Biomedical subjects

E J Grayhack

Publications and source records attributed to E J Grayhack.

8 recordsLinked to original sources

PubMed2001

Genomic analysis of biochemical function.

Establishing the linkage between an individual biochemical activity and the gene(s) specifying that activity has been facilitated by advances in mass spectrometry and affinity purification methods. In addition, a genomic protein array has been produced in yeast by fusing each yeast open reading frame to glutathione-S-transferase, thus linking each protein with its cognate gene. Purification and biochemical assay of pools of glutathione-S-transferase-open-reading-frame proteins allows analysis of the entire proteome for biochemical activities, followed by simple deconvolution to identify the responsible open reading frame. An alternative method to analyze large sets of proteins is the use of protein microarrays in which over 10,000 individual proteins can be immobilized and assayed on a single slide.

Animals↗

PubMed1999

A biochemical genomics approach for identifying genes by the activity of their products.

For the identification of yeast genes specifying biochemical activities, a genomic strategy that is rapid, sensitive, and widely applicable was developed with an array of 6144 individual yeast strains, each containing a different yeast open reading frame (ORF) fused to glutathione S-transferase (GST). For the identification of ORF-associated activities, strains were grown in defined pools, and GST-ORFs were purified. Then, pools were assayed for activities, and active pools were deconvoluted to identify the source strains. Three previously unknown ORF-associated activities were identified with this strategy: a cyclic phosphodiesterase that acts on adenosine diphosphate-ribose 1"-2" cyclic phosphate (Appr>p), an Appr-1"-p-processing activity, and a cytochrome c methyltransferase.

Adenosine Diphosphate Ribose↗

PubMed1997

Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated.

Acid Phosphatase↗

PubMed1992

The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site.

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

Base Sequence↗

PubMed1987

Transcription antitermination by phage lambda gene Q protein requires a DNA segment spanning the RNA start site.

The gene Q protein of phage lambda is a transcription antiterminator that modifies RNA polymerase near the phage late gene promoter and thereby causes antitermination at distant sites. To define the site of action of Q protein, we have reconstructed the regulatory system on plasmids that allow the intracellular concentration of Q protein to be regulated, and that allow the effect of Q protein on transcription from variant promoter segments to be measured in vivo and in vitro. We show that DNA sequences essential for Q protein-mediated antitermination span the RNA start site, but do not extend beyond nucleotide 18 of the late RNA coding region. We also show that the modification that permits antitermination persists while RNA polymerase passes at least two terminators in vivo and in vitro.

Bacteriophage lambda↗

PubMed1985

Phage lambda gene Q antiterminator recognizes RNA polymerase near the promoter and accelerates it through a pause site.

The positive regulator encoded by phage lambda gene Q is a transcription antiterminator that affects RNA polymerase initiating at the phage late gene promoter, but not at other promoters. We show that this nucleotide-sequence-specific interaction of Q protein and RNA polymerase can occur while the enzyme is pausing after 16 nucleotides of the late gene transcript have been made. Furthermore, Q protein chases RNA polymerase from this early pause site, so that it both recognizes the enzyme and changes its transcription properties at this site. We suggest that the ability of Q-modified RNA polymerase to escape this pause reflects the change that allows it to go through terminators. We also show that NusA protein is required for efficient Q protein activity in vitro.

Bacterial Proteins↗

PubMed1983

Purification of the bacteriophage lambda late gene regulator encoded by gene Q.

The product of bacteriophage lambda gene Q is a transcription antiterminator that activates phage late gene expression. We report a method to purify the lambda Q protein to near homogeneity. We have followed during purification both activity of Q protein to provoke in vitro synthesis of the lambda late protein endolysin in the DNA-dependent protein-synthesizing system, and radioactivity in a polypeptide that we show by genetic criteria to be lambda Q. These co-purify throughout the procedure.

Bacteriophage lambda↗

PubMed1982

The phage lambda Q gene product: activity of a transcription antiterminator in vitro.

Phage lambda gene Q encodes a positive regulator of phage late gene expression. We show that purified Q gene protein functions as a transcription antiterminator in vitro. It acts at the terminator for lambda 6S RNA, a small constitutive transcript that precedes the late genes, thus opening transcription into the entire late gene region. It is active in a purified transcription system consisting otherwise of only RNA polymerase, L factor and DNA containing the region encoding the 6S RNA. Q protein thus acts directly on the transcription complex to alter its response to transcription terminators.

Bacteriophage lambda↗