretch is centered at position -31 with respect to the transcription initiation site, and a potential CCAAT box is centered at position -138. Several elements that are homologous to regulatory elements of other plant genes have been identified in the flanking regions of hpr-A.

Alcohol Oxidoreductases↗

PubMed1989

Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber.

A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a lambda gt11 cDNA library made by reverse transcription of poly(A)+ RNA from cucumber cotyledons. In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A)+ RNA and subsequent immunoprecipitation with HPR antiserum. Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity. RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development. This closely resembles the pattern seen for HPR-specific translatable mRNA. DNA blot analysis indicated that a single HPR gene is likely present per haploid genome. Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa. The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E. coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway. Statistical analyses show that this similarity is significant (z greater than 10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

Alcohol Oxidoreductases↗

PubMed1986

Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila.

The Drosophila melanogaster Gart locus, known from previous work to encode the enzyme activity phosphoribosylglycinamide formyltransferase (GART), specifies two alternatively processed mRNAs and two proteins. We introduced the entire Gart locus into a Drosophila tissue culture cell line in which the locus is active. The resulting cell clones contained numerous copies of the locus and overproduced both mRNAs and both expected proteins, thus markedly facilitating analysis of these molecules. We assayed extracts of the clones for the activities of 10 enzymes important for de novo purine synthesis and found that, in addition to GART, two other purine pathway activities, phosphoribosylamine-glycine ligase (phosphoribosylglycinamide synthetase, GARS) and phosphoribosylformylglycinamidine cyclo-ligase (phosphoribosylaminoimidazole synthetase, AIRS), are similarly overproduced. All three activities are present together on the larger overproduced protein. A smaller protein appears to possess only GARS activity. Therefore, alternative mRNA processing can allow cells to produce enzyme activities in forms that are either linked or unlinked to other activities.

Acyltransferases↗

PubMed1983

A Drosophila metabolic gene transcript is alternatively processed.

We have determined the organization and transcription of a Drosophila DNA segment coding for the purine pathway enzyme, GAR transformylase. Because the transcript encoding this enzyme is very rare, we used a novel method for exon mapping without isolating a cDNA. Our results suggest a long polypeptide having multiple domains, with GAR transformylase at the COOH terminus preceded by an extensive repeat. Part of the same DNA segment specifies a shorter transcript, which consists of the same 5' exons but lacks the last three 3' exons. A polyadenylation signal within an intron allows this single gene to encode two polypeptides. We hypothesize that alternative processing of a transcript is a basis for channeling metabolic intermediates into two different pathways.

Acyltransferases↗