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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. 142 73

The DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3'-->5' exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.
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PMID:Characterization of the nucleotide sequence of the Lymantria dispar nuclear polyhedrosis virus DNA polymerase gene region. 146 55

Transcriptional regulation of the bacteriophage T4 late genes requires the participation of three DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45, and that act at an enhancer-like site. Transcriptional activation by these DNA replication proteins also requires the function of an RNA polymerase-bound coactivator protein that is encoded by T4 gene 33 and a promoter recognition protein that is encoded by T4 gene 55. Transcriptional activation in DNA constructs, in which the enhancer and a T4 late promoter can be segregated on two rings of a DNA catenane, has now been analyzed. The ability of an interposed DNA-binding protein to affect communication between the enhancer and the promoter was also examined. Together, these experiments demonstrate that this transcription-activating signal is conveyed between its enhancer and a T4 late promoter by a DNA-tracking mechanism. Alternative activation mechanisms relying entirely on through-space interactions of enhancer-bound and promoter-bound proteins are excluded.
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PMID:A transcriptional enhancer whose function imposes a requirement that proteins track along DNA. 159 72

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.
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PMID:HIV-1 reverse transcriptase: structure predictions for the polymerase domain. 170 98

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently. DNA strand elongation continued past most chloroadenine (ClA) insertion sites but resulted in shorter chains than when dATP was inserted. Phage T4 DNA polymerase incorporated CldATP least efficiently; Klenow fragment of Escherichia coli DNA polymerase I and modified T7 DNA polymerase (Sequenase) showed intermediate ability to utilize the analogue. Incorporation of several consecutive ClA residues into the replicating strand dramatically reduced the ability of Sequenase, Klenow fragment, and T4 DNA polymerases to continue strand elongation. In the absence of the corresponding normal deoxyribonucleoside triphosphate during DNA synthesis, ClA was frequently misincorporated as thymine, cytosine, or guanine by both AMV RT and HIV-1 RT but rarely, if at all, by Klenow fragment, Sequenase, and T4 DNA polymerase. Except T4, for most DNA polymerases, CldATP at 10-20-fold molar excess over dATP was not a strong competitive inhibitor of dATP, as judged by the amount of strand extension and polymerase pause sites during DNA synthetic reactions. Our results indicate that the degree of strand extension in the presence of CldATP, the number and location of polymerase pause sites, and the amount of misincorporation of the analogue are both polymerase- and sequence-dependent.
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PMID:Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases. 170 19

A primase-reverse-transcriptase of Halobacterium halobium was purified by column chromatography on DEAE-cellulose, hydroxyapatite and carboxymethyl-cellulose, followed by sedimentation on a glycerol gradient. The enzyme is a multifunctional enzyme containing reverse transcriptase. DNA polymerase and RNase H activities and does not require a performed primer to initiate DNA synthesis. Using a single-stranded DNA as template, this enzyme synthesizes oligonucleotides (8-12 bases) that can be used a primer by Escherichia coli DNA nucleotidyltransferase I (DNA polymerase I, Klenow fragment). Two polypeptides of 67 and 57 kDa were found after 14750-fold purification of the enzyme.
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PMID:Reverse transcriptase in archaebacteria. Purification and characterization of a primase-reverse-transcriptase complex from Halobacterium halobium. 170 56

A small-scale plasmid preparation is described that is useful for a variety of procedures from double-stranded sequencing to in vitro transcription. No specialized equipment or reagents are required. The preparation of plasmid DNA does not require the use of RNase; instead the larger RNAs are precipitated with 2.5 M ammonium acetate. The resulting plasmid DNA is used routinely for double-stranded sequencing with the Klenow fragment of DNA polymerase and has been used for generating deletions with exonuclease III. In addition, the plasmid DNA has been used to generate transcripts with T7 RNA polymerase that translate well in reticulocyte lysate.
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PMID:A small-scale plasmid preparation yielding DNA suitable for double-stranded sequencing and in vitro transcription. 170 92

Extracts derived from Albizia amara were found to demonstrate activity in a recently developed hplc system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. All four isolates interacted with calf thymus DNA and were generally cytotoxic with a battery of cultured mammalian cells. As determined with Salmonella typhimurium strain TM677, isolates X1 and X3 were bactericidal, but not mutagenic. Isolate X1 was found to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. With DNA polymerase, the reaction was shown to be inhibited in a manner that was competitive with respect to DNA. In addition, isolate X1 inhibited each of the following: platelet aggregation, human lymphocyte transformation, phorbol-ester-induced chemiluminescence with human granulocytes, and cyclooxygenase activity. Detection of these alkaloids on the basis of their interaction with DNA exemplifies the validity of this approach.
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PMID:Biological activity of novel macrocyclic alkaloids (budmunchiamines) from Albizia amara detected on the basis of interaction with DNA. 172 78

We have amplified herpes simplex virus type 1 (HSV-1) DNA sequences from individual latently infected mouse trigeminal ganglia by polymerase chain reaction (PCR) assays. This report presents two useful modifications in the PCR technique. The first involves the use of two sets of closely spaced, oppositely oriented oligonucleotide primers and two rounds of 20-40 PCR cycles, first with the more widely spaced outer primers and then with the internal nested primers. This method enhanced the sensitivity of PCR detection as shown by assays of HSV-1 sequences in human brain. The second modification was designed to detect selectively HSV-1 sense or anti-sense RNA transcripts when both are present by adding a single primer during an initial reverse-transcriptase-mediated cDNA synthesis reaction. After destruction of the RNA template, standard PCR is initiated by the addition of the second primer and thermus aquaticus DNA polymerase (Taq). We show here applications of both of these modifications to amplify HSV-1 sequences from nervous system tissue.
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PMID:Application of polymerase chain reaction assays to studies of herpes simplex virus latency. 185 Nov 47

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57


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