Endogenous RNA Polymerase Research Articles

Specific interference of metalaxyl with endogenous RNA polymerase activity in isolated nuclei fromPhytophthora megasperma f. sp.medicaginis

The systemic fungicide metalaxyl preferentially inhibits [3H]uridine incorporation into RNA by mycelium ofPhytophthora megasperma f. sp.medicaginis. Even at high concentrations of metalaxyl inhibition is not complete but circa 80%. Neither uptake of [3H]uridine nor its conversion into UTP is inhibited, indicating that interference with RNA synthesis takes place. Synthesis of RNA that lacks poly(A) sequences is more affected than that of poly(A)+ RNA. Metalaxyl has no effect on the activity of RNA polymerases present in mycelial extracts fromPhytophthora nor on that of polymerases I and II that have been partially purified with a procedure involving precipitation with polyethyleneimine, selective elution of RNA polymerases from the polyethyleneimine precipitate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. RNA polymerase II in mycelial extracts is half-maximally inhibited by α-amanitin at concentrations below 0.01 ¼g/ml. Both metalaxyl and α-amanitin inhibit endogenous RNA polymerase activity of isolated nuclei ofPhytophthora. According to their sensitivity to metalaxyl and α-amanitin, three types of endogenous activity can be distinguished: (a) an α-amanitin-sensitive type, the activity of which is stimulated by ammonium sulfate; (b) an α-amanitin-insensitive but metalaxyl-sensitive type; and (c) a type insensitive to both metalaxyl andα-amanitin. The first type of activity is characteristic of RNA polymerase II; the identity of the latter two remains to be elucidated. Metalaxyl andα-amanitin do not have any effect on free nuclear polymerases when assayed at a concentration of 50 mM ammonium sulfate with poly[d(A-T)] as exogeneously added template in the presence of actinomycin D to inhibit endogenous RNA polymerase activity. At 250 mM ammonium sulfate the free polymerase activity becomes α-amanitin sensitive but remains metalaxyl insensitive. Metalaxyl apparently inhibits RNA synthesis by specific interference with template-bound andα-amanitin-insensitive RNA polymerase activity. Endogenous polymerase activity of nuclei isolated from a metalaxyl-resistant mutant ofP. megasperma f. sp.medicaginis is not inhibited by metalaxyl, indicating that interference with RNA synthesis is the primary action of metalaxyl and that modification of the target site may lead to resistance.

Modulation of RNA Polymerase Activities in the Intestine of Adult Rats by Dietary Sucrose

The effect of sucrose feeding on endogenous intestinal RNA polymerase activities and on chromatin structure was studied in rats. Adult rats were given a 70% sucrose solution for 15 hours following a 48-hour starvation period. Comparison was made with rats starved for 63 hours and with ad libitum nourished animals. Chromatin-bound RNA polymerase I activity was significantly reduced by starvation. Sucrose feeding provoked a significant rise in the activity, but the level found in the nourished rats was not reached. The free poly[d(A-T)]-dependent RNA polymerase I activity of the sucrose-fed rats exceeded that of the starved and the nourished animals. Chromatin-bound RNA polymerase II activity was enhanced most markedly by sucrose feeding. The balance between the chromatin-bound and free enzymes was shifted towards the chromatin-bound state when compared to the starved and nourished rats. Starvation caused a reduction in the size of oligonucleosomes but sucrose feeding restored almost entirely the original pattern obtained in the nourished animals. These results reflect modifications in the structure of chromatin after sucrose feeding. The present report demonstrates that the adaptive processes triggered in the intestine by dietary sucrose are associated with changes in gene expression.

Effect of Nucleosides on Nucleologenesis and Nucleolar Activity in Onion Root Meristems

Brief treatments of the roots of Allium cepa L. bulbs with either 5×10-5 or 5×10-4 M adenosine stimulate nucleologenesis and accelerate chromatin decondensation in post-mitotic nuclei of meristem cells. However, at one order of magnitude higher (5×10-3 M adenosine), both nucleologenesis and chromatin decondensation are depressed. Neither of these two processes is altered when roots are treated with other ribonucleosides (guanosine, cytidine, uridine, at 5×10-4 M). In addition to the cytological effects, the stimulatory doses of adenosine produce an important enhancement of the «in vivo» 3H-uridine incorporation into total RNA in the meristems. Determinations of 3H-UTP incorporation in meristems made permeable, under conditions which preferentially measure endogenous RNA polymerase I activity, reveal a slight increase in the number of nascent rRNA chains in such adenosine-pretreated roots.

Transcriptional control of the fibronectin gene in chick embryo fibroblasts transformed by Rous sarcoma virus.

Rous sarcoma virus transformed-chick embryo fibroblasts contain decreased amounts of the adhesive protein fibronectin that results from a lowering of fibronectin mRNA levels (Fagan, J.B., Sobel, M.E., Yamada, K.M., de Crombrugghe, B., and Pastan, I. (1981) J. Biol. Chem. 256, 520-525). In the present study, nuclei isolated from chick embryo fibroblasts (CEF) and Rous sarcoma virus transformed-chick embryo fibroblasts (RSV-CEF) were used to measure relative rates of fibronectin RNA synthesis. [32P]RNA was synthesized by preinitiated endogenous RNA polymerases in nuclei, and the radioactive RNA was subsequently hybridized to DNA fragments spanning approximately 40 kilobase pairs (approximately 85%) of the fibronectin gene. Transformation of CEF by RSV resulted in a 7- to 8-fold reduction in the amount of 32P-labeled nuclear RNA hybridizing with fibronectin DNA sequences when the nuclei were incubated for either 3.5 or 45 min. These results indicate that decreased transcription and not reduced RNA stability plays a major role in lowering fibronectin mRNA levels in RSV-CEF. When nuclear RNA was transcribed in the presence of Sarkosyl, diminished RNA synthesis was still observed suggesting that reduced fibronectin transcription in RSV-CEF is not due to a block in RNA chain elongation. Our data indicate that transcriptional regulation of the fibronectin gene is an important mechanism of lowering steady state levels of fibronectin mRNA in RSV-CEF.

Transcriptional activity in previtellogenic oocyte germinal vesicles from Xenopus laevis

We have isolated germinal vesicles from previtellogenic Xenopus oocytes on a scale suitable for biochemical studies. The organelles were active in RNA synthesis, mainly mediated by endogenous RNA polymerase I. Nascent transcripts were 5-18S in size and complementary to DNA sequences present at intermediate reiteration frequency (several hundred copies per haploid genome). Hybridization to cloned 5S and 40S ribosomal genes demonstrated that the in vitro transcripts contained the latter, but not the former sequences in detectable amounts. The germinal vesicles also exhibited an ability to take up and retain UTP and a UTP-binding activity, probably protein(s), was extractable from the organelles with 0.35 M NaCl.

Region-specific initiation of mouse mammary tumor virus RNA synthesis by endogenous RNA polymerase II in preparations of cell nuclei.

Adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were used to demonstrate initiation of mouse mammary tumor virus (MMTV) RNA in preparations of whole nuclei from control and glucocorticoid-treated MMTV-infected rat hepatoma tissue culture cells. RNA chains initiated in the cell-free reaction retain a thiol group at the 5' end and can be separated from thiol-free RNA chains by chromatography on mercury-Sepharose. The abundance of MMTV sequences was determined by nucleic acid hybridization with filter-bound DNA representing four different regions of the MMTV genome. About six times more MMTV RNA is initiated with GTP beta S than with ATP beta S. Most of the cell-free initiation of MMTV RNA occurs within or very near a 380-nucleotide section of the proviral long terminal repeat that is the presumptive site of transcription initiation in vivo. The sensitivity of MMTV RNA initiation and synthesis to alpha-amanitin and actinomycin D are characteristic of DNA-directed transcription by RNA polymerase II. Nuclei from glucocorticoid-treated cells initiate approximately 10 times more MMTV RNA than nuclei from control cells.

Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II

A new fractionation procedure separates a wholecell HeLa extract into three components required for accurate in vitro transcription. One component (Spl) is a promoter-specific factor required for transcription of the SV40 early and late promoters, but not for transcription of other promoters we have tested. The second component (Sp2) is a general factor required for transcription of the SV40 promoters and a series of others, including the adeno-virus 2 major late promoter, the human β-globin promoter and the avian sarcoma virus promoter. The third component is a fraction containing the endogenous RNA polymerase II. When SV40 and adenovirus templates were present simultaneously in an in vitro transcription reaction, addition of the Spl factor stimulated SV40 early promoter transcription 40-fold, and inhibited adenovirus major late promoter transcription by 40%. This finding suggests that Spl is involved in promoter selection, and is not merely a general transcription stimulatory factor.

Characterization and transcription of bovine thyroid chromatin

1. 1. Thyroidal chromatin, isolated from bovine thyroid nuclei, has been characterized and the endogenous RNA polymerase activity was studied. 2. 2. The template activity of chromatin was investigated for exogenous homologous RNA polymerase I and II. 3. 3. The influence of chromosomal proteins on the transcription process was described.

Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei of Xenopus laevis oocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei of Xenopus laevis oocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 mini-chromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2-5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2-5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS-minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

HMG-proteins 1 and 2 are required for transcription of chromatin by endogenous RNA polymerase

Summary Chromatin prepared from isolated mouse myeloma cell nuclei faithfully transcribes RNA using endogenous RNA polymerase. Extraction of the chromatin with 0.35M KC1 greatly reduces the ability of the chromatin to transcribe RNA, but RNA synthesis is restored by adding back the extracted material. The major proteins extracted under these conditions are the high mobility group proteins — HMG 1 and 2. When purified HMG proteins 1 and 2 were added back to the extracted chromatin preparations total RNA synthesis was stimulated 3 to 5 fold. The synthesis of the RNA polymerase III products, 5S rRNA and tRNA precursors, was also stimulated the same amount.

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.

Transcription of Simian Virus 40 chromosomes in an extract of HeLa cells.

Simian Virus 40 (SV40) chromosomes were incubated with a concentrated extract of HeLa cells containing RNA polymerase II and other factors involved in transcription. SV40-specific RNA was synthesized. In the absence of HeLa cell extract the synthesis of labeled RNA by endogenous RNA polymerase in the chromosome preparations amounted to less than one tenth of that when the HeLa cell extract was present. Incubation with the HeLa extract increased the amount of Sarkosyl-resistant (i.e., transcribing) RNA polymerase on the SV40 chromosomes. When restriction endonucleases able to cut SV40 DNA were added to transcription reactions containing SV40 chromosomes and the HeLa cell extract, RNAs of discrete length were produced. The RNAs were identified as correctly initiated run-off transcripts of the early and late genes. Most of the RNA synthesized by the HeLa extract from SV40 chromosomes was from the region of the late genes, whereas transcription of purified SV40 DNA was from both the early and the late regions, with the early transcripts predominating.

Role of chromatin phospholipids on template availability and ultrastructure of isolated nuclei

The influence of phospholipid vesicles has been tested on isolated nuclei by evaluating the endogenous DNA-dependent RNA polymerase and the chromatin ultrastructure. Negatively charged phosphatidylserine liposomes have a stimulating effect on RNA synthesis, while the vesicles obtained with the neutral sphingomyelin, phosphatidylethanolamine and phosphatidylcholine, and the acidic phosphatidylinositol, are inhibitory. The enhancement of transcription by phosphatidylserine seems to be due to both elongation and initiation of RNA transcripts and very likely depends on interactions of the lipid with the template rather than with the enzymes. The morphological analysis indicates deep changes of the chromatin organization, mainly concerning the size and distribution of the fibers, without any variation of the nuclear volume. The rearrangement of the chromatin could account for the variations of the template availability for RNA synthesis induced by the vesicles and might be associated with changes of the nuclear matrix.

Transcriptional activation and structural alteration of wheat chromatin during germination and seedling‐growth

The chromatin was isolated from embryos or young seedlings of wheat, Triticum aestivum L. cv. Mukakomugi, and the template activity, the endogenous RNA polymerase activity, the susceptibility to DNase 11 and polylysine binding capacity were assayed. Both transcriptional activities of chromatin were very low for the first 12 h of germination, increased from 12 to 24 h, and then increased markedly during the next 48 h of post‐germination. During the 72 h of germination, the template activity and the endogenous RNA polymerase activity increased 10‐fold and 40‐fold, respectively, while they decreased from 72 h to 96 h of seedling growth. Seventy‐two h seedling chromatin was more susceptible to endonuclease and was bound with more polylysine than the germ chromatin. These results suggest that the structure of the chromatin available for transcription changes during seed germination and seedling‐growth.

Differentiation between Broccoli Necrotic Yellows Virus and Lettuce Necrotic Yellows Virus by their Transcriptase Activities

SUMMARY An endogenous RNA-dependent RNA polymerase has been demonstrated in broccoli necrotic yellows virus (BNYV) particles. The enzyme requires a temperature of 26 °C, a pH value of 7.5, 0.5% NP40 and 4 mm-Mg2+ for maximum synthesis of RNA; Mn2+ inhibited the activity. Addition of EDTA to the reaction mixture did not stimulate the activity in the presence of higher amounts of Mg2+. The in vitro transcription products of BNYV hybridized to the virus genome, but not to that of lettuce necrotic yellows virus (LNYV). The transcriptase activity was abolished by centrifugation on a glycerol gradient after treating the virus with NP40 in a high salt solution; combining the pellet and the upper part of the gradient restored the activity. Cross-complementation of BNYV and LNYV transcription could not be obtained by heterologous combination of the pellet and supernatant fractions of the two viruses.

Activation of the major Drosophila heat-shock genes in vitro

Inactive Drosophila heat-shock genes of isolated diploid nuclei can be induced to a transcriptionally active state by exposure to cytoplasmic extracts from heat-shocked Drosophila cells. No effect was observed on histone gene transcription, and extracts from non-heat-shocked cells were ineffective. The factor in the cytoplasmic extract has been partially purified and characterized. It is protease-sensitive and heat-labile. A striking change accompanies in vitro activation that permits transcription by E. coli RNA polymerase of the chromatin 5′-distal to the structural genes at the 87A and 87C heat-shock gene loci; we have previously observed a similar change after in vivo heat-shock induction. That this change occurred in the absence of endogenous RNA polymerase II activity suggests that these changes may reflect the earliest event in gene activation. Inasmuch as activation also took place after histone H1 depletion, this histone does not appear to be essential for this step of gene activation.

Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli.

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or CO2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5'-triphosphates or deoxyribonucleoside 5'-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, and dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.