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)

Using the mouse anti-human retinoblastoma gene product (pRB) monoclonal antibody, PMG3-245, pRB was detected immunohistocytochemically in human hepatocellular carcinoma (HCC) tissues and a human HCC cell line, designated OCUH-16, recently established in our laboratory. This antibody reacted with human pRB and yielded a single band of approximately 110 kd from cultured OCUH-16 cells. The granules that stained for pRB were found mostly in the HCC cell nuclei, with a few granules observed in the rough endoplasmic reticulum by electron microscopy. Most of the stained granules were located in the euchromatin-rich areas. The percentage of OCUH-16 cells that expressed pRB or DNA polymerase alpha (DNA-PA) decreased over time as the number of OCUH-16 cells increased. The number of HCC cells that stained for pRB in the biopsy specimens from 11 patients varied and pRB expression was well maintained in early and advanced HCC. The level of pRB expression did not correlate with the differentiation of HCC cells or the clinical prognosis. The expression of pRB statistically correlated with that of DNA-PA (P < .01; r = .92). Some sinusoidal cells also stained for pRB. These findings imply that large deletions in the pRB gene are rare in the initiation or promotion of HCC. The correlation between pRB and DNA-PA may suggest that stained pRB participates in the proliferation of both HCC and non-HCC cells.
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PMID:Expression of the retinoblastoma gene product in human hepatocellular carcinoma. 753 39

The retinoblastoma gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To analyze the expression of retinoblastoma gene product in the process of liver regeneration and the initiation of hepatocellular carcinoma, we studied immunohistochemically the expression of retinoblastoma gene product and DNA polymerase alpha (DPA) in 33 patients with various liver diseases. Only a few hepatocytes positive for retinoblastoma gene product were found in undamaged, nonregenerating liver tissues, whereas many hepatocytes positive for retinoblastoma gene product were detected in specimens of regenerating liver obtained from patients with acute or chronic liver diseases. Similarities were found between distribution patterns of hepatocytes positive for retinoblastoma gene product and those of hepatocytes positive for DPA, and a highly significant positive correlation was found between the number of hepatocyte nuclei stained for retinoblastoma gene product per 1000 nuclei examined (R-LI) and the number of hepatocyte nuclei stained for DPA per 1000 nuclei examined (D-LI) in tissues obtained from patients with nonmalignant liver disease. Hepatocellular carcinoma cells positive for DPA were detected in the 14 hepatocellular carcinoma specimens tested. In ten of these specimens, hepatocellular carcinoma cells positive for retinoblastoma gene product were found but not in the other four. For all hepatocellular carcinoma specimens, R-LI was proportional to D-LI. Thus in both nonmalignant and malignant liver, retinoblastoma gene product increased in proportion to proliferation of hepatocytes or hepatocellular carcinoma cells.
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PMID:Immunohistochemical analysis of retinoblastoma gene product (pRB) expression in malignant and non-malignant liver diseases. 787 33

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.
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PMID:Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein. 790 59

A variety of studies have now implicated the cellular transcription factor E2F as a key participant in transcription control during the cell growth cycle. Although the recent isolation of molecular clones encoding proteins that are components of the E2F activity (E2F1 and DP-1) provides an approach to defining the specific involvement of E2F in these events, definitive experiments remain difficult in the absence of appropriate genetic systems. We have now identified a Drosophila equivalent of E2F1 that we hope will allow an eventual genetic approach to the role of E2F in cellular regulatory events. A cDNA clone was isolated from a Drosophila cDNA library by using a probe containing sequence from the E2F1 DNA binding domain. The sequence of the clone, which we term drosE2F1, demonstrates considerable homology to the human E2F1 sequence, with over 65% identity in the DNA binding region and 50% identity in the region of E2F1 known to interact with the retinoblastoma gene product. A glutathione S-transferase-drosE2F1 fusion protein was capable of binding specifically to an E2F recognition site, and transfection assays demonstrated that the drosE2F1 product was capable of transcription activation, dependent on functional E2F sites as well as sequences within the C terminus of the protein. Finally, we have also identified E2F recognition sequences within the promoter of the Drosophila DNA polymerase alpha gene, and we demonstrate that the drosE2F1 product activates transcription of a test gene under the control of this promoter. We conclude that the drosE2F1 cDNA encodes an activity with extensive structural and functional similarity to the human E2F1 protein.
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PMID:Functional properties of a Drosophila homolog of the E2F1 gene. 811 98

E2F has been implicated in growth control because of its association with the retinoblastoma protein and the presence of E2F binding sites in the promoters of several growth-regulated genes. Proteins that bind to an E2F site have been cloned from human and mouse cells. However, these two proteins (human E2F1 and mouse DP-1) are quite different in sequence. We have now cloned a mouse cDNA encoding a protein 86% identical to the human E2F1 protein. The mouse E2F1 cDNA encodes a 430-amino-acid protein with a predicted molecular weight of 46,322 and detects mRNAs of 2.7 and 2.2 kb. Using primers complementary to sequences in the mouse E2F1 3' untranslated region, we mapped the mouse E2F1 gene to chromosome 2, near the Agouti and c-src loci. To understand the role of the different E2F family members in the growth of mouse NIH 3T3 cells, we examined the levels of E2F1 and DP-1 mRNAs in different stages of the cell cycle. Since the levels of E2F1 but not DP-1 mRNA correlated with changes in transcription from the dhfr promoter, we examined whether E2F1 could activate various growth-regulated promoters. We found that E2F1 could activate some (dhfr, thymidine kinase, and DNA polymerase alpha) but not all (thymidylate synthase, cad, and c-myc) of these promoters. On the basis of changes in levels of E2F1 and its ability to transactivate growth-regulated promoters, we propose that E2F1 may mediate growth factor-initiated signal transduction.
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PMID:Cloning, chromosomal location, and characterization of mouse E2F1. 811 19

Temperature-sensitive (ts) cell cycle mutant mouse cell, tsFT20, is deficient in DNA polymerase alpha activity to initiate DNA replication at replicon origins. Here, we analyzed phenotypes concerning growth control genes in the arrested tsFT20 cells. Analysis of cyclins showed that expression levels of cyclin D1, which is essential for G1/S transition, remarkably decreased in the mutant cells after temperature up-shift. Further we examined phosphorylation states of retinoblastoma protein (pRB) in the cells. Though the tsFT20 cells arrested in G1/S-S phase at nonpermissive temperature (Eki et al., (1990) J. Biol. Chem. 265 26-33), a large proportion of pRB was found as an underphosphorylated growth-suppressive form in the arrested cells. In revertant cell lines of tsFT20, pRB was not underphosphorylated even at nonpermissive temperature. The pRB underphosphorylation occurred later than the decrease of mRNA levels of cyclin D1, thus the underphosphorylation may be caused by the decrease in amount of cyclin D1 protein. These results indicated that the mutational inactivation of DNA polymerase alpha evokes phenotypes in which the inhibitory machinery of G1/S transition has been turned on.
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PMID:Temperature-sensitive mutation of DNA polymerase alpha induces growth-suppressive phenotypes involving retinoblastoma protein and cyclin D1. 852 29

The expression of DNA polymerase alpha (pol alpha) was studied in human fibroblast lines W138 (fetal lung) and GM3529 (skin, established from a 66 yr old donor), and their Simian virus 40 (SV40) large tumor antigen (TAg)-transformed corollaries, 2RA and 2-1 respectively. Both SV40-transformed and pSV3.neo (SV40-derived plasmid)-transformed cells express TAg, a virally encoded protein not expressed by the normal parent cell lines. Northern blot hybridization studies showed increased recovery of pol alpha mRNA from transformed cells compared with normal cells. This increase was correlated with increased pol alpha mRNA transcription as determined by nuclear run-on assays. Northern blot analyses also showed an increase in the instability of translationally active pol alpha mRNA in transformed cells. The results suggest that TAg, in addition to its dsDNA binding, pol alpha binding, retinoblastoma protein binding and helicase activities, may be involved either directly or indirectly in regulation of the steady state mRNA levels of pol alpha at the transcriptional level in both fetal and aged donor-derived transformed fibroblasts.
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PMID:Differential expression of DNA polymerase alpha in normal and transformed human fibroblasts. 864 60

The TFDP genes encode a family of transcription factors that can form heterodimers with E2F family proteins in vivo. The E2F-TFDP transcription factors are major regulators of genes that are required for the progression of S-phase, such as DHFR and DNA polymerase alpha, and they play a critical role in cell cycle regulation and differentiation. The retinoblastoma tumor suppressor protein has been shown to induce growth arrest by binding to E2F-TFDP and repressing its activity. Two human TFDP genes have been cloned, namely TFDP1 and TFDP2 (or DP1 and DP2). In the present study, we identified genomic clones of TFDP1, its pseudogene TFDP1P and TFDP2, and we mapped them to chromosome 13q34, 1q32.3, and 3q23, respectively. Chromosomal abnormalities involving regions 13q34 and 3q23 have been reported in certain lymphomas and other diseases associated with loss of cell cycle regulation, and the involvement of the TFDP transcription factors remains to be elucidated.
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PMID:Genomic cloning and chromosomal assignment of the E2F dimerization partner TFDP gene family. 902 91

Computer analysis of a conserved domain, BRCT, first described at the carboxyl terminus of the breast cancer protein BRCA1, a p53 binding protein (53BP1), and the yeast cell cycle checkpoint protein RAD9 revealed a large superfamily of domains that occur predominantly in proteins involved in cell cycle checkpoint functions responsive to DNA damage. The BRCT domain consists of approximately 95 amino acid residues and occurs as a tandem repeat at the carboxyl terminus of numerous proteins, but has been observed also as a tandem repeat at the amino terminus or as a single copy. The BRCT superfamily presently includes approximately 40 nonorthologous proteins, namely, BRCA1, 53BP1, and RAD9; a protein family that consists of the fission yeast replication checkpoint protein Rad4, the oncoprotein ECT2, the DNA repair protein XRCC1, and yeast DNA polymerase subunit DPB11; DNA binding enzymes such as terminal deoxynucleotidyltransferases, deoxycytidyl transferase involved in DNA repair, and DNA-ligases III and IV; yeast multifunctional transcription factor RAP1; and several uncharacterized gene products. Another previously described domain that is shared by bacterial NAD-dependent DNA-ligases, the large subunits of eukaryotic replication factor C, and poly(ADP-ribose) polymerases appears to be a distinct version of the BRCT domain. The retinoblastoma protein (a universal tumor suppressor) and related proteins may contain a distant relative of the BRCT domain. Despite the functional diversity of all these proteins, participation in DNA damage-responsive checkpoints appears to be a unifying theme. Thus, the BRCT domain is likely to perform critical, yet uncharacterized, functions in the cell cycle control of organisms from bacteria to humans. The carboxyterminal BRCT domain of BRCA1 corresponds precisely to the recently identified minimal transcription activation domain of this protein, indicating one such function.
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PMID:A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. 903 68

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G0 or early G1, and is capable of binding to and potentiating the activity of DNA polymerase alpha (pol alpha). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A. Because of these requirements, a TAg homologous protein could be expected to contain epitopes with amino acid sequences similar to those of TAg at critical functional sites, such as ATP, pRb and pol alpha binding sites. TAg and a putative cellular homolog of TAg, DNA pol alpha accessory protein (alpha AP), were compared for pRb and pol alpha interaction, and for immunological identity. The analyses utilized immunoaffinity-purified TAg and pRb from a baculovirus expression system, and DNA pol alpha/primase and alpha AP chromatographically isolated from a mouse lymphocytic leukemia cell line. Monoclonal antibodies specific for the pol alpha or pRb binding sites on TAg interacted with alpha AP strongly enough to be employed for immunoaffinity purification of alpha AP. Anti-pRb and anti-TAg reciprocally coimmunoprecipitated pRb bound to TAg and pRb bound to alpha AP. The functional consequences of pol alpha interaction with TAg or alpha AP in the presence or absence of pRb was determined using pol alpha nucleotide incorporation assays. alpha AP exhibited the capacity to stimulate pol alpha activity, a capacity which was diminished in the presence of pRb. Lastly, TAg and alpha AP independently co-purified with pol alpha through a multi-step chromatographic protocol. These data indicate that a pol alpha accessory protein, alpha AP, exhibits functional and immunological similarities to SV40 TAg, suggest that alpha AP is involved in regulation of the initiation of DNA synthesis, and support the proposal that alpha AP may be a normal cell protein homologous to SV40 large T antigen.
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PMID:A DNA polymerase alpha accessory protein exhibits structural and functional similarities to SV40 large tumor antigen. 906 21


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