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)

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.
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PMID:Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum. 285 70

A cDNA library was efficiently synthesized from mouse neuroblastoma poly(A)+RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3'-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed Pst I cut pBR322. E. coli K12 RR1 cells were transformed and 2.5-5 X 10(6) transformants per microgram cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blunt ends to be tailed synchronously. This allowed a higher transformation efficiency without loss of 5'-sequences. (2) Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.
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PMID:High-efficiency cloning of DNA sequences complementary to mouse neuroblastoma polyadenylated RNA. 286 33

Transcriptional regulation of the delta-aminolevulinic acid synthetase gene of Rhizobium meliloti was investigated under conditions of normal vegetative growth and during symbiosis with the legume host alfalfa. S1 nuclease mapping and DNA sequence analysis indicated that transcription originates from two sites separated by 238 base pairs. A deletion analysis of the putative promoter regions P1 and P2, corresponding to the proximal and distal RNA start sites, was carried out with Bal-31 nuclease. Promoter function was monitored as beta-galactosidase activity after fusing the deletions to lac Z and introducing them into Rhizobium on a broad host range plasmid. The data obtained suggest that both regions function equivalently as promoters. The DNA sequences of P1 and P2 show considerable homology in the region between -35 and the start of transcription. Both contain a -35 region that is analogous to the consensus E. coli promoter sequence, while the -10 region is dissimilar. No resemblance was found between either P1 or P2 and the promoter regions of genes under general nitrogen control.
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PMID:Analysis of the 5' regulatory region of the gene for delta-aminolevulinic acid synthetase of Rhizobium meliloti. 299 20

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Monoclonal antibody F7-26 was obtained by fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with DNA treated by nitrogen mustard (HN2). Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. Two-parameter analysis for the antibody binding and DNA content showed no binding of antibody to replicating DNA in control cells. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Binding of monoclonal antibody specific for the determinants expressed on ssDNA to the cells treated with alkylating agents may be attributed to local DNA denaturation. Potentiation of L-phenylalanine mustard cytotoxicity by buthionine sulfoximine or hyperthermia was accompanied by increased antibody binding to cellular DNA. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.
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PMID:Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody. 303 52

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.
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PMID:Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12. 330 36

We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes. Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence. Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used. Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium.
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PMID:Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter. 341 Mar 21

The xylABC promoter (OP1), located on the TOL plasmid of Pseudomonas putida contains sequences homologous to the conserved regions found in nitrogen fixation (nif) promoters and in other promoters subject to nitrogen control. XylA-lac fusions were constructed in order to monitor expression from the OP1 promoter in Escherichia coli. Transcription was activated in the presence of the heterologous regulatory genes ntrC or nifA from Klebsiella pneumoniae as well as by the homologous P. putida regulatory gene xylR. In all cases activation was also dependent on the ntrA gene, whose product has been implicated as a specific sigma factor for ntr activatable operons. The 5' ends of xylA mRNA, detected by S1 nuclease mapping of in vivo transcripts, were identical in strains containing xylR, ntrC or nifA as transcriptional activators. However, activation of the K. pneumoniae nifL or nifH promoters by xylR was not detected.
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PMID:The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. 352 Feb 41

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.
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PMID:Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene. 353 23

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.
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PMID:Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells. 378 Aug 72

We sequenced the 274-nucleotide intercistronic glnA-glnL region of Escherichia coli to localize regulatory regions postulated from genetic evidence. The transcriptional start of glnLp, identified by S1 nuclease mapping, preceded the structural gene by 32 bases. NR1, the glnG gene product and a repressor of glnLp, protected from DNase digestion a region of DNA between -12 and +15 from the transcriptional start. A mutation rendering glnLp insensitive to NRI was within the protected region in a-TGCA- sequence found in all nitrogen-regulated operons, providing evidence for involvement of this sequence in interactions with NRI. We also observed in the intercistronic region a potential rho-independent terminator preceding glnLp and a sequence previously found in other intercistronic regions.
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PMID:Identification and regulation of the glnL operator-promoter of the complex glnALG operon of Escherichia coli. 614 34


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