Methylation and Breakdown of Microsomal and Soluble Ribonucleic Acid from Rat Liver by Diazomethane

179

1. The following methods for hydrolysis of methyl-(14)C-labelled RNA, and for chromatographic isolation and determination of the products, were investigated: enzymic digestion to nucleosides at pH6 or 8; alkaline hydrolysis and conversion into nucleosides; hydrolysis by acid to pyrimidine nucleotides and purine bases, or completely to bases; chromatography on Dowex 50 (NH(4) (+) form) at pH6 or 8.9, or on Dowex 50 (H(+) form), or on Sephadex G-10. 2. The suitability of the various methods for determination of methylation products was assessed. The principal product, 7-methylguanosine, was unstable under the conditions used for determinations of nucleosides. 3- and 7-Methyladenine and 3- and 7-methylguanine are best determined as bases; 1-methyladenine and 3-methylcytosine can be isolated as either nucleosides or bases; O(6)-methylguanine is unstable under the acid hydrolysis conditions used and can be determined as the nucleoside; 3-methyluracil was detected, but may be derived from methylation of the ionized form of uracil. 3. Differences between the patterns of methylation of RNA and homopolyribonucleotides by the N-methyl-N-nitroso compounds and dimethyl sulphate were found: the nitroso compounds were able to methylate O-6 of guanine, were relatively more reactive at N-7 of adenine and probably at N-3 of guanine, but less reactive at N-1 of adenine, N-3 of cytosine and probably at N-3 of uridine. They probably reacted more with the ribose-phosphate chain, but no products from this were identified. 4. The possible influences of these differences on biological action of the methylating agents is discussed. Nitroso compounds may differ principally in their ability to induce miscoding in the Watson-Crick sense by reaction at O-6 of guanine. Both types of agent may induce miscoding to a lesser extent through methylation at N-3 of guanine; both can methylate N atoms, presumably preventing Watson-Crick hydrogen-bonding. N-Methyl-N-nitrosourea can degrade RNA, possibly through phosphotriester formation, but this mechanism is not proven.

Section: Assessment Of Chromatographic Proceduressupporting

confidence: 91%

Section: Assessment Of Chromatographic Proceduresmentioning

confidence: 99%

Methylation of ribonucleic acid by the carcinogens dimethyl sulphate, N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine. Comparisons of chemical analyses at the nucleoside and base levels

Lawley

Shah

1972

179

“…The latter process would be expected to cause fission of polyribonucleotides at neutral pH (Brown, Magrath & Todd, 1955); although in DNA, which lacks the 2'-hydroxyl group, such instability of phosphotriester groups would not necessarily result (Brown & Todd, 1955). Ludlum (1967Ludlum ( , 1969 reported that di-(2chloroethyl)methylamine and ethyl methanesulphonate did degrade polyribonucleotides at neutral pH; Kriek & Emmelot (1963) and Holy & Scheit (1966) found degradation of RNA or oligonucleotides respectively after extensive alkylation in vitro by diazomethane. The action of dimethylnitrosamine in vivo on polyribosomes of rat liver was interpreted by Mizrahi & DeVries (1965) as showing degradation of mRNA; whereas from analogous experiments Villa-Trevino (1967) concluded that the inhibition of protein synthesis could be caused by methylation of mRNA, but not necessarily by its degradation.…”

mentioning

confidence: 99%

The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage μ2

Shooter

Edwards

Lawley

1971

Bacteriophage mu2 is inactivated by both mono- and di-functional sulphur mustards at relatively low extents of alkylation. No degradation of alkylated RNA was detected. Cross-linking of RNA to protein was observed with the difunctional agent, but this reaction was only a minor contribution to the inactivation. Analyses of the reaction products in bacteriophage RNA showed that, at the mean lethal doses, more than one mono-alkylation of guanine had occurred but the sum total of other types of RNA alkylation was close to a single event. The results therefore suggest that inactivation results from the mono-alkylation of adenine or cytosine. In experiments with the difunctional agent cross-linking of RNA bases or of RNA to protein also prevented replication, the existence of these reactions accounting for the greater sensitivity of the bacteriophage to this agent.

“…been discussed by various authors (Kriek & Emmelot, 1963;Magee & Barnes, 1967;Villa-Trevino, 1967;Vernie et al, 1971 Distance from rotor centre (cm) Fig. 5.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of protein synthesis by N-methyl-N-nitrosourea in vivo

Kleihues

Magee

1973

1. The intraperitoneal injection of N-methyl-N-nitrosourea (100mg/kg) caused a partial inhibition of protein synthesis in several organs of the rat, the maximum effect occurring after 2-3h. 2. In the liver the inhibition of protein synthesis was paralleled by a marked disaggregation of polyribosomes and an increase in ribosome monomers and ribosomal subunits. No significant breakdown of polyribosomes was found in adult rat brains although N-methyl-N-nitrosourea inhibited cerebral and hepatic protein synthesis to a similar extent. In weanling rats N-methyl-N-nitrosourea caused a shift in the cerebral polyribosome profile similar to but less marked than that in rat liver. 3. Reaction of polyribosomal RNA with N-[(14)C]methyl-N-nitrosourea in vitro did not lead to a disaggregation of polyribosomes although the amounts of 7-methylguanine produced were up to twenty times higher than those found after administration of sublethal doses in vivo. 4. It was concluded that changes in the polyribosome profile induced by N-methyl-N-nitrosourea may reflect the mechanism of inhibition of protein synthesis rather than being a direct consequence of the methylation of polyribosomal mRNA.