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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The staphylococcal nuclease A gene has been successfully cloned and overexpressed in E. coli under the transcriptional control of the bacteriophage lambda PRPL promoters regulated by the temperature sensitive repressors. The SDS-PAGE analysis demonstrates that the nuclease A is produced to the extent of as much as 60% of the total cellular protein. The N-terminal analysis of the nuclease A shows that the amino terminal formyl methionine residue of the enzyme is precisely processed. The recombinant nuclease A with full activity is finally obtained after appropriate solubilization--denaturation and renaturation treatment. The conformational identity of the renatured nuclease A in different conditions is also studied by using hydrophobic interaction chromatography on a phenyl-superose HR5/5 column.
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PMID:High-level expression of staphylococcal nuclease A in Escherichia coli. 799 69

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA. 803 23

Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.
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PMID:Overexpression and purification of avian ovomucoid third domains in Escherichia coli. 847 48

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit.
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PMID:Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor 2-associated 67-kDa protein (p67). 849 45

Telomeres of vertebrate chromosomes terminate with a short 5'-d(TTAGGG)-3' single-stranded overhang that can form in vitro tetrahelical structures. Here we describe a new protein from rat hepatocyte nuclei designated quadruplex telomere-binding protein 42 (qTBP42) that tightly binds 5'-d(TTAGGG)n-3' and 5'-d(CCCTAA)n-3' single-stranded and tetraplex forms of 5'd(TTAGGG)n-3'. The thermostable qTBP42 was isolated from boiled nuclear extracts and purified to near homogeneity by successive steps of column chromatography on DEAE-cellulose, phosphocellulose, and phenyl-Sepharose. A subunit molecular size of 42.0 +/- 2.0 kDa was determined for qTBP42 by Southwestern blotting and SDS-polyacrylamide gel electrophoresis of the protein and its UV cross-linked complex with labeled telomeric DNA. A native size of 53. 5 +/- 0.9 kDa, estimated by Superdex copyright 200 gel filtration, suggests that qTBP42 is a monomeric protein. Sequences of five tryptic peptides of qTBP42 contained motifs shared by a mammalian CArG box-binding protein, hnRNP A/B, hnRNP C, and a human single-stranded telomeric DNA-binding protein. Complexes of qTBP42 with each complementary strand of telomeric DNA and with quadruplex forms of the guanine-rich strand had 3.7-14.6 nM dissociation constants, Kd, whereas complexes with double-stranded telomeric DNA had up to 100-fold higher Kd values. By associating with tetraplex and single-stranded telomeric DNA, qTBP42 increased their heat stability and resistance to digestion by micrococcal nuclease.
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PMID:Purification and characterization of qTBP42, a new single-stranded and quadruplex telomeric DNA-binding protein from rat hepatocytes. 902 Jan 72

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.
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PMID:Archaeal nucleosomes. 935 1

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.
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PMID:Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system. 1083 3

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.
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PMID:Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity. 1127 37

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.
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PMID:Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. 1142 31

Residual dipolar couplings reflect the orientation of vectors between pairs of magnetic nuclei relative to a unique set of molecular axes. Thus, unlike NOEs and scalar couplings, dipolar couplings provide access to long-range structural information. A prerequisite for measurement of these NMR parameters is imposition of a weak net alignment, most simply by forcing the macromolecules to tumble in an asymmetric environment that restricts some orientations more than others. In this report, several denatured forms of staphylococcal nuclease are aligned by using compressed and stretched polyacrylamide gels, a nonionic type of lipid bilayer disk or bicelle, and a liquid crystalline phase formed by a cationic lipid. All three types of media can be used at high urea concentrations. While polyacrylamide gels and bicelles produce similar alignment tensors through steric interactions, a liquid crystalline phase of cetylpyridinium bromide aligns denatured nuclease along a different set of axes, presumably through electrostatic effects. The analysis of residual dipolar couplings collected with two different alignment tensors may permit the calculation of ensembles of conformations. The dipolar couplings observed for staphylococcal nuclease denatured with urea, by low pH or by deletion of residues from both termini, suggest that all denatured forms share a common "topology", one which has been shown previously to be native-like. Although SDS/nuclease complexes give sharp and disperse (1)H-(15)N correlation spectra, only small couplings are observed in strained polyacrylamide gels.
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PMID:Molecular alignment of denatured states of staphylococcal nuclease with strained polyacrylamide gels and surfactant liquid crystalline phases. 1186 48


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