Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure.

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.     

Poly(A) polymerases of rat liver nuclei. Purification and specificity.

Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120-130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the poly-adenylation by adding 80-90 more AMP units to the polyadenylated end of chromosomal RNA.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

DNA-Dependent RNA Polymerases from Artemia salina

Embryos and larvae of the brine shrimp, Artemia salina , provide a useful biological system for biochemical studies of animal development. Dormant encysted embryos can be cultured readily in the laboratory to provide large quantities of free-swimming nauplius larvae. The rate of synthesis of all classes of RNA in swimming larvae declines markedly between 24 and 72 h after immersion of dormant embryos in sea water. Nuclei were isolated from 24–72 h larvae and RNA polymerase activity was measured under conditions in which the nuclei remained intact. Total RNA polymerase activity of isolated nuclei decreased in parallel with RNA synthesis in vivo. RNA polymerases were solubilized from nuclei and fractionated by chromatography on DEAE-cellulose. The levels of both RNA polymerases I and II also decreased in parallel with RNA synthesis in vivo. The specific activity of highly purified RNA polymerase II was determined by comparison of enzyme activity with the mass of RNA polymerase II subunits displayed on SDS gels. The specific activities of RNA polymerase II preparations from 24 and 72 h larvae were identical. The number of polymerase II molecules was estimated from the mass of the subunits. The number of molecules per nucleus declined from 20,000 at 24 h to 3500 at 72 h.     

Human chorionic gonadotropin increases chromatin solubility in isolated bovine and human luteal nuclei

We have previously demonstrated that bovine and human luteal nuclei contain human chorionic gonadotropin/luteinizing hormone (hCG/LH) receptors and that these gonadotropins can directly stimulate nuclear membrane enzyme activity (nucleoside triphosphatase) involved in messenger ribonucleic acid (mRNA) transport from the nucleus to the cytoplasm. The present studies were undertaken to investigate the effect or hCG on chromatin solubility, reflecting perhaps synthesis and transport of RNA, in isolated bovine and human luteal nuclei. hCG increased chromatin solubility in a concentration-dependent manner. This hCG effect is either blocked or substantially reduced by the addition of hCG antiserum; denatured hCG had no effect and cyclic adenosine 3',5'-monophosphate could not mimic the hCG response. hCG had no effect on chromatin solubility in bovine liver or kidney nuclei and hormones other than hCG, human LH, or the beta subunit of hCG had no effect on chromatin solubility in bovine luteal nuclei, demonstrating the tissue and hormone specificity of the response. These findings further strengthen the concept of direct gonadotropin regulation of nuclear functions of luteal cells.     

Vesicular stomatitis virus infection reduces the number of active DNA-dependent RNA polymerases in myeloma cells.

Infection of mouse myeloma (MPC-11) cells with vesicular stomatitis virus resulted in rapid loss in activity of cellular RNA polymerases associated with nuclear chromatin. No RNA polymerase inhibitor could be detected in extracts of infected cell nuclei. Reconstitution experiments with solubilized RNA polymerases dissociated from chromatin of infected and uninfected cells demonstrated that vesicular stomatitis viral infection did not affect the ability of the polymerases to function on endogenous or exogenous templates; nor did infection alter the template capability of the chromatin. Measurement of the number of actively growing RNA chains revealed that infected cell nuclei contained fewer active polymerase units; however, the rates of RNA chain elongation were the same in nuclei from infected and uninfected cells. Quantitation of the number of polymerase units active in nuclear chromatin revealed that the alpha-amantin-sensitive polymerase II was more severely reduced by viral infection than were polymerases I and III.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.