Reaction of nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) with tobacco mosaic virus and its RNA.

"Nitrosoguanidine" (N-methyl-N'-nitro-N-nitrosoguanidine) acts on polynucleotides in neutral aqueous solution, methylating guanine in the 7-position, and, to a much lesser extent, adenine. In 67 percent dimethylformamide the effect on the purines decreased, and what seems to be 3-methyl-cytosine appeared. In formamide solution no base changes were detected. Nitrosoguanidine had little mutagenic effect on tobacco mosaic virus RNA in water; it had greater effect in dimethylformamide, and even greater in formamide; it was a strong mutagen only when it acted on intact tobacco mosaic virus particles. Thus, neither the methylation of guanine nor the cytosine modification represents the main mutagenic event.

Further studies of the mixed acetals of nucleosides.

We reported in 1988 on a new nucleoside modification reaction: the exocyclic amino groups of (d)adenosine and (d)cytidine react rapidly at ambient temperature with acetaldehyde and alcohols to give stable mixed acetals (N-ethylethoxy-acetal). NH2 + O = CH(CH3) + ROH-->NH-CH(CH3)-O-R + H2O. Here we report in detail on the occurrence of this reaction in very dilute aqueous solution (ie under biological conditions), on its mechanism and kinetics, on the mixed acetal formation with other aldehydes and other nucleic acid components, and on the question of whether these adducts are mutagenic.

RNA-dependent RNA polymerases of plants.

The existence of RNA-dependent RNA polymerases (EC 2.7.7.48) in plants has been definitely proven by their isolation in pure form from cucumber and tobacco in our laboratory and from cowpea at Wageningen. These enzymes are single-chain proteins of 100-130 kilodaltons. They show clear physical and biochemical differences characteristic for a given plant species, even when their amounts in the plants were greatly increased prior to isolation by infection with the same virus. The role of these enzymes in plant physiology remains unknown.

Nucleoside adducts are formed by cooperative reaction of acetaldehyde and alcohols: possible mechanism for the role of ethanol in carcinogenesis.

The exocyclic amino groups of ribonucleosides and deoxyribonucleosides react rapidly at ambient temperature with acetaldehyde and alcohols to yield mixed acetals [--NH--CH(CH3)OR]. Nucleotides and nucleoside di- and triphosphates also react. Depending on the nucleoside used and on the relative amounts of aldehyde, alcohol, and water, preparative reactions reach equilibrium with yields up to 75% in a few hours. The structures have been confirmed by fast atom bombardment MS and proton NMR. Half-lives at 37 degrees C have been determined, and maximum stability is in the pH range of 7.5-9.5. In the absence of alcohol, acetaldehyde-nucleoside adducts could be isolated at 4 degrees C, but these were too unstable to characterize except for their UV spectra, also at 4 degrees C. Ethanol is often present in human blood and tissues, and acetaldehyde is its initial metabolic product, as well as being formed by many other metabolic processes. Both chemicals have separately been implicated in carcinogenic and other cytopathologic processes, but no cooperative mechanism has been proposed. The reactions reported here are of biological concern because they also occur in dilute aqueous solution. These findings supply a mechanism by which ethanol can be covalently bound to nucleic acids under physiological conditions.

Biological consequences of incorporation of 5-fluorocytidine in the RNA of 5-fluorouracil-treated eukaryotic cells.

Treatment of HeLa cells with 5-fluoro-[3H]uracil leads to the incorporation into cellular RNA of 5-fluorocytidine to the extent of about 0.2% of the 5-fluorouridine incorporated. In tobacco mosaic virus RNA produced in tobacco leaves this ratio is one order of magnitude lower. Copolymers of cytidylic with 5-fluorocytidylic acids show unchanged template activity with E. coli RNA polymerase, but slightly altered messenger activity in the wheat germ system, compared to poly(C), and it is suggested that some of the biological consequences of 5-fluorouracil treatment of living cells and organisms may be attributed to this mechanism.

Identification of the 5' end of Rous sarcoma virus RNA.

The 5'-terminal triphosphate of the 35S RNA isolated from Rous sarcoma virus is blocked by 7-methylguanosine in 5' linkage with the penultimate nucleoside which is methylated in the 2'-O-ribose position, a type of endgroup found in all animal mRNAs investigated during the past year. The specific nuclease-resistant terminal fragment of RSV RNA has the structure 7mG5'ppp5'GmpCp-. This finding supports the belief that RNA of Rous sarcoma virus represents a (+) strand messenger which may be directly translated upon infection.

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.

Tryptophan analogues form adducts by cooperative reaction with aldehydes and alcohols or with aldehydes alone: possible role in ethanol toxicity.

Previous work showed that the exocyclic amino groups of nucleic acid components react quickly at ambient temperature with acetaldehyde and ethanol to yield mixed acetals [R-NH-CH(CH3)-O-C2H5]. We now find that the same type of reaction occurs readily with the nitrogen of 3-substituted indoles (e.g., indole-3-acetic acid and N-acetyltryptophan), analogues of the amino acid tryptophan. In contrast, unsubstituted indole reacts very rapidly at the carbon in ring position 2 or 3 with acetaldehyde to form bis(indolyl)ethane without ethanol entering into the reaction. Product structures have been confirmed by fast atom bombardment MS and 1H NMR. The former reaction occurs optimally in 30-50% aqueous solution below pH 4. It also proceeds more slowly and with reduced yields in aqueous media at more neutral pH. This reaction may be of biological concern, as it supplies a mechanism for protein modifications with possible toxic effects in human tissues where ethanol is metabolized.

Lack of specificity of virus-stimulated plant RNA-dependent RNA polymerases.

RNA-Dependent RNA polymerase preparations from tobacco and cowpea plants, either uninfected, or infected with tobacco necrosis or cowpea mosaic virus, were tested for their capability to bind radioactive viral or other RNAs or polynucleotides. The limited binding of up to 30% of all RNAs tested was found to be completely nonspecific in regard to any infecting virus, and it was nonspecifically diminished by excess of other unlabeled RNAs. These data are in line with our previous conclusion that plant viral RNAs are replicated by host enzymes. The data also speak against the hypothesis that these enzymes might be modified as a consequence of viral infection.

Effect of introduction of small alkyl groups on mRNA function.

Treatment of RNA with dimethyl sulfate methylates only nitrogens, preferentially the 7 position of guanosine, whereas treatment with ethylnitrosourea ethylates mainly oxygens, preferentially the phosphodiester groups. Two plant viral mRNAs were modified with these two reagents at levels of 4-28 alkylations per molecule. The ability of alkylated RNAs to stimulate amino acid incorporation in the wheat germ system was somewhat diminished by both types of modification, but the predominant protein made, as ascertained by polyacrylamide gel electrophoresis, was the typical gene product of the respective mRNA. These data suggest that random alkylations, mostly of either the guanosine N-7s or the phosphates, do not interfere with peptide chain elongation, but that the ability to initiate translation properly is affected by these substitutions, presumably through their effect on the conformation of the RNAs.