The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

Shooter, K. V.; Howse, Ruth; Merrifield, R.

doi:10.1042/bj1370313

Cited by 42 publications

(4 citation statements)

References 13 publications

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“…It has long been presumed that ribosephosphotriesters are too labile to be isolated from alkyl RNA (reviewed by Singer, 1975), and direct proof of their formation, based on their isolation, has been lacking (Shooter et al, 1974a). The experimental data in this section identifies the major product of ethylnitrosourea treated TMV-RNA as ethyl phosphotriesters which are relatively stable, releasing ethanol to varying extents only upon hydrolysis (Table V).…”

Section: Resultsmentioning

confidence: 99%

Specificity of different classes of ethylating agents toward various sites in RNA

TSinger¹,

Fraenkel‐Conrat²

1975

Biochemistry

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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.

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Section: Resultsmentioning

confidence: 99%

Specificity of different classes of ethylating agents toward various sites in RNA

TSinger¹,

Fraenkel‐Conrat²

1975

Biochemistry

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“…Our previous study (Miller et al, 1974) showed that complementary ribooligonucleotides form complexes with tRNA with binding constants 8 to 20 times higher than corresponding oligodeoxyribonucleotides. Since phosphotriester derivatives of ribooligonucleotides are readily hydrolyzed in aqueous solution (Shooter et al, 1974;Singer and Fraenkel-Conrat, 1975), the 2'-0-methylribooligonucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, was prepared. In this analogue, the methylated 2'-hydroxyl groups are prevented from participating in cleavage of the phosphotriester moiety, while the basic conformational features of the ribonucleotide-type backbone are maintained (Alderfer et al, 1974).…”

Section: Discussionmentioning

confidence: 99%

Effects of a trinucleotide ethyl phosphotriester, G^mp(Et)G^mp(Et)U, on mammalian cells in culture

Miller¹,

Braiterman²,

Ts’o³

1977

Biochemistry

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The nonionic 2'-O-methyribooligonucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, is complementary to the...ApCpC...sequence found in the amino acid accepting stem of most tRNAs and the anticodon region of tRNAgly and to the threonine codon of mRNA. Gmp(Et)Gmp(EtU forms hydrogen-bonded complexes with the amino acid accepting stem of tRNApheyeast and unfractionated tRNA Escherichia coli under physiological salt conditions at 37 degrees C as determined by equilibrium dialysis. The extent of phenylalanine aminoacylation of tRNApheE.coli is inhibited 39% by Gmp(Et)Gmp(Et)U at 37 degrees C in solution. The triester is resistant to hydrolysis by serum nucleases and cell lysates. The triester is readily taken up by transformed Syrian hamster fibroblasts growing in monolayer. Within the cell, the triester is deethylated to give the trinucleotide species Gmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU and is also hydrolyzed to dimeric and monomeric units. Treatment of transformed fibroblasts in monolayer with 25 micronM Gmp(Et)Gmp(Et)U results in a 40% inhibition of cellular protein synthesis with a concurrent slight increase in cellular RNA synthesis during the first 4 h. After 4 h, the rate of cellular protein synthesis begins to recover while RNA synthesis returns to that of the control. Our biochemical studies suggest that inhibition of cellular protein synthesis might be expected if Gmp(Et)Gmp(Et)UGmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU, which have been taken up by or formed within the cell, physically bind to tRNA and mRNA and inhibit the function of these nucleic acids. The reversible inhibition of protein synthesis may be a consequence of further degradation of the trinucleotide species within the cell as well as to an increase in supply of RNA molecules involved in protein synthesis. The growth of the transformed fibroblasts is inhibited during the first 24 h of incubation with 25 micronM Gmp(Et)Gmp(Et)U after which growth proceeds at a normal rate. In cloning experiments, the number and size of colonies formed by the transformed fibroblasts after 5 days exposure to 25 micronM triester is decreased by 50% relative to untreated controls. The temporary inhibition of cell growth may reflect the transitory inhibition of cellular protein synthesis caused by the triester.

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“…Trimethyl Phosphate: The nucleotide (20 mg -100 mg) was dissolved in a mixture of TBAF (10 eq) in THF and trimethyl phosphate (0.25 -2 ml) and the solution was stirred at room temperature for up to 24 h. Solvents were removed at reduced pressure and the residue was dissolved in chloroform which was subsequently washed with water (4X). The chloroform was evaporated and the residue was treated with Dowex 50WX8 (Na+ form) resin in Table 3 Nucleoside Equivalents Time Products (%) TBAF uridine 10 18 h N3-MeU (100%) adenosine 10 18 h N6-MeA (12), N6-DMeA (12) adenosine 20 48 h N6-MeA (18), N6-DMeA (55) cytidine 10 68 h N3-MeC (58), N3,N4-DMeC (7) cytidine 20 68 h N3-MeC (15), N3,N4-DMeC (84) guanosine '10 18 h N2-MeG (70) + 3 others unidentified ethanol :water (1:1) for 15 h. The solvent was removed by filtration and evaporated. The residue was treated with 80% HOAc for 30 min at 85°C (to remove trityl groups when present) and the residue was applied directly to Whatman 3MM preparative paper sheets developed in solvent A or B'.…”

Section: Xphl3mentioning

confidence: 99%

Fluoride ion catalyzed alkylation of nucleic acid derivatives using trialkyl phosphates, dialkyl sulfates and alkyl methanesulfonates

Ogilvie¹,

Beaucage²,

Gillen³

et al. 1979

Nucl Acids Res

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Trimethyl phosphate, dimethyl and diethyl sulfate and methyl and ethyl methanesulfonate all give high yields of alkylation on purines and pyrimidines in the presence of tetrabutylammonium fluoride. Trimethyl phosphate produces near quantitative yields of diesters of nucleic acids but gives virtually no triester formation. The alkyl sulfates produce very high yields of triesters of nucleic acids including cyclic phosphates while the alkyl methanesulfonates are intermediate in reactivity. It was observed that in the absence of fluoride ion the dialkylsulfates gave reasonable yields of thymidine monosulfates.

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The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

Cited by 42 publications

References 13 publications

Specificity of different classes of ethylating agents toward various sites in RNA

Specificity of different classes of ethylating agents toward various sites in RNA

Effects of a trinucleotide ethyl phosphotriester, G^mp(Et)G^mp(Et)U, on mammalian cells in culture

Fluoride ion catalyzed alkylation of nucleic acid derivatives using trialkyl phosphates, dialkyl sulfates and alkyl methanesulfonates

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The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

Cited by 42 publications

References 13 publications

Specificity of different classes of ethylating agents toward various sites in RNA

Specificity of different classes of ethylating agents toward various sites in RNA

Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture

Fluoride ion catalyzed alkylation of nucleic acid derivatives using trialkyl phosphates, dialkyl sulfates and alkyl methanesulfonates

Contact Info

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Resources

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Effects of a trinucleotide ethyl phosphotriester, G^mp(Et)G^mp(Et)U, on mammalian cells in culture