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)

Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.
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PMID:Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP. 19 61

Two types of the long form of the rat prolactin receptor have been identified by two independent groups. One type of receptor has a consensus sequence for a putative ATP/GTP binding site in the cytoplasmic domain, while the other does not. Since this site would confer kinase activity to the prolactin receptor and represent an important element in the process of signal transduction, we wanted to clarify this discrepancy. To that end, three procedures capable of detecting a point mutation were performed: Southern analysis of reverse transcribed polymerase chain reaction (PCR) products, allele-specific PCR, and S1 nuclease analysis. All results clearly indicated that the sequence for the putative ATP/GTP binding site does not exist in the prolactin receptor.
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PMID:Absence of a putative ATP/GTP binding site in the rat prolactin receptor. 137 4

Mammalian ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin ADP-ribosyltransferase activity, were grouped into three classes based on deduced amino acid sequence. Human ARF 1, a class I ARF, is identical with its bovine counterpart, has a distinctive pattern of tissue and developmental expression, and is encoded by a approximately 1.9-kilobase mRNA. ARF 1 cDNAs were isolated from a human fibroblast cDNA library; one arose via an alternative polyadenylation signal (AA-TACA) 84 nucleotides 5' to the polyadenylation signal (AATAAA) used in the 1815-base pair cDNA. The polyadenylation signals, their respective locations, and the surrounding nucleotide sequences are conserved in human and rat. The human ARF 1 gene, with four introns, spans approximately 16.5 kilobases. Exon 1 (46 base pairs) contains only untranslated sequence. Translation initiates in exon 2, which encodes the sequence GXXXXGK involved in phosphate binding (GTP hydrolysis). The sequence DVGG is encoded in exon 3, and NKQD, which is involved in the interaction with the guanine ring, is interrupted following the codon for Q by intron 4. The carboxyl-terminal 53 amino acids and greater than 1110 base pairs of 3'-untranslated region are encoded in exon 5. Primer extension and mung bean and S1 nuclease mapping indicated multiple transcription initiation sites and were consistent with Northern analyses. The 5'-flanking region has a high GC content but no TATA or CAAT box, as found in housekeeping genes. In addition, the two human class I ARF genes, ARF 1 and ARF 3, have similar exon/intron organizations and use GC-rich promoters.
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PMID:Characterization of the human gene encoding ADP-ribosylation factor 1, a guanine nucleotide-binding activator of cholera toxin. 157 40

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.
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PMID:Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript. 246 43

Transcription of the spo0B gene and genes downstream of it was investigated by S1 nuclease protection experiments. The spo0B gene was transcribed from a single promoter, and this transcript extended through a gene, obg, coding for a 47,668 Mr protein. Transcription of this operon ended in a stem-loop structure. The sequence of the deduced obg protein contained a region with homology to known GTP-binding proteins in the nucleotide-binding regions. The amino-terminal portion of this protein showed homology to mammalian collagen, suggesting a structural role. The purified obg protein was shown to bind [alpha-32P]GTP in vitro. Several attempts to inactivate the obg gene were unsuccessful, indicating that the obg gene product was essential for growth. The possible function of this protein and its relationship to RAS-like proteins and sporulation was discussed. Immediately downstream of the obg gene were two genes involved in phenylalanine biosynthesis, pheB and pheA. The pheA gene coded for monofunctional prephenate dehydratase, on the basis of the high homology of the deduced amino acid sequence to prephenate dehydratases of bacterial origin. The sequence of the pheB gene product was not homologous to chorismate mutase, and its function remains unknown. Transcription of the phe genes was shown to begin at the stem-loop structure between obg and pheB. The possibility was entertained that phe gene transcription arises from processing or antitermination of the spo0B transcript.
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PMID:The Bacillus subtilis spo0B stage 0 sporulation operon encodes an essential GTP-binding protein. 253 15

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.
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PMID:Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA. 271 42

We isolated and characterized human genomic clones encompassing the gene for the alpha-subunit of Gi2, a GTP-binding signal transduction protein abundantly expressed in myeloid cells. The gene is divided into 9 exons and spans 23.5 kb. Exons 2, 6 and 7 encode putative guanine nucleotide-binding domains that are highly conserved among GTP-binding proteins. A polyadenylation signal located within exon 9 predicts an mRNA size (approximately 2.3 kb) several hundred bases longer than that of published cDNAs, and consistent with the size seen on RNA blot hybridization. Primer extension and S1 nuclease analysis determined a major and several minor transcriptional start sites. The first exon and 5' flanking region are highly G + C rich, contain several GC boxes (SP1 transcription factor binding sites), a CAAT box, and lack a TATA box. The presumptive promoter region is thus similar to that of ras and other widely expressed genes.
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PMID:Cloning and characterization of the human gene for the alpha-subunit of Gi2, a GTP-binding signal transduction protein. 283 12

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.
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PMID:The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus. 302 62

A region that forms the S1 nuclease site in Co1E1 DNA is shown to code for an in vitro transcript, called S1 RNA-B, which contains a 5'-terminal GTP residue that exhibits noncomplementarity with the template's DNA sequence. The synthesis of S1 RNA-B initiates four bases upstream from the start point for S1 RNA-C. The initial four bases in S1 RNA-B and S1 RNA-C are identical. The relative synthesis of S1 RNA-B to S1 RNA-C is sensitive to the concentration of GTP, a substrate that is required for elongation past the +4 position in S1 RNA-C. Dinucleotides that are expected to only initiate synthesis of S1 RNA-C yield two transcripts that appear to initiate from the S1 RNA-C and S1 RNA-B start sites. In vitro studies involving other Co1E1 transcripts, RNA-B and RNA-C, provide similar observations concerning the noncomplementary initiation phenomenon. A model involving transcriptional slippage is suggested to explain the noncomplementary initiation phenomenon. The model proposes that the cycling reaction of Escherichia coli RNA polymerase produces tetranucleotides that are transposed to nearby upstream sequences for priming transcription.
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PMID:Synthesis of in vitro Co1E1 transcripts with 5'-terminal ribonucleotides that exhibit noncomplementarity with the DNA template. 302 12

We characterized the in vivo transcripts of two Cellulomonas fimi genes, the cenA gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4) and the cex gene, which encodes an extracellular exo-beta-1,4-glucanase (EC 3.2.1.91). By Northern blot analysis, cenA mRNA was detected in C. fimi RNA preparations from glycerol- and carboxymethyl cellulose-grown cells but not from glucose-grown cells. In contrast, cex mRNA was detected only in the preparations from carboxymethyl cellulose-grown cells. Therefore, the transcription of these genes is subject to regulation by the carbon source provided to C. fimi. By nuclease S1 protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, 5' termini were found 51 and 62 bases before the cenA translational initiation codon and 28 bases before the cex translational initiation codon. S1 mapping with unlabeled DNA probes and C. fimi RNA which had been isolated in vivo but which had been 5' labeled in vitro with guanylyltransferase and [alpha-32P]GTP confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. Comparative analysis of the DNA sequences immediately upstream of the initiation sites of the cenA and cex mRNAs revealed a 30-base-pair region where these two sequences display at least 66% homology. S1 mapping was also used to locate the 3' termini of the cenA and cex transcripts. Three 3' termini were found for cenA messages, whereas only one 3' terminus was identified for cex mRNA. The transcripts of both genes terminate in regions where their corresponding DNA sequences contain inverted repeats.
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PMID:Regulation, initiation, and termination of the cenA and cex transcripts of Cellulomonas fimi. 380 71


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