RNA and protein metabolism in the particulate fractions of the gametophytes of bracken fern during growth in red and blue light

Summary The metabolism of RNA and protein in the gametophytes of bracken fern (Pteridium aquilinum) is affected by the quality of light in which they are grown. When sporelings were grown as two-dimensional gametophytes in blue light, particulate fractions separated from the sporelings exhibited greater incorporation of uridine-³H and leucine-³H into RNA and protein, respectively, than those from sporelings grown as one-dimensional protonema in red light. After various periods of exposure of gametophytes to red or blue light in the presence of uridine-³H, the nuclei-rich fraction showed the highest specific activity in RNA, and irrespective of incubation time, blue light was more effective than red light. The possibility that enhanced synthesis of RNA in the nucleus in response to blue light is significantly related to the morphological growth pattern of the gametophytes, is discussed.     

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.     

Selective inhibition by arabinosylcytosine of oligo(U)-containing RNA synthesis in germinating wheat embryos

Arabinosylcytosine (araC) was found to influence the pattern of RNA labelling in early wheat embryos. Although the rate of uridine-¹⁴C incorporation into total RNA remained unchanged, specific radioactivity of an oligo(U)-containing subclass of nonpolyadenylated RNA was markedly lower (50%) in araC-treated (1.5 mM, 1 h) than in control embryos. The inhibitory effect was stronger for nuclear than for cytoplasmic subpopulation of these RNA species. h labelling of all other RNA fractions, including bulk nonpolyadenylated RNA as well as poly(A)-containing RNA of both nuclear and cytoplasmic origins, did not respond to the presence of araC.     

NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

The incorporation of 5-³H-uridine and 5-³H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-³H and cytidine-³H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G₁ in both the nucleus and the reconstituting nucleolus. Thereafter, through the G₁ and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G₁ or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells.     

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia

Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues (Golosow and Grobstein, 1962), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al., 1964). We have shown that this fraction also stimulates the incorporation of thymidine-³H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen.The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine-³H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine-³H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly.MF also affected RNA and protein synthesis. The rate of leucine-³H incorporation into protein and uridine-¹⁴C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.     

Autoradiography of tritiated uridine in shoot apices ofChenopodium rubrum

The incorporation of uridine-5-³H into shoot apices ofChenopodium rubrum, plants was studied using autoradiography. The evaluation of the rate of incorporation into the nucleolus and the extranucleolar part of the nucleus as a function of the total radioactivity in the apex yields quantitative data on the distribution of labeling in these parts of the nucleus. Incubation of intact germinating plants in uridine-³H makes it possible to carry out chase experiments. Curves of uridine incorporation into the nucleolus and the extranucleolar part of the nucleus were obtained which demonstrated a non-linear course of incorporation. When incubating with uridine from 30 to 120 min the nucleolar/extranucleolar ratio of labelling was found to increase from 2 to 3. In chase experiments this ratio changed within three days from 3 to 1. Interpretation of these results in view of the function of RNA localized in different parts of the nucleus is discussed.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Comparative effects of 1, 10-phenanthroline and 1, 7-phenanthroline on DNA and RNA synthesis in mouse spleen

When 1, 10-phenanthroline at 10^?4 mole/kg was administered intraperitoneally to C3H mice, a significant decrease of (³²P) Na₂HPO₄ incorporation into splenic DNA and RNA was noted within 15 min. The same dose or higher was required to significantly inhibit the incorporation of (5-³H) uridine and (methyl-³H) thymidine into splenic nucleic acid. 1, 10-phenanthroline also decrease the incorporation of the ³²P into DNA and RNA in 6C3HED ascites tumor. 1, 7-phenanthroline, a non-chelating analogue at 10^?4 mole/kg, less effectively altered the rate of the ³²P incorporation into splenic nucleic acid within 15 min, but significantly inhibited the incorporation within 1 hr.     

Changes in nucleic acid metabolism in barley seedlings during vernalization

Changes in nucleic acid metabolism of barley seedlings duringvernalization were investigated using thymidine-³H and uridine-³H. DNA content increased in the early germination stage from the1st to 3rd week in vernalized seedlings. In unvernalized seedlings,the most rapid increase was found in the late germination stage.RNA content in the vernalized seedlings increased after 1 weekand reached maximum level after 3 weeks of vernalization treatment.The unvernalized seedlings had a comparatively high contentat 2 days' germination which then gradually increased. Much thymidine-³H was incorporated into DNA and uridine-³H intoRNA fractions in the seedlings during early vernalization. Onthe contrary, without vernalization, heavy incorporation ofthymidine-³H was delayed during the late germination stage.Incorporation of uridine-³H showed a linear increase. A more detailed distribution of thymidine-³H and uridine-³Hin the nucleic acids was examined by methylated albumin-coatedkieselguhr column chromatography. A considerable amount of theincorporated uridine-³H was found in the tenaciouslybound ribonucleicacid (TB-RNA) in the vernalized seedlings. (Received January 18, 1973; )     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

Hormonal effects on RNA synthesis by stage 6 oocytes of Xenopus laevis.

RNA synthesis has been studied in “large” oocytes of Xenopus laevis, both as a function of time after injection of females with human chorionic gonadotropin (HCG) and in relation to the induction of maturation with progesterone in vitro. Rates of RNA synthesis were measured by analyzing the kinetics of incorporation of exogenous [³H]guanosine, and microinjected [³H]- or [¹⁴C]GTP, into acid-precipitable material, coupled with measurements of precursor pool specific activity. The kinetics of incorporation into RNA of injected precursor are biphasic, indicating the synthesis of both stable and unstable RNA species. Estimates of the total rate of synthesis (stable and unstable) were derived from fitting a linear function to data over the first 60–90 min, while a linear function fit to the data beyond 90 min represented largely the synthesis of stable RNA species.Exposure of oocytes to progesterone had no effect on initial synthetic rates, but maturing oocytes synthesized stable RNA at 1.4–1.6 times the rate in control oocytes. A comparison of data obtained with oocytes from unstimulated (no prior HCG treatment) and HCG-stimulated females indicated that HCG has no substantial effect on rates of RNA synthesis. The significance of continued RNA synthesis in large full grown oocytes is discussed.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Mitochondrial RNA and protein synthesis in enucleated African green monkey cells

The behavior of extramitochondrial protein synthesis and of mitochondrial RNA and protein synthesis was examined in the cytoplasts of African green monkey kidney cells (TC-7 subline) at different times following enucleation by cytochalasin B. The rate of incorporation of [³H]isoleucine into protein of the soluble cytoplasmic fraction decreased in an approximately exponential fashion, with a half-life of about five hours, during the first 26 hours after enucleation. Discrete mitochondrial 16 S, 12 S and 4 S RNA components were identified among the products of cytoplast RNA synthesis. The rates of [³H]uridine incorporation into the 16 and 12 S RNA components as well as into total RNA declined progressively after enucleation to a barely detectable level by the 20th hour. By contrast, the rate of chloramphenicol-sensitive [³H]isoleucine incorporation into protein (due to mitochondrial protein synthesis) did not undergo a substantial decline for at least 20 hours in TC-7 cytoplasts; instead, a reproducible transient stimulation occurred in the first hours following enucleation. The products of mitochondrial protein synthesis pulse-labeled in nucleated cells and in cytoplasts 24 hours after enucleation exhibited similar electrophoretic profiles.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

In vitro studies of cerebral cortical RNA and nucleotide metabolism in hypothyroidism.

—Thyroid hormone deficiency induced during the neonatal period in the rat, resulted in an enhanced incorporation of [2-¹⁴C]uridine and [8-¹⁴C]adenosine in vitro into cerebral cortical RNA at 25 days of age. An examination of the acid-soluble pool constituents separated by polyethyleneiminecellulose TLC, revealed that all phosphorylated derivatives were more highly labelled compared to controls. These differences were not apparent at a lower incubation temperature (4°C). When the average specific activity of precursor pool ATP labelled from adenosine was utilized for the calculation of the rate of RNA synthesis, no change was observed in hypothyroidism. The results are compatible with a maturational-dependent increase in nucleoside transport and rate of phosphorylation in hypothyroidism which is reflected in the stimulated incorporation into cerebral RNA. The apparent normal rate of RNA synthesis coupled with a diminished cellular RNA concentration in thyroid hormone deficiency, suggests an increased RNA turnover. Experiments with actinomycin D revealed no apparent difference in the rate of decay of rapidly-labelled (nuclear) RNA. The possibility is discussed that the processing of nuclear RNA, the formation of stable ribosomal complexes and events at the translational level are subject to modification in developing hypothyroid rats.     

The pattern of methylation of RNA in peripheral nerve of the chick during development

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14-day embryos, 17-day embryos and 3-day- old chicks was incubated with [methyl-³H]methionine or with [2-¹⁴C]uridine and [methyl-³H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14-day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3-day-old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3-day-old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly-labelled RNA of the 17-day embryonic nerve represented a transitional state between that of the 14-day embryos and that of the 3-day-old chicks. The 17-day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse-chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA-like RNA were synthesized.     

Effects of the rat renotropic system on 14C-uridine incorporation into RNA and RNA precursors.

We used the incorporation of ¹⁴C-uridine into RNA of incubating kidney fragments from normal control rats to evaluate RNA metabolism. Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-operatively stimulate ¹⁴C-uridine incorporation into RNA significantly more than sera from sham-operated rats (sham). Differently, sera from uni and sham rats have little influence on specific activities of endogenous uridine nucleotide pool in renal fragments. Renal extracts were obtained by homogenizing kidneys in saline. Extracts from kidneys of uni and sham rats 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of uridine pools. Correcting for the latter dilution demonstrates that kidney extracts alone have little effect on ¹⁴C-uridine incorporation into RNA. We then followed the results when these sera and extracts were combined. Compared to fragments incubating in sham sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances ¹⁴C-uridine into renal RNA, whether or not results are corrected for changes in the specific activities of the uridine pools. We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor in the remaining kidney. The tissue factor can only form an excitor to ¹⁴C-uridine incorporation into RNA when serum is present. The rat renotropic system that enhances incorporation of ³H-thymidine into DNA also can stimulate ¹⁴C-uridine incorporation into renal RNA.     

The incorporation of uridine-3H into the shoot apices of photoperiodically induced and non-induced plants ofChenopodium rubrum L.

The influence of photoperiodic induction on the incorporation of uridine-³H into the shoot apices ofChenopodium rubrum was studied using the technique of autoradiography. No increase in uridine incorporation was detected either during induction lasting three days or immediately after its termination. Pyroninophylia likewise did not rise. However, changes in uridine incorporation related to morphogenetic activity during leaf formation and later during differentiation of inflorescences were well marked. The distribution of label in the nucleus immediately after three inductive cycles shows the ratio of extranucleolar to nucleolar incorporation to be higher in non-induced control plants than in induced ones. Data from literature pointing to an activation of RNA synthesis during transition to flowering are discussed and compared with other systems where ontogenetic changes are accompanied by marked changes in RNA synthesis. It is assumed that the activation of RNA synthesis after induction is connected mainly with the activation of growth. However, inChenopodium rubrum photoperiodic induction proceeds together with limited growth and without activation of RNA synthesis.     

Changes in RNA-, DNA- and proteinsynthetic activity during the formation of analfin processes in ethisterone-treated females of Oryzias latipes

The incorporations of uridine-³H, thymidine-³H, and leucine-³H were studied in the process-forming regions of the anal-fin rays of the ethisterone-treated females of Oryzias latipes. The activity of alkaline phosphatase was also studied. The increased incorporation of uridine-³H was detected between 12 and 24 hours of ethisterone treatment, attaining the maximum at 24 hours. The percentage of thymidine-³H labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of leucine-³H was found to increase during the first 12 hours, attaining a constant level at 24 hours. An additional increase in incorporation of leucine-³H took place at 60 hours, the incorporation coming up to the maximum at 72 hours. In the horny substance secreted by the scleroblast mass, grains in the autoradiograph were detected at and after 72 hours. Alkaline phosphatase activity was manifested between 48 and 72 hours. These results seem to correspond to the histological changes, such as the appearance of the precursor cells of scleroblasts at 48 hours, the formation of scleroblast mass during the next 24 hours, and the initiation of horny substance secretion at 72 hours.