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)

A series of active elongation complexes of the phage T7 RNA polymerase were obtained through stepwise walking of the polymerase along an immobilized DNA template. Transcripts were radiolabeled at the 16th to 18th residues, and a photocross-linkable 4-thio-UMP was separately incorporated at the 22nd, 24th, 32nd, and 38th residues. Such complexes (up to 51 nucleotides) produced by the incorporation of one nucleotide at a time were isolated and individually subjected to long wave UV cross-linking. Only when the cross-linker was positioned at the 3'-end (-1) of the elongating RNA and 8 nucleotides upstream (-9), was the RNA substantially cross-linked to the polymerase, regardless of how far it was from the 5'-end of the transcripts. Linkage of the 3'-end residue was mapped to the Thr(636)-Met(666) region, which contains nucleotide-binding sites. The -9 residue was cross-linked to the Ala(724)-Met(750) region rather than to the N-terminal region. These two contacts were maintained throughout the elongation complexes and reveal a route of nascent RNA through the T7 RNA polymerase in elongation complexes.
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PMID:Two site contact of elongating transcripts to phage T7 RNA polymerase at C-terminal regions. 1105 70

Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase. Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (approximately 120 kDa) of the protein complex. We assigned all of the (13)C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques. The result of (13)C-carbonyl NMR experiment on [(13)C'-Met]-CRP in the presence of both cyclic AMP and RNA polymerase alpha subunit showed that the two proteins interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP. The results also showed the effectiveness of the selective labeling and (13)C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules.
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PMID:Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by (13)C-carbonyl NMR. 1143 80

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.
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PMID:Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2. 1152 94

The rhabdoid cell, which is typically observed in malignant rhabdoid tumor (MRT) and other malignant neoplasms, has an eosinophilic cytoplasm containing a spheroid perinuclear inclusion body. This distinct cell is known to act as a highly aggressive indicator in many types of malignant tumors and is characterized by aggregates of intermediate filaments, comprising both vimentin and cytokeratin (CK) 8, which is mainly expressed in simple-type epithelium such as liver and intestine. To clarify the cause of the inclusion body formation, we analyzed the alteration of the complete human CK8 gene (KRT 8: 1724 base pairs) in seven samples of MRT (three from frozen materials and four from cultured cell lines) by reverse-transcriptase polymerase chain reaction, followed by direct sequencing. In addition, the two cell lines, Huh7 and HeLa, which lacked rhabdoid feature, six pediatric malignant tumors, including three cases of primitive neuroectodermal tumor (PNET) and three of Wilms' tumor; and 15 normal liver tissue (as a control) were also analyzed. All MRT samples had missense mutations in the human KRT 8 gene, i.e., Arg89 --> Cys (5/7); Arg --> Cys251 (3/7); Glu267 --> Lys (6/7); Ser290 --> Ile, Met; (7/7) and Arg301 --> His(4/7), none of which was detected in any control samples. Among these mutations, the most noteworthy findings were that Arg89 belongs to the H1 subdomain of the head domain and that Arg251 belongs to the short nonhelical linker segment, or L1-2. Both these mutations are noted for their relationships to lateral protofilament-protofilament interactions. In addition, Ser290 has been previously reported to be a phosphorylation site, which has been recognized to play an important role in filament organization, leading to conformational change of the CK8 filaments. In conclusion, mutated codons of CK8 gene in MRT were located in the important region involved in the conformational change of intermediate filament.
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PMID:Mutation analysis of human cytokeratin 8 gene in malignant rhabdoid tumor: a possible association with intracytoplasmic inclusion body formation. 1185 May 43

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

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.
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PMID:[Use of the real-time RT-PCR method for investigation of small stable RNA expression level in human epidermoid carcinoma cells A431]. 1452 Aug 71

The level of 5S rRNA and tRNAi(Met)1 synthesized by RNA polymerase III was investigated in human epidermoid carcinoma cells A431 at different physiological states: low and high proliferation and apoptosis. The real-time RT-PCR method using SYBR Green I was applied to measure certain RNA species in total cellular RNA. The share of 5S rRNA was practically the same in slowly and actively proliferating A431 cells, but increased about 2.5-fold in apoptotic cells. The share of initiator tRNAi(Met)1 in actively proliferating and apoptotic cells was 1.5-2.0 times higher than in slowly proliferating cells. Our results suggest a possible existence of special mechanisms regulating RNA polymerase III-directed transcription from different type promoters in accordance with the physiological state of the cell.
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PMID:Small stable RNA level depends on the physiological state of the cell. 1534 89

Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3' flanking region (rs165599)--both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val--had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3' SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a predominant factor that determines higher COMT activity in the prefrontal cortex, which presumably leads to lower synaptic dopamine levels and relatively deleterious prefrontal function.
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PMID:Functional analysis of genetic variation in catechol-O-methyltransferase (COMT): effects on mRNA, protein, and enzyme activity in postmortem human brain. 1545 4

Liver regeneration depends on timely restoration of cellular mass while orchestrating structural matrix remodeling. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the extracellular matrix (ECM) turnover and, more recently, the processing of growth factors and cytokines. We have previously demonstrated that TIMP-1 inhibits preneoplastic hepatocyte proliferation by attenuating growth factor bioavailability. In the present study, we examined the role of TIMP-1 in de novo hepatocyte cell division during liver regeneration. Comprehensive real-time reverse-transcriptase polymerase chain reaction analyses of regenerating livers revealed significant inductions in the messenger RNA of TIMP-1, TIMP-3, TIMP-4, MMP-2, MMP-9, MMP-13, MMP-14, and MMP-24, while MMP-15 expression was significantly reduced. Induction of TIMP-1 occurred during the peak of hepatocyte DNA synthesis. Studies using genetically altered mice revealed that TIMP-1 loss of function accelerated hepatocyte cell cycle progression. This finding was demonstrated by earlier expression of cyclin D1, proliferating cell nuclear antigen, and phosphorylated histone H3, which mark the G(1)-S, S, and M phase, respectively. Conversely, TIMP-1 gain of function delayed cell cycle progression. MMP activity was increased in the absence of Timp-1. Examination of hepatocyte growth factor (HGF), and its receptor Met, both of which provide a mitogenic signal for hepatocyte division, showed increased HGF activity in Timp-1(-/-)-regenerating livers. HGF is released from the ECM and is proteolytically processed to its active form. Active HGF was elevated in Timp-1(-/-) mice, leading to increased immunostaining of phosphorylated Met as well as activation of a downstream effector, p38. In conclusion, TIMP-1 is a novel negative regulator of HGF activity during liver regeneration.
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PMID:Metalloproteinase inhibitor TIMP-1 affects hepatocyte cell cycle via HGF activation in murine liver regeneration. 1572 41

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.
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PMID:Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal. 1578 Aug 78


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