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)

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.
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PMID:Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II. 55 98

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).
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PMID:RNA synthesis in isolated nuclei of the dinoflagellate Crypthecodinium cohnii. 57 93

The transcription of freshly prepared nuclei and lysates from rat liver is stimulated by exogenous RNA polymerase B from calf thymus to an insignificant extent only. This also holds for chromatin isolated from nuclei lysates by separation on a Sepharose 4 B column. After removal of histone H1 by pretreatment with 0.2 M ammonium sulphate no further stimulation by added RNA polymerase has been found. If, however, the incubation time was extended, or the nuclei had been kept frozen at -20 degrees C for some time before use, a significant increase in RNA synthesis by the added RNA polymerase was obtained. In the freshly prepared nuclei as well as in the undamaged chromatin the template capacity was highly restricted. This can be seen from the fact that after pretreatment of the chromatin with 0.4 M ammonium sulphate the RNA synthesis was stimulated about 13fold.
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PMID:Transcriptional capacity of rat liver nuclei and of isolated chromatin at different ammonium sulphate concentrations. 70 21

RNA polymerase activity has been measured in liver and brain of C57BL-6J mice to determine if a change in enzyme activity can be correlated with decreasing survivorship of the animals. The RNA polymerases in tissue homogenates were solubilized by treatment with a buffer of high ionic strength and resolved by DEAE-Sephadex chromatography. Enzyme activity was quantitated by measuring the incorporation of [3H]UMP into RNA using heat denatured calf thymus DNA as the template. Statistically significant differences in polymerase activities were not observed in liver tissue from 18-, 25-, and 29-month-old animals or in brain tissue from 23- to 31-month-old animals. These age groups span the period of most rapid decrease in survivors,ip in our colony of mice (from 93% to 16%). The evidence indicates that changes in liver or brain RNA polymerase activities are not correlated with the rapid decrease in survivorship of these animals.
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PMID:RNA polymerase activities in liver and brain tissue of aging mice. 74 26

The paper deals with the comparative investigation of initiation and in vitro RNA synthesis on DNA template by E. coli RNA polymerase and B-form of calf thymus RNA polymerase. It was shown in hybridization experiments that in the range of Cot values between 10(2) and 10(4) RNA synthesized by calf thymus RNA polymerase was hybridized with homologous DNA more effectively than RNA synthesized by E. coli RNA polymerase. No differences were observed in the case of low Cot values. RNA chains synthesized by calf thymus RNA polymerase contained in average about 300-600 nucleotides per chain as determined in the kinetic experiments with ATP-gamma-32P and GTP-gamma-32P. These values are in average 5-10 times lower than in the case of bacterial enzyme. The data presented show that calf thymus and E. coli RNA polymerases initiate the RNA synthesis at apparently different sites of calf thymus DNA. The results obtained make the possibility of specific transcription of eucaryotic DNA by bacterial RNA polymerase to be doubtful.
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PMID:[Transcription of DNA by RNA polymerases of E. coli and calf thymus]. 76 44

Polar organic compounds, including DMSO, increase RNA synthesis on isolated chromatin by E. coli RNA polymerase and RNA polymerase II from calf thymus. Transcription is stimulated on chromatin from Friend-virus-infected erythroleukemia cells and from various other sources. Using procedures which inhibit specifically the formation of a stable initiation complex, it is shown that the stimulation does not result from an increase in initiation of both E. coli and the eukaryotic RNA polymerase. After separation of chromatin into template active and inactive fractions, DMSO increases RNA synthesis by a factor of about 1.5 using the template inactive fraction, while stimulation of transcription on the template active portion is lower (factor of 1.2). It is suggested that the effect on RNA synthesis is mediated by a weakening of the apolar interactions between histones in chromatin subunits, releasing transcription partially from the constraints imposed by histones.
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PMID:Stimulation of transcription on chromatin by polar organic compounds. 78 22

The 1400 base pair repeat produced by digestion of calf satellite I DNA (phi = 1.714 g/cm3) with EcoRI, was cloned in E. coli. The hybrid plasmid (pGM 214) which contains the ColE1-Ap vector (pSF 2124) and the 1400 base pair fragment replicates stably in E. coli and can be amplified by chloramphenicol treatment. No clone was found in which more than one "repeat unit" of the satellite I DNA was present in the chimaera plasmid. Digestion of the original satellite I and the plasmid pGM 214 with R-SmaI shows that the satellite DNA replicated in E. coli is cleaved by the restriction endonuclease SmaI whereas the original satellite I DNA from calf thymus is not, suggesting that the satellite I contains a large amount of modified cytosine or guanosine, probably 5-methyl-cytosine. R-EcoRI* produces a number of fragments with the satellite I in the range of 300 base pairs to 1400 base pairs. A physical map of pGM 214 (and pSF 2124) with R-EcoRI, R-HincII, R-HindIII, R-SmaI, R-BamI and R-EclI was constructed. The 1400 base pair "repeat unit" in the pGM 214 is efficiently transcribed in vitro by purified RNA polymerase, starting from a pSF 2124 promoter. The restriction enzyme EclI produces a 350 base pair repeat with calf satellite II (phi = 1,722 g/cm3), whereas the satellite I is not cut by this enzyme.
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PMID:Cloning of calf thymus satellite I DNA in Escherichia coli. 79 69

A purification procedure for RNA polymerase from uninfected and phage SP01-infected Bacillus subtilis is presented. The RNA polymerase purified from B. subtilis 10 min after infection with wild type phage SP01 is resolved into two major fractions (B, C) and one minor fraction (A) by calf thymus DNA-cellulose chromatography. Fraction C is indistinguishable from RNA polymerase from uninfected cells with respect to transcription specificity (both before and after phosphocellulose chromatography). Fraction B yields, on subsequent phosphocellulose chromatography, an enzyme (B-P) whose properties distinguish it from the host RNA polymerase. Enzyme B-P preferentially transcribes SP01 DNA and selectively forms rapidly initiating complexes with SP01 DNA but not with heterologous DNA. The SP01 RNA synthesized by Enzyme B-P includes, as previously reported, a large proportion of asymmetrical middle viral RNA. Host RNA polymerase holoenzyme synthesizes asymmetrical early viral RNA, while host core polymerase synthesizes symmetrical RNA that is complementary to early, middle, and late in vivo viral RNA and contains a preponderance of antimessenger. The subunit composition of Enzyme B-P is identical to host core polymerase with respect to the beta,beta', and alpha subunits and two additional components of mr equals 9,500 and 11,000 that we observe in all preparations of RNA polymerase. In addition, Enzyme B-P has two subunits of mr equals 13,000 and 28,000, which are synthesized after phage infection. On heterologous template, Enzyme B-P and host core polymerase have comparable activities. On these templates, addition of host initiation factor, sigma, restores full activity to Enzyme B-P as well as to host core polymerase. Sigma also modifies the activity of Enzyme B-P on SP01 DNA, restoring some asymmetrical early RNA transcription while retaining some asymmetrical middle RNA transcription.
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PMID:RNA polymerase from phage SP01-infected and uninfected Bacillus subtilis. 80 88

A purification procedure is described by which we obtained DNA-dependent RNA polymerase B (or II) from third-instar larvae of Drosophila melanogaster in essentially pure form. The enzyme is similar to the analogous polymerases from other eukaryotes in its enzymic and structural properties. It preferentially transcribes DNAs containing single-stranded regions, and it is inhibited by low amounts of the toxin alpha-amanitin; 50% inhibition occurs at an alpha-amanitin concentration of 0.03 mug/ml. Dodecylsulfate-polyacrylamide gel electrophoresis resolves the purified Drosophila polymerase B into ten polypeptides with molecular weights as follows: 1 (174000), 2 (137000), 3 (34000), 4 (22000), 5 (18000), 6 and 7 (16000), 8 (15000), and 9 and 10 (less than 15000). The relative amounts of polypeptides 1-4 were constant at molar ratios of approximately 1:1:1:2 in different preparations of the enzyme, while the amounts of polypeptides 5-10 showed more variation. An antiserum directed against the Drosophila RNA polymerase B inhibited the activity in vitro of the B enzymes from Drosophila, yeast, and calf thymus. However, only the Drosophila enzyme gave a precipitin reaction with the antiserum. When the antiserum was added to Drosophila RNA polymerase B at different stages of the purification, the resulting precipitates were found to contain nearly constant proportions of seven of the ten polypeptides present in the purified enzyme.
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PMID:RNA polymerase B from Drosophila melanogaster larvae. Purification and partial characterization. 81 97

A comparative radioautographic study of the RNA precursors incorporation on polytene chromosomes of Drosophila in vivo in the cells of salivary glands, and in vitro during incubation of E.coli RNA polymerase on slides with fixed chromosomes was performed.--The pattern of in vivo 3H-uridine incorporation on different sections of the chromosomes drastically differed from the in vitro 3H-UTP incorporation which seems to be much more related to DNA content of the individual small sections. In both cases puffing of the loci resulted in the increase of RNA synthesis but in vitro only 2-3 fold and in vivo much more. Hence, RNA synthesis in vitro was unspecific and did not reflect the in vivo RNA synthesis.--On the other hand, E.coli RNA polymerase completely mimics in vitro the dosage compensation phenomenon making twice as much RNA on one X-chromosome of males (1X2A) as on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females and super-females (3X2A), and the intermediate amount of RNA on each of X-chromosomes of intersexes (2X3A). It is suggested that the differences in the in vitro template activity of X-chromosomes of cells with different X:A ratio are due to different extent of condensation of their deoxyribonucleoprotein (DNP). Yet, both male and each of female X-chromosomes bind the same amount of thymus histone FI labelled with fluorochrome which indicates that they contain the same amount of "open" regions with exposed chromosomal DNA accessible to external proteins.--On the basis of these observations a hypothesis is put forward which suggests that RNA transcription in animal chromosomes is regulated at two levels by different mechanisms; the first one controls the extent of condensation of DNP of genetic loci and determines their competence to the second mechanism which involves the action of gene-specific activator proteins. According to this hypothesis the phenomenon of dosage compensation of sex-linked genes is due to decondensation of DNP of male X-chromosome which renders its loci twice as responsive to activators as compared to the same loci in females.
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PMID:Comparison of in vivo and in vitro RNA synthesis on polytene chromosomes of Drosophila. 81 77


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