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)

Genome-length complementary DNA (cDNA) transcripts were synthesized in vitro by using purified virions of avian myeloblastosis virus. Moloney murine leukemia virus, and clone 124 mouse sarcoma virus. The size of the genomelenth cDNA transcripts was measured on either alkaline sucrose gradients or alkaline agarose gels. The longest cDNA transcripts synthesized by using avian myeloblastosis virus, Moloney murine leukemia virus, and clone 124 mouse sarcoma virus were 7, 9 and 6 kilobases (kb), respectively. The in vitro system used was capable of synthesizing double-stranded DNA, but the plus strands (same polarity as the viral RNA) were only 0.5 to 1.5 kb long. Lone Moloney murine leukemia virus cDNA transcripts were used as templates to synthesize the second plus strand. Essentially two strategies were employed as follows. (i) The 3' ends of the cDNA transcripts were extended by addition of 50 to 100 dAMP residues by terminal deoxynucleotidyl transferase. The (dA)n-tailed cDNA transcripts were used as templates along with an oligomer of dT as primer and Escherichia coli DNA polymerase to synthesize the plus strands. (ii) DNase-digested calf thymus DNA was used to prime the synthesis of plus strands on long cDNA with E. coli DNA polymerase I. In both cases, the synthesis of the plus strands was monitored by increased resistance of the cDNA templates to single-strand-specific S1 nuclease. The double-stranded DNA was fractionated on neutral sucrose gradients. Analysis of the double-stranded DNA synthesized by using oligo(dT) primer showed the plus strands to be about 5 to 6 kb long, whereas the plus strands synthesized by using DNase-digested calf thymus DNA primers were only 0.3 to 0.5 kb long. Double-stranded DNA synthesized by either method has an average size of 6 x 10(6) daltons. Double-stranded DNA was also synthesized by using cDNA transcripts as templates without the addition of any primers. In this case, the plus strands were covalently linked to the template strand and were not representative of the whole parent strand.
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PMID:Genome organization of RNA tumor viruses. I. In vitro synthesis of full-genome-length single-stranded and double-stranded viral DNA transcripts. 20 13

The structural modification of double-stranded circular DNA of simian virus 40 and plasmid ColE1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. Stepwise hydrolysis with endonuclease S1 and DNase followed by DNA base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of DNA, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. Of the two synthetic double-stranded polymers, poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), bound to the diol-epoxide, only the former showed a marked hydrolysis after endonuclease S1 treatment, whereas binding occurred 24-fold more on the latter.
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PMID:Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. 27 58

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

Telomeric heterochromatin can be demonstrated in Allium cepa chromosomes when root tip squashes are subjected to a C-banding procedure (treatment with saturated barium hydroxide for 10 min, followed by 1 h in phosphate buffer at 60 degrees C). Acridine orange (A0) staining indicated that the chromosomal DNA was denatured by the alkaline treatment and that it renatured within the first 3-7 min in the hot buffer. The DNA of the telomeres reannealed somewhat faster than the rest of the chromosomal DNA, but the AO staining suggested that all chromosomal DNA was double stranded after 7 min in buffer. Digestion of the chromosomes with a single strand specific nuclease, DNase S1, followed by Feulgen staining, demonstrated that the AO staining gives a somewhat misleading picture of the extent of DNA denaturation and renaturation. The S1 nuclease results showed that the chromosomal DNA was completely denatured by the alkaline treatment, but that a fraction of the DNA reannealed during the deionized water wash that preceded the incubation in hot buffer. Neither controls nor chromosomes subjected to the complete C-banding procedure were affected by S1 nuclease digestion, demonstrating that virtually all of the chromosomal DNA was double stranded both before and after the C-banding process. These results, along with the fact that the appearance of the bands was unaffected when the buffer incubation was performed at high (80 degrees C) or low (40 degrees C) temperature, indicated that differential DNA denaturation and renaturation is unlikely to be responsible for C-banding in this species.
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PMID:Differential rates of DNA denaturation and renaturation in situ in relation to the C-banding of Allium cepa chromosomes. 75 82

The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression.
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PMID:Characterization of the rat pulmonary surfactant protein A promoter. 160 51

Abnormal tubulofilamentous particles were identified by electron microscopy using a simple touch negative staining technique from brains of mice infected with four strains of the scrapie agent. Treatment by three proteolytic enzymes and subsequent treatment with DNase and mung bean nuclease of grids prepared from the infected animals confirmed previous observations that the tubulofilamentous particles observed in scrapie-effected brains are complex structures. The core of the tubulofilamentous particle scrapie-associated fibrils was revealed by treatment with SDS. Treatment with proteolytic enzymes and subsequent treatment with DNase or mung bean nuclease or S1 nuclease also revealed typical and transitional stages of scrapie-associated fibrils. However, treatment with RNase A had no effect. The data suggest that nucleic acid is a single-stranded DNA protected by a protein coat.
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PMID:Evidence of ssDNA in tubulofilamentous particles: their relationship to scrapie-associated fibrils. 167 41

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

In vitro studies have demonstrated that the estrogen receptor (ER) can bind to the rat PRL estrogen response element (ERE) located 1700 basepairs upstream of the transcriptional start site. However, the mechanism by which the receptor-DNA complex influences the activity of RNA polymerase located in the promoter region is not understood. To begin investigating this process, we developed cell lines derived from GH3 cells that contain steroid-responsive bovine papillomavirus minichromosomes. Within these minichromosomes is a hybrid gene composed of the 5' flanking region of the PRL gene, driving the expression of the Tn5 gene. The episomal PRL DNA sequences responded to 17 beta-estradiol (E2) by increasing the rate of Tn5 gene transcription. Nucleosome mapping experiments using micrococcal nuclease demonstrated that nucleosome-like structures were assembled on the minichromosome in an ordered array separated by 150-200 basepairs of DNA. Novel S1 nuclease as well as DNase-I-hypersensitive sites in the chromatin of the promoter and distal regulatory regions of the episomal PRL gene were detected by indirect end-labeling studies. The nuclease hypersensitive sites in the distal region containing the ERE were modified after treatment of the cells with either E2 or the antiestrogen 4-hydroxytamoxifen. However, only E2 treatment of cells resulted in an increase in the nuclease hypersensitivity of the promoter region and induced gene expression, while antiestrogen treatment had no effect on either parameter. This suggests that complex interactions between factors located at the distal and proximal regulatory regions ultimately determine the transcriptional response of the PRL gene to E2.
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PMID:An interaction between the 5' flanking distal and proximal regulatory domains of the rat prolactin gene is required for transcriptional activation by estrogens. 196 74

RNA from IdUrd-treated P3HR1 cells was used for the construction of a cDNA library and screened with B95-8 EBV DNA BamHI fragment B and G probes. One clone, BG9, containing a 1.7 kb cDNA insert was further studied. Complete DNA sequence analysis revealed that BG9 encompassed the B95-8 EBV DNA sequences from nucleotide 120,747 to nucleotide 122,412 and corresponded to the BGLF5 open reading frame of the EBV DNase gene. Comparison of the sequences of BG9 with that of published B95-8 EBV DNA indicated that there were 14 different bases which results in 7 amino acid residue changes. The product of in vitro transcription/translation of a subclone, pGEM-BG9, contained the EBV DNase activity and a 52 kDa protein was immunoprecipitated from the in vitro translation products using serum from a patient with nasopharyngeal carcinoma which contained a high level of anti-DNase activity. Northern hybridization of P3HR1 RNA with the BG9 probe revealed a complex pattern of transcription in this region. Subgenomic DNA fragments were then used to map these RNA species to the B95-8 EBV DNA sequence. The result of S1 nuclease analysis indicated that a DNase ORF containing transcript sized 2.0 kb is initiated at nucleotide 122,435 +/- 1 and terminated at nucleotide 120,741 of the EBV genome.
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PMID:Molecular characterization of a cDNA clone encoding the Epstein-Barr virus (EBV) DNase. 217 60

To gain insight into the normal controls mediating expression of the c-Ki-ras protooncogene, we have identified DNA sequence elements within its promoter that are essential for transcriptional activity. Transient expression assays using the bacterial chloramphenicol acetyltransferase gene were used initially to localize regions directing primary promoter function. Stepwise deletion of 5' promoter sequences resulted in a gradual decrease in the ability to drive transcription of the reporter gene, suggesting that this promoter is composed of multiple cis-acting elements. Gel mobility-shift and DNase protection studies involving a 166-base-pair DNA fragment allowed the identification of protein-binding sites corresponding to these multiple regulatory elements. One element demonstrating particular transcriptional influence exists as a homopurine/homopyrimidine-rich region that in vitro exhibits S1 nuclease sensitivity and binds at least one nuclear protein. Data from competition binding experiments suggest that this nuclear factor may be influential in the regulation of other essential growth-control genes as well.
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PMID:An S1 nuclease-sensitive homopurine/homopyrimidine domain in the c-Ki-ras promoter interacts with a nuclear factor. 218 46


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