Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To obtain cDNAs for analysis of mucin gene transcription in rat models of human disease, we screened a rat intestinal cDNA library in lambda ZAPII using an upstream non-tandem repeat cDNA fragment of the human MUC 2 gene (Gum, J., Hicks, J., Toribara, N., Rothe, E., Lagace, R., and Y., K. (1992) J. Biol. Chem. 267, 21375-21383). Three cDNAs, 1-1, 8-1, and 21-1, were isolated. A translation start site was found in cDNA 21-1. Combined nucleotide sequence for the three cDNAs contained an open reading frame spanning 4546 base pairs. This amino-terminal sequence contains a non-tandem repeat domain enriched in cysteine (1391 residues) followed by an irregular tandem repeat domain (122 residues). Identity with the human gene is about 80% in the non-tandem repeat domain and about 38% in the irregular tandem repeat domain. Primer extension and S1 nuclease protection analysis indicate a transcription start site at 28 base pairs upstream of translation initiation. Northern analysis showed expression of cognate RNA in the intestine and airway but not heart and spleen. The cDNAs have been used to isolate the gene promoter, the structure of which should yield clues to the regulation of mucin expression in rat models of human disease.
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PMID:Molecular cloning of the amino-terminal region of a rat MUC 2 mucin gene homologue. Evidence for expression in both intestine and airway. 802 37

A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.
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PMID:S1 nuclease: immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site. 861 49

The dsz gene cluster of Rhodococcus erythropolis IGTS8 comprises three genes, dszA, dszB, and dszC, whose products are involved in the conversion of dibenzothiophene (DBT) to 2-hydroxybiphenyl and sulfite. This organism can use DBT as the sole sulfur source but not as a carbon source. Dsz activity is repressed by methionine, cysteine, Casamino Acids, and sulfate but not by DBT or dimethyl sulfoxide. We cloned 385 bp of the DNA immediately 5' to dszA in front of the reporter gene lacZ of Escherichia coli. We showed that this region contains a Rhodococcus promoter and at least three dsz regulatory regions. After hydrazine mutagenesis of this DNA, colonies that were able to express beta-galactosidase in the presence of Casamino Acids were isolated. Sequencing of these mutants revealed two possible regulatory regions. One is at -263 to -244, and the other is at -93 to -38, where -1 is the base preceding the A of the initiation codon ATG of dszA. An S1 nuclease protection assay showed that the start of the dsz promoter is the G at -46 and that transcription is repressed by sulfate and cysteine but not by dimethyl sulfoxide. The promoter encompasses a region of potential diad symmetry that may contain an operator. Immediately upstream of the promoter is a protein-binding domain between -146 and -121. Deletion of this region did not affect repression, but promoter activity appeared to be reduced by threefold. Thus, it could be an activator binding site or an enhancer region.
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PMID:Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8. 893 95

The bld mutants of Streptomyces coelicolor A3(2) are blocked at the earliest stage of sporulation, the formation of aerial hyphae, and are pleiotropically defective in antibiotic production. Using a phage library of wild-type S. coelicolor DNA, we isolated a recombinant phage which restored both sporulation and antibiotic production to strains carrying the single known bldD mutation. Nucleotide sequence analysis of a 1.3-kb complementing subclone identified an open reading frame, designated bldD, encoding a translation product of 167 amino acid residues. Nucleotide sequence analysis of the bldD-containing fragment amplified from the chromosome of a bldD mutant strain revealed a point mutation changing a tyrosine residue at amino acid position 62 to a cysteine. Although a comparison of the BldD sequence to known proteins in the databases failed to show any strong similarities, analysis of the BldD sequence for secondary structural elements did reveal a putative helix-turn-helix, DNA recognition element near the C terminus of the protein. A comparison of bldD transcript levels in the bldD+ and bldD mutant strains using both Northern blot analysis and S1 nuclease protection studies showed vast overexpression of bldD transcripts in the mutant, suggesting that BldD negatively regulates its own synthesis. High-resolution S1 nuclease mapping identified the transcription start point as a G residue 63 nucleotides upstream from the bldD start codon and 7 nucleotides downstream from -10 and -35 sequences resembling E. coli-like streptomycete promoters.
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PMID:The bldD gene of Streptomyces coelicolor A3(2): a regulatory gene involved in morphogenesis and antibiotic production. 951 25

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.
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PMID:Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity. 1508 52


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