Mouse Brain DNA-Dependent RNA Polymerases After Chronic Morphine Treatment

Abstract: Chronic morphine pellet implantation was found to decrease the specific activity of two forms of mouse brain RNA polymerase I and to alter the requirements of Mg²⁺ and Mn²⁺ for the activities of RNA polymerases II and III. DNA-dependent RNA polymerases were partially purified from small dense nuclei isolated from brains of naive and morphine tolerant-dependent mice, and three RNA polymerases were separated on a DEAE-Sephadex A-25 column. The three fractions, referred to as peak I, peak II, and peak III, were studied, characterized, and identified as being RNA polymerases I, II, and III, respectively. Chronic-morphine pellet implantation resulted in a lower specific activity of RNA polymerase I, but the specific activities of RNA polymerases II and III were not affected. This effect was prevented by preimplantation of a naloxone pellet and thus was narcotic-specific. Chronic morphine treatment lowered the concentration of Mg²⁺ required for optimal activity of RNA polymerase II and elevated the Mn²⁺-Mg²⁺ activity ratios of RNA polymerases II and III. A second DEAE-Sephadex A-25 column chromatography of the peak I RNA polymerase was carried out, revealing five component activity peaks. Two of these contained lower specific activities as a result of chronic morphine pelletimplantation. These specific changes in RNA polymerase function in morphine tolerance-dependence may be associated with the elevated chromatin template activities, altered chromatin phosphorylation, and elevated rates of cell-free translation that have been reported by others.     

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.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

DNA-dependent RNA polymerases from Artemia salina. Subunit structure of polymerase II.

RNA polymerase II from larvae of the brine shrimp, Artemia salina, was highly purified by two cycles of DEAE-cellulose chromatography followed by centrifugation through discontinuous sucrose gradients. Gradient fractions were subjected to elctrophoresis is polyacrylamide gels containing sodium dodecyl sulfate. The subunit structure of RNA polymerase II was determined by quantitative comparison of the polypeptides and enzyme activity present in each gradient fraction. The enzyme contains one copy of each of four subunits with estimated molecular weights of 170,000, 130,000, 36,000 and 24,000. The total molecular weight agrees well with the molecular weight estimated for the native enzyme by density gradient centrifugation.     

Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.     

Messenger RNA for ribosomal proteins in dormant and developing Artemia salina embryos

Ribosomal proteins from Artemia salina have been separated in a two-dimensional acrylamide gel system and assigned to the small and large ribosomal subunits. Poly(A)-containing RNA was prepared from dormant cysts and from polysomes of 30 min, 1 h, 5 h and 12 h embryos and hatching larvae. The mRNA from different stages was translated in a wheat-germ lysate and its template activity for ribosomal proteins was analyzed. It was observed that mRNA activity for ribosomal proteins is stored in the cytoplasm of dormant cysts and that it is found associated with polysomes of 30 min and of later stages.     

Protein synthesis in Artemia salina

It is thought that eucaryotic elongation factor eEF-Ts catalyzes the replacement of GDP for GTP on eucaryotic elongation factor eEF-Tu. We have found that eEF-Ts displays a strong nucleoside diphosphate phosphotransferase activity. This transferase activity resides in a dimer molecule of a subunit molecular weight close to 30,000. The transfosforylating activity of eEF-Ts results in a stimulatory effect of ATP, GTP, UTP and CTP on protein synthesis provided that GDP is present. The specificity for guanine nucleotides in protein synthesis resides only in eEF-Tu.     

Evidence of a store of informational RNA in encysted embryos of Artemia salina

RNA prepared from dormant cysts and developmental stages of the brine shrimp Artemia salina stimulated the incorporation of ¹⁴C-leucine into polypeptide by a cell-free Escherichia coli system. Preparations from cysts were about as active as those from hatching embryos or nauplii. When analysed by density gradient centrifugation the activity of cyst RNA showed a heterodisperse distribution, not quantitatively related to the absorbance profile. These results and evidence from similar experiments with crude ribosome preparations indicated that the contribution of 18S and 28S ribosomal RNA to the template-like activity was fairly limited. The experiments suggest that RNA with latent messenger activity is present in Artemia cysts during the resting stage.     

The sequence of the 5S ribosomal RNA of the crustacean Artemia salina

The primary structure of the 5 S rRNA isolated from the cryptobiotic cysts of the brine shrimp Artemia salina is pACCAACGGCCAUACCACGUUGAAAGUACCCAGUCUCGUCAGAUCCUGGAAGUCACACAACGUCGGGCCCGGUCAGUACUUGGAUGGGUGACCGCCUGGGAACACCGGGUGCUGUUGGCAU _OH.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.     

Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.