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)

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
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PMID:Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts. 628 36

We have demonstrated faithful transcriptional initiation of cloned Xenopus rRNA genes upon injection into Xenopus oocytes. This observation has been made possible by the use of an S1 nuclease assay that is both sensitive and quantitative. In order to detect rRNA synthesis from the injected template above the large background of rRNA endogenously present in oocytes, the divergence of ribosomal DNA sequences between two Xenopus species was utilized. Cloned X. laevis ribosomal DNA was injected into the nuclei of X. borealis oocytes. Total oocyte RNA was then isolated and hybridized to a radioactive DNA probe that over laps the 5' end of X. laevis rRNA; endogenous rRNA of the X. borealis oocytes does not hybridize to the probe. RNA/DNA hybrids were treated with S1 nuclease and protected fragments were sized by polyacrylamide gel electrophoresis. RNA made from the injected rDNA protects the same region of probe as does authentic X. laevis precursor rRNA. Thus, transcription appears to initiate on the cloned, microinjected X. laevis rDNA at the same site as is used in vivo. This synthesis is not impaired by coinjection of an amount of alpha-amanitin sufficient to inhibit RNA polymerase II and III; therefore the reaction is mediated by RNA polymerase I. The amount of transcription may be reproducibly quantitated and we have varied a number of parameters in order to maximize transcriptional expression of the injected rDNA. Eight independently isolated X. laevis rDNA clones as well as several subcloned initiation regions of these genes are all accurately transcribed at approximately equal efficiency. This assay should facilitate analysis of several aspects of rRNA transcription, including deleniation of the Xenopus RNA polymerase I promoter location.
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PMID:Accurate transcription of cloned Xenopus rRNA genes by RNA polymerase I: demonstration by S1 nuclease mapping. 628 99

During simian virus 40 viral maturation, a series of modifications occur which alter the composition of viral nucleoprotein complexes. As a consequence, the chromatin that is extracted from extracellular simian virus 40 virions exhibits properties that are similar to those of transcriptionally active eucaryotic chromatin. The influence of this chromatin structure on specific RNA initiation by RNA polymerase II was examined by using the in vitro HeLa cell extract of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980). The 5' ends of RNA transcripts were positioned by the "run-off" assay, in which transcripts extend from the initiation site to termination sites created by restriction cleavage and by S1 nuclease analysis, using DNA probes labeled at their 5' termini. Two major early RNA transcripts, which originated at map positions 5,240 +/- 10 and 5,145 +/- 10, and two major late RNA transcripts, which originated at map positions 325 +/- 10 and 185 +/- 10, were identified. Transcripts were initiated with comparable relative efficiencies at the same 5' site when either purified DNA or chromatin was used as the template. Our results suggest that extracellular simian virus 40 virion chromatin modifications do not regulate simian virus 40 promoter selection but function to increase the accessibility of RNA promoter sequences to RNA polymerase II and allow efficient elongation of the RNA chain after the initiation event.
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PMID:Accurate transcription of simian virus 40 chromatin in a HeLa cell extract. 629 28

Drosophila genomic DNAs containing a chromosomal locus 87C1 70,000-dalton heat shock protein gene, the locus 79B actin gene, and the 88F actin gene have been used as templates in an in vitro HeLa transcription system. RNA polymerase II-dependent transcription initiates from specific sites on the heat shock protein gene and the 79B actin gene. The locations of the transcription start sites were determined by two types of experiments: sizing of RNA runoff transcripts and S1 nuclease mapping of the 5' terminus of the in vitro transcripts. Transcription initiates at or near the in vivo initiation site of the heat shock protein gene and initiates at or near a site 14 nucleotides downstream of the in vivo start site of the 79B actin gene. The addition of the 79B actin, 88F actin, or heat shock protein templates to a HeLa extract transcription reaction precluded the transcription of a second template subsequently added. The exclusion occurs rapidly, within 15 s, is not dependent on transcription, and is only partially resistant to high concentrations of the second added template. We propose that stable protein-promoter complexes play an important role in maintaining exclusive transcription of the first template added in vitro.
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PMID:In vitro transcription of Drosophila actin and 70,000-dalton heat shock protein genes. 631 67

Accurate and highly efficient (80%) splicing of a single mRNA precursor to processed products was achieved using HeLa cell extracts to synthesize and process RNA in vitro from recombinant plasmids containing specific DNA segments from adenovirus 2 (Ad2) and the nondefective adenovirus-simian virus 40 (Ad+2ND1) hybrid. One plasmid, pRID, contains a segment of Ad2 DNA spanning chromosome map coordinates 75.9-83.4. The other plasmid, pRW9, contains the analogous viral region from Ad+2ND1. RNA synthesis from pRID in vitro occurs for more than 60 min and is directed by RNA polymerase II. RNA products consistent in size with the expected precursor and the two processed mRNAs are made. RNA blot hybridization analyses showed that these products are complementary to the Ad2 insert in the plasmid and that the appropriate intervening sequence was absent from the smallest processed mRNA. Comparison of the splice patterns of RNA made in vitro to those of RNAs taken from infected cells using the nuclease S1 technique demonstrated the accuracy of intron removal.
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PMID:Efficient coupled transcription and mRNA splicing in vitro using plasmids derived from early region 3 of adenovirus 2 and a nondefective adenovirus-simian virus 40 hybrid. 632 37

We examine the influence of the immunoglobulin locus on the expression of the translocated c-myc oncogene in mouse plasmacytomas. The level of c-myc RNA was 30- 35-fold greater in tumor cells than in normal, quiescent B cells. Mitogen stimulation of the lymphocytes with lipopolysaccharide induced a 15-fold increase in c-myc expression per cell to a level that was similar to that in the transcription of the translocated c-myc gene involved initiation from sequences in the first c-myc intron. Abundant RNA transcripts were also found from the noncoding strand of the c-myc intron in most tumor lines. S1 nuclease mapping was used to locate the intronic sequences that are used to initiate the tumor-specific c-myc RNAs. Six different initiation sites within the intron were mapped, none of which have the TATA sequence usually associated with eucaryotic RNA polymerase II promoters. The noncoding strand transcripts were also found to initiate in the c-myc intron. Transcription of the c-myc coding strand was independent of the position of the translocation breakpoint, even when the heavy chain switch and constant regions were deleted.
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PMID:Transcriptional activation of the translocated c-myc oncogene in mouse plasmacytomas: similar RNA levels in tumor and proliferating normal cells. 632 72

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is alpha-amanitin sensitive (1 micrograms/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.
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PMID:Transcription of a trout protamine gene in vitro: the effects of alteration of promoters. 632 91

We have used a soluble in vitro RNA polymerase II transcription system to define the site of initiation of Moloney murine leukemia viral RNA synthesis. Molecularly cloned integrated and unintegrated Moloney murine leukemia virus DNAs were used as templates. The 5' ends of in vitro transcripts and virion RNA of Moloney murine leukemia virus were compared by nuclease S1 protection experiments. Our results indicate that viral sequences upstream of the in vivo cap site are implicated in the transcription of viral RNA and that the 5' end of an in vitro transcript derived from an integrated Moloney murine leukemia virus clone corresponds to the 5' end of viral genomic RNA.
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PMID:Identification of a RNA polymerase II initiation site in the long terminal repeat of Moloney murine leukemia viral DNA. 694 80

Sea urchin (psammechinus miliaris) H2A histone genes shown to be promoter mutants from oocyte injection experiments were tested for their ability to initiate transcription in vitro. Circular templates were transcribed with HeLa cell extracts, and the transcripts were assayed by mung bean or S1 nuclease mapping of the 5' ends. The transcripts of the H2A mutants produced in vitro were qualitatively similar and, in most cases, identical to those seen in oocyte injection experiments, but quite large quantitative differences were observed for some H2A mutant genes. Both the T-A-T-A box and far upstream sequences residing in the modulator segment E [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 7102--7106] were found to be essential for maximal transcription in vitro. Deletion of either of these sequence elements reduced transcription to 20%. A similar reduction in the amount of H2a transcripts was found when a T-A-T-A-to-T-A-G-A point mutant was tested in vitro. Essential far upstream sequences were mapped between nucleotides -139 and -111, 5' to the initiation site of transcription. In the standard run-off transcription test using restriction fragments, the effects of these sequences could be mimicked by free DNA ends, suggesting that the function of this in vitro upstream sequence might be to provide an entry side for RNA polymerase II.
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PMID:Delimitation of far upstream sequences required for maximal in vitro transcription of an H2A histone gene. 695 85

We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase. The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5. It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids. The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping. Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site. No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence. The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene.
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PMID:Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. 715 87


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