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)

Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200(gag-pol)) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag proteins recognized Pr200(gag-pol). Pr200(gag-pol) contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145(pol)), 135,000 (Pr135(pol)), and 125,000 (Pr125(pol)) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80(pol)) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [(3)H]methionine showed that p80(pol) was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80(pol)), similar in size to mature viral p80(pol), was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80(pol). Pulse-chase studies showed that Pr80(pol), Pr125(pol), and Pr135(pol) were stable polypeptides, whereas Pr200(gag-pol) and Pr145(pol) were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200(gag-pol) occurred for a short time in the absence of protein synthesis.
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PMID:Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein. 7 22

The RNase-T1-resistant oligonucleotides of two Prague Rous sarcoma viruses with temperature-sensitive (ts) DNA polymerases (DNA nucleotidyltransferases), termed ts LA 337 and 335 of one leukosis virus, RAV-6, and 20 of their recombinant progeny have been mapped relative to the 3' poly (A) terminus of the viral RNA. The resulting oligonucleotide maps have been ocrrelated with markers of the four known viral genetic elements encoded in the RNA of 10,000 nucleotides. In accord with previous results recombinant RNAs contained (i) oligonucleotides characteristic of the src gene, coding for sarcoma formation, between the poly(A) end and 2000 nucleotides and (ii) olignucleotides characteristic of the env gene, coding for the envelope glycoprotein, between 2500 and 5000 nucleo tides from the poly(A) end. (iii) A cluster of four oligonucleotides that mapped between 6000 and 8000 nucleotides from the 3' poly(A) end of each RNA was shared by both parental viruses and all recombinants. Since all other map segments of our recombinants failed to segregate with the ts- or wild-type markers of the parental DNA polymerase gene (pol), it was concluded that the ts pol lesion maps in this RNA segment. (iv) The 5' segment of each recombinant RNA contained a cluster of four to five oligonucleotides whose parental origin correlated with an electrophoretic marker of one of the parental virion proteins, p27, a major product of the viral gag gene. The gene order 5'-gag-pol-env-src-poly(A) is consistent with our data.
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PMID:Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants. 18 81

High molecular weight RNA (35S) isolated from avian myeloblastosis virus directs the cell-free synthesis of two prominent polypeptides of 180,000 and 76,000 molecular weight. The latter polypeptide has previously been identified as the precursor to the group-specific antigens of the virus ("gag" proteins) [Vogt, V. M., Eisenman, R. & Diggelmann, H. (1975) J. Mol. Biol. 96, 471-493]. Two-dimensional tryptic peptide analyses of the [35S]methionine-labeled peptides demonstrate that the 180,000-dalton product is a polyprotein that can account for all the peptides of the avian myeloblastosis virus DNA polymerase (DNA nucleotidyltransferase, EC 2.7.7.7) and those of the gag viral proteins. This is direct confirmation of the genomic order of the viral structural genes, placing the polymerase gene adjacent to the 5'-proximal gag gene of the virus. Furthermore, our findings suggest that the primary polymerase gene product is the beta subunit of the enzyme. These results are discussed in relation to the proposed structural gene map for the avian retraviruses and suggest a model for the in vivo processing of the viral polymerase.
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PMID:Cell-free synthesis of the precursor polypeptide for avian myeloblastosis virus DNA polymerase. 20 Sep 40

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.
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PMID:Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. 156 78

We have been studying the role of human cytomegalovirus (HCMV) as a potential cofactor in human immunodeficiency virus (HIV)-related disease. The clinical relevance of HCMV is highlighted by the fact that it is a principal viral pathogen in patients with AIDS and is known to infect the same cells as HIV. In this study, we focused on the molecular interactions between HIV and HCMV in human fibroblasts and in the human glioblastoma/astrocytoma-derived cell line U373 MG, cells which can be productively infected by both viruses. Because these cells are CD4-, we used HIV pseudotyped with a murine amphotropic retrovirus as described previously (D. H. Spector, E. Wade, D. A. Wright, V. Koval, C. Clark, D. Jaquish, and S. A. Spector, J. Virol. 64:2298-2308, 1990). Initial studies showed that when cells were preinfected with HIV (Ampho-1B) for 5 days and then superinfected with HCMV, HIV antigen production dropped significantly in the coinfected cells but continued to rise in cells infected with HIV (Ampho-1B) alone. HCMV production, however, was unaffected by the presence of HIV. Further analysis showed that HIV steady-state RNA levels and gag and env protein production were also inhibited in the presence of HCMV. The transcriptional inhibition of HIV was particularly surprising in view of the previous results of several other laboratories as well as our own that HCMV infection stimulates HIV long terminal repeat-chloramphenicol acetyltransferase (LTR-CAT) expression in transient expression assays. To investigate this further, we transfected the HIV LTR-CAT construct into either uninfected cells or cells which had been preinfected with HIV. The cells were infected with HCMV 24 h posttransfection and assayed for CAT gene expression at 48 h after HCMV infection. Although there was some stimulation of the LTR-CAT in cells that were dually infected by HIV and HCMV, it was 16-fold less than that in the cells infected only with HCMV. This suggests that in the presence of the HIV infection, the stimulation of the HIV LTR-CAT gene by HCMV is significantly reduced. Experiments with UV-irradiated HCMV and the HCMV DNA polymerase inhibitor ganciclovir showed that HCMV transcription is necessary for the reduction in HIV production to occur; however, replication of the HCMV genome or any events which take place after DNA replication are not necessary. These results, coupled with the observation that inhibition is usually first seen between 8 and 24 h after HCMV infection, suggest that an HCMV early protein is involved in repression of HIV.
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PMID:Human cytomegalovirus inhibits human immunodeficiency virus replication in cells productively infected by both viruses. 165 86

S-Antigen (S-Ag) is a well characterized 45,000 m.w. photoreceptor cell protein. When injected into susceptible animal species, including primates, it induces an experimental autoimmune uveitis, a predominantly T cell-mediated autoimmune disease of the retina and uveal tract of the eye, and of the pineal gland. In this study we found an amino acid sequence homology between a uveitopathogenic site of S-Ag, several viral proteins and one additional nonviral protein. An experimental autoimmune uveitis and pinealitis was induced in Lewis rats with these different synthetic peptides, corresponding to the amino sequence of hepatitis B virus DNA polymerase, gag-pol polyprotein of Baboon endogenous virus and gag-pol polyprotein of AKV murine leukemia virus and potato proteinase inhibitor IIa, which contain three or more consecutive amino acids identical to peptide M in S-Ag. Lymph node cells from rats immunized with either peptide M or the different synthetic peptides showed a significant degree of cross-reaction. Mononuclear cells from monkeys (Macaca fascicularis) immunized with peptide M also showed significant proliferation when incubated with either peptide M or synthetic peptides as measured by in vitro lymphocyte mitogenesis assay using [3H]TdR. Based on our findings we conclude that a viral infection may sensitize the mononuclear cells that can cross-react with self proteins by a mechanism termed molecular mimicry. Tissue injury from the resultant autoantigenic event can take place in the absence of the infectious virus that initiated the immune response.
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PMID:Molecular mimicry between a uveitopathogenic site of S-antigen and viral peptides. Induction of experimental autoimmune uveitis in Lewis rats. 168 49

The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants.
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PMID:Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity. 171 22

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.
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PMID:Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes. 299 63

Chicken myeloblasts transformed by avian myeloblastosis virus (AMV) in the absence of nondefective helper virus (termed nonproducer cells) were found to release a defective virus particle (DVP) that contains avian tumor viral gag proteins but lacks envelope glycoprotein and a DNA polymerase. Nonproducer cells contain a Pr76 gag precursor protein and also a protein that is indistinguishable from the Pr180 gag-pol protein of nondefective viruses. The RNA of the DVP is 7.5 kilobases (kb) long and is 0.7 kb shorter than the 8.2-kb RNAs of the helper viruses of AMV, MAV-1 and MAV-2. Comparisons based on RNA.cDNA hybridization and mapping of RNase T1-resistant oligonucleotides indicated that DVP RNA shares with MAV RNAs nearly isogenic 5'-terminal gag and pol-related sequences of 5.3 kb and a 3'-terminal c-region of 0.7 kb that is different from that found in other avian tumor viruses. Adjacent to the c-region, DVP RNA contains a contiguous specific sequence of 1.5 kb defined by 14 specific oligonucleotides. Except for two of these oligonucleotides that map at its 5' end, this sequence is unrelated to any sequences of nondefective avian tumor viruses of four different envelope subgroups as well as to the specific sequences of fibroblast-transforming avian acute leukemia and sarcoma viruses of four different RNA subgroups. The specific sequence of the DVP RNA is present in infectious stocks of AMV from this and other laboratories in an AMV-transformed myeloblast line from another laboratory, and it is about 70% related to nucleotide sequences of E26 virus, an independent isolate of an AMV-like virus. Preliminary experiments show DVP to be leukemogenic if fused into susceptible cells in the presence of helper virus. We conclude that DVP RNA is the leukemogenic component of infectious AMV and that its specific sequence, termed AMV, may carry genetic information for oncogenicity. Thus we have found here a transformation-specific RNA sequence, unrelated to helper virus, in a highly oncogenic virus that does not transform fibroblasts.
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PMID:Genetic structure of avian myeloblastosis virus, released from transformed myeloblasts as a defective virus particle. 615 39

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.
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PMID:Avian retrovirus pp32 DNA-binding protein. I. Recognition of specific sequences on retrovirus DNA terminal repeats. 629 95


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