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)

Biochemical and immunological properties of structural and non-structural polypeptides of the human simplex viruses (HSV1 and HSV2) and four related herpesviruses of non-human primates [Herpesvirus simiae (B virus), H. cercopithicus (SA8), H. saimiri 1 (HVS 1), and H. ateles 1 (HVA 1)] were compared. Using a radioimmunoassay (RIA), the presence of antigenic determinants shared among all six viruses was demonstrated. The relative degree of antigenic cross-reactivity among these viruses was further assessed by competition RIA. Antigenically, HSV 1 and HSV 2 were most closely related to each other although both SA 8 and B virus were also very closely related to HSV 1. Considerably less cross-reactivity existed between either HVS 1 or HVA 1 and the other four primate herpesviruses. Cross-hybridization between simian and human herpesvirus genomes demonstrated that extensive homology exists between each of the simian viruses and both HSV1 and HSV 2. Viral polypeptides bearing common antigenic determinants were identified by immune precipitation of infected cell polypeptides and by immunoblotting. Among the polypeptides of HSV which were recognized by antisera to simian viruses were the VP 5 and p40 proteins, both of which are structural components of the virion nucleocapsid. Using recombinant plasmids containing sequences of the HSV 1 VP5, p40, DNA polymerase, major DNA binding protein, and TK enzyme genes, homologous sequences were detected in all four simian viruses. Together, these results demonstrate that HSV 1, HSV 2, SA 8, and B virus form a closely related sub-group of the primate herpesviruses; HVS 1 and HVA 1 are also related to the other four primate herpesviruses, albeit more distantly.
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PMID:Simian alphaherpesviruses and their relation to the human herpes simplex viruses. 255 32

We have constructed a map of the genes encoded by a 23,000-nucleotide-pair region of herpes simplex virus type 1. This region, defined by the three adjacent EcoRI fragments N (map coordinates 0.298 to 0.315), F (0.315 to 0.421), and M (0.421 to 0.448), has previously been shown by genetic analysis to contain the genes for thymidine kinase, nucleocapsid protein p40, glycoprotein B, DNA-binding protein, and DNA polymerase. We report the identification and mapping of RNAs defining 13 viral genes encoded by the region 0.298 to 0.448. The transcriptional pattern shows families of overlapping messages, similar to those observed in other regions of the viral genome. We also isolated mutants representing four distinct complementation groups and physically mapped several of the mutations to regions within EcoRI fragment F by marker rescue. Mutations representing complementation groups 1-9 (glycoprotein B), 1-1 (DNA-binding protein), and 1-3 (DNA polymerase) were mapped to coordinates 0.361 to 0.368 to 0.411, and 0.411 to 0.421, respectively. A fourth previously undefined complementation group was mapped to the region between glycoprotein B and DNA-binding protein. Comparing the transcription mapping with marker rescue data suggests that the genes for glycoprotein B, DNA-binding protein, DNA polymerase, and nucleocapsid protein p40 are expressed as 3.3-, 4.2-, 4.3- or 4.2- or both, and 2.4-kilobase mRNAs, respectively.
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PMID:Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45. 619 14

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.
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PMID:Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. 768 3

The divalent nickel ion (Ni2+) is one of several metal ions that can substitute for Mg2+ in the activation of DNA polymerases in vitro, but usually with very low efficiency. We have purified and partially characterized a Ni(2+)-binding protein (p40) from HeLa cell extracts that can specifically enhance the polymerase activity of DNA polymerase alpha (pol alpha) and other DNA polymerases in response to Ni2+. This protein, with a molecular mass of 40 kDa, is a single stranded DNA binding protein that binds to a M13 DNA template-primer with an optimum stoichiometry of approximately 90 equiv of protein per equiv of DNA template and enhances the affinity of pol alpha for the primer-template. In the presence of Ni2+, p40 exhibits an increased affinity for DNA. The p40 increased by 3- to 6-fold the rates at which pol alpha and the Klenow fragment of Escherichia coli DNA polymerase I (KF) replicate different DNA templates in response to Ni2+. The low processivity of Ni(2+)-activated pol on primed M13 ssDNA was also enhanced by the presence of p40. The rates of Ni(2+)-dependent replication by inherently more processive enzymes, DNA polymerase delta and T4 DNA polymerase, were not significantly increased by p40 when M13 ssDNA was used as a template; however, p40 did increase the activity of T4 polymerase on an activated calf thymus DNA template. The protein did not stimulate Mg(2+)-activated DNA replication.
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PMID:A single stranded DNA binding protein isolated from HeLa cells facilitates Ni2+ activation of DNA polymerases in vitro. 799 74

p37 and p40 are two cloned gene products of the five-subunit human cellular DNA replication factor activator 1 (A1) protein complex (also called replication factor C). Here, we describe the solubilization, purification, and characterization of these two proteins that were overproduced in Escherichia coli. Using a nitrocellulose filter binding assay, we demonstrated that the purified A1 p37 protein associated with DNA preferentially at the primer terminus, a property resembling that of the A1 complex. We also show that in the presence of relatively high levels of salt, the recombinant p37 protein alone activated DNA polymerase epsilon but not polymerase delta in catalyzing the elongation of DNA chains. The p40 protein specifically associated with cellular p37 and proliferating-cell nuclear antigen (PCNA) present in HeLa cell cytosolic extract. The addition of purified p40 protein abolished the in vitro polymerase delta-catalyzed DNA elongation reaction dependent on both PCNA and A1. However, this inhibition was reversed by excess polymerase delta, suggesting a specific interaction between the polymerase and the p40 protein. Thus, while p37 binds DNA at the primer end and has a specific affinity for pol epsilon, p40, which binds ATP, interacts with PCNA and pol delta. These activities are essential for the DNA elongation reactions that lead to the synthesis of leading-strand DNA and the maturation of Okazaki fragments.
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PMID:The subunits of activator 1 (replication factor C) carry out multiple functions essential for proliferating-cell nuclear antigen-dependent DNA synthesis. 809 61

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.
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PMID:In vitro reconstitution of human replication factor C from its five subunits. 869 48

The quantitative capacity of the reverse transcription-polymerase chain reaction (RT-PCR) is generally underestimated. In this study, PCR and RT-PCR products were amplified from serially diluted DNA and RNA templates, respectively, using a 35-cycle PCR. In the approximate 30- to 100-fold range of template input above the lower limit of detection, herpes simplex virus ICP27 RT-PCR product yield was dependent on the logarithm of template mRNA input (r2 = 0.99). Likewise, regression analysis indicated that yields of interleukin-12 p40, herpes simplex virus DNA polymerase, and interferon-gamma PCR products were dependent on the logarithm of template DNA input over 40- (r2 = 0.98), 60- (r2 = 0.96), and 100-fold (r2 = 0.99) ranges, respectively. This quantitative relationship appears to derive from the competition for reactants between specific PCR products and nonspecific primer-dimers that occurs at limiting concentrations of template. Although primer-dimers are not generally considered a common feature of PCR, 30 of 32 primer pairs tested in this study produced primer-dimer amplification in the absence of template. Because the coefficient of variation in replicate PCRs was typically 10-20% in the linear range, the precision of PCR was sufficient to measure 4-fold differences in template concentration. Thus, with statistically adequate sample numbers, an appropriate standard curve, and the inherent quantitative capacity of the method, differences in the abundance of a mRNA species are measurable by 35-cycle RT-PCR.
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PMID:The inherent quantitative capacity of the reverse transcription-polymerase chain reaction. 988 74

We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNgamma, IL-16) were expressed at variable amounts. IFNgamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.
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PMID:Quantitative real-time PCR for the measurement of feline cytokine mRNA. 1058 8

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.
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PMID:Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus. 1092 93

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.
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PMID:Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. 1113 25


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