STUDIES ON RAPIDLY LABELLED NUCLEAR RNA OF RAT BRAIN

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with [³H]uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by [methyl-³H] methionine. The other two fractions (Q2 RNA and TD RNA) are DNA-like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA-like RNA behave differently. Initially, the label in the DNA-like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA-like RNA during the first 30 min; however, unlike DNA-RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA-like RNA. It appears that the labelling of ribosomal RNA relative to DNA-like RNA is more rapid in liver than in brain.     

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

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

Methylation reactions and the phytoalexin response in alfalfa suspension cultures

 In order to determine why the activated methyl cycle is up-regulated in plants undergoing defence responses to fungal pathogens we have monitored the utilisation of methyl groups derived from methionine in cell-suspension cultures of alfalfa (Medicago sativa L.) treated for various times with fungal elicitor, by carrying out a parallel labelling study with [³⁵S]methionine and [methyl-³H]methionine. The distribution of the two radiolabels among the medium, soluble cellular components and cell wall was then determined. In the absence of elicitor the utilisation of the two radiolabels was similar. However, in the presence of the elicitor the total incorporation of radioactivity from [methyl-³H]methionine into metabolites was far greater than from [³⁵S]methionine, indicating that the methyl label had been utilised in methylation reactions. Elicitor treatment resulted in up to a sixfold increase in the use of ³H-methyl groups in the methylation of hydrophobic metabolites. In the period 0–24 h after elicitor treatment, increased methylation was directed largely into the synthesis of the isoflavonoid phytoalexin medicarpin and related metabolites. Newly synthesized phytoalexins were exported into the medium, while a significant proportion of the medicarpin accumulating in the cell in the early stages of elicitation was derived from the hydrolysis of its respective conjugate. Elicitor treatment also modified the incorporation of ³H-methyl groups into the cell wall. Between 0 and 24 h after elicitor treatment the methylation of pectin in the cell wall declined. After 24 h, pectin methylation recovered and was associated with an increase in the methylation of other wall-bound polysaccharide components. Since no other major metabolic sink for the increased methylation was determined we conclude that the increased activity of the activated methyl cycle during defence interactions in alfalfa is required to support phytoalexin synthesis and cell wall modifications. Received: 1 August 1996 / Accepted: 24 October 1996     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.     

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-³H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-³H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.     

Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G₁ phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-¹⁴C] methionine and [methyl-³H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G₁ phase. In both conditions it suppressed ³H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.     

Non-uniform distribution of methylatable CCGG sequences on human chromosomes as shown by in situ methylation

We carried out in situ methylation of human chromosomes with the HpaII methylase using [³H]methyl-S-adenosyl-l-methionine as the methyl group carrier. Autoradiographs localising [³H]methyl groups show methylatable CCGG sequences in the R-bands as well as in the short arms of the acrocentric chromosomes that include ribosomal DNA. The strongest labelling was observed over a subset of R-bands, including T-bands. Since methylatable CCGG sequences are representative of the unmethylated fraction of DNA, we suggest that differences in the degree of DNA methylation could be involved in the structure and function of chromosomal bands.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

A Simple Radioenzymatic Method to Measure Picogram Amounts of DOPA in Brain and Biological Fluids

A simple radioenzymatic method for the determination of DOPA is described. The method is based on the conversion of DOPA to 3-O-[methyl-³H]DOPA by catechol-O-methyltransferase in the presence of S-adenosyl-[methyl-³H]methionine and purification of the labelled product by Sephadex G10 and Dowex 50 W × 4 ion exchange resin. The method has been applied to the assay of endogenous DOPA in different brain areas and to measuring DOPA accumulation after inhibition of aromatic amino acid DOPA decarboxylase.     

Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Characteristics of Protein Carboxyl Methylation in the Rat Hypothalamus

Abstract: The formation of methyl-labeled S-adenosylmethionine (AdoMet) and methyl esters of endogenous methyl-acceptor proteins (MAPs) was studied in a synaptosomal preparation from the rat hypothalamus labeled with L-[methyl-³H]methionine. Incubation of synaptosomes with l -[methyl-³H]methi-onine resulted in a rapid labeling of the AdoMet pool and a less rapid formation of ³H-methyl-MAPs. Accumulation of ³H-methyl-MAPs was linear over a 30-min period. The effects of various inhibitors of AdoMet-dependent trans-methylation reactions on the formation of carboxylmethylated MAPs were examined. When hypothalamic synaptosomes were preincubated with l -[methyl-³H]methionine and subsequently incubated for 30 min in the presence of S-adenosyl-l -homocysteine (AdoHcy, 100 μm ), ³H-methyl-MAP formation was inhibited by approximately 70%. 100 μm -l -homocysteine thiolactone (HTL) as well as 100 μm -3-deazaadenosine (c³Ado) also caused a 60–70% inhibition of ³H-methyl-MAP formation; the combination of both c³Ado and HTL produced a slightly but not significantly greater inhibition than either agent alone. 10 μm -adenosine or 10 μm -HTL each produced an approximately 40% inhibition of ³H-methyl-MAP formation: the inhibitory effect of the two agents in combination was additive. Sinefungin and A9145C, potent inhibitors of bovine adrenomedullary protein carboxyl methylase, had no effect on ³H-methyl-MAP formation in hypothalamic synaptosomes at concentrations up to 1 mM. However, these compounds were potent inhibitors of ³H-methyl-MAP formation in lysed synaptosomes incubated with [³H-methyl]AdoMet. These results demonstrate that hypothalamic synaptosomes are capable of methio-nine activation and protein carboxyl methylation.     

A comparative PET study using different 11C-labelled amino acids in Walker 256 carcinosarcoma-bearing rats

In Walker 256 carcinosarcoma-bearing rats, the dynamic distribution of l-[1-^11C]tyrosine, l-[methyl-¹¹C]methionine, l-[1-¹¹C]methionine and d-[1-¹¹C]methionine has been measured by PET. An equivalent tumor-imaging potential was observed for each of the three l-amino acids. Thirty minutes after injection, the tumors accumulated 57% (P < 0.01) more ¹¹C-activity from l-[1-¹¹C]methionine than from l-[methyl-¹¹C]methionine. At the same point of time, the livers showed a 33% (P < 0.001) higher ¹¹C-uptake with l-[methyl-¹¹C]methionine than with l-[1-¹¹C]methionine. The dynamic tissue data are in agreement with the findings in experiments with ¹⁴C-analogs.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Vorstufen der ribosomalen RNA in frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary Six high molecular weight, rapidly labelled RNA species were detected in freely suspended callus cells of Petroselinum sativum by means of isotope labelling and electrophoretic separation in agarose-polyacrylamide gels. On the basis of their migration in the latter the RNA species were calculated to have the following molecular weights: 2.9×10⁶, 2,4×10⁶, 1.9×10⁶, 1.4×10⁶, 1.0×10⁶ and 0.75×10⁶ daltons. Thus they can clearly be distinguished from the two ribosomal RNA species (1.3×10⁶ and 0.7×10⁶ daltons). During incubation of the cells with [³H]methyl-methionine as a methyl donator all six components incorporated radioactivity rapidly. With [³H]nucleosides or [³H]orotic acid as precursors the 2.9×10⁶ and the 2.4×10⁶ daltons RNA were labelled within 10 min, while the other high molecular weight species appeared after about 20 min of labelling.Prolongation to 45–120 min resulted in accumulation of radioactivity preferentially in the 1.4×10⁶ and 0.75×10⁶ daltons RNA and in the ribosomal RNA species. The results of cell fractionation experiments provide evidence that these rapidly labelled high molecular weight RNA species are synthesized in the cell nucleus. The kinetics of their synthesis together with the other data obtained strongly support the suggestion that these RNA species function as precursors in the processing of ribosomal RNA. The possible mechanism of this process is discussed.

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Verwendete Abkürzungen EDTA Äthylendiamintetraessigsäure - DNase Desoxyribonuclease - Imp./min epm - MAK methyliertes Albumin an Kieselgur - POPOP 1,4- bis (4-Methyl-5-Phenyloxazol)-Benzol - PPO 2,5-Diphenyloxazol - RNase Ribonuclease - S Sedimentationskoeffizient in Svedberg-Einheiten - SDS Natriumdodecylsulfat - TPE Tris-Phosphat-EDTA-Puffer - Tris Tris-(hydroxymethyl)-aminomethan - Upm rpm