Regulation of tyrosine aminotransferase messenger ribonucleic acid in rat liver. effect of cycloheximide on messenger ribonucleic acid turnover

Tyrosine aminotransferase messenger ribonucleic acid (mRNA) activity in rat liver was rapidly increased 3-6-fold following in vivo administration of hydrocortisone acetate, dibutyryladenosine cyclic 3',5'-phosphate, or the protein synthesis inhibitor cycloheximide. Treatment with the steroid hormone or cyclic nucleotide in combination with cycloheximide resulted in levels of tyrosine aminotransferase mRNA 10-20-fold greater than control values. These changes in mRNA activity were not accompanied by changes in albumin mRNA or total liver template activity. The rapid decline in tyrosine aminotransferase mRNA activity following cordycepin inhibition of de novo RNA synthesis was prevented by cycloheximide treatment. This protection was not observed when pactamycin was substituted for cycloheximide, demonstrating that the inhibition of protein synthesis per se was not responsible for the stabilization of tyrosine aminotransferase mRNA. Based upon the effects of cycloheximide and pactamycin on rat liver polysome structure, it is concluded that the cycloheximide-mediated increase in tyrosine aminotransferase mRNA activity is the result of stabilization of the mRNA molecule which renders the message less susceptible to inactivation and degradation in the cytoplasm. The action of cycloheximide is very specific for tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and probably several other mRNAs that code for minor liver proteins that turn over rapidly in response to hormonal or metabolic stimuli.     

Stimulation of tyrosine aminotransferase activity by dl-alpha-methyltryptophan.

DL-alpha-Methyltryptophan (alphaMeTrp), a synthetic analogue of tryptophan, has been found to be a potent inducer of hepatic tyrosine aminotransferase activity in the adrenalectomized rat. alphaMeTrp is inactive in vitro. Unlike the action of other known inducers (tryptophan, hydrocortisone, adenosine cyclic 3:5-monophosphate, and glucagon), maximal stimulation of enzyme activity occurs only 16 to 30 hours after alphaMeTrp administration and the activity is still elevated at 96 hours. Only the L isomer of alphaMeTrp is active, and addition of a hydroxyl group to position 5 of the indole ring renders an inactive compound. The induction can be prevented by actinomycin D or cycloheximide but not galactosamine. Administration of alphaMeTrp together with hydrocortisone produced an additive stimulation of enzyme activity. alphaMeTrp given along with glucagon or adenosine cyclic 3:5-monophosphate caused a further but not additive increase in enzyme activity. Tryptophan given along with alphaMeTrp promoted no extra stimulation whatsoever. These data indicate that alphaMeTrp and tryptophan may act via a common pathway which in part requires RNA synthesis. Other enzymes, namely alanine and aspartate aminotransferase, ornithine aminotransferase, ornithine carbamoyltransferase, serine dehydratase, and histidine ammonialyase, were not affected by treatment of rats with alphaMeTrp.     

Transcriptional and translational control of the biphasic increase in ornithine decarboxylase activity in liver

The requirements for protein and RNA synthesis for each of the two increases in liver ornithine decarboxylase activity after the injection of unoperated rats with a solution containing glucagon and after 70% hepatectomy were studied with cycloheximide and actinomycin D. Protein synthesis is required for both increases whereas RNA formation is essential for the first elevation only. The second increase appears to be dependent upon RNA that is made during the period of the first rise in activity.The two rises in the decarboxylase activity may be caused by different stimuli. After the injection of the mixture with glucagon, the first elevation is accompanied by increases in hepatic tyrosine aminotransferase activity and in the rate of transport into liver cells of the model amino acid, α-aminoisobutyrate. Neither an increase in the aminotransferase activity nor in amino acid uptake occurs, however, during the period of the second elevation in the decarboxylase activity.     

Induction of particulate and soluble isoenzymes of tyrosine aminotransferase by hydrocortisone in the liver of rats as a function of age.

The induction of soluble cytoplasmic (c-), and particulate mitochondrial (m-) and nuclear (n-) isoenzymes of tyrosine aminotransferase (TAT) by hydrocortisone in the liver of 6-, 35- and 76- week old rats was studied. In contrast to the earlier reports, both the particulate isoenzymes (m- &; n-TAT) are induced by hydrocortisone. This induction is actinomycin D sensitive. The degree and pattern of induction of the three isoenzymes of TAT vary with age. The possibility of separate regulatory mechanisms for the synthesis of the three isoenzymes is discussed.     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

Contribution of a change in mRNA half-life to the accumulation of the tissue-specific S-100 protein during postnatal development of the mouse brain

The S-100 protein accumulates rapidly in the mouse brain between 15 and 21 days of postnatal development. The accumulation of this protein is brought about mainly by an increased rate of its synthesis. The present study focuses on attempts to determine if a change in the half-life of messenger RNA (mRNA) is involved in bringing about the increased rate of synthesis of the S-100 protein. Utilizing the inhibition of RNA synthesis by actinomycin D, we were able to show that the halflife of mRNA increases concurrently with an increase in the rate of synthesis of the S-100 protein. Utilization of actinomycin D does present hazards in interpretation of results on mRNA stability; experiments were performed to determine if the results obtained were due to side effects of the drug. As far as could be determined, the possible side effects of actinomycin D did not affect our results.     

Origin of the actinomycin D insensitive RNA species in Aedes albopictus cells.

In the presence of actinomycin D or a combination of actinomycin D and either camptothecin or alpha-amanatin. Aedes albopictus cells synthesize a variety of single stranded RNA species. These actinomycin D resistant species are ethidium bromide sensitive and they are present in the cell cytoplasm in an RNase resistant structure which has the sedimentation and buoyant density characteristics of mitochondria. Twelve actinomycin D insensitive RNA species can be detected by electrophoresis in 7M urea and 11 of these bind to oligo(dT)-cellulose. An identical set of oligo(dT)-cellulose binding RNA species is obtained when A. albopictus cells are labeled in the presence of camptothecin alone. The actinomycin D insensitive RNA species which bind to oligo(dT)-cellulose hybridize to mitochondrial DNA. These data indicate that the actinomycin D insensitive RNA species have a mitochondrial origin and are not associated with the replication of an inapparent contaminating virus.     

De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

Synthesis of protein and RNA for initiation and growth of the protonema during germination of bracken fern spore

Cycloheximide inhibited initiation and elongation of the protonemal cell during germination of the spores of bracken fern. Incorporation of ¹⁴C-leucine into protein was also profoundly affected by the drug. Concentration of actinomycin D sufficient to inhibit incorporation of ³Huridine into heavy RNA fractions of spores did not prevent initiation of the protonema, but inhibited its subsequent elongation. Protein synthesis during initiation and growth of protonema was not appreciably sensitive to actinomycin D. As in the case of rhizoid initiation, protein synthesis necessary for initiation of protonema during germination appears to involve preformed messenger RNA.     

On the regulation of tyrosine transaminase, glutamatic dehydrogenase and aspartic transaminase in Tetrahymena

Aspartic transaminase, tyrosine transaminase, lactic dehydrogenase, and glutamic dehydrogenase were studied in Tetrahymena pyriformis in order to gain a better understanding of the control of the entrance and exit of metabolic intermediates to and from the major carbohydrate pathways. Glucose decreased the activity of aspartic transaminase, tyrosine transaminase and glutamic dehydrogenase but not lactic dehydrogenase. Actinomycin D (6 and 12 μg/ml) blocked the decrease in glutamic dehydrogenase and aspartic transaminase activity caused by glucose; 12 μg/ml partially prevented the decrease in tyrosine transaminase activity. Actinomycin D alone had little effect on enzyme activity. Uracil incorporation into RNA was doubled by 6 μg/ml actinomycin D, a concentration which did not alter the RNA content of the cells. At 12 μg/ml this drug caused a small decrease in RNA spec. act. Cycloheximide at 10 μg/ml, a concentration which inhibited protein synthesis by 70%, caused a three-fold increase in aspartic transaminase and a two-fold increase in glutamic dehydrogenase. In the presence of both cycloheximide and glucose, the drug effect predominated. Thus both actinomycin D and cycloheximide blocked the glucose-induced decrease in enzyme activity. These results suggest that the levels of aspartic transaminase, glutamic dehydrogenase, and probably tyrosine transaminase are regulated at least in part by a degradative control system.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

The hydrating effect of lathyrogenic compounds on chick-embryo cartilage in vivo

1. Injection of 0.16mug. of actinomycin D into pupae of the beetle Tenebrio molitor L. results in the development of modified adults in which the head and thorax are essentially adult while the abdomen and wings remain pupal-like. It is suggested that the messenger RNA for the development of head and thorax is present in the animal from the first day of pupation. 2. Injection of 0.16mug. of actinomycin D brings about 51-67% inhibition of labelled uridine incorporation into RNA. 3. When thymus DNA is mixed with actinomycin D before injection into pupae the latter develop into normal adults. This protection does not occur when DNA and actinomycin D are injected separately. 4. The inhibition of incorporation of labelled uridine into RNA by actinomycin is diminished to some extent when DNA and actinomycin D are injected separately and abolished if they are injected together. 5. Inhibition of RNA synthesis by actinomycin D in vitro is fully reversible. DNA or deoxyguanosine can reverse the effect of actinomycin D. 6. Incorporation of labelled glycine into protein is not affected by actinomycin D injection during the first 6 days of pupation. On the seventh day it becomes diminished in control pupae but this effect is prevented by actinomycin D. It is suggested that the template for protein synthesis is stable during the first 6 days of metamorphosis and that on the seventh day there is a qualitative change in the protein synthesized on the template.     

Flagellar synthesis in Salmonella typhimurium: requirement for ribonucleic acid synthesis

The micro-complement-fixation assay has been demonstrated to be a sensitive assay for flagella which occur in nanogram amounts. By use of this assay, it was found that flagellar synthesis occurs during starvation of Salmonella typhimurium for tryptophan, an amino acid not present in flagellar protein. Under these conditions net ribonucleic acid (RNA) synthesis was reduced to approximately 10% of the control rate. Less than 1 mug of actinomycin D per ml further reduced RNA synthesis to less than 1% of the control rate in a culture sensitized by prior treatment for 5 min at 37 C with 5 x 10(-4)m ethylenediaminetetraacetate in 0.33 m tris(hydroxymethyl)aminomethane-chloride (pH 8.0). In the presence of actinomycin D, no synthesis of flagellar protein could be detected. Analysis of fractions of RNA separated by zone centrifugation indicated that actinomycin D reduces the production of template RNA as well as of ribosomal RNA. This suggests that in S. typhimurium the production of flagellar protein requires the concomitant synthesis of RNA. There is no evidence that a stable messenger RNA specific for flagellar synthesis is present.     

Selective inhibition by sodium butyrate of glucocorticoid-induced tyrosine aminotransferase synthesis in hepatoma tissue-cultured cells

Sodium butyrate in a 5 mM concentration prevents the induction of tyrosine aminotransferase in hepatoma culture cells, without affecting the basal level of the enzyme. This effect is reversible immediately after the removal of butyrate, or after a lag, if butyrate was present for more than 2 h. Neither the amount of cellular RNA nor the rate of total RNA synthesis were affected by sodium butyrate. Furthermore, butyrate does not inhibit protein synthesis: [35S]methionine incorporation into proteins, measured in a reticulocyte lysate system, shows no significant difference between the translation capacity of the RNAs from butyrate-treated cells and from dexamethasone-induced or uninduced cells. Nevertheless, when tyrosine aminotransferase was isolated from the translation products by its specific antiserum and analyzed by gel electrophoresis, we observed that the amount of the enzyme synthetized in the presence of RNAs from dexamethasone/butyrate-treated cells was strongly diminished relative to that synthesized in the presence of RNA from dexamethasone-induced cells. These experiments indicate that the treatment of the cells with butyrate decreases the activity of the specific messenger RNA for tyrosine aminotransferase to a level close to the basal level.