The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.     

Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.     

PERSISTENCE OF ALTERED RNA SYNTHESIS IN RAT CEREBRAL CORTEX 12h AFTER A SINGLE ELECTROCONVULSIVE SHOCK

The effect of electroshock (ECS) on RNA synthesis in nuclei and cytoplasm of rat cerebral cortex was examined using a double label technique by intraventricular injection of [³H] and [¹⁴C]orotate. At t h after ECS, the incorporation into nuclear RNA was 80% of the control rate and the appearance of newly synthesized RNA in the cytoplasm was only 27.6%. Analysis on composite polyacrylamide-agarose gels of purified RNA showed that the ³H/¹⁴C ratio of each gel slice slowly increased with decreasing M.W. of the RNA. This has been interpreted as an inhibition in the rate of processing of nuclear RNA. When the nuclear RNA was subjected to denaturation with 50% dimethyl sulphoxide (DMSO) this effect was enhanced. In a similar experiment, rats were injected, treated to ECS and killed 12 h later. The overall incorporation into nuclear and cytoplasmic RNA was increased to 174%, and 137.5% respectively. Analysis on gels showed very little variation in the ³H/¹⁴C ratio of the steady state levels of nuclear RNA. They compared well with a control experiment where rats were injected with [³H] and [¹⁴C]orotate as described above but no ECS was applied to the [¹⁴C] labelled animals. However a 1 h pulse label given 11 h after ECS treatment revealed that the rate of incorporation into nuclear RNA still showed a decrease of 81% of the control. The nuclear RYA fractionated on gels clearly showed that the inhibition of the processing rate of nuclear RNA was still occurring. This effect was again magnified on denaturation of the RNA with DMSO. This suggests that ECS may disturb RNA metabolism in nervous tissue for much longer periods than previously realised.     

Stored mRNA in sporangiospores of the fungus Mucor racemosus.

When introduced into nutrient medium under air, the asexual sporangiospores of Mucor racemosus germinated with 5 to 8 h, culminating with the emergence of germ tubes. We found that sporangiospores increased 20% in dry weight during the first 60 min of germination, indicating a high degree of synthetic activity. Sucrose density gradient analysis of spore extracts revealed that the percentage of ribosomes associated with mRNA increased from 22.5% in dormant spores to 85% within 10 min after the addition of medium and remained at this level for at least 3 h. L-[14C]leucine was immediately incorporated at a rapid rate into protein of a leucine auxotroph, whereas [3H]uracil or [32P]phosphate was incorporated into RNA at a significant rate only 20 min after the addition of medium. This newly synthesized RNA occurred in polysomes only after 30 min had passed. Pool synthesized RNA occurred in polysomes only after 30 min had passed. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from dormant spores by oligodeoxythymidylic acid-cellulose chromatography and was found to comprise 3.3% of the total cellular RNA. Sucrose density gradient centrifugation revealed the polyadenylated RNA to be heterodisperse in size, ranging from 6S to 20S. It was concluded that M. racemosus sporangiospores contain preformed mRNA which is translated commencing immediately upon the addition of nutrient medium.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO

Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30-minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin.     

The action of cordycepin on nascent nuclear RNA and poly(A) synthesis in regenerating liver.

Following a 5 min pulse of [5- 3H]orotic acid via the protal vein, the specific radioactivity of non-poly(A)heterogeneous nuclear RNA (HnRNA) reaches a peak at 12 h after partial hepatectomy. In contrast, poly(A)-HnRNA was maximally elevated only at 2 h after operation. After intraportal injection of cordycepin (3'-deoxyadenosine) 1 min before [5-3H]orotic acid, a dose-dependent inhibition of nuclear HnRNA and rRNA occurred. Fractionation of HnRNA on poly(U)-Sepharose following 20 mg/kg of cordycepin revealed that a 65% reduction occurred in the labeling of poly(A)-HnRNA while non-polyactivity of UTP in control and cordycepin-treated animals indicated no significant alterations in these parameters. Assessment of poly(A) size using poly(A)-HnRNA annealed with oligo(dT)10 as template primer for Escherichia coli DNA polymerase I, showed that 20 mg/kg of cordycepin inhibited nuclear polyadenylylation by 43%; no alteration in the binding of poly(A)-HnRNA to Millipore filters occurred at this dose of cordycepin. These results indicate that cordycepin is a non-selective inhibitor of nuclear RNA and poly(A)synthesis in regenerating rat liver.     

Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity

The role of endogenous regucalcin in the regulation of ribonucleic acid (RNA) synthesis activity in the nucleus of normal and regenerating rat livers was investigated. Nuclear RNA synthesis was measured by the incorporation of [(3)H]-uridine 5'-triphosphate into the nuclear RNA in vitro. The presence of regucalcin (0.25 or 0.5 microM) in the reaction mixture caused a significant decrease in nuclear RNA synthesis of normal rat liver. alpha-Amanitin (10(-8)-10(-6) M), an inhibitor of RNA polymerase II and III, decreased significantly nuclear RNA synthesis activity. The effect of regucalcin (0.25 microM) in decreasing nuclear RNA synthesis activity was not seen in the presence of alpha-amanitin (10(-6) M). The calcium chloride (10 microM)-increased nuclear RNA synthesis activity was significantly suppressed by the addition of regucalcin (0.25 microM). RNA synthesis activity was significantly enhanced in the nuclei of regenating rat liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly inhibited in the presence of PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M). Western analysis of the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy showed a significant increase in regucalcin protein as compared with that of sham-operated rats. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in nuclear RNA synthesis activity of normal rat liver. This increase was completely blocked by the addition of regucalcin (1.0 microM). The effect of anti-regucalcin monoclonal antibody (50 ng/ml) in increasing nuclear RNA synthesis activity was significantly enhanced in the nuclei of regenerating liver obtained at 24, 48, or 72 h after partial hepatectomy. This enhancement was significantly suppressed by the addition of alpha-amanitin (10(-6) M), PD98059 (10(-5) M), staurosporine (10(-6) M), or vanadate (10(-3) M) in the reaction mixture. The present study demonstrates that endogenous regucalcin has a suppressive effect on the enhancement of RNA synthesis activity in the nucleus of regenerating rat liver with proliferative cells.     

Nuclear translocation of estrogen-receptor complex and stimulation of RNA synthesis by estrogens of different biological potencies in the female rat pituitary

Stimulation of RNA synthesis and of nuclear translocation of estrogen-receptor complexes was investigated in isolated nuclei of anterior pituitaries of castrated female rats after injection with estrogens of different biological potencies. The assay system for the estimation of total RNA synthesis was validated and data suggest that incorporation of [3H]UMP into acid-precipitable material is consistent with RNA synthesis. An increase in RNA synthesis was seen 30 min after application of either 17 beta-estradiol, estriol or 1,3-diacetyl-17 alpha-ethinyl-7 alpha-methyl-1,3,5,(10)estratriene-17,3-ol (DMEE). RNA synthesis was maximal 90 min after estrogen application. Thereafter, RNA synthesis decreased slowly and reached pretreatment levels 3, 8 and 30 h after application of estriol, 17 beta-estradiol and the diacetyl derivative of ethinyl-estradiol, respectively. All estrogens were found to stimulate rapidly nuclear translocation of estrogen-receptor complexes. Peak levels of nuclear receptor contents were reached 30 min after administration of estrogens. A concomitant depletion of cytosol receptor levels was noted. Nuclear retention of estrogen-receptor complexes paralelled duration of enhanced RNA synthesis and correlated with biological potencies of the steroids. Data of present experiments combine to suggest that long-term nuclear retention is a requisite for expression of biological activity of estrogens at the anterior pituitary. Furthermore, the degree of biological activity seems to be associated with duration of stimulation of RNA synthesis, amount of estrogen-receptor complexes translocated to the nucleus, and duration of nuclear retention.     

Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma

Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([³H]adenosine/[¹⁴C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Comparative studies of the effects of galactosamine and actinomycin D on nuclear ribonucleoprotein particles from rat liver

Nuclear ribonucleoprotein particles of 75S were obtained from rat liver nuclei after mild sonication and isotonic salt extraction only when the preparation was carried out in the presence of a cytosolic ribonuclease inhibitor. Particles of 38S were isolated in the absence of inhibitor. The 38S nuclear ribonucleoprotein (nRNP) particles showed a protein/RNA ratio of 8, and a buoyant density of 1.39 g/ml in cesium chloride solution. They were further characterized by the pattern of their proteins on sodium dodecylsulfate (SDS)-acrylamide gel electrophoresis. Incorporation of [³H]cytidine into nuclear RNA was reduced to approx. 20% of controls 3 and 6 h after administration of galactosamine or actinomycin D. However, when [³H]cytidine was administered 30 min prior to the drugs a decrease of radioactivity in 38S nRNP particles to 43 and 81% of controls was found after 3 h. The yield of 38S particles 3 h after galactosamine or actinomycin D dropped to 41% and 78% of controls, and after 6 h to 43 and 70%, respectively. Six hours after galactosamine or actinomycin D treatment, the protein to RNA ratio increased to 13.3 and 9.1. No significant changes in protein patterns 3 h after treatment with galactosamine or actinomycin D were observed. Possible mechanisms, such as impaired transport of 38S nRNP particles after actinomycin D treatment or increased loss of particles due to a defective nuclear membrane after galactosamine administration are discussed.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.