The In Vitro Synthesis of RNA within the Rat Nodose Ganglion Following Vagotomy

Abstract: This report describes the application of an in vitro labelling procedure for the evaluation of changes in the uptake and incorporation of tritiated nucleotides into RNA of the rat nodose ganglion following crush injury of the cervical vagus nerve. Significant changes in the incorporation into 28S, 18S and 4S RNA were observed at 3 and 9 days after injury which confirms and extends our previous in vivo observations where [³²P]orthophosphate was used as the precursor. An early stimulation in the uptake of nucleotides, which was maximal at 2 days after injury, was also observed. Evidence is presented which indicates that this data reflects a real increase in RNA synthesis within the injured tissue concomitant with an increase in the uptake of nucleotide precursors which may reflect an increase in the nucleotide pool size. The transient nature of the rRNA synthetic responses and their occurrence prior to the peak of the chromatolytic changes suggest that there may be a shift in the distribution of ribosome types resulting in qualitative changes in protein production rather than an overall increase in protein synthesis resulting from an increased ribosome population.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Metabolism of glycosaminoglycans in CCl4-induced liver regeneration

The incorporation of [₃₅S]-SO₄ into glycosaminoglycans of liverin vivo and in in liver slices and into the glycosaminoglycans associated with the hepatic plasma membrane of rats at different periods after a heavy dose of CC1₄ have been studied. The incorporation of [35S]-SO4 into total glycosaminoglycans decreased to as low as 40% of the control at 24 h after the administration of CC14 and later on increased reaching a maximum on the 4th day. The amount of [35S]-SO4 incorporation into heparan sulphate was also reduced to about 40% of control at 12–24 h after the onset of injury and increased thereafter reaching a maximum on the 4th day. There was only a partial reduction in the synthesis of chondroitin sulphate in the early stage of injury and then it steadily increased reaching about 3 times the control level on 4–6 days. The [³⁵S]-SO4-incorporation into dermatan sulphate, after a slight initial decrease remained at the control levels. On the 8th day after the CCl₄-induced liver injury, the rate of [³⁵S]-SO₄-incorporation was almost equal to that in normal controls. The incorporation of [³⁵S]-SO₄ into hepatic plasma membrane glycosaminoglycans showed a similar change decreasing to about 35% of control at 24h followed by an increase, reaching normal levels on the 4th day after the administration of CC1₄. About 90% of the plasma membrane glycosaminoglycans was found to be heparan sulphate. The yield of plasma membrane from normal and CCl₄-induced regenerating liver was found to be similar and therefore the results obtained were not due to difference in the yield of the membrane preparation. The data also indicate that there was no difference in the degree of sulphation. The significance of these changes in the metabolism of sulphated glycosaminoglycans particularly plasma membrane heparan sulphate in tissue regeneration has been discussed.     

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.     

Posttranslational Protein Modification by Amino Acid Addition in Regenerating Optic Nerves of Goldfish

Previous experiments have demonstrated that 4S RNA, (tRNA), is transported axonally during the reconnection and maturation of regenerating optic nerves of goldfish. The present experiments were performed to determine if tRNA is transported axonally during elongation of these regenerating nerves and whether, as has been demonstrated in other systems, it participates in posttranslational protein modification (PTPM). [3H]Uridine was injected into both eyes of fish with intact optic nerves and 0, 2, 4, or 8 days after bilateral optic nerve cut. Fish were killed 2 days after injection, and [3H]RNA was isolated from retinae and nerves by phenol extraction and ethanol precipitation. [3H]RNA was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the percentage of [3H]4S RNA remained constant in all retinal and control nerve samples, regenerating nerves showed a twofold increase by 6 days after injury, suggesting that [3H]4S RNA is transported axonally in regenerating nerves as early as 6 days after injury. In other experiments, the 150,000-g supernatant of optic nerves was analyzed for incorporation of 3H-amino acids into proteins. No incorporation of 3H-amino acid was found in the soluble supernatant, but when the supernatant was passed through a Sephacryl S-200 column (removing molecules less than 20,000 daltons), [3H]Arg, [3H]Lys, and [3H]Leu were incorporated into proteins. This posttranslational addition of amino acids was greater (1.4-5 times for Lys and 2-13 times for Leu) in regenerating optic nerves than nonregenerating nerves, and the growing tips of regenerating nerves incorporated 5-15 times more [3H]Lys and [3H]Leu into proteins than did the shafts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo.

Biosynthetic processes related to the production of vitellogenin (yolk precursor protein) have been examined in the fat body of adult female Locusta migratoria. Vitellogenin-producing capacity was assayed by incubation of fat body with [³H]leucine, followed by precipitation from the medium with specific antiserum. In normal development, vitellogenin synthesis began at about Day 7 after emergence and became maximal at about Day 13, when this protein accounted for 60% of the total fat body protein output. The production of other proteins increased to a lesser extent, becoming maximal at about Day 6. The incorporation of uridine into fat body RNA rose to a maximum at Day 8, which coincided with a marked increase in tissue RNA content. The DNA content in adult female fat body approximately doubled between Days 3 and 8. Vitellogenin synthesis, and the increases in RNA and DNA, were prevented by removal of the corpora allata (the source of juvenile hormone). In allatectomized locusts, vitellogenin synthesis was induced by JH or an analog, ZR-515. Applied topically in acetone, these gave steep dose-response curves, half-maximal at 75 and 150 μg, respectively. After a single treatment with ZR-515, fat body vitellogenin production rose slowly during 48 hr, then steeply to a maximum at 72 hr, but after decay of this effect during 10 days, a second application of ZR-515 induced renewed synthesis with little initial lag. Hormone treatment produced a smaller increase in the output of other proteins, and an increase in incorporation into RNA which preceded the major rise in vitellogenin synthesis. Male fat body produced little or no vitellogenin. These results are consistent with action of JH at the gene level.     

Incorporation of tritiated adenosine into mouse ovum RNA

The total RNA of ovulated mouse ova has been examined by polyacrylamide gel electrophoresis. The amount of RNA present in the two main peaks observed, 28 S and 18 S ribosomal RNA, has been estimated as 0.20 ng.The RNA of ovulated mouse ova was labeled by exposure of growing mouse oocytes to adenosine-8-³H in vivo. For this purpose a small volume of a concentrated solution of the precursor was injected into the ovarian bursa, and ova were collected by superovulation at various subsequent times. The major growth phase of the oocyte is known to lie between 20 and 6 days before ovulation. Significant incorporation into egg RNA was observed when bursal injection was performed between 19 and 7 days, but not between 5 days and 1 day before ovulation.The types of labeled RNA in ova ovulated at five intervals between 19 and 7 days after bursal injection of adenosine-8-³H or uridine-5,6-³H were analyzed by polyacrylamide gel electrophoresis. The distribution of label on the gels demonstrated that the bulk of the label appeared in ribosomal RNA and transfer RNA. In addition labeled heterogeneous RNA was estimated to represent 10–15% of the total incorporation.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.     

Rate of RNA synthesis and tRNA end-labeling during early development of Phaseolus

Summary Axes of Phaseolus vulgaris cease synthesis of RNA during the maturation stage of embryogeny. During the imbibition phase of germination RNA synthesis resumes after the axes reach a normal water content. In the first hour of inbibition a very low rate of incorporation of ³H-adenosine into is RNA found, and the primary site of incorporation is the-CCA end of tRNA. At later stages of germination tRNA end-labeling accounts for a minor fraction of adenosine incorporation. The rate of RNA synthesis increases after initiation of axis elongation to a maximal rate at 18 h of germination. ATP pool-size and specific activity vary over a several-fold range during development, an important consideration in determining the rate of RNA synthesis.Supported by a grant from the National Science Foundation (GB 8709) to M. E. Clutter and I. M. Sussex and by a National Science Foundation Predoctoral Fellowship (V. W.). Submitted to the Graduate School, Yale University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

EFFECT OF HAEMORRHAGE ON THE RAPID AXONAL TRANSPORT OF NEUROHYPOPHYSIAL PROTEINS OF THE RAT

Following injection of [³⁵S]cysteine into the region of the supraoptic nucleus male rats were subjected to haemorrhage and the radioactivity of the supraoptic nucleus and neurohypophysial proteins was measured at various time intervals after injection. Following haemorrhage the incorporation of [³⁵S]cysteine into supraoptic nucleus proteins increased. Evidence was obtained for a lag period of 1 to 2 h for the supraoptic nucleus proteins to become available for axonal transport. As judged from the time of arrival of labelled material in the neurohypophysis, haemorrhage did not change the rapid rate of axonal transport (190 mm/day). At 15 min following bleeding, the radioactivity in fraction A (a neurophysin) of the neurohypophysis was reduced, which indicated a release of this rapidly transported protein. During the following 15 min an increase in the protein-bound radioactivity of the neural lobe occurred which exceeded that in controls. This is taken as evidence for increased axonal transport in response to haemorrhage.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

RNA metabolism in chick embryo skin]

Explants from 7, 8, 9, 11, 13-day chick embryonic skin incorporating (3H) Uridine for different periods 1 hr, 3 or 4 hr and a chase with actinomycin) are studied with respect to free (F) or membrane bound (B) cytoplasmic polysomes and to RNA extracted from them. Polysome specific activity decreases at older stages but the amount of polysomes increases due to increased protein synthesis. At each stage B polysomes are less abundant but more radioactive than F polysomes. RNA extracted from each kind is analysed on sucrose gradients: one half of each fraction is precipitated by TCA to estimate total radioactivity, the other is retained on millipore at high salt concentration to estimate radioactivity in messenger-like RNAs due to their poly-A sequences. The pattern of the labelling of the different fractions of RNA changes with the length of incorporation, the stages of explants and the kind of polysomes (F or B); at 11-13 days the incorporation is slow, radioactivity is low and distributed among several peaks of poly-A RNA; at 7-8 days the incorportion is rapid, dispersed throughout the gradient; at 9 days, a midway stage, incorporation is particularly high into 12S and 24S fractions from B RNA. In the 5 studied stages the labelling of this 12S occurs early, remains for a longer time and cannot be chased. These observations suggest stability of the 12S RNA. Since, in 14-day chick embryos, feather keratin m RNA has been shown to sediment at 12S and although our experiments have been done with total skin because this differentiating tissue is the site of extensive interactions between dermis and epidermis, they suggest that this 12S RNA is the actual keratin m RNA and might be synthesised some days before the onset of keratin synthesis. Its template ability will be investigated at earlier stages.     

Increased phenylalanine incorporation in regenerating skeletal muscle grafts

Skeletal muscle regenerates following grafting, but little is known about protein synthesis and its regulation during regeneration. We determined the sequence of changes in protein synthesis in rat extensor digitorum longus (EDL) muscle by the measurement of phenylalanine (Phe) incorporation into muscle protein at various times after grafting. Compared with control EDL, Phe incorporation in grafts doubled in 1 day, was four- to eight-fold greater from days 2 to 10 after grafting, and then subsided. Tissue mass (wet weight) increased rapidly from days 7 to 20 in EDL grafts. The maximal increase in protein synthesis occurred 7-10 days after grafting, whether or not the nerve was left intact. Autoradiography indicated that incorporated radioactivity was associated with regenerating muscle fibers on day 10. Deficiencies of insulin, pituitary or testicular hormones, or chronic in vivo administration of insulin, growth hormone, testosterone, or tri-iodothyronine did not substantially alter the elevation in incorporation of the Phe into muscle protein 10 days after grafting. The breakdown of EDL protein, measured in vitro simultaneously with protein synthesis, was increased five-fold, and overall protein degradation was elevated six-fold 10 days after grafting. These findings indicate that Phe incorporation is rapidly elevated following grafting of the EDL, and that by days 7-10 reflects synthesis in regenerating muscle fibers. The increase in protein synthesis associated with muscle regeneration at this time appears to be independent of innervation and anabolic hormones.     

The mechanism of regulation of fibroblastic cell replication. II. Participation of the nucleoli

Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar ⁴ cells and in the levels of uptake of ³H-uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso-nucleolinar type. The intensity of nucleolar labeling by ³H-uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre- and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar ³H-uridine incorporation. The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis. During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Germination of phaseolus vulgaris I. Resumption of axis growth

Growth of the excised axis of Phaseolus vulgaris L. (var. White Marrowfat) begins after a 7-hour incubation in buffer or water at 26°. Growth, as measured by axis elongation or fresh weight increase, is linear for at least 8 hours with a resultant fresh weight increase of approximately 65%. Cell elongation begins 4 or 5 hours prior to cell division and 5 or 6 hours prior to radicle protrusion in the intact seed.

The initiation of axis elongation is apparently dependent on synthesis of RNA and protein. Both actinomycin D and puromycin inhibit the initiation of elongation. Actinomycin I) inhibits the incorporation of ATP-8-C¹⁴ into axis RNA and C¹⁴-leucine into protein, while puromycin inhibits the incorporation of C¹⁴-leucine into axis protein.

The respiratory rate of the axes increases sharply at about the time of initiation of cell elongation. Dinitrophenol initially increases O₂ uptake by the axes, but at the end of 15 hours the rates of O₂ uptake by control or dinitrophenol-treated axes are approximately the same.

     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.