Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).
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PMID:The stringent response in Streptomyces coelicolor A3(2). 171 Mar 11

Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.
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PMID:Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product. 218 Sep 35

The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations. We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor. Termination at all but the 421 position depends on rho factor. In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site. These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs. Several stop points also correspond to 3' end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly. Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.
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PMID:Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene. 247 37

Cathepsin G is a 26,000-Da serine protease that is found in the azurophil granules of neutrophils and monocytes. The cathepsin G gene is expressed at high levels in U937 promonocytic cells, but is down-regulated with phorbol-induced differentiation. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic fibroblast library using cathepsin G cDNA, and obtained two lambda clones that contained the cathepsin G locus. The cathepsin G gene spans 2.7 kilobase pairs of genomic DNA and consists of 5 exons and 4 introns. The genomic organization of cathepsin G is similar to that of human neutrophil elastase, rat mast cell protease II, murine adipsin, and murine cytotoxic T-cell serine proteases, with protease catalytic residues located near the borders of exons 2, 3, and 5. Using in situ hybridization techniques, we localized cathepsin G to chromosome 14q11.2, a site that is near the alpha/delta T-cell receptor complex. Cathepsin G transcription is abolished in U937 nuclei with 2 micrograms/ml alpha-amanitin, indicating that this gene is probably transcribed by RNA polymerase II. The 5' end of the cathepsin G gene was defined by primer extension and S1 nuclease protection assays. A TATA box is found at position -29, and a CAAT box is found at -69 with respect to the transcription initiation site. Having defined the genomic structure and chromosomal location of cathepsin G, we are now attempting to identify the DNA elements in or near this gene that mediate its tissue and development-specific pattern of expression.
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PMID:Genomic organization and chromosomal localization of the human cathepsin G gene. 256 62

The gene (pai1) encoding human plasminogen activator inhibitor, PAI-1 was cloned from a lambda EMBL3 genomic library and was found to span approx. 12 kb and to contain eight introns. Primer extension and S1 nuclease analyses both showed the transcription start point to be located 142 nt upstream from the start codon. The 5'-flanking region was sequenced and found to contain a TATA box, but no CAAT sequence. When a fragment containing 730 nt of 5'-untranslated region was placed upstream from a promoterless cat gene, it was shown to function as a promoter when transfected into COS cells. Northern-blot analysis was consistent with low level expression of the endogenous pai1 gene in COS cells. When the pai1 gene structure was compared to those of other members of the serine-protease-inhibitor encoding gene family, little conservation of intron positions was observed.
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PMID:Structure and expression of the human gene encoding plasminogen activator inhibitor, PAI-1. 261 14

A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.
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PMID:Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts. 277 4

Thrombomodulin is an endothelial membrane anticoagulant protein that is a cofactor for protein C activation. We have evaluated the expression of thrombomodulin in cultured mouse hemangioma cells before and after treatment with phorbol myristate acetate (PMA), an agent that stimulates protein kinase C. We also isolated a cDNA encoding 481 amino acids of mouse thrombomodulin and the entire 3'-untranslated portion of its mRNA. The deduced amino acid sequence of mouse thrombomodulin is similar to those determined for human and bovine thrombomodulin. An S1 nuclease protection assay was used to measure thrombomodulin mRNA in hemangioma cells. The half-life for thrombomodulin mRNA was 8.9 +/- 1.8 h (S.D.) in cells treated with actinomycin D. Treatment with PMA had no effect on thrombomodulin mRNA levels. Thrombomodulin turnover was evaluated by immunoprecipitation of [35S]methionine-labeled thrombomodulin. The t1/2 was 19.8 +/- 3.9 h (S.D.); PMA treatment decreased the t1/2 to 10.9 +/- 1.1 h (S.D.) while increasing the rate of synthesis to a maximum of 190% of control. Protein C cofactor activity on hemangioma cells was reduced 35 +/- 4% by treatment with PMA within 30 min. This decrease was associated with a parallel decline in cell surface thrombomodulin antigen and with enhanced phosphorylation of thrombomodulin on serine residues. We conclude that thrombomodulin is phosphorylated in response to treatment of hemangioma cells with PMA which leads to decreased protein C cofactor activity and both increased degradation and synthesis of thrombomodulin.
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PMID:The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells. 284 23

Seminal vesicle F gene, encoding an androgen-regulated serine-rich structural protein of the rat copulatory plug, has been sequenced together with 5' and 3' flanking regions. The intron/exon arrangement of the gene deduced from restriction maps was confirmed. The major and possible minor transcriptional start points were located by primer extension analysis and S1 nuclease mapping. A published nucleotide sequence for seminal vesicle S gene which also encodes an androgen-regulated protein of the copulatory plug has been extended to allow comparison of F and S genes. The considerable sequence homology between the two genes confirms their evolutionary relatedness. Homology is especially high in their promoter regions and their transcriptional start points are identical. They share several regions of dyad symmetry including one just upstream of the promoter. The upstream regions of F and S genes were compared with those of five other androgen-responsive rodent genes in an attempt to identify common sequence motifs that might be involved in hormonal regulation of gene expression.
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PMID:Sequence organisation of rat seminal vesicle F gene: location of transcriptional start point and sequence comparison with six other androgen-regulated genes. 298 4

During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross-hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3'-flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process.
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PMID:Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids". 299 55

lambda gt11 cDNA libraries derived from human brain were screened with oligonucleotide probes for recombinants that code for alpha subunits of G signal transduction proteins. Eleven alpha s clones were detected with both probes and characterized. Four types of alpha s cDNA were cloned that differ in nucleotide sequence in the region that corresponds to amino acid residues 71-88. The clones differ in the codon for alpha s amino acid residue 71 (glutamic acid or aspartic acid), the presence or absence of codons for the next 15 amino acid residues, and the presence or absence of an adjacent serine residue. S1 nuclease protection experiments revealed at least two forms of alpha s mRNA. A mechanism for generating four species of alpha s mRNA by alternative splicing of precursor RNA is proposed.
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PMID:Human cDNA clones for four species of G alpha s signal transduction protein. 302 54


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