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 degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.
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PMID:Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques. 24 20

A procedure is described for the isolation of highly purified heavy-chain immunoglobulin mRNAs from a variety of mouse plasmacytomas (IgA, IgG, and IgM producers). The use of fresh tissue and the rapid isolation and direct extraction of membrane-bound polyribosomes were found to be essential in obtaining large quantities of undegraded heavy-chain mRNAs. The individual mRNAs were purified by two cycles of oligo(dT)-cellulose chromatography, sodium dodecyl sulfate--sucrose gradient centrifugation, and electrophoresis on 98% formamide containing polyacrylamide gels. When added to a cell-free protein-synthesizing system from wheat germ, the MPC-11 gamma2b and H2020 alpha heavy-chain mRNAs efficiently directed the synthesis of a predominant product of 55 000 molecular weight, while the synthesis of a 70 000 dalton protein in addition to other lower molecular weight polypeptides were observed with MOPC 3741 mu mRNA. All of these proteins were immunoprecipitable with class-specific heavy-chain antisera, and in the case of the gamma2b in vitro products good correspondence in a comparative trypsin--chymotrypsin fingerpring with in vivo labeled gamma2b heavy chain was observed. The gamma2b and a alpha heavy-chain mRNAs possessed a chain length of approximately 1800 nucleotides and the mu mRNA a size of approximately 2150 nucleotides when examined under stringent denaturation conditions. The purities of the alpha, gamma2b, and mu mRNAs were estimated to be 60--80%, 50--70%, and 50--83%, respectively, on the basis of their hybridization rates with cDNA probes in comparison to mRNA standards of known complexity. Heavy-chain mRNAs of the same class isolated from different mouse strains (Balb/C or NZB) display no detectable sequence differences in cross hybridization experiments, even though the cDNA--mRNA hybrids are submitted to stringent S1 nuclease digestion. These results indicate that allotypic determinants represent only a minor fraction of the heavy-chain constant region sequence in the mouse.
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PMID:Isolation, purification, and properties of mouse heavy-chain immunoglobulin mRNAs. 41 5

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.
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PMID:A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA. 42 57

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.
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PMID:Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali. 49 24

The bean rust fungus, Uromyces appendiculatus, undergoes thigmotropic differentiation to produce infection structures. Six differentiation-specific genes have been isolated and one, INF24, has been characterized [Bhairi et al., Gene 81 (1989) 237-243]. Here, we report the structure of a second gene, INF56, which was subcloned on a 2.6-kb fragment and sequenced. The location of the 1.0-kb INF56 transcript was determined by S1 nuclease protection and primer extension. A TATA box was found 38 bp upstream and a CAAT box 130 bp upstream from the major transcription start point (tsp). The gene contains two open reading frames: ORF2 is nested within ORF1; they share a 67-bp intron. ORF1 encodes a 14.1-kDa polypeptide which has an amino acid sequence rich in Gly, Pro and Ser. It has sequence similarity to a functional domain (V2) of mammalian cytokeratin type II. ORF2 encodes a 10.1-kDa polypeptide which is rich in Pro. It shares similarity with the cell-surface recognition region of chicken fibronectin. Hybrid selection and in vitro translation of the INF56 mRNA yielded two polypeptides of 15.5 and 23 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. INF56 is constitutively expressed at a low level, but the abundance of its steady-state transcript is upshifted 4.5 h after spore hydration during the period that infection structures are formed.
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PMID:Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus. 154 77

The immediate-early (IE) gene (IR1 gene) of equine herpesvirus 1 (EHV-1) encodes a single, spliced 6.0-kb mRNA during cytolytic infection. However, under early (in the presence of phosphonoacetic acid) and late (8 h postinfection; no metabolic inhibitors) conditions, in addition to the 6.0-kb IE mRNA, a 4.4-kb early (E) mRNA is transcribed from the IE gene region beginning at approximately 4 h postinfection. To map and characterize the 4.4-kb E mRNA and the protein product of this early gene (IR2 gene), Northern (RNA) blot hybridization, S1 nuclease, primer extension, and in vitro transcription and translation analyses were used. The data from RNA mapping analyses revealed that the 4.4-kb E IR2 mRNA (i) maps at nucleotides 4481 to 635 within each of the inverted repeats of the short region and thus is encoded by sequences that lie entirely within the IE gene, (ii) is transcribed in the same direction as the IE mRNA, initiating at nucleotide 4481, which lies 25 bp downstream of a putative TATA-like sequence and 1,548 bp downstream of the transcription initiation site of the IE mRNA, and (iii) is 3' coterminal with the IE mRNA which terminates at nucleotide 635 of the inverted repeats. The IR2 open reading frame was inserted into the transcription expression vector pGEM-3Z, and the RNA transcribed from this construct (pGEM44) was shown to be a 4.2-kb transcript that contained all IR2 sequences. In vitro translation of the 4.2-kb RNA yielded a major protein of approximately 130 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. This protein corresponds to the predicted IR2 product of 1,165 amino acids that would be in frame with the major IE polypeptide (IE1 = 200 kDa; 1,487 amino acids) and thus would be a 5'-truncated form of the IE1 polypeptide. The presence and potential role of the IR2 gene embedded within the IR1 gene increase the complexity of the regulation of the IE gene region during various stages of a productive infection.
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PMID:An early gene maps within and is 3' coterminal with the immediate-early gene of equine herpesvirus 1. 164 93

The gene encoding a Marek's disease virus (MDV) pp38 phosphoprotein has been identified, sequenced, and localized to the BamHI H fragment to the left of the putative MDV origin of replication. The open reading frame was defined by sequencing of a lacZ-pp38 fusion protein gene from a lambda gt11 expression library. The entire open reading frame is 290 amino acids long and codes for a protein with a calculated molecular weight of 31,169, compared with the size of 38 kDa of the phosphorylated form estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. S1 nuclease protection analysis showed that the pp38 gene is transcribed leftward as an unspliced mRNA. On the basis of transcriptional mapping studies, the pp38 transcript is predicted to be about 1.8 kb in length without a poly(A) sequence. Its promoter-enhancer region overlaps that of the major rightward BamHI H 1.8-kb transcript implicated in tumor induction. This region contains Oct-1, Sp1, and CCAAT motifs as well as the putative origin of replication. The pp38 protein is the only presently known antigen that is consistently associated with the transformation state. It may play a significant role in MDV transformation.
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PMID:Structural analysis and transcriptional mapping of the Marek's disease virus gene encoding pp38, an antigen associated with transformed cells. 165 57

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reclassification of Treponema hyodysenteriae and Treponema innocens in a new genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov. 170 92

Molecular methods have been applied to analyze the expression of the Alcaligenes eutrophus poly(3-hydroxybutyrate) (PHB) synthase gene (phbC). The translational initiation codon was identified by analysis of the amino acid sequence of a PHB synthase-beta-galactosidase fusion protein. This protein was purified to almost gel electrophoretic homogeneity by chromatography on DEAE-Sephacel and on aminophenyl-beta-D-thiogalactopyranoside-Sepharose from cells of A. eutrophus which harbored a phbC'-'lacZ fusion gene. A sequence (TTGACA-18N-AACAAT), exhibiting striking homology to the Escherichia coli sigma 70 promoter consensus sequence, was identified approximately 310 bp 5' upstream from the translation initiation codon. An S1 nuclease protection assay mapped the transcription start point of phbC 6 bp downstream from this promoter. The location of the promoter was confirmed by analyzing the expression of active PHB synthase in clones of E. coli harboring 5' upstream deletions of phbC ligated to the promoter of the lacZ gene (lacZp) in a Bluescript vector. Plasmids do181 and do218, which were deleted for the first 108 or 300 bp of the phbC structural gene, respectively, conferred the ability to synthesize large amounts of different truncated PHB synthase proteins to the cells. These proteins contributed to approximately 10% of the total cellular protein as estimated from sodium dodecyl sulfate-polyacrylamide gels. The modified PHB synthase encoded by plasmid do181 was still active. Clones in which the lacZp-'phbC fusion harbored the complete phbC structural gene plus the phbC ribosome binding site did not overexpress PHB synthase.
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PMID:Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. 198 16

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


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