The detection of sequence variation, for which DNA sequencing has emerged as the most sensitive and automated approach, forms the basis of all genetic analysis. Here we describe and illustrate an algorithm that accurately detects and genotypes SNPs from fluorescence-based sequence data. Because the algorithm focuses particularly on detecting SNPs through the identification of heterozygous individuals, it is especially well suited to the detection of SNPs in diploid samples obtained after DNA amplification. It is substantially more accurate than existing approaches and, notably, provides a useful quantitative measure of its confidence in each potential SNP detected and in each genotype called. Calls assigned the highest confidence are sufficiently reliable to remove the need for manual review in several contexts. For example, for sequence data from 47-90 individuals sequenced on both the forward and reverse strands, the highest-confidence calls from our algorithm detected 93% of all SNPs and 100% of high-frequency SNPs, with no false positive SNPs identified and 99.9% genotyping accuracy. This algorithm is implemented in a software package, PolyPhred version 5.0, which is freely available for academic use.

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.

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 glucosecontaining medium is similar to that present in wildtype 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.Proteins that regulate transcription are themselves regulated in a variety of ways. Post-translational mechanisms, including covalent alterations such as phosphorylation/dephosphorylation, are common ways of controlling the activity of transcription factors that mediate environmental influences where the response is rapid or transient (1).In the yeast Saccharomyces cerevisiae, Adr1 is the principal transcriptional activator of the glucose-repressible alcohol dehydrogenase (ADH2) gene (2-4). Adr1 also regulates the expression of genes involved in glycerol metabolism (5) and in peroxisome function and biogenesis (6, 7). These ADR1-regulated genes are not expressed when yeasts are growing in the presence of glucose, but are turned on when glucose is exhausted.At the ADH2 promoter, activation of gene expression requires the binding of two monomers of Adr1 to a 22-base pair dyad-symmetric sequence designated UAS1.1 Two Cys 2 -His 2 -type zinc fingers and the region immediately preceding the fingers, make up its DNA binding domain, designated ABD (8 -10). Adr1 appears to contain multiple transcription activation domains (9,11,12) that interact with components of a histone acetyltransferase complex, TFIIB, and components of TFIID (13,14). A regulatory region of Adr1, responsible for glucose repression, has not been identified (11,12). Glucose repression in yeast involves multiple mechanisms. The best understood of these requires three genes: MIG1, encoding a DNA-binding protein, TUP1, and SSN6. A Tup1-Ssn6 complex is recruited by Mig1 to the promoter of glucose-repressed genes, where it blocks transcription (15-17). Re...

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.Activation domains of transcription factors transmit signals to the transcriptional machinery of a cell to ensure proper gene expression. When tethered to a DNA binding domain, either covalently or via protein-protein interaction, they ensure that signals are transmitted to appropriate genes to activate their transcription. Although the specificity of gene expression is determined primarily by the DNA binding domain of transcription factors, activation domains can contribute to this specificity (1-4).Activation domains function by contacting other proteins that are components of the transcriptional machinery (5). The proteins contacted by activation domains include various subunits of TFIID, including TATA-binding protein itself and TATA-binding protein-associated factors, and other general transcription factors such as TFIIB, TFIIH, and members of adaptor or mediator complexes. By contacting these proteins, they recruit RNA polymerase II to the promoter and facilitate initiation and elongation of transcription (5-11).ADR1 encodes a post-translationally regulated transcription factor in the yeast Saccharomyces cerevisiae that activates the expression of the glucose-repressed ADH2 gene as well as genes involved in glycerol metabolism and peroxisome biogenesis (12-23). As with many eukaryotic transcription factors, Adr1p contains multiple domains. The ADR1 DNA binding domain (ABD) 1 is encompassed by amino acids ϳ70 -165 (16). A nuclear targeting signal is found in the first 21 amino acids of the protein (24). Four transcr...