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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.
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PMID:The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme. 7 Apr 28

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function. 169 96

RNA tumor viruses contain a characteristic RNA-dependent DNA polymerase (reverse transcriptase) which has been thought to be related to the induction of leukemia by this virus. A disturbance in a zinc-dependent enzyme system was first postulated to account for the demonstrated differences in zinc metabolism of normal and leukemic leukocytes [Vallee et al. in (1949) Acta Unio. Int. Contra Cancrum 6, 869 and (1950) Acta Unio. Int. Contra Cancrum 6, 1102]. In order to investigate the relationship between zinc and the initiation of leukemia in chickens by avian myeloblastosis virus, we have examined the metalloenzyme nature of its reverse transcriptase. The present data show that this protein is a zinc metalloenzyme demonstrating the postulated relationship between zinc and a leukemic process. Paucity of purified enzyme generated the design of a novel system of analysis incorporating microwave-induced emission spectrometry combined with gel exclusion chromatography. It provides precision, reproducibility, and remarkable limits of detection on mul samples containing 10(-12) to 10(-14) g-atoms of metal, and is thus orders of magnitude more sensitive than other methods. The chromatographic fraction with highest enzymatic activity contains 1.8 x 10(-11) g-atoms of zinc per 1.6 mug of protein, corresponding to either 1.8 or 2.0 g-atoms of zinc per mole of enzyme for a molecular weight previously determined either as 1.6 or 1.8 x 10(5). Copper, iron and manganese are absent, i.e., at or below the limits of detection, 10(-13) to 10(-14) g-atoms. Agents known to chelate zinc inhibit the enzyme, while their nonchelating isomers do not. The data underline the participation of zinc in nucleic acid metabolism and bear importantly upon the lesions that accompany leukemia and zinc deficiency.
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PMID:RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus: a zinc metalloenzyme. 413 17

Melanoma cells often display a multidrug-resistant phenotype, but the mechanisms involved are largely unknown. We have studied here the recently identified transport-associated proteins, MRP and LRP, and the well-known drug resistance marker P-glycoprotein using a panel of 16 human melanoma cell lines and 71 benign and malignant melanocytic tissue samples. By flow cytometry and immunohistochemistry, expression of P-glycoprotein was not detectable on the protein level in the 10 cell lines analyzed, although by reverse transcriptase polymerase chain reaction, MDR-1 gene expression was demonstrated in 2 of 10 cell lines. In addition, immunohistology revealed P-glycoprotein expression in only 1 of 71 melanocytic lesions. In contrast, MRP was detected in a subset of melanoma cell lines by reverse transcriptase polymerase chain reaction and immunohistology (4 of 10). LRP expression was observed in 8 of 10 melanoma cell lines by immunochemistry and in 10 of 10 by reverse transcriptase polymerase chain reaction. Furthermore, MRP was detected immunohistologically in almost 50% of primary and metastatic melanoma specimens, although no significant differences were found between metastases taken before or after chemotherapy. Expression of LRP was detected in a subset of nevi with nevus cells exhibiting up to 25% positive LRP reactivity. In 13 of 21 primary melanomas and 23 of 37 metastases, more than 25% of tumor cells were stained by the LRP-56 monoclonal antibody. Particularly in the group of metastases with more than 50% of LRP-positive cells, 7 of 11 of the metastases had been previously exposed to chemotherapeutic drugs. Although the expression of membrane transport proteins may explain only the chemoresistance toward lipophilic, natural compounds and not resistance against alkylating agents, the lack of P-glycoprotein expression after chemotherapeutic treatment and the significant expression of MRP and LRP in melanoma cells provide first insights into the drug-resistant phenotype in melanoma. Additional studies analyzing the role of MRP and LRP in chemoresistance of melanoma are warranted.
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PMID:Membrane transport proteins associated with drug resistance expressed in human melanoma. 749 78

Human malignant melanoma is characterised by unresponsiveness to conventional chemotherapy. Melanoma-derived cell lines are often markedly chemoresistant, suggesting that cellular mechanisms mediate the multidrug resistance (MDR) phenotype. The multidrug resistance-associated protein (MRP) is a drug transporter protein associated with resistance to a broad spectrum of lipophilic drugs. To investigate whether MRP is involved in intrinsic drug resistance of human melanoma, we analysed expression and functional activity of MRP as well as its impact on chemoresistance in 40 melanoma cell lines (35 established by us from primary and metastatic lesions and 5 obtained from international sources), as well as in one dysplastic naevus-derived cell line and in normal melanocytes. By reverse transcriptase-polymerase chain reaction various levels of MRP mRNA were detected in all melanoma cell lines, and by immunoblot the corresponding protein in a high percentage of them. Functional activity of MRP was assayed by analysing cellular accumulation of 3H-daunomycin (3H-DM) and calcein in response to MRP-modulators by beta-spectrometric and fluorescence-activated cell sorter analysis, respectively. Probenecid (PRO), N-ethylmaleimide (NEM) and benzbromarone (BB) moderately (< or = 1.43-fold) but significantly enhanced intracellular accumulation of MRP substrate probes corresponding to MRP expression. Moreover, the sensitivity of melanoma cell lines to daunomycin (DM) and doxorubicin (DOX), but not to vinblastine (VBL), etoposide (VP-16) and cisplatin (CDDP), analysed by an MTT-based survival assay, were inversely correlated with MRP-gene expression. Our results imply that MRP may be a component of the intrinsic chemoresistance phenotype characteristic of human malignant melanoma.
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PMID:Possible role of the multidrug resistance-associated protein (MRP) in chemoresistance of human melanoma cells. 909 73

Monoclonal antibody 57B specifically detects MAGE-3 gene protein expression. MAGE-derived peptides are recognized by CD8+ T cells and applied in immunotherapy. We examined formalin-fixed, paraffin-embedded tissue of 61 melanoma (primary, n = 40; metastatic, n = 21) and 46 control cases (junctional, dermal, compound, Spitz, Reed, and balloon-cell nevi) by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase method after antigen retrieval. Immunoreactivity was rated positive at 20 positive cells per tumor or more. Staining pattern was homogeneous, scattered, or focal. All control samples and internal controls were immunonegative. Staining with monoclonal antibody 57B showed a specificity of 100% with a sensitivity of 44%. Immunopositivity (overall, 44% of melanomas) increased along with tumor, node, and metastasis stage; pT1 showed 13%, pT2 22%, pT3a 29%, pT3b 45%, pT4 100%, pTxN1 60%, and pTxNxM1a 63% of samples positive. The staining pattern was homogeneous on pT1 to pT3a tumors, homogeneous or focal in pT3b and pT4a, and homogeneous, focal, or scattered in pTxN1 and pTxNxM1a. The frequency of immunopositivity relates well to data on mRNA expression using reverse transcriptase polymerase chain reaction in a subgroup analyzed by both methods. Monoclonal antibody 57B can be used to allow profiling of melanomas using routine archival tissue, when considering immunotherapeutic approaches involving MAGE-3-derived epitopes.
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PMID:MAGE-3 immunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution. 940 5

The gene MTS1 encodes p16INK4, an inhibitor of cyclin-dependent kinase 4, and is frequently deleted, mutated, or silenced by promoter methylation in melanoma cells and in the germline of familial melanoma patients. Although MTS1 may thus be the candidate melanoma suppressor gene that maps to chromosome 9p21, it is not clear how dysfunction at that locus temporally relates to melanoma progression. To further test its role in sporadic melanoma, the expression of p16INK4-protein and -mRNA was characterized in melanomas and melanocytic nevi by immunocytochemistry and in situ reverse transcriptase-polymerase chain reaction. Histologic tissue sections were immunolabeled with anti-p16INK4 antibody for 108 melanocytic lesions, including common and atypical nevi, in situ melanomas, primary invasive melanomas, and metastatic tumors. A subset of the lesions was analyzed for expression of p16INK4-mRNA, employing forward and reverse intron-bridging primers for reverse transcriptase-polymerase chain reaction amplification of the transcript corresponding to exons 1 and 2 of MTS1. Strong immunolabeling was detected in the melanocytes of common nevi and of nevi with architectural disorder and cytologic atypia. By digital image analysis, in contrast, labeling intensity decreased significantly and progressively in the melanocytes of in situ, invasive, and metastatic melanomas. Results from the in situ reverse transcriptase-polymerase chain reaction analysis were confirmatory, showing a strong signal in the melanocytic nevi but progressive signal attenuation with increasing stage of melanoma. These data indicate correlation between gradual loss of expression of the MTS1 locus and progression of melanoma, further supporting an emerging role for the gene in the malignant transformation of melanocytes. The failure to demonstrate reduced expression in nevi suggests either that these lesions are not an early stage in melanoma development, in contrast to prevailing assumptions, or that loss of p16INK4 function is not an initiating event in melanocyte transformation.
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PMID:Expression of the tumor suppressor gene product p16INK4 in benign and malignant melanocytic lesions. 962 Mar 1

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT-PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.
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PMID:DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases. 969 34

Recently p73, a novel p53 homologous tumour suppressor gene, has been cloned and mapped to chromosome 1p36. Like p53, important functions of p73 in controlling the cell cycle and programmed cell death have been described. Loss of p73 has been demonstrated in neuroblastomas and its involvement in tumorigenesis has been suggested to occur in other neuroectodermal cancers. Since genetic alterations at the tumour suppressor locus 1p36 have been also identified in malignant melanomas, we investigated the expression of p73 in a panel of nine different human melanoma cell lines, 17 melanocytic naevi, 17 primary malignant melanomas and 20 metastases by reverse transcriptase polymerase chain reaction (PCR) and Southern blotting. We observed significant p73 mRNA expression in all the cell lines and tissue specimens except one benign melanocytic naevus and one melanoma metastasis. Sequencing the PCR fragments of nine melanoma cell lines derived from primary tumours and five metastases over the entire p73 DNA binding domain revealed wild-type sequences in all cases. In summary, we conclude that loss of p73 mRNA expression or mutations in the p73 DNA binding domain do not represent common genetic events involved in the pathogenesis of malignant melanomas.
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PMID:Loss of expression or mutations in the p73 tumour suppressor gene are not involved in the pathogenesis of malignant melanomas. 991 12

Normal human melanocytes have been shown to respond to the signal peptide endothelin by increased proliferation and melanin formation. Contradictory findings, however, have been reported about which of the two endothelin receptors (EDNRA or EDNRB) is expressed in normal melanocytes and melanoma cells. Moreover it was not clear whether malignant cells differ from their normal precursors in this respect. Screening a melanocyte cDNA library for genes downregulated in melanomas identified clones specific for EDNRB. Northern blots proved that the corresponding mRNA is generally expressed in cultures of human cutaneous melanocytes and congenital melanocytic nevus cells. In 16 of 17 melanoma cell lines, however, the expression of EDNRB mRNA was strongly downregulated. EDNRA was only weakly expressed and detectable by northern blotting in 12 of 17 cultures of benign melanocytic cells and four of 17 melanoma cell lines. Nested reverse transcriptase-polymerase chain reaction proved several melanoma cell lines to be completely negative for EDNRA expression. Gene deletion as the cause of missing endothelin receptor expression was ruled out by genomic Southern blots. Receptor binding assays confirmed RNA data revealing 1.6 x 105 endothelin-1 binding sites per cell for a melanocyte culture and between 8.7 x 104 and 400 sites per cell for melanoma cell lines. Expression of pigmentation genes coding for tyrosinase, TRP-1 and TRP-2 correlated positively with that of EDNRB but negatively with EDNRA expression. EDNRB but not EDNRA expression is therefore typical for melanocytic cells, and downregulation of EDNRB seems to be an important characteristic of melanoma cells possibly related to malignancy or apoptosis.
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PMID:Downregulation of endothelin B receptor in human melanoma cell lines parallel to differentiation genes. 1038 40


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