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)

The single-stranded DNA binding protein RP-A is required in SV40 DNA in vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replication in vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase alpha-primase complex.
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PMID:Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. 133 80

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.
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PMID:Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA. 135 40

The proliferative capacity of brain-tumor cells was analyzed in vitro and in situ using monoclonal antibody (MAb) against deoxyribonucleic acid (DNA) polymerase alpha. For the in vitro studies, two cultured human glioma cell lines were investigated using MAb against DNA polymerase alpha, the MAb Ki-67, a serum against proliferating cell nuclear antigen (PCNA/cyclin), bromodeoxyuridine (BUdR), and an anti-BUdR MAb. During exponential growth of the cells, the percentage of polymerase alpha-positive cells (the "polymerase alpha score") ranged from 72.0% to 77.1%, the Ki-67-positive cells (the "Ki-67 score") ranged from 43.4% to 59.4%, the PCNA/cyclin-positive cells from 30.9% to 41.4%, and the BUdR labeling index from 28.6% to 39.3%. For the in situ studies, tissue from 60 human brain tumors and from two normal human brains was investigated and the polymerase alpha scores and Ki-67 scores were compared. In normal brain tissue, no immunostaining was found by either method. In brain tumors, both the polymerase alpha scores and the Ki-67 scores correlated with the histological grade of malignancy. Polymerase alpha scores were generally higher than Ki-67 scores in the same specimen, especially in malignant brain tumors. These findings suggest that immunostaining of DNA polymerase alpha is a convenient and important new method by which to estimate the cellular proliferation rate of brain tumors. Polymerase alpha scores may be closer to the growth fraction of the individual tumor than the MAb Ki-67 or other scores.
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PMID:Immunohistochemical demonstration of DNA polymerase alpha in human brain-tumor cells. 196 2

Several nuclear and surface proteins are expressed in varying amounts in the different phases of the cell cycle. For some of them the coding gene is not known and changes in their expression could simply be secondary to changes in the proliferative activity of the population. Other proteins are oncogene products, probably having a direct regulatory function in cell proliferation, differentiation and malignant transformation. Studying these proteins may both permit a better understanding of the mechanisms regulating proliferation and differentiation and provide kinetic parameters for describing the cell cycle. Based on antibodies against these proteins, bivariate flow cytometry (FCM) is able to quantitate their expression simultaneously with DNA distribution. This allows protein expression to be related precisely with each cell cycle phase in populations having different proliferative activity. Further advantages of bivariate FCM are that few cells are required for the analysis and the percentage of cells expressing the (onco) gene product can be determined. Several cellular proteins have been investigated with bivariate FCM, and the data are reviewed. Some proteins not coded by oncogenes (such as cyclin, the Ki-67 reactive antigen and DNA polymerase alpha) are expressed in cycling, but not in G0 cells and are of special interest for the kineticist, since they could identify cells which are able to initiate DNA synthesis, i.e. those representing the "growth fraction" of the population. Statin, on the contrary, is apparently expressed only in G0 cells. The expression of some proteins coded by oncogenes, such as p53 and the c-myc product is high in proliferating G1 cells and decreases with differentiation. The expression of the c-ras product is not strictly related to cell cycle phases and increases with differentiation. Technical improvements (allowing, for example, the monitoring of the changes in protein expression following the microinjection of a protein-blocking substance into the cells and the inclusion of phenotype markers into the analysis) will expand the role of bivariate FCM for these research works.
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PMID:Cell cycle-related proteins and flow cytometry. 214 99

A panel of murine hybridoma cell lines which produce antibodies against polypeptides present in human placental DNA polymerase delta preparations was developed. Eight of these antibodies were characterized by virtue of their ability to inhibit DNA polymerase delta activity and immunoblot the 170-kDa catalytic polypeptide. Six of these eight antibodies inhibit DNA polymerase delta but not DNA polymerase alpha, showing that the two proteins are distinct. However, the other two monoclonal antibodies inhibited both DNA polymerase delta and alpha activities, providing the first evidence that these two proteins have a structural relationship. In addition to antibodies against the catalytic polypeptide we also identified 11 antibodies which recognize 120-, 100-, 88-, 75-, 62-, 36-, and 22-kDa polypeptides in DNA polymerase delta preparations, suggesting that these proteins might be part of a replication complex. The antibody to the 36-kDa polypeptide was shown to be directed against proliferating cell nuclear antigen/cyclin. These antibodies should prove useful for studies aimed at distinguishing between DNA polymerases alpha and delta and for the investigation of the functional roles of DNA polymerase delta polypeptides.
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PMID:Immunochemical studies of DNA polymerase delta: relationships with DNA polymerase alpha. 247 16

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.
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PMID:Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells. 256 36

We have investigated the gene expression of PCNA (Proliferating Cell Nuclear Antigen)/cyclin in rat tissues and the R3230AC mammary tumor. The steady-state mRNA level of PCNA/cyclin in a tissue is related to the proliferation of the tissue. The observation was confirmed with the results from the studies of the immunoblotting analyses and the DNA polymerase activity measurements. Furthermore, an overexpression of PCNA/cyclin was found in the R3230AC mammary tumor, which is accompanied by an altered PCNA/cyclin gene structure detected with the Southern blot analysis.
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PMID:Gene expression of PCNA/cyclin in adult tissues and the R3230AC mammary tumor of rat. 256 93

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.
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PMID:Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. 257 94

Aspects of the regulation of DNA replication and mitosis have been studied using a cell-free extract of Xenopus eggs. The extract is characterized by repeated cycles of DNA replication and mitosis, which are accompanied by periodic synthesis and degradation of cyclins as well as fluctuations in the level of Histone H1 kinase activity. DNA replication in this system is dependent upon the formation of a nucleus. However, while nuclear structures are clearly required for initiation, a complete nuclear membrane does not appear to be necessary. Indirect immunofluorescence and DIC microscopy indicate that nuclear reformation from chromosomes occurs asynchronously around individual chromatids. Lamin polymerization, biotin-11-dUTP incorporation and association of polymerases with chromatin occur before membrane formation is complete. S phase nuclei are typified by the co-distribution of both anti-DNA polymerase alpha and anti-PCNA antibodies as discrete spots of fluorescence which align the chromatin. However, as DNA replication is terminated, PCNA fluorescence fades and DNA polymerase alpha dissociates from the chromatin and is redistributed throughout the nucleoplasm. By inhibiting DNA replication with aphidicolin, both DNA polymerase alpha and PCNA remain associated with the chromatin throughout prolonged incubation. Under these conditions mitosis is delayed by up to 70 min, although both the general rate of protein synthesis and more importantly the rate of cyclin synthesis and histone kinase activation are unaffected. Upon nuclear envelope breakdown and lamin dispersal, cyclins degrade; however, no chromosomes are formed, and both PCNA and DNA polymerase alpha remain associated with the chromatin. Also, histone kinase activity is maintained at elevated levels.
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PMID:DNA replication and cell cycle control in Xenopus egg extracts. 257 47

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.
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PMID:Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways. 264 71


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