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)

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,
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PMID:Organization of sequences of avian globin mRNA. 6 81

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.
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PMID:Nucleic acid hybridization using DNA covalently coupled to cellulose. 16 82

Varying the concentration of Triton X-100, a nonionic detergent used to promote the DNA polymerase activity of Rous sarcoma virus in an endogenous reaction, showed a very sharp peak at about 0.02% (vol/vol) for optimal DNA synthesis. The yield of DNA at this concentration of Triton exceeded yields obtained at concentrations above the optimum by a factor of 2-5 for the 90-min reaction. At optimal Triton concentration, about 1-7% of the DNA made in the absence of actinomycin and about 4-10% of the DNA made in the presence of actinomycin was 2.5 X 10(6) daltons or greater, as estimated by formamide polyacrylamide gel electrophoresis and by alkaline sucrose gradient sedimentation. No large DNA was obtained at higher than optimal Triton concentrations. The large DNA molecules were rendered totally resistant to single-strand specific nuclease S1 after hybridization to an excess of viral RNA. It was concluded that at optimal detergent concentration, the viral DNA polymerase can synthesize full-size DNA transcripts of viral RNA.
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PMID:In vitro synthesis of full-length DNA transcripts of Rous sarcoma virus RNA by viral DNA polymerase. 17 81

Superhelical covalently closed circular replicative form DNA (RF I) of coliphage M13 appears as a relaxed molecule that has a base-unpaired region in the form of a bubble (100 to 200 base pairs long) seen in electron micrographs when spread in the presence of formaldehyde and formamide or after pretreatment with glyoxal. S1 endonuclease, specific for single-stranded DNA, converts superhelical M13 RF I DNA, but not nonsuperhelical M13 RF I to a significant extent, into unit-length linear molecules by sequential nicking of two strands. The locations of S1 nuclease-susceptible sites and glyoxal-fixed base-unpaired regions were both related to the five A-T-rich regions in M13 RF DNA. While S1 nuclease does not show preference for any of these sites, glyoxal-fixed bubbles occur predominantly at the major A-T-rich region in M13 RF DNA.
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PMID:Base-unpaired regions in supercoiled replicative form DNA of coliphage M13. 32 5

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

Nine transcripts complementary to mouse L cell mitochondrial DNA have been detected, sized and mapped to restriction fragments using the method of Berk and Sharp (1977). RNA isolated from L cell mitochondria was hybridized to 32P-labeled, cloned L cell mitochondrial DNA restriction fragments in 70% formamide under conditions 5 degrees C above the melting temperature of the DNA-DNA duplex, but approximately 15 degrees C below the melting temperature of the RNA-DNA duplex. The heteroduplexed material was then treated with the single-strand-specific nuclease S1, whick cleaves the single-stranded DNA not protected by RNA-DNA duplex formation into oligonucleotides and leaves intact 32P-labeled, single-stranded DNA replicas complementary to the transcripts. The single-stranded DNA replicas were then resolved and sized by alkaline agarose gel electrophoresis. Hybridization to strand-separated, 32P-labeled L cell mitochondria DNA restriction fragments under the same conditions showed that all nine transcripts hybridized exclusively to the heavy strand (H strand) of restriction fragments isolated as the dense strand from alkaline CsCl gradients, indicating that all stable transcripts 300 bases or longer detected by this technique originate from genes on the H strand. The two most abundant transcripts homologous to mitochondrial DNA map adjacent to the origin of replication. This result is consistent with map positions assigned to the large and small mitochondrial ribosomal RNAs isolated from Xenopus laevis and HeLa cells. Six of the other seven transcripts map continuously in approximately 40% of the genome. Only one transcript of 950 bases maps in the first quadrant of the genome as defined by the origin and direction of mitochondrial DNA replication, and it does not lie within the D loop region. The genetic function of the remaining 75% of this region of the genome is yet to be determined.
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PMID:The transcription map of mouse mitochondrial DNA. 66 30

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

Foldback DNA is defined by its rapid, concentration-independent renaturation, consistent with intramolecular base pairing of inverted repeat sequences. Foldback DNA, isolated from renatured mouse main band DNA by hydroxyapatite chromatography, is spread for electron microscopy by the formamide isodenaturing technique. A large fraction of the molecules can be recognized as intramolecular "hairpins"--structures in which complementary sequences on a single DNA strand form base-paired "stem" regions analogous to tRNA stems. The stem regions of the hairpins have a wide distribution of lengths, averaging about 1000 base pairs. About 60% of the stem regions terminate in single-stranded loops, ranging from 400 to many thousands of nucleotides in length, while 40% of the hairpins do not have discernible loops. There are about 40,000 hairpin-forming sequences in the main band portion of the mouse haploid genome. They appear to be either clustered in groups or confined to about one third of the DNA, rather than uniformly or randomly distributed. Another large fraction of the molecules seen in foldback DNA consists of linear structures, some of which are probably also hairpins. The electron microscopic results, along with simple theoretical considerations, make possible a better interpretation of our previous studies of the yield and S1 nuclease resistance of mouse foldback DNA.
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PMID:An electron microscopic study of mouse foldback DNA. 115 97

Reannealed hybrid molecules of wild-type bacteriophage lambda DNA were prepared in aqueous solutions of formamide at a variety of NaCl concentrations at both room temperature ( 22 degrees C) and 37 degrees C. Treatment of the hybrid DNA molecules with the single-strand-specific nuclease S1 from Aspergillus oryzae followed by alkaline sucrose gradient sedimentation was used to monitor the extent and fidelity of hybridization. The optimal renaturation conditions at room temperature were found to be: 50% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Optimal conditions at 37 degrees C were: 32% formamide, 35-55 mM NaCl and 10 mM Tris-HCl (pH 8.5) at 20-25 mug DNA/ml. Under these conditions approximately 85-90% of the input single-stranded DNA (molecular weight 1.5 X 10(7)) was rendered S1-nuclease-resistant within 8 h at room temperature and 5 h at 37 degrees C. Neither Mg2+ nor spermidine appeared to have an effect on either the extent or fidelity of duplex formation. Experiments performed with excess enzyme and with lambda/lambda imm 434 heteroduplex hybrids suggested that the hybrid that the hybrid DNA molecules formed under optimal conditions contained no, or only short (less than 1%), mismatched regions.
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PMID:Renaturation of bacteriophage lambda DNA. Determination of the optimal renaturation conditions using a single-strand-specific DNase and alkaline-sucrose-gradient assay system. 124 79

In the filamentous cyanobacterium Anabaena variabilis, specialized cells called heterocysts occur in a regular pattern along the filament and are the sites of nitrogen fixation. We used two different types of DNA-excess RNA hybridization techniques to estimate the number of genes expressed in recently differentiated, mature heterocysts. In the first, RNA and DNA were incubated in a phosphate buffer at 60 degrees C, and the hybrids were separated from the unhybridized material by hydroxylapatite chromatography. In the second, the nucleic acids were incubated at 50 degrees C in a buffer containing 50% formamide, and the fraction of DNA in duplexes was assayed by S1 nuclease digestion. Both techniques revealed that approximately 65% of the A. variabilis genome was expressed in vegetative cells and 45% of the genome was expressed in heterocysts. Two experiments were conducted to estimate the number of heterocyst-specific mRNA transcripts. In one, hybridization of heterocyst RNA to a null DNA probe (DNA not transcribed in vegetative cells) revealed that heterocyst-specific transcripts were encoded by 25% of the DNA sense strand, representing approximately 1,000 genes (assuming each to be 1,500 nucleotides in length). The second approach, in which total cell DNA was hybridized to a mixture of heterocyst and vegetative cell RNA, indicated that 14.7% of the DNA sense strand, or about 600 genes, was transcribed exclusively in the heterocyst. The remaining 900 to 1,300 transcripts present in the heterocyst appeared to be constitutively produced in both vegetative cells and heterocysts. The heterocyst-specific transcripts were present in abundant copies in the cell, while transcripts that occurred in both cell types were present at much lower frequency.
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PMID:Estimation of gene expression in heterocysts of Anabaena variabilis by using DNA-RNA hybridization. 242


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