Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells). Dissociation between nuclear iodothyronine receptor concentration and growth hormone mRNA synthesis during the deoxyribonucleic acid synthesis phase of the cell cycle

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labeling with [3H]leucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehyde-treated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p less than 0.005 (from 13,430 +/- 609 to 19,150 +/- 1160 cpm/10(6) cells/2 h) and increased GH mRNA, p less than 0.001 (from 7.2 +/- 1.2 to 14.5 +/- 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cell populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/10(6) cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.     

Effect of cycloheximide on protein and ribonucleic acid synthesis in cultured human lymphocytes

1. Phytohaemagglutinin stimulates the transformation into blast cells of human lymphocytes incubated in vitro. This transformation is accompanied by an increase in the incorporation of [(14)C]leucine into protein and [(3)H]uridine into RNA. 2. The incorporation of [(14)C]leucine by cultures grown in the presence or absence of phytohaemagglutinin is inhibited to the same extent by cycloheximide, a known inhibitor of protein synthesis. 3. Lymphocytes grown without phytohaemagglutin synthesize mainly non-ribosomal RNA. [(3)H]Uridine incorporation by these cells was increased by cycloheximide. 4. Lymphocytes incubated with phytohaemagglutinin begin to synthesize substantial quantities of ribosomal RNA. Under these conditions [(3)H]uridine incorporation was partially inhibited by cycloheximide. This inhibition is shown to be largely a result of inhibition of the synthesis of ribosomal RNA.     

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.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Studies on the inhibition of protein synthesis by selenodiglutathione.

Amino acid incorporation in a cell-free system derived from rat liver has previously been found to be inhibited by GSSeSG (selenodiglutathione). In the present experiments the effect of GSSeSG on protein synthesis in 3T3-f cells, on growth and protein synthesis in Escherichia coli, and on amino acid incorporation in a cell-free system derived from E. coli, was studied. GSSeSG inhibits the incorporation of [3H]leucine into protein by 3T3-f cells. This inhibition cannot be reversed by removing GSSeSG and is correlated with the uptake of GSSeSG. Sodium selenite (Na2SeO3) and oxidized glutathione had no inhibitory effect in this system. [3H]Uridine or [3H]thymidine incorporation into RNA or DNA was not inhibited, indicating that the primary action of GSSeSG was on protein synthesis. GSSeSG did not influence the growth of E. coli in a synthetic medium, although enhanced amino acid incorporation was observed. In the cell-free system derived from E. coli, amino acid incorporation was not changed by GSSeSG, indicating that elongation factor G, in contrast to elongation factor 2 of mammalian cell systems, is not blocked by GSSeSG.     

Comparative inhibition of nuclear RNA synthesis in cultured mouse leukemia L1210 cells by adriamycin and 4'-epi-adriamycin

Adriamycin and 4'-epi-adriamycin were compared as to their effect on nRNA synthesis. 4'-Epi-adriamycin was a more effective inhibitor than the parent compound of RNA synthesis as measured by incorporation of [3H]-uridine. Adriamycin inhibited all three species of nRNA (ribosomal, non-poly(A)hnRNA, poly(A)hnRNA) to approximately the same extent. 4'-Epi-adriamycin on the other hand inhibited the nRNA species in the following order: non-poly(A)hnRNA greater than ribosomal RNA greater than poly(A)hnRNA. The inhibitory effects of both drugs on incorporation of uridine into RNA were reversible at low concentrations (5 microgram/ml).     

On the use of alpha-amanitin as a transcriptional blocking agent in mouse embryos: a cautionary note

We have tested the effect of alpha-amanitin at 10, 50 and 100 micrograms/ml, on precursor uptake and incorporation into poly(A)+ RNA and poly(A)- RNA of mouse embryos on days 2, 3 and 4 of gestation. Embryos were pretreated with the inhibitor for 2 hr, then labeled for 2 hr in its continued presence. RNA fractions were separated by affinity chromatography on oligo(dT)-cellulose. alpha-Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 micrograms/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 micrograms/ml, was effective in all stages in substantially suppressing incorporation into poly(A)+ RNA within 2 hr. Longer treatments increased the level of suppression to a maximum of about 80%. Incorporation into poly(A)- RNA was suppressed to roughly the same extent. Despite previously reported data, it cannot be assumed that alpha-amanitin at concentrations less than 100 micrograms/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos.     

Potassium Requirement for Synthesis of Macromolecules in Bacillus subtilis Infected with Bacteriophage 2C

A mutant of Bacillus subtilis 168 (strain 168 KW), defective in its ability to concentrate K(+) from low levels in the growth medium, was used to study the role of K(+) in the development of phage 2C. Both the final burst size and the duration of the rise period depended on the K(+) concentration in the medium. During normal infection (in the presence of K(+)), host deoxyribonucleic acid (DNA) synthesis stopped. The synthesis of host messenger ribonucleic acid (RNA) continued throughout infection, albeit at a steadily decreasing rate. The synthesis of ribosomal RNA and its subsequent incorporation into mature ribosomes also proceeded. In contrast to these findings, host DNA and messenger RNA synthesis were not inhibited in cells infected in the absence of K(+). Only "early" phage messenger RNA was synthesized under these conditions of infection. Phage DNA synthesis was dependent on K(+) irrespective of the requirement for this cation in protein synthesis.     

Sequence relationships between adenovirus 2 early RNA and viral RNA size classes synthesized at 18 hours after infection.

Synthesis of cytoplasmic viral RNA was studied during infection of cultured human (KB) cells with adenovirus 2. At 6 h, before viral DNA synthesis began 5% of the poly(A)-containing RNA hybridized to viral DNA; by 12 h and at later times more than 80% was virus specified. At 18 h after infection, four major size classes of cytoplasmic viral RNA were identified among the poly(A)-containing molecules. These size classes migrated as 27S, 24S, 19S, and 12 to 15S in polyacrylamide gels. The three larger size classes could also be identified in denaturing formamide gels. Hybridization of the 27S, 24S, and 19S viral RNAs was not inhibited by RNA harvested from cells at early times in infection. Therefore, these three major RNAs must code for late viral proteins. Hybridization of the 12 to 15S RNA was partially inhibited by RNA from cultures harvested at early times, suggesting that in this size class some of the RNA labeled at 18 h codes for early viral proteins.     

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.     

Leishmania spp.: effect of inhibitors on growth and on polyamine and macromolecular syntheses

Ethidium bromide, pentamidine isethionate, and MGBG [methylglyoxal-bis (guanylhydrazone)] inhibited the uptake of radioactive putrescine by leishmanial (Leishmania spp.; Leishmania tropica major; Leishmania mexicana; Leishmania donovani) promastigotes and interfered with their polyamine synthesis. Inhibition was apparent as early as 1 hr after adding these drugs to the parasites at growth-inhibiting concentrations. Ethidium bromide also inhibited the incorporation of radioactive uracil into leishmanial RNA at growth-inhibiting concentrations, while DNA synthesis was inhibited by ethidium bromide at high concentrations after a lag period. MGBG inhibited the synthesis of leishmanial DNA and RNA at growth-inhibiting concentrations.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells I. Partial Purification and Characterization

Poly(A) polymerase activity is induced during vaccinia virus infection of HeLa cells. The enzyme is maximally induced at 3.5 h postinfection. Partial purification frees the preparation of RNase activity and RNA polymerase activity. ATP is the substrate for poly(A) synthesis. A small amount of poly(A) is produced from added adenosine diphosphate due to the production of ATP by an adenylate kinase present in the preparation. The incorporation of ATP into poly(A) is dependent on divalent cations (Mg²⁺ or Mn²⁺) and is not inhibited by UTP, CTP, or GTP. Poly(U) stimulates ATP incorporation; poly(A) and poly(C) have little effect on ATP incorporation, and poly(dT) is extremely inhibitory. RNA prepared from HeLa cells and from the partially purified poly(A) polymerase (the enzyme preparation contains endogenous RNA [Brakel and Kates]) stimulates ATP incorporation by poly(A) polymerase which was subjected to DEAE-cellulose chromatography. RNase's, pancreatic and T₁, inhibit the production of poly(A). DNase has little effect. Poly(U) is able to stimulate poly(A) production in the presence of T₁ RNase.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

Effects of 3′deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: Analysis of response to aldosterone

Summary Previous studies have shown that aldosterone increases transepithelial active Na⁺ transport after a latent period of about 60 min and incorporation of³H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na⁺ transport measured as short-circuit current (scc). The incorporation of³H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of³H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na⁺ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.