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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)

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.
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PMID:Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease. 90 77

Superhelical [3-H]DNA (replicative form I, RFI) of bacteriophage phiX174 slowly but spontaneously took up 32-P-labeled homologous single-stranded fragments at 4 degrees. Uptake was accelerated by heating to 75 degrees. RFI did not take up single-stranded fragments derived from DNA of Escherichia coli or from separated strands of phage lambda. Uptake was inhibited by low concentrations of ethidium bromide. Relaxed circular phiX174 DNA did not take up homologous fragments. Per molecule of RFI, the complexes contained as much as 90 nucleotide residues of homologous fragment. The 32-P-lebeled fragments were largely resistant to digestion by exonuclease I, and were not displaced by heating complexes at 60 degrees for 1 min in 16 mM or 100 mM NaCl. Under comparable conditions of temperature and salt all of the fragments were displaced from complexes in which at least one phosphodiester bond was cleaved by pancreatic DNase, but a significant fraction of the fragments was retained in complexes that were relaxed by digestion with S1 nuclease. These observations are interpreted to mean that S1 nuclease digested the plus (viral) strand of the recipient RF at the site of uptake in some instances. Transfection of E. coli by heterozygous complexes produced recombinant progeny, thereby showing that genetic information can be transferred from the fragment of plus strand to progeny plus strands. We propose that both uptake of a third strand by superhelical DNA and the action of nucleases on the resulting complex may simulate early steps in genetic recombination.
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PMID:Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination. 109 67

Chromatography on BD-cellulose columns with a salt gradient and formamide separates cellular DNA into two fractions (fraction I eluted within the salt gradient, fraction II with formamide), the proportions of these two fractions (ca. 2:1) being similar for DNA from a number of eucaryotic organisms. Yeast DNA was chosen for a detailed study of the mode of fractionation. Several physicochemical parameters, binding to nitrocellulose filters, sensitivity towards nuclease S1, labelling properties in vivo, and hybridization properties of the two DNA fractions were compared. It was shown that both fractions are native DNA and that the fractionation does not depend on the size or the (G + C) content of the DNA. Fraction I DNA contains only a small portion of molecules having single-stranded ends. Fraction II DNA is a heterogeneous population, containing molecules with peculiar structural characteristics: (a) It contains DNA molecules with single-stranded ends and/or gaps sensitive to nuclease S1; labeling experiments suggested that these are molecules undergoing repair and replication. (b) Another portion of fraction II is molecules sensitive to nuclease S1 in regions which are not single-stranded. (c) A third portion is DNA which, after treatment with nuclease S1, is still strongly bound to the resin. Indications that the segregation may be due to the presence of specific DNA sequences comes from the above experiments and from the finding that fraction I DNA is enriched in ribosomal genes and fraction II DNA in tRNA genes.
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PMID:Characteristics of DNA fractionated on benzoylated DEAE-cellulose. 110 98

Treatment of cells or nuclei with bleomycin induces DNA lesions. We detect the presence of lesions as the release of fragments from bulk DNA when cells (or nuclei) are lysed in dilute alkali. To further characterize the lesions we have altered experimentally the average nucleosome repeat length and probed the lysate with nuclease S1 in order to remove single-stranded DNA. In salt-incubated nuclei with short average nucleosome repeat length (140-145 base pairs) (and also with long nucleosome-free stretches of DNA) one can induced fewer DNA lesions in the nucleosome-containing DNA as compared to nuclei with 190-195 base pairs average nucleosome repeat length. Hence the ability of bleomycin to induce DNA lesions is dependent on nucleosome repeat length.
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PMID:Bleomycin-induced DNA lesions are dependent on nucleosome repeat length. 168 3

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.
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PMID:Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1. 232 56

Atrial natriuretic factor (ANF) is a group of peptides, originally isolated from the cardiac atria, that have a number of important effects on blood pressure, renal function, and salt balance. In the current study, expression of the ANF gene in certain extra-atrial tissues of the rat has been examined by radioimmunoassay of extracted ANF protein and by blot-hybridization, nuclease S1 analysis, and primer-extension analysis of the ANF mRNA. ANF peptides and mRNA were detected in cardiac ventricles, lung, and pituitary gland at levels generally less than or equal to 1% those of cardiac atria. The ANF transcripts in extra-atrial tissue appear to be very similar to those synthesized in the atria. They are polyadenylylated, are equivalent in overall length (950-1050 nucleotides), and have identical 5' termini. A secondary transcription start site mapping approximately 80 base pairs upstream from the primary start site is employed in atria and to a lesser extent in other tissues. The ANF transcript is present throughout the cardiac ventricles from apex to base and in the septum as well as the ventricular free walls. The transcript is more prevalent in the left ventricle and interventricular septum than in the right ventricle. Immunocytochemistry using various anti-rat ANF antibodies localized ANF immunoreactivity to the atrial myocytes; the ventricular myocytes, particularly along the endothelial surface of the ventricular chamber; perialveolar cells in the lung; and the gonadotropin-producing cells of the pituitary. The data indicate that the capacity for ANF gene expression extends beyond atrial tissue, albeit at much reduced levels, and may suggest alternative, perhaps paraendocrine, functions for the peptide in these tissues.
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PMID:Extra-atrial expression of the gene for atrial natriuretic factor. 242 40

An improved method for mapping RNA transcript boundaries by the nuclease protection technique is presented. This method exploits the large (greater than 20 degrees C) difference in the thermal stability of RNA:DNA and DNA:DNA duplexes in concentrated chaotropic salt solutions. At 45 degrees C in 3.0 M sodium trichloroacetate RNA:DNA hybridization is very efficient but DNA:DNA duplexes remain completely denatured. For many applications, this solvent system can eliminate the need to prepare probes that are free of competing or irrelevant DNA molecules. Fifty- to 100-fold more RNA:DNA hybridization is observed when reassociation is performed in 3.0 M sodium trichloroacetate than in solutions containing high concentrations of formamide. A comparison of the use of S1 nuclease or mung bean nuclease suggests that mung bean nuclease can produce more precise and less ambiguous nuclease protection patterns.
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PMID:Use of sodium trichloroacetate and mung bean nuclease to increase sensitivity and precision during transcript mapping. 243 1

The effect of hypertonic conditions on RNA synthesis in cultured chick embryo cells was examined. The appearance of newly synthesized 28 S, 18 S, and 4 S and 5 S RNA into the cytoplasm was found to be decreased by hypertonic conditions. The appearance of newly synthesized poly(A)+ RNA into the cytoplasm was also found to be depressed. To examine the behavior of a specific mRNA, nuclear and cytoplasmic levels of procollagen alpha 2(I) mRNA were measured during high salt treatment. While nuclear levels of this mRNA were found to increase, those of the cytoplasm fell markedly. S1 nuclease digestion studies of an intron flanked by two exons revealed that the pro alpha 2(I) collagen nuclear RNA that accumulated under hypertonic conditions was spliced. The nuclear accumulation of mRNA appears therefore to be due to a hypertonic block of nuclear-cytoplasmic transport, and not to an inhibition of RNA splicing.
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PMID:Changes in ribosomal RNA, poly(A)+ RNA, and collagen alpha 2(I) mRNA synthesis and processing during hypertonic treatment of cultured chick embryo cells. 244 98

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.
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PMID:A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein. 252 Dec 17

We have observed changes in the chromatin structure of a human histone gene promoter that may be functionally related to variations in transcription during the cell cycle. A detailed analysis of the chromatin structure of a cell cycle-dependent human H4 histone gene and its flanking sequences was performed using DNase I, S1 nuclease, and restriction endonucleases. This gene was previously shown to have a DNase I- and S1-sensitive site for which the boundaries varied with the cell cycle, and we have now precisely mapped these modifications. During S phase, the entire coding region of this gene and the 5'-flanking region up to approximately -600 base pairs are sensitive to both DNase I and S1, while during mitosis/G1, accessibility to these enzymes is greatly decreased in regions from -250 to -600 base pairs and downstream of +100 base pairs. DNase I- and S1-hypersensitive sites in the proximal promoter region (which contains two sites of protein-DNA interaction as well as sequence elements necessary for the correct initiation of transcription) are present throughout the cell cycle, as is an additional site sensitive to both DNase I and S1, located at -700 to -800 base pairs. Restriction enzyme analysis confirmed the general openness of the promoter region and relative insensitivity of the 3'-flanking region, while salt wash experiments indicated several discrete sites in the promoter that are candidates for regulatory interactions. The chromatin structure of the proximal promoter region of this H4 gene is different during early S phase when it is maximally transcribed, as indicated by the ability of a high salt wash to render this region inaccessible to the restriction enzyme MspI only at this time of the cell cycle.
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PMID:Fine mapping of the chromatin structure of a cell cycle-regulated human H4 histone gene. 291 Aug 51


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