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)

Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.
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PMID:Nucleotide sequence and promoter-specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene. 790 77

A plasmid carrying the 5' flanking region of the mouse proliferating-cell-nuclear-antigen (PCNA) gene or DNA polymerase beta gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild-type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase beta gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase beta gene promoter, we identified the unique 10-bp palindromic sequence (-24 to -15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21-bp upstream from the transcription-initiation site.
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PMID:Differential effect of p53 on the promoters of mouse DNA polymerase beta gene and proliferating-cell-nuclear-antigen gene. 790 18

We and others have described methods to label specific nucleic acid sequences in fixed cells by reverse in situ transcription (IST). They are simple alternatives to the tedious steps of in situ hybridization with labeled probes. We have favored use of thermostable DNA polymerases after heat denaturation of template secondary structure, accompanied by synthesis of cDNA from an annealed primer, but the approach has been limited by the low reverse transcriptase (RT) activity of Taq polymerase and delayed detection methods. We have improved the technique by the use of recombinant Thermus thermophilus (rTth) DNA polymerase and fluorescein-12-dUTP (FIST). Jurkat T lymphocytes were stimulated with ionomycin + phorbol myristate acetate to produce interleukin-2 (IL-2) mRNA in vitro overnight. They were cytospun onto slides and fixed in 70% ethanol + 30% DEPC-treated water, acetone, and air-dried. The slides were placed on a temperature-controlled heating block, and the cell spot was covered with a plastic coverslip. The temperature was raised to 95 degrees C, and 5-10 microliters of modified Perkin-Elmer/Cetus rTth RT reaction mix was injected under the edge of the coverslip. Each 10 microliters of mix in DEPC-water contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MnCl2, 1 mM dithiothreitol, 10 U placental ribonuclease inhibitor, 0.125 mM dA,C,GTPs, 0.1 mM fluorescein-12-dUTP, 2 U rTth DNA polymerase, and 4 pM 22-mer oligonucleotide primer, which spanned the second intron of IL-2. After 3 min at 95 degrees C, 1 min at 50 degrees C and 10 min at 72 degrees C, the slides were washed in 0.5 x phosphate-buffered saline, pH 7.0, at 42 degrees C, in 70% ethanol, 100% ethanol, and air-dried. The cells were mounted in antifade solution (2% n-propyl gallate in 70% glycerol), and could be viewed immediately by fluorescence microscopy. Image analysis showed that stimulated Jurkat cells were brighter than uninduced controls or those treated with RNase or without polymerase or primer. FIST appears to be useful for the detection of specific mRNAs in single cells.
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PMID:In situ transcription with Tth DNA polymerase and fluorescent nucleotides. 798 81

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.
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PMID:A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro. 812 85

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, O.C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25 degrees C and to temperatures as high as 100 degrees C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.
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PMID:Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes. 838 89

rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.
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PMID:Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities. 838 13

In the presence of Mg2+ ions, polynucleotide phosphorylase (PNPase, EC 2.7.7.8) is known to synthesize RNA-like polymers using ribonucleoside-5'-diphosphate (NDP) substrates but to be unable to utilize deoxyribonucleoside substrates. Our experiments show that when MgCl2 is replaced by FeCl3, PNPase becomes able to synthesize deoxyheteropolymers using deoxyribonucleoside-5'-diphosphates (dNDPs). The deoxyheteropolymer formed from the four dNDPs is degraded by pancreatic DNase, but not by RNase, and is readily used as a template by DNA-dependent DNA polymerase. Synthesis of this DNA-like polymer is accomplished de novo without the help of any primer or preexisting template. What is more, dA/dG and dC/dT ratios of polymers synthesized by different bacterial PNPases closely match ratios found in DNA of the bacterial species the enzyme came from.
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PMID:De Novo Synthesis of DNA-Like Molecules by Polynucleotide Phosphorylase In Vitro 866 1

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.
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PMID:Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication. 895 95

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.
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PMID:ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase. 900 81

A simplified technique for the detection of transcripts from a defined promoter is described. After reverse transcription, a PCR target sequence is selectively added to the 3' end of cDNA strands by DNA polymerase extension directed by an oligonucleotide template. Those cDNA molecules that do not have ends within a few nucleotides of the promoter start site are not extended and thus are excluded from subsequent amplification. Even when amplified products are visualized by ethidium bromide staining of agarose gels, this method requires only 1% of the RNA usually needed for detection of mRNA by standard RNase protection utilizing radiolabeled probes. In contrast to direct detection of cDNA by PCR, this procedure restricts amplification to a narrow subset of transcripts even when other overlapping colinear transcripts are present. We call this detection procedure specific amplification of cDNA ends (SPACE).
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PMID:Simple method for selective amplification of cDNA from a defined promoter. 904 2


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