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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA was isolated from mouse brain after in vivo gamma-ray irradiation, treated with endonuclease S1 from Aspergillus oryzae if necessary, and analysed further by alkaline and neutral sucrose gradient centrifugation. In parallel, its template activity was determined by DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay as described previously. Similar experiments were performed with cultured mouse leukaemia cells (L5178Y) irradiated in vitro at 0 degrees C. Irradiation induced single- and double-strand breaks in the DNA of the brain with a yield of 1.0 and 0.1 break per 10(12) dalton per rad (100 eV/break and 770 eV/break), respectively. The yield of single-strand breaks in the brain was lower than that found in the cultured cells, whereas the yield of double-strand breaks was found to be almost the same in both cases. Treatment of irradiated DNA with single-strand-specific S1 endonuclease gave rise to further breaks detected on neutral sucrose gradient analysis. The yield of these breaks was also higher in the brain compared to the cultured cells. The increase per unit dose in the template activity of the DNA from the brain was found to be five times as much as that found in the cultured cells. Then, the average number of deoxyribonucleotides incorporated per break was determined on DNA which had experienced different treatments. The value for the brain DNA irradiated in vivo was found to be five times as much as that found for DNA treated with pancreatic deoxyribonuclease and 10 times as much as those found for DNA from the cultured cells and isolated brain nuclei irradiated in vitro at 0 degrees C. Thus, in vivo irradiation seemed to induce gaps with 3'-OH terminals in addition to simple breaks with or without 3'-OH terminals found in the cultured cells. Radiation-induced single-strand breaks and 3'-OH terminals in the DNA of the brain were repaired following irradiation. Approx. 20--40% of the terminals or breaks induced were, however, remaining at 3 h or more after irradiation, depending on the dose administered.
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PMID:Induction and repair of strand breaks and 3'-hydroxy terminals in the DNA of mouse brain following gamma irradiation. 71 24

A DNA probe specific for sequences complementary to globin RNA has been prepared in vitro from globin complementary DNA (cDNA). Globin cDNA was used as the template for the synthesis of a complementary strand (ccDNA) by avian myeloblastosis virus DNA polymerase. After treatment with single strand-specific S1 nuclease, double-stranded globin DNA was denatured in the presence of a vast excess of globin RNA. After hybridization of the cDNA with globin RNA, the ccDNA was isolated by hydroxyapatite chromatography. The ccDNA probe hybridized efficiently to globin cDNA, but not at all to globin RNA. This probe should prove useful in assessing the asymmetry of gene expression in cell-free transcriptional systems.
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PMID:In vitro synthesis of a DNA probe for antisense globin sequences. 88 69

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.
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PMID:Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases. 114 Dec 22

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.
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PMID:Regulation of the Epstein-Barr virus DNA polymerase gene. 131 4

The myb proto oncogene product (c-Myb) is a transcriptional regulator and its expression and function are tightly linked to the cellular entry into S phase and DNA synthesis. It has been shown [Venturelli, D., Travali, S. & Calabretta, B. (1990). Proc. Natl. Acad. Sci. USA, 87, 5963-5967] that inhibition of T-cell proliferation by a myb antisense oligomer is accompanied by down-regulation of DNA polymerase alpha expression. To examine whether the transcription of the DNA polymerase alpha gene is directly regulated by c-Myb, we have identified and characterized the 5' regulatory region of the human DNA polymerase alpha gene. Two major and several minor transcription start sites were identified by nuclease S1 mapping. DNA sequence analysis showed that the promoter region contains no TATA box, one CCAAT box and putative Ap-1, AP-2 and E2F binding sites. In DNAase I footprinting, the bacterially expressed c-Myb protected six sites in the 5' flanking region of the human DNA polymerase alpha gene. However, c-Myb did not activate the DNA polymerase alpha gene promoter in a co-transfection assay. Our results suggest that an unknown factor(s) is required for the c-Myb-induced activation of the DNA polymerase alpha gene promoter, or c-Myb does not directly activate this promoter.
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PMID:The c-myb proto-oncogene product binds to but does not activate the promoter of the DNA polymerase alpha gene. 140 40

The conservation of the herpesvirus DNA polymerases has allowed cross-hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMV EcoRI fragment B which spans the HindIII site separating HindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the PstI site in HindIII fragment D to a BglII site in HindIII fragment H. Initiating in HindIII fragment D and extending into HindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3' coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites in HindIII fragment D. By primer extension, we were able to map precisely the 5' end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF.
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PMID:Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. 171 83

We have investigated the transcriptional activity of the 60.1- to 68.3-map-unit region of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Twelve transcripts mapping to this region were expressed at various times during infection. An early 4.0-kb transcript, potentially coding for a 143-kDa peptide essential for viral DNA replication, was maximally abundant at 6 h postinfection (p.i.). Transcripts of 0.5, 1.1, 1.4, 2.1, and 3.1 kb were most abundant at 12 h p.i., while two large transcripts of 5.2 and 6.8 kb were expressed maximally at 24 h p.i. In the presence of cycloheximide, and in ts8-infected cells at the nonpermissive temperature, only the 4.0-kb RNA was expressed. Northern (RNA) blot analysis using DNA subfragments from the EcoRI D fragment as probes suggested that many of the transcripts overlapped. Strand-specific cRNA probes revealed that the majority of the RNAs were transcribed in the counterclockwise direction. S1 nuclease and primer extension analysis were used to map the 5' ends of transcripts coded within the 60.1- to 64.8-map-unit region. Mapping of the 3' ends of the 1.1-, 4.0-, 5.2-, and 6.8-kb transcripts suggested that these RNAs were all coterminal at their 3' ends. A minicistron was found between the early 4.0-kb transcription start site and the predicted ATG start codon of the p143 gene. Several similar sequence motifs were identified in the promoter regions of the p143 gene and the AcMNPV DNA polymerase gene.
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PMID:Transcription analysis of the EcoRI D region of the baculovirus Autographa californica nuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene. 173 Nov 6

We have determined the transcriptional organization of the Escherichia coli dnaX gene, the structural gene for both the gamma and tau subunits of DNA polymerase III holoenzyme. By S1 nuclease protection and primer extension mapping of transcripts encoding the dnaX products, one primary promoter of dnaX has been identified that initiates transcription 37 nucleotides upstream from the first codon. dnaX resides in an operon with two recently sequenced genes, orf12, encoding an unidentified product, and recR, the structural gene for a protein involved in the recF pathway of recombination. Under conditions of balanced growth, a very small amount of transcription from the upstream apt promoter (less than 5%) contributes to the expression of tau and gamma, too low for apt to be considered to be on an operon with dnaX, orf12, and recR are transcribed from an independent promoter as well as from the dnaX promoter, providing a mechanism for orf12 and recR to be regulated independent of dnaX. Transcription of the dnaX-orf12-recR operon is terminated upstream from the previously characterized heat shock gene htpG. The dnaX and orf12-recR promoters, cloned into a promoter detection vector, efficiently direct the expression of the downstream reporter gene, lacZ. These results extend our knowledge of the genetic and transcriptional organization of this region of the E. coli chromosome. The transcriptional organization has been defined as follows: apt, dnaX-orf12-recR, htpG. All of these genes are transcribed in the clockwise direction and only dnaX, orf12 and recR are contained in the dnaX operon.
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PMID:Transcriptional organization of the Escherichia coli dnaX gene. 187 Jan 25

Salt-induced and supercoiling-induced B-Z junctions in pWR756, a plasmid containing (GC)16, were probed with bisulfite-methoxyamine, a modification reagent specific for single-stranded nucleic acids. The modification sites were analyzed with S1 nuclease and the modified cytosines were determined from termination sites of DNA chain elongation by DNA polymerase. The results showed that most accessible cytosines are the same for both types of B-Z junctions.
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PMID:Probing salt-induced and supercoiling-induced B-Z junctions in DNA by bisulfitemethoxyamine. 210 85

The properties and sources of all known class-I, class-II and class-III restriction endonucleases (ENases) and DNA modification methyltransferases (MTases) are listed and newly subclassified according to their sequence specificity. In addition, the enzymes are distinguished in a novel manner according to sequence specificity, cleavage position and methylation sensitivity. Furthermore, new nomenclature rules are proposed for unambiguously defined enzyme names. In the various Tables, the enzymes are cross-indexed alphabetically according to their names (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174, and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the ENases include relaxed specificities (integrated within Table II), the structure of the generated fragment ends (Table III), interconversion of restriction sites (Table IV) and the sensitivity to different kinds of DNA methylation (Table V). Table VI shows the influence of class-II MTases on the activity of class-II ENases with at least partially overlapping recognition sequences. Table VII lists all class-II restriction endonucleases and MTases which are commercially available. The information given in Table V focuses on the influence of methylation of the recognition sequences on the activity of ENases. This information might be useful for the design of cloning experiments especially in Escherichia coli containing M.EcodamI and M.EcodcmI [H16, M21, U3] or for studying the level and distribution of site-specific methylation in cellular DNA, e.g., 5'- (M)CpG-3' in mammals, 5'-(M)CpNpG-3' in plants or 5'-GpA(M)pTpC-3' in enterobacteria [B29, E4, M30, V4, V13, W24]. In Table IV a cross index for the interconversion of two- and four-nt 5'-protruding ends into new recognition sequences is complied. This was obtained by the fill-in reaction with the Klenow (large) fragment of the E. coli DNA polymerase I (PolIk), or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments [K56, P3].(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3). 217 84


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