Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

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

The biosynthesis of transfer RNA in insects. II. Isolation of transfer RNA precursors from the posterior silk gland of Bombyx mori.

The occurrence of precursors to tRNA in the post-polysomal fraction of the posterior silk gland of Bombyx mori was demonstrated by pulse-chase labeling and DNA-RNA hybridization competition experiments. These precursors had molecular sizes ranging from 4S to 5S on polyacrylamide gel electrophoresis. Analysis of the incorporation of the methyl group from [methyl-14C]methionine revealed that a radioactive peak on polyacrylamide gel appeared in the 4.5S region during brief labeling. This suggested that some methylation occurred at the 4.5S precursor step.     

Effects of sinefungin on rRNA production and methylation in the yeast Saccharomyces cerevisiae

The antifungal agent, Sinefungin (SF), has been shown to be an inhibitor of transmethylation reactions. We report here the effects of SF on the production and methylation of rRNA in the yeast, Saccharomyces cerevisiae. Under conditions of SF treatment which have been shown to affect the regulation of cell proliferation in this yeast, pulse-chase labeling experiments using [methyl-3H]methionine and [3H]uracil indicated that methyl incorporation into rRNA during a short labeling period was inhibited, and stable 18 S rRNA production was differentially decreased. Other experiments quantitating modified nucleotides in newly produced rRNA showed that stable molecules were methylated. Taken together, these results suggest that SF slows methylation of rRNA, and is associated with differential loss of undermethylated 18 S rRNA species.     

Methylated messenger RNA in mouse kidney.

Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.     

Insulin in methionine and homocysteine kinetics in healthy humans: plasma vs. intracellular models

Methionine is a sulfur-containing amino acid that is reversibly converted into homocysteine. Homocysteine is an independent cardiovascular risk factor frequently associated with the insulin resistance syndrome. The effects of insulin on methionine and homocysteine kinetics in vivo are not known. Six middle-aged male volunteers were infused with L-[methyl-2H3,1-13C]methionine before (for 3 h) and after (for 3 additional hours) an euglycemic hyperinsulinemic (150 mU/l) clamp. Steady-state methionine and homocysteine kinetics were determined using either plasma (i.e., those of methionine) or intracellular (i.e., those of plasma homocysteine) enrichments. By use of plasma enrichments, insulin decreased methionine rate of appearance (Ra; both methyl- and carbon Ra) by 25% (P < 0.003 vs. basal) and methionine disposal into proteins by 50% (P < 0.0005), whereas it increased homocysteine clearance by approximately 70% (P < 0.025). With intracellular enrichments, insulin increased all kinetic rates, mainly because homocysteine enrichment decreased by approximately 40% (P < 0.001). In particular, transmethylation increased sixfold (P < 0.02), transsulfuration fourfold (P = 0.01), remethylation eightfold (P < 0.025), and clearance eightfold (P < 0.004). In summary, 1) physiological hyperinsulinemia stimulated homocysteine metabolic clearance irrespective of the model used; and 2) divergent changes in plasma methionine and homocysteine enrichments were observed after hyperinsulinemia, resulting in different changes in methionine and homocysteine kinetics. In conclusion, insulin increases homocysteine clearance in vivo and may thus prevent homocysteine accumulation in body fluids. Use of plasma homocysteine as a surrogate of intracellular methionine enrichment, after acute perturbations such as insulin infusion, needs to be critically reassessed.     

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.     

Radiolabelling of Mycobacterium avium oligosaccharide determinant and use in macrophage studies

Internal radiolabelling procedures were used to radiolabel the oligosaccharide determinant of the glycopeptidolipids (GPL) from serovars 4 and 20 of the Mycobacterium avium complex. Mycobacteria were cultured in the presence of [6-3H]fucose, [2-3H]mannose or [methyl-3H]methionine, after which radiolabelled native lipid was extracted and distribution of radioactivity in native and deacetylated lipid was determined by thin-layer chromatographic methods. Incorporation of radiolabel was confirmed by examining acid hydrolysates of purified GPL for 3H-labelled sugars on cellulose thin-layer plates. Least incorporation of radiolabel into GPL was observed with [6-3H]fucose, whereas better incorporation was obtained with [2-3H]mannose and [methyl-3H]methionine. Use of [methyl-3H]methionine resulted in the radiolabelling of the methylated sugars in both the oligosaccharide determinant and the 3,4-di-O-methylrhamnose located at the terminus of the peptide core. Use of [2-3H]mannose resulted in the incorporation of radioactivity into the oligosaccharide determinant as 2-O-methylfucose, found in the GPL of both serovars 4 and 20. GPL radiolabelled with [2-3H]mannose were subsequently examined in macrophage cultures and found to be relatively inert to degradation by those phagocytic cells. These results substantiate earlier findings with the GPL of serovar 20 and indicate that these mycobacterial components may play a role in pathogenesis.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

Studies on myelin-basic-protein methylation during mouse brain development.

The synthesis and methylation in vivo of myelin basic protein (MBP) during the mouse brain development has been investigated. When mice ranging in age from 13 to 60 days were injected intracerebrally with L-[methyl-3H]methionine, the incorporation of radioactivity into MBP isolated from youngest brain was found to be the highest and declined progressively in mature brains. This pattern of radioactivity incorporation was inversely correlated with the total amount of MBP in the brains, suggesting a higher ratio of MBP methylation to synthesis in younger brain. To differentiate the relative rate of protein synthesis and methylation, animals were given intracerebral injections of a L-[methyl-3H]methionine and L-[35S]methionine mixture and the ratio of 3H/35S (methylation index) was determined. The ratios in the isolated MBP fractions were higher than those of 'acid extracts' and 'breakthrough' fractions, with a maximal ratio in the youngest brain. This high ratio was well correlated with the higher protein methylase I (PMI) activity in younger brains. The MBP fractions were further separated on SDS/polyacrylamide-gel electrophoresis into several species with apparent Mr ranging from 32,400 to 14,500. The results indicated that each protein species accumulated at a characteristic rate as a function of age. The high-Mr (32,400) species was predominant in younger brain, whereas the smaller MBP was the major species in older brain tissue. The importance of this developmental pattern of MBP synthesis and methylation is discussed in relation to PMI activity.     

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.     

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.     

Cytoplasmic location of undermethylated messenger RNA in Novikoff cells.

Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Synthesis of nucleic RNA in hydroxythiamine-induced vitamin B1 deficiency

24 hours after administration of hydroxythiamine (vitamin B1 antimetabolite) in a dose of 40 mg per 100 g of body weight, the incorporation of (2(14)C)-orotic acid and (methyl-14C)-methionine into total nuclear RNA decreases. The content of RNA and the specific radioactivity of cytoplasmic UTP and methionine pools remain unchanged. Thermal phenol fractionation of nuclear RNA reveals a decrease in a predominant synthesis of nucleolar RNA, while that of heterogeneous nuclear RNA, tRNA, and 5S-RNA remains unaffected.     

DcrA, a c-type heme-containing methyl-accepting protein from Desulfovibrio vulgaris Hildenborough, senses the oxygen concentration or redox potential of the environment.

The amino acid sequence of DcrA from Desulfovibrio vulgaris Hildenborough, a strictly anaerobic, sulfate-reducing bacterium, indicated homology with the methyl-accepting chemotaxis proteins from enteric bacteria (A. Dolla, R. Fu, M. J. Brumlik, and G. Voordouw, J. Bacteriol. 174:1726-1733, 1992). The homology is restricted to the cytoplasmic C-terminal signaling domain. The periplasmic N-terminal sensor domain was found to contain a unique sequence, CHHCH, corresponding to a consensus c-type heme binding site. A pretreated, DcrA-specific polyclonal antiserum, generated against DcrA protein overproduced in Escherichia coli, was used for immunoprecipitation of 35S-labeled DcrA from D. vulgaris and Desulfovibrio desulfuricans G200(pJRFR2), a transconjugant that overexpresses functional DcrA. Labeling of the latter with the heme precursor 5-amino-[4-14C]levulinic acid, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography, confirmed the presence of c-type heme, while labeling with L-[methyl-3H]methionine in the absence of protein synthesis confirmed that DcrA is a methyl-accepting protein. The base liability of the incorporated radioactivity indicated methyl ester formation like that occurring in the methyl-accepting chemotaxis proteins of enteric bacteria. L-[methyl-3H]methionine labeling of D. desulfuricans G200(pJRFR2) under different conditions indicated that methyl labeling of DcrA decreased upon addition of oxygen and increased upon subsequent addition of the reducing agent dithionite. These results indicate that DcrA may serve as a sensor of oxygen concentration and/or redox potential.     

Incorporation of Intracisternally Administered l-[methyl-3H]Methionine and S-Adenosyl-l-[methyl-3H]-Methionine into Rat Brain Phospholipids

The incorporation of intracisternally injected L-[methyl-3H]methionine [( 3H]Met) or S-adenosyl-L-[methyl-3H]methionine (Ado[3H]Met) into rat brain AdoMet and phospholipid pools was examined. When [3H]Met was administered, both AdoMet and phospholipid pools were labeled. However, exogenously injected Ado[3H]Met did not serve as a substrate for phospholipid-N-methyltransferases. It was concluded that only Ado[3H]Met formed in situ was utilized to methylate phospholipids and that this process was initiated on the cytoplasmic side of the membrane. The apparent biological half-life in brainstem of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine formed from [3H]Met was 1.4 and 1.7 days, respectively. The half-life of phosphatidylcholine could not be determined due to interference from peripheral sources.     

Further investigation of methylation on sickle erythrocyte membranes

The methylation of erythrocyte membrane proteins has been investigated with fractionated reversible and irreversible sickle erythrocytes to better understand conflicting results obtained from two laboratories (Green and Kalra (6), Ro et al. (1). When subpopulations of intact erythrocytes obtained by two different separation methods (33% bovine serum albumin and Stractan II gradient centrifugations) were incubated with L-[methyl-3H] methionine at pH 7.2 and 37 degrees C, membranes from both reversible and irreversible sickle erythrocyte populations showed about half the [3H]methyl group incorporation than that observed in normal erythrocytes. In addition, this difference in the level of methylation between normal and sickle cells was maintained during the entire course of a 2-hr incubation utilizing S-adenosyl-L-[methyl-3H]methionine, the immediate in vivo methyl donor.     

Regulation of species-specific ribosomal RNA in a somatic hybrid cell.

When 13B hamster-mouse hybrid cells are harvested either right after 4 h of incubation with [me-3H]methionine or following 26 h of "chase" with excess non-radioactive methionine, in both cases about half of the labeled cytoplasmic rRNA is of hamster type. It had been previously shown in this laboratory (Eliceiri, G.L. (1973) Biochim. Biophys. Acta 312, 737-741) that when [3H]uridine was the radioactive precursor about 80% of the labeled cytoplasmic rRNA was of hamster type after a short incubation, and about half after a long incubation. It is postulated that a temporary difference in the specific acitivity of [3H]UTP in possibly segregated mouse and hamster types of nucleoli might account for these results. The master/mouse ratio of cytoplasmic rRNA in hybrid 13B is similar in free and in membrane-bound ribosomes, and in ribosomes of sparse (rapidly growing) cell populations and of confluent (slowly growing) cells.