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)

High-level expression of the full-length human retinoic acid receptor (RAR) alpha and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RAR alpha protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a Kd of 2.1 x 10(-10) M.
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PMID:Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor. 185 Aug 32

The mouse B2 element is a moderately repetitive nt sequence of 180 bp transcribed by RNA polymerase III (Pol III) at high levels in embryonic and transformed cells. The B2 sequence is present in either orientation within the noncoding regions of a number of genes transcribed by RNA polymerase II (Pol II). We sought to determine if the small B2 transcripts generated by Pol III are natural antisense RNA molecules which might hybridize to complementary sequences present within Pol II transcripts. Chimaeric reporter genes encoding Escherichia coli gpt were constructed containing a B2 repeat in either orientation within the 5'- or 3'-untranslated regions. These constructs were transfected into embryonal carcinoma (EC) cells and expression of the reporter gene was analysed in EC cells and retinoic acid-treated EC cells, which contain high and low levels of small B2 RNAs, respectively. Although the B2 sequences affected expression of the reporter gene, these effects did not appear to be due to hybridization of the small B2 RNA to the reporter transcripts. The presence of B2 sequences near a Pol II-transcribed gene can alter expression of that gene in a position- and orientation-dependent manner, suggesting these repetitive elements may be cis-acting regulators of gene expression.
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PMID:The rodent B2 sequence can affect expression when present in the transcribed region of a reporter gene. 201 66

B2 genes are short repeated sequences which are transcribed by RNA polymerase III. Abundant transcripts accumulate in embryonic and transformed cells, but transcripts are rare or absent from normal differentiated cell types. During retinoic acid-induced differentiation of P19 embryonal carcinoma cells, an early transient increase in B2 RNA levels is followed by a rapid drop in expression. The marked changes in B2 RNA levels are most likely due to transcriptional modulation since B2 RNA stabilities are unaffected by differentiation. At least four short-lived B2 RNAs with apparent lengths of 150, 180, 240, and 500 nucleotides were characterized. The two larger RNAs are polyadenylated and are more stable in cells. A cDNA of a B2 gene was isolated which was over 99% identical to the consensus sequence. This B2 cDNA can be transcribed in human cells and yields at least two distinct transcripts. We propose a model for B2 RNA metabolism which describes transcription, posttranscriptional modification and processing, and nucleocytoplasmic transport.
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PMID:Synthesis and processing of small B2 transcripts in mouse embryonal carcinoma cells. 237 Aug 62

We have examined RNA synthesis by nuclei isolated from testes of rats of varying vitamin A status. Nuclei from retinol-deficient animals showed substantially decreased RNA synthesis by polymerase II when compared to nuclei from normal animals. Within 4 hours after oral administration of retinyl acetate (as the source of retinol) to deficient animals, RNA synthesis by polymerase II had significantly increased. Administration of retinoic acid had a similar but lesser effect. Nucleoside analysis after alkaline hydrolysis of the RNA synthesized by the endogenous polymerase II suggested that the increased activity was due to a greater number of actively transcribing polymerase II molecules on the DNA. Further, when the template capacity of testicular chromatin isolated from deficient and retinyl acetate refed animals was compared, the number of sites recognized by E. coli RNA polymerase was increased twofold after retinyl acetate administration. We conclude that these retinol-induced changes in transcription are due at least in part to changes in chromatin structure.
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PMID:Vitamin A status affects chromatin structure. 242 69

Changes of nucleolar organizer region in HL-60 cells after treated with retinoic acid (RA) were studied with techniques of silver-staining nucleolar organizer region (Ag-NOR) in metaphase karyotypes, Brachet's reaction and with our improved TEM techniques for studying silver-stained active nucleolar organizer region (Ag-aNOR) in interphase nucleoli. Number of Ag-NOR in HL-60 cells is 4.5/cell on average. The Ag-NOR number of cells treated with RA showed no remarkable difference from that of control group. Ag-aNOR number treated with RA was reduced obviously as compared with that of control group. Meanwhile, the changes of nucleolus number showed by Brachet's reaction were in accordance with those of Ag-aNOR. Therefore, it may be concluded: (1). Though the number of active rRNA genes did not changed after the differentiation of HL-60 cells induced by RA, their expression was clearly inhibited: (2). The relationship between the changes of Brachet-No and Ag-aNOR is in positive correlation (r = 0.98, p less than 0.01). EM examination of Ag-aNOR of HL-60 cells reveals that Ag-protein (RNA polymerase I) only presented in fibrillar centers (FC) and the dense fibrillar components (DFC) of nucleolus. In addition, in control group, large amount of Ag-protein, FC, DFC and granular components (GC) were observed, and there were many large nucleoli in a nucleus, meanwhile, the cells of the treated group tended to be mature, with a decrease in the amount of Ag-protein, FC, DFC and GC accordingly, and the nucleoli reduced both in size and number significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Studies on changes in nucleolar organizer region of human promyelocytic leukemia cells (HL-60) treated with retinoic acid]. 262 98

The murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.
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PMID:Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor. 268 57

Our understanding of how vitamin D mediates biological responses has entered a new era. It is now clear that the bulk of the biological responses supported by vitamin D occur as a consequence of its metabolism to its daughter metabolite 1 alpha,25-dihydroxyvitamin D3 (a steroid hormone). The fact that 1,25(OH)2D3 receptors are ubiquitous in tissue distribution opens the possibility for unforeseen biological functions of the vitamin D endocrine system. For example, 1,25(OH)2D3 serves as an immunoregulatory hormone and a differentiation hormone besides its classical role in mineral homeostasis. The avian 1,25)OH)2D3 receptor has recently been cloned and shown to be a member of the nuclear transacting receptor family that includes estrogen, progesterone, glucocorticoid, thyroxine (T3), aldosterone, and retinoic acid receptors. We have compiled an extensive number of RNA polymerase II-transcribed genes that are regulated by 1,25(OH)2D3. Classification of these genes on functional grounds identifies and formulates the several genetic circuits or biochemical systems in which 1,25(OH)2D3 plays an essential regulatory role. These systems include genes that govern oncogene and lymphokine expression as well as those involved in mineral homeostasis, vitamin D metabolism, and regulation of a set of replication-linked genes (c-myc, c-myb, and histone H4), which are critical for rapid cellular proliferation. An integrated analysis of the combinations of genetic circuits regulated by 1,25(OH)2D3 suggests that they may be collectively tied to a DNA replication-differentiation switch.
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PMID:1,25(OH)2-vitamin D3 receptors: gene regulation and genetic circuitry. 284 48

In the present study the effect of all-trans-retinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (1) The tumor mince was incubated with 1 microM RA for 30 min at 30 degrees C; the RNA polymerase activity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 25 degrees C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the addition of RA-CRABP into the reaction mixture did not alter the enzyme activity. These results suggest that alteration of RNA polymerase activity may be an essential step in the retinoid action in mammary tissues.
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PMID:Effect of all-trans-retinoic acid on nuclear RNA polymerase activity in chemically-induced rat mammary tumors. 318 28

The insulin-like growth factor (IGF) regulatory system has a major impact on bone physiology. Among the modulators of IGFs, a family of structurally related proteins, the IGF-binding proteins (IGFBPs), have been shown to either potentiate or inhibit IGF actions on bone growth. However, the regulation of IGFBP expression in bone cells is not completely understood. In the present study, the expression of IGFBP-5 was analyzed in primary osteoblastic cells (Ob cells) isolated from 22-day-old fetal rat calvariae. Treatment of Ob cells with either IGF-I or all-trans-retinoic acid (RA) caused a time- and dose-dependent increase in IGFBP-5 messenger RNA (mRNA) levels, as determined by Northern blot analysis. Stimulation of IGFBP-5 mRNA was obtained at 100 nM IGF-I between 6 and 16 h (2- to 2.5-fold) and 100 nM RA between 16 and 24 h (3- to 4-fold). Concomitant treatment of Ob cells with IGF-I and RA revealed an additive effect and a 5- to 7-fold increase in IGFBP-5 mRNA levels after 16-24 h. The effect of IGF-I and RA and their combination on IGFBP-5 transcripts was similar in confluent and subconfluent cultures of Ob cells. IGF-I and RA did not change IGFBP-5 mRNA stability in Ob cells after transcription arrest with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole. IGF-I and RA at 100 nM elevated the levels of IGFBP-5 heterogenous nuclear RNA, measured by reverse transcription-polymerase chain reaction. The effect was similar to that observed on mRNA levels. IGFBP-5 from rat Ob cells appeared as a single band of 31 kilodaltons in both the conditioned medium and the extracellular matrix as determined by Western immunoblots. IGF-I and RA, both at 100 nM, increased IGFBP-5 by 2- to 3-fold after 24 h. In conclusion, IGF-I and RA modify the synthesis and secretion of IGFBP-5 in rat Ob cells through pathways that may involve increased transcription and elongation and/or altered processing of heterogenous nuclear RNA. Our data suggest that IGFBP-5 may play a role in the osteoblastic-differentiated function regulated by IGF-I and RA.
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PMID:Insulin-like growth factor (IGF) I and retinoic acid induce the synthesis of IGF-binding protein 5 in rat osteoblastic cells. 753 61

We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.
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PMID:Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells. 768 82


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