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)

We studied the induction and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) in the ribosomal RNA genes (rDNA) in cultured hamster and human cells. In these genes, which are transcribed by RNA polymerase I, we found no evidence for transcription-coupled repair. The induction of CPDs was heterogeneous in rDNA due to nucleotide sequence: it was lower on the transcribed strand than on the nontranscribed strand and slightly lower in the coding region than in the nontranscribed spacer. Nevertheless, no dramatic difference in CPD induction was observed between rDNA and the dihydrofolate reductase (DHFR) gene. In Chinese hamster ovary cells, we observed no removal of CPDs from either rDNA strand within 24 h after UV irradiation. In these experiments, we did observe efficient repair of the transcribed, but not the nontranscribed, strand of the DHFR gene, in agreement with published results. In human cells, repair of rDNA was observed, but it showed no strand preference and was slower than that reported for the genome overall. No significant differences in repair were observed between restriction fragments from transcribed and nontranscribed regions or between growth-arrested and proliferating human cells, with presumably different levels of transcription of rDNA. We conclude that the modest level of rDNA repair is accomplished by a transcription-independent repair system and that repair is impeded by the nucleolar compartmentalization of rDNA. We discuss the possibility that recombination, rather than repair, maintains the normal sequence of rDNA in mammalian cells.
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PMID:Lack of transcription-coupled repair in mammalian ribosomal RNA genes. 839 97

Intergenic regions of polycistronic pre-mRNAs of trypanosomatid protozoans are the sites of two processing reactions: polyadenylation of the upstream gene and trans-splicing of the capped miniexon to the downstream gene. Their close proximity and the lack of consensus motifs at poly(A) sites led us to test whether poly(A) site selection is governed by the location of the downstream splice acceptor in the DHFR-TS locus of Leishmania major. Whenever the position of the downstream splice site was altered, the poly(A) site was shifted 400-500 nucleotides upstream of the new splice site. In contrast, when the wild-type poly(A) site was eliminated, the downstream splice site was unaffected, and polyadenylation was maintained 200-500 nucleotides upstream of the splice site. In a second set of experiments, T7 RNA polymerase expressed in Leishmania was used to direct the synthesis of artificial pre-RNAs in vivo whose expression was found to require the presence of a downstream splice acceptor. We conclude that poly(A) site selection in Leishmania is specified by the position of the downstream splice acceptor and propose a scanning model for poly(A) site selection after splice site recognition.
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PMID:Coupling of poly(A) site selection and trans-splicing in Leishmania. 850 37

Herpesvirus saimiri, an oncogenic gamma herpesvirus of primates, is the only eukaryotic virus that carries the entire metabolic gene set for a complex biochemical synthesis. Every element of the thymidine synthesis gene cascade is present in the virus, and their function is probably related to the uniquely high A + T content of the genome. Although one member of the gene set, dihydrofolate reductase (DHFR), is mapped in a region required for oncogenesis, very little is known of the expression and function of this gene in transformed cells. We report the expression of the DHFR sequence on a novel, unique tricistronic transcript in virally transformed tumor cells. The DHFR sequence is the first open reading frame on a 5.3 kb minor transcript. Alpha-amanitine sensitivity indicates that it is an RNA polymerase II transcript, and since it is also polyadenylated it appears to be a functional, relatively unstable (half-life 3 hr) mRNA. Initiation of transcription uniquely overlaps with the HSUR3 small RNA gene. Expression of the small transcript appears to be alpha-amanitine resistant, implicating polymerase III transcription. Together with the remarkably low-level expression of HSUR3 in tumor cells, the data may indicate transcription interference between two different RNA polymerases, with unusual overlapping regulation and initiation.
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PMID:A polycistronic transcript in transformed cells encodes the dihydrofolate reductase of herpesvirus saimiri. 856 Jul 76

Camptothecin is a widely used anti-tumor drug that specifically inhibits DNA topoisomerase I. It is believed that topoisomerase I participates in the process of transcription by relaxing torsional stress induced in the duplex DNA by the elongating RNA polymerase. We have assessed the effects of camptothecin on RNA polymerase II transcription from the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Using in vivo [3H]uridine pulse labeling and in vitro nuclear run-on techniques to estimate relative rates of transcription, it was found that camptothecin stimulated RNA synthesis from promoter-proximal sequences of the DHFR gene, while transcription from promoter-distal sequences was reduced. Furthermore, camptothecin caused a significant accumulation of RNA polymerases in the 5'-end of the DHFR gene. The effect of camptothecin on transcription was reversible, resulting in a wave of RNA synthesis recovery in a 5' to 3' direction through the DHFR gene following a chase with camptothecin-free medium. We conclude that camptothecin stimulates initiation but inhibits elongation of the RNA polymerase II transcribed DHFR gene.
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PMID:The anti-cancer drug camptothecin inhibits elongation but stimulates initiation of RNA polymerase II transcription. 856 33

A series of cyano- and carboxyborane adducts of cyclohexylamines and toluidines were shown to be cytotoxic towards suspended single cell tumors. The carboxyborane adducts of cyclohexylamine were more potent than the cyanoborane adducts of cyclohexylamine or any of the toluidine derivatives. A number of the compounds were active at 8 mg/kg/day i.p. in the Ehrlich ascites carcinoma screen in vivo. The mode of action study with N-methylcyclohexylaminecyanoborane 10 in L-1210 lymphoid leukemia cells showed that RNA synthesis was markedly reduced followed by DNA synthesis. Purine de novo synthesis was suppressed at PRPP-amido transferase, IMP dehydrogenase, and dihydrofolate reductase enzyme sites. The agent also interfered with DNA template activity causing reduction of DNA polymerase alpha, and RNA polymerase I, II and III activities. The d[NTP] pools were marginally reduced while DNA viscosity was reduced and DNA fragmentation occurred.
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PMID:Synthesis and cytotoxicity of amine-borane adducts of cyclohexylamines and toluidines. 858 54

Photoactivated psoralen was used to probe region-specific chromatin structure in Chinese hamster ovary (CHO) cells. Specifically, the chromatin structure of six regions within the dihydrofolate reductase (DHFR) gene was probed with photoactivated psoralen in cells cultured in such ways as to differentially express the DHFR gene. Cells were irradiated with X-rays prior to the psoralen photocross-linking reaction in order to eliminate the influence of any DNA torsional tension on the psoralen binding and the sequence-specificity of psoralen binding was adjusted for. It was found that a region encompassing the promoter of the serum-regulated DHFR gene was about 50% more accessible to psoralen photocross-linking in serum-stimulated cells and about 90% more accessible in serum-starved cells than the other five regions of the DHFR gene analyzed and the genome overall. Treating serum-stimulated cells with the RNA polymerase II transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) or the topoisomerase I inhibitor camptothecin reversed the elevated accessibility of the DHFR promoter region. These results suggest that the accessible chromatin structure of the DHFR promoter is not dependent on serum-stimulated poising of the gene for transcription, but may reflect the ability of the RNA polymerase to clear the promoter.
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PMID:Effect of differential gene expression on the chromatin structure of the DHFR gene domain in vivo. 867 2

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

Pneumocystis carinii has been shown to cause extra-alveolar infections in humans, but the lack of a reproducible animal model has hindered the elucidation of mechanisms of P. carinii dissemination. In the present study, PCR and the immunosuppressed rat model were used to gain further insight into the dissemination of P. carinii organisms in extrapulmonary (EP) tissues. Primer sequences specific to major surface glycoprotein (MSG) and dihydrofolate reductase (DHFR) were used to detect P. carinii in lungs and EP tissues. Sprague-Dawley rats were grouped into two classes: one group included rats that had primary episodes of pneumocystosis and the other group included rats that had undergone treatment for P. carinii infection and that had second episodes of pneumocystosis. PCR analysis with MSG primers with tissues obtained from both groups of rats showed the presence of P. carinii DNA in adrenal tissue, bone marrow, blood, and heart, kidney, liver, lymph node, spleen, and thyroid tissues. Reverse transcriptase-PCR (RT-PCR) analysis was carried out with DHFR primers with lung, spleen, heart, kidney, and liver tissues from both groups of rats. Only those tissues that showed a positive PCR result and hybridization signal for the MSG gene were used for the RT-PCR experiments. RT-PCR analysis showed that the P. carinii DHFR gene is actively transcribed in these tissues, thereby indicating the presence of viable P. carinii organisms in EP tissues. Our observations suggest that P. carinii dissemination is influenced by factors other than P. carinii chemotherapy and that heavy organism load and destruction of lung tissue may contribute to the dissemination of P. carinii. The study provides an animal model that can be used for further investigations of the causes of EP pneumocystosis.
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PMID:Identification of extrapulmonary Pneumocystis carinii in immunocompromised rats by PCR. 878 65

The human CSB gene, mutated in Cockayne's syndrome group B (partially defective in both repair and transcription) was previously cloned by virtue of its ability to correct the moderate UV sensitivity of the CHO mutant UV61. To determine whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome, the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61 and a CSB transfectant of UV61 were compared. In each cell line, formation and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) were measured in the individual strands of the actively transcribed DHFR gene and in a transcriptionally inactive region downstream of DHFR. AA8 cells efficiently remove CPDs from the transcribed strand, but not from either the non-transcribed strand or the inactive region. There was no detectable repair of CPDs in any region of the genome in UV61. Transfection of the human CSB gene into UV61 restores the normal repair pattern (CPD removal in only the transcribed strand), demonstrating that the DNA repair defect in UV61 is homologous to that in Cockayne's syndrome (complementation group B) cells. However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has independent roles in DNA repair and RNA transcription pathways.
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PMID:The human CSB (ERCC6) gene corrects the transcription-coupled repair defect in the CHO cell mutant UV61. 881 Oct 84

In vitro DNA-dependent RNA transcription using bacteriophage T3 RNA polymerase may be rendered hypermutagenic by employing biased nucleoside triphosphate (NTP) concentrations and manganese cations. Using the E. coli R67 plasmid-encoded dihydrofolate reductase (DHFR) gene as target substitution rates approaching 4 x 10(-2) per base per reaction could be achieved, on a par with hypermutagenic reverse transcription. In all cases the majority of substitutions was that expected from the NTP pool bias. The addition of manganese ions increased the frequency of mutations, particularly the proportion of transversions. Functional DHFR hypermutants with up to 8% amino acid substitutions were readily obtained from a single reaction which, given the unique mutation matrix allows exploration of sequence space complementary to that accessed by other hypermutagenic protocols.
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PMID:Hypermutagenic in vitro transcription employing biased NTP pools and manganese cations. 904 46


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