Secondary methylation of yeast ribosomal precursor RNA.

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.     

Partial base-methylation and other structural differences in the 17 S ribosomal RNA of sycamore cells during growth in cell culture.

Sycamore (Acer pseudoplatanus L.) cytoplasmic rRNA was investigated in rapidly dividing cells, cells starting mitosis after the lag phase of growth (4 days) induced by deconditioning of the culture medium and also in growth-arrested cells from 10 day-old cultures deprived of exogenous auxin (i.e. exponential, early exponential and 2,4-dichlorophenoxyacetic acid (2,4-D)-deprived cultures). rRNA was extracted and purified from mixed 14C-labelled exponential cultures and 3H-labelled early exponential cultures. A 14C-labelled exponential culture and a 3H-labelled 2,4-D-deprived culture were analyzed in the same way. The 17 S rRNA molecules from both early exponential and 2,4-D-deprived cultures displayed a lower electrophoretic mobility on polyacrylamide gels than those from exponential cultures. Alkaline and acid hydrolysates of purified 17 S rRNA labelled on the phosphate groups or the methyl groups were analyzed on ion-exchange resins. There was no change in the extent of ribose methylation of the molecule from the three different cultures. However, the base methylation of the 17 S rRNA was decreased in early exponential cultures and in 2,4-D-deprived cultures. Part of the molecules synthesized in early exponential cultures specifically lacked 7-methylguanine, N6-methyladenine and N6,N6-dimethyladenine. The possible significance of these changes in the 17 S rRNA were discussed.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.     

Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in vivo with methyl methanesulphonate and NN-dimethylnitrosamine

1. rRNA was isolated from rat liver at short intervals after the intraperitoneal injection of [(14)C]methyl methanesulphonate (50mg/kg) or NN-di[(14)C]methylnitrosamine (2mg/kg). These doses were chosen to minimize the effects of toxicity. 2. The following methods of hydrolysis of [(14)C]methylated rRNA were employed: enzymic digestion to nucleosides at pH8; alkaline hydrolysis and conversion into nucleosides; acid hydrolysis to bases. 3. The methylation products were analysed by chromatography on columns of Dowex-50 (H(+) form) and Dowex-50 (NH(4) (+) form). 4. With both methylating agents the principal product of methylation was 7-methylguanine. Differences were obtained, however, in the molar proportions of the minor bases 3-methylcytosine, 1-methyladenine and 7-methyladenine. Methylation at the O-6 position of guanine was a significant feature of rRNA obtained from the NN-di[(14)C]methylnitrosamine-treated animals but was not detected in rRNA after treatment with [(14)C]methyl methanesulphonate.     

Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat.

Two hypermodified, alkali-stable dinucleotide sequences, each containing a base modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M.W. (1974) Biochemistry 13, 5453-5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-psi p and psi m-Ap, where Cm=O2'-methylcytidine and psi m-O2'-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not the both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that psi m-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-psi p is localized in wheat cytosol 26S rRNA. The presence of psi m in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M.S.N. & Maden, B.E.H. (1976) J. Mol. Biol. 101, 235-254).     

Transfer RNA methyltransferase activity in paramecium aurelia.

The tRNA methyltransferases from Paramecium aurelia were investigated. The effects of varying the Mg2+ and NH4+ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum tRNA methyltransferase activity was observed at pH 7.8 and 37 degrees C. The Mg2+ optimum occurred at 0.66 mM in the absence of NH4+ while the NH4+ optimum occurred at 100 mM in the absence of Mg2+. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, N2-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of tRNA in P. aurelia is similar to that observed in other eukaryotes.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

5-Methylcytosine in Chlorelle pyrenoidosa DNAs.

The presence of 5-methylcytosine in Chlorella pyrenoidosa (strain 211/8b) DNA's has been investigated by means of paper chromatography and thermal chromatography on hydroxyapatite. It has been shown that nuclear DNA contains 3.5 mol% 5-methylcytosine whereas no significant amount of this base can be detected in chloroplast DNA. The thermal chromatography of nuclear DNA labelled from [6-3H]- or [Me-14C] methionine lead us to conclude that the 5-methylcytosine content is directly proportional to the G + C content of the various DNA fractions. The existence of methylated sequences in DNA is postulated and the biological function of the 5-methylcytosine is discussed.     

The effect of thioacetamide on the maturation of high-molecular-weight ribonucleic acid in tumour cells

1. Although thioacetamide treatment of Krebs II ascites-tumour cells did not markedly affect the rate of RNA synthesis in vivo, it caused the formation of an unusual single-stranded RNA component sedimenting at approx. 26s. 2. The maturation process leading to the formation of methylated RNA was examined by following the kinetics of incorporation into RNA of radioactivity from [G-(3)H]uridine and l-[Me-(14)C]methionine. In treated and untreated tumour cells extensive methylation was observed, not only of the ribosomal RNA species, but also of their precursors, especially the precursor species sedimenting at 35s. 3. Evidence is also presented to suggest that methylation of low-molecular-weight RNA species occurs both in the nucleus and in the cytoplasm of these tumour cells. 4. Thioacetamide did not appear to have an effect on RNA methylation in vivo, and in thioacetamide-treated cells the 26s RNA accumulated within the nucleus, where it was methylated. 5. It is postulated that the 26s RNA is most likely to arise as a result of a fault in the scission process that gives rise to the ribosomal RNA components from their high-molecular-weight precursors.     

Sites of methylation of DNA bases by the action of organophosphorus insecticides in vitro

Methylation in vitro of DNA by three methyl-14C-labelled organophosphorus insecticides has been studied. The ability of methylbromphenvinphos, methylparathion and malathion to methylate N-7 of guanine in DNA can be expressed as 100:40:15. Among the methylation products, no O6-methylguanine, a known mutagen, was found. Both in the reaction with dsDNA and with ssDNA 7-methyl-guanine was the main methylation product. However, all methyl derivatives of adenine (3-methyladenine, 1-methyladenine and 7-methyladenine) constituted about 40% and 50% of all methylation products in the case of dsDNA and ssDNA, respectively. The only methyl derivative of pyrimidine we have identified was 3-methylcytosine. In the case of dsDNA 3-methylcytosine appeared in small amounts but in the alkylated ssDNA 3-methylcytosine C constituted about 20% of all alkylation products.     

N4-methylcytosine as a minor base in bacterial DNA.

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.     

ALKYLATION OF RAT BRAIN NUCLEIC ACIDS BY N-METHYL-N-NITROSOUREA AND METHYL METHANESULPHONATE

Abstract— Alkylation of rat brain nucleic acids in vivo was measured after a single intravenous injection (1 mmol/kg body wt.) of N -[¹⁴C]methyl- N -nitrosourea and [¹⁴C]methyl methanesulphonate. The main product with both compounds was 7-methylguanine, The extents of methylation on this position in DNA and RNA were similar with methylnitrosourea but methyl methanesulphonate produced twice as much 7-methylguanine in DNA as in cytoplasmic RNA. Brain DNA from rats treated with labelled methylnitrosourea contained radioactive O ⁶-methylguanine, accounting for about 12 per cent of the radioactivity present as 7-methylguanine and cytoplasmic RNA contained about half this amount of O ⁶-methylguanine. Neither DNA nor cytoplasmic RNA from methyl methanesulphonatetreated rats contained any detectable O ⁶-methylguanine. Treatment with both compounds resulted in varying small amounts of methylation of other nucleic acid bases including 1-methyladenine, 3-methyladenine and 3-methylcytosine. The possible relevance of alkylation of brain nucleic acids to the induction of brain tumours is discussed.     

The influence of pH on arsenazo III.

None of the methods already reported for elimination of pectins from rRNA extracts allowed the complete removal of methylated polysaccharides from methyl-labeled cytoplasmic 17 and 26 S rRNA preparations of sycamore (Acer pseudoplatanus L.) cells. An improved procedure for purifying large amounts of higher plant cytoplasmic rRNA labeled on the methyl groups was investigated. Bulk cellular RNA from sycamore cells incubated for 24 to 36 h with methyl-labeled methionine was extracted at 4°C by the phenol-extraction procedure. Most of the pectic compounds (that accounted for about 30% of the total label of RNA extracts) was selectively precipitated, before the 66% ethanol precipitation of nucleic acid, by bringing the deproteinized aqueous layer to 10% ethanol ?0.15 m sodium acetate. Cytoplasmic rRNA, 17 and 26 S, were isolated by repeated sucrose gradient sedimentations and further chromatographed on a methylated albumin kieselgurh (MAK) column. The old-fashioned MAK chromatography proved to be very useful for elimination of residual pectins, since these compounds eluted in the void volume of the column. This purification procedure gave in a reproducible way cytoplasmic 17 and 26 rRNA virtually free of any labeled DNA, mRNA, plastid rRNA, and pectic compounds.     

Comparative study of DNA methylation in three unicellular eucaryotes.

We have analyzed the nature/content of methylated bases in the nuclear DNA of three unicellular eucaryotes. The pattern of methylation was different for each of the three organisms studied: Saccharomyces cerevisiae contained only 5-methylcytosine; Tetrahymena pyriformis contained only N6-methyladenine; and Chlamydomonas reinhardi contained both modified bases.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).