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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The splicing of a procaryotic precursor RNA transcribed from the T4 phage thymidylate synthase (td) gene with SP6 RNA polymerase was investigated in vitro. The intron excision-cyclization reaction increased progressively to 60 degrees C. Exon ligation, though barely detectable at the lower temperatures, was greatly enhanced at 60 degrees C. Both reactions required Mg2+. The addition of guanosine to the 5' end of an intron-exon II intermediate via a 3',5'-phosphodiester bond was essential for the ligation of exon I to exon II. The added guanosine and the first intron-encoded uridine are subsequently lost as a dinucleotide from the 5' end during cyclization of the linear form of the excised intron RNA. Exon ligation is intramolecular and occurs more readily in the nascent RNA molecule (cotranscriptionally) than in the finished transcript (posttranscriptionally). These data and the identification of various structural elements (P, Q, R, S, E, E') in the td intron that are found typically in eucaryotic class I introns firmly establish the td intron as the first example of class I intron of procaryotic origin.
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PMID:Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene. 303 75

The expression of a number of genes was measured in P1798 cells treated for various periods of time with 0.1 microM dexamethasone. Thymidine kinase (TK) activity decreased under these conditions with 50% inhibition achieved within approximately 8 h. Decreased TK activity was associated with reduced abundance of TK mRNA. Analysis of nuclear transcription indicated that this was attributable to a decrease in the number of RNA polymerase II molecules engaged in transcription of the TK gene. With respect to TK, there was an overall correlation between enzyme activity, mRNA, and nuclear transcription. The data are consistent with the hypothesis that glucocorticoid inhibition of expression of TK is primarily due to inhibition of transcription. Transcription of the TK gene was also reduced by greater than 90% after inhibition of protein synthesis for 6 h. This suggests that transcription of this gene requires a protein of short biological half-life. It is proposed that this hypothetical transcription factor is regulated by glucocorticoids. The amount of thymidylate synthase and dihydrofolate reductase remained constant for at least 24 h in dexamethasone-treated P1798 cells. Dihydrofolate reductase mRNA likewise remained constant. However, the mRNA encoding thymidylate synthase decreased 80-90% within 24 h. The mRNA encoding ornithine decarboxylase also decreased. In neither case did this appear to be primarily due to inhibition of transcription of the respective genes. The abundance of the mRNAs encoding hypozanthine-guanine phosphoribosyl transferase and phosphoglycerate kinase did not decrease in dexamethasone-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glucocorticoid regulation of the genes encoding thymidine kinase, thymidylate synthase, and ornithine decarboxylase in P1798 cells. 339 44

cDNAs encoding the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max were isolated and sequenced. The 1794 base full length cDNA contains a single open reading frame of 1593 bases. The predicted size of the encoded protein is 530 amino acids with a molecular weight of 59,707. The protein has two domains: a 226 residue DHFR domain in the N-terminus, which is over 30% identical to human DHFR or the DHFR domain of protozoal DHFR-TS, and a 304 residue thymidylate synthase (TS) domain, which is over 60% identical to eukaryotic TS enzymes. The whole protein sequence is greater than 75% identical to DHFR-TS sequences from two other plants, Daucus carota and Arabidopsis thaliana. The sequence of two tryptic peptides obtained from DHFR preparations matched the predicted amino acid sequence, one peptide lying in the DHFR domain and the other in the TS domain. These results indicate that DHFR and TS exist in a bifunctional polypeptide in Glycine max. The coding region of the cDNA was inserted downstream of the T7 promoter and translation initiation signals in the vector pET-3a. This construct (pDR-TS) was transformed into Escherichia coli BL21 (DE) [plysS] which produces T7 RNA polymerase upon induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of the bifunctional enzyme was confirmed by detection of both DHFR and TS activities. The purified enzyme has a subunit molecular mass of 60 kDa. This is the first report of expression of a plant DHFR-TS cDNA.
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PMID:Cloning, nucleotide sequence and expression of the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max. 774 62

In this report, we describe the use of two human colon carcinoma cell lines, HCT-8 and HT-29, as potential models to study DNA- and RNA-directed cytotoxicity due to 5-fluorouracil (FUra) exposure by flow microfluorimetric analysis of DNA cell content. The sensitivity of the HT-29 line (EC50 = 0.9 microM) to FUra was somewhat greater than that of the HCT-8 line (EC50 = 4 microM), but each presented a dramatically different DNA histogram after exposure to FUra. In HCT-8, an unexpected and nearly complete disappearance of cells in S-phase occurred, whereas in HT-29 the expected accumulation of cells at the G1-S border was observed. The absence of HCT-8 cells in S-phase also occurred as a result of two RNA polymerase inhibitors: actinomycin D and dichloro-D-ribofuranosylbenzimidazole. However, an accumulation of cells in S-phase was observed in the presence of 5-fluorodeoxyuridine. These results suggest that in the HCT-8 cell line, FUra predominantly causes an RNA-related toxicity. By comparison, the rate of formation of 5-fluorodeoxyuridine monophosphate, the increased dUMP pool size, and low thymidylate synthase activity in the HT-29 line are consistent with its greater susceptibility to DNA-directed toxicity. Further evidence was seen in the prevention of FUra cytotoxicity by thymidine in HT-29, but not in HCT-8 cells. Similarly, Leucovorin synergized the action of FUra in HT-29 but not in HCT-8. Enzymatic correlates supporting these observations are seen in the greater activity of uridine kinase than thymidine kinase (20:1) in HCT-8 cells compared with that in HT-29 cells (4:1).
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PMID:Aberrant cell cycle inhibition pattern in human colon carcinoma cell lines after exposure to 5-fluorouracil. 787 61

Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.
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PMID:Structure--activity relationships of the didemnins. 870 12

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.
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PMID:Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction. 898 25

Studies of mouse models of tuberculosis (TB) infection have indicated a central role for MHC class I-restricted CD8+ T cells in protective immunity. To define antigens and epitopes of Mycobacterium tuberculosis (MTB) proteins that are presented by infected cells to CD8+ T cells, we screened 40 MTB proteins for HLA class I A*0201-binding motifs. Peptides that bound with high affinity to purified HLA molecules were subsequently analyzed for recognition by CD8+ cytotoxic T lymphocytes. We identified three epitopes recognized by CD8+ T cells from patients recovering from TB infection. Those three epitopes were derived from three different antigens: thymidylate synthase (ThyA(30-38)), RNA polymerase beta-subunit (RpoB(127-135)), and a putative phosphate transport system permease protein A-1 (PstA1(75-83)). In addition, CD8+ T cell lines specific for three peptides (ThyA(30-38), PstA1(75-83), and 85B(15-23)) were generated from peripheral blood mononuclear cells of normal HLA-A*0201 donors. These CD8+ T cell lines specifically recognized MTB-infected macrophages, as demonstrated by production of IFN-gamma and lysis of the infected target cells. Finally, CD8+ cytotoxic T lymphocytes reduced the viability of the intracellular MTB, providing evidence that CD8+ T cell recognition of MHC class I-restricted epitopes of these MTB antigens can contribute to effective immunity against the pathogen.
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PMID:Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. 1103 87

Chilo iridescent virus (CIV), the type species of the genus Iridovirus, a member of the Iridoviridae family, is highly pathogenic for a variety of insect larvae. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity and strategy of the CIV genome was elucidated by the analysis of the complete DNA nucleotide sequence of the viral genome (212,482 bp) using cycle sequencing by primer walking technology. Both DNA strands were sequenced independently and the average redundancy for each nucleotide was found to be 1.85. The base composition of the viral genomic DNA sequence was found to be 71.37% A+T and 28.63% G+C. The CIV genome contains 468 open reading frames (ORFs). The size of the individual viral gene products ranges between 40 and 2432 amino acids. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs were nonoverlapping. The comparison of the deduced amino acid sequences to entries in protein data banks led to the identification of several genes with significant homologies, such as the two major subunits of the DNA-dependent RNA polymerase, DNA polymerase, protein kinase, thymidine and thymidylate kinase, thymidylate synthase, ribonucleoside-diphosphate reductase, major capsid protein, and others. The highest homologies were detected between putative viral gene products of CIV and lymphocystis disease virus of fish (LCDV). Although many CIV putative gene products showed significant homologies to the corresponding viral proteins of LCDV, no colinearity was detected when the coding strategies of the CIV and LCDV-1 were compared to each other. An intriguing result was the detection of a viral peptide of 53 amino acid residues (ORF 160L) showing high homology (identity/similarity: 60.0%/30.0%) to sillucin, an antibiotic peptide encoded by Rhizomucor pusillus. Iridovirus homologs of cellular genes possess particular implications for the molecular evolution of large DNA viruses.
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PMID:Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus. 1144 71

Bacteriophage phiKZ is a giant virus that efficiently infects Pseudomonas aeruginosa strains pathogenic to human and, therefore, it is attractive for phage therapy. We present here the complete phiKZ genome sequence and a preliminary analysis of its genome structure. The 280,334 bp genome is a linear, circularly permutated and terminally redundant, A+T-rich double-stranded DNA molecule. The phiKZ DNA has no detectable sequence homology to other viruses and microorganisms, and it does not contain NotI, PstI, SacI, SmaI, XhoI, and XmaIII endonuclease restriction sites. The genome has 306 open reading frames (ORFs) varying in size from 50 to 2237 amino acid residues. According to the orientation of transcription, ORFs are apparently organized into clusters and most have a clockwise direction. The phiKZ genome also encodes six tRNAs specific for Met (AUG), Asn (AAC), Asp (GAC), Leu (TTA), Thr (ACA), and Pro (CCA). A putative promoter sequence containing a TATATTAC block was identified. Most potential stem-loop transcription terminators contain the tetranucleotide UUCG loops. Some genes may be assigned as phage-encoded RNA polymerase subunits. Only 59 phiKZ gene products exhibit similarity to proteins of known function from a diversity of organisms. Most of these conserved gene products, such as dihydrofolate reductase, ribonucleoside diphosphate reductase, thymidylate synthase, thymidylate kinase, and deoxycytidine triphosphate deaminase are involved in nucleotide metabolism. However, no virus-encoded DNA polymerase, DNA replication-associated proteins, or single-stranded DNA-binding protein were found based on amino acid homology, and they may therefore be strongly divergent from known homologous proteins. Fifteen phiKZ gene products show homology to proteins of pathogenic organisms, including Mycobacterium tuberculosis, Haemophilus influenzae, Listeria sp., Rickettsia prowazakeri, and Vibrio cholerae that must be considered before using this phage as a therapeutic agent. The phiKZ coat contains at least 40 polypeptides, and several proteins are cleaved during virus assembly in a way similar to phage T4. Eleven phiKZ-encoded polypeptides are related to proteins of other bacteriphages that infect a variety of hosts. Among these are four gene products that contain a putative intron-encoded endonuclease harboring the H-N-H motif common to many double-stranded DNA phages. These observations provide evidence that phages infecting diverse hosts have had access to a common genetic pool. However, limited homology on the DNA and protein levels indicates that bacteriophage phiKZ represents an evolutionary distinctive branch of the Myoviridae family.
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PMID:The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. 1191 76

The novel antitumor compound NC-190 strongly inhibited the growth of FM3A cells with an IC50 of 0.019 microg/ml (0.042 microM) when cultured with NC-190 for 48 h. NC-190 potently suppressed DNA synthesis, with 90% inhibition observed at 0.1 microg/ml of NC-190. RNA and protein syntheses were also suppressed under the same conditions, but to a lesser extent. We then measured the cellular enzymatic activities of DNA polymerase alpha, RNA polymerase, thymidine kinase, thymidylate synthase and Leu-tRNA synthetase of FM3A cells cultured with or without NC-190. Of these 5 enzymes, the activity of thymidine kinase was most strongly suppressed by NC-190, by 77%. Although NC-190 did not directly inhibit the activitiy of thymidine kinase in a cell-free system, expression of mRNA of thymidine kinase was suppressed by 75% in NC-190-treated cells. These results indicate that NC-190 can suppress the expression of the gene for thymidine kinase and the inhibition of thymidine kinase contributes to the inhibition of cell growth by NC-190 together with the inhibition of topoisomerase II.
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PMID:The topoisomerase II-inhibitor NC-190 reduces the level of thymidine kinase mRNA in murine tumor cells. 1463 16


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