QUANTITATIVE ANALYSIS OF NUCLEIC ACID ALKYLATION IN VITRO AND IN CELL CULTURE
Abstract
Chemical modification of polycytidylic acid with methyl methane-sulfonate has been studied by ('13)C NMR of the intact polymer, ('1)H NMR of the polymer, and ('1)H NMR, high-pressure liquid chromatography, and secondary-ion mass spectrometry (SIMS) of the nucleosides following enzymatic degradation. The only site of modification is at the N-3 position of cytidine. The extent of modification is quantitatively determined by ('14)C incorporation, integration analysis of NMR signals, HPLC peaks, and SIMS, using {('2)H(,3)-methyl} 3-methylcytidine as an internal reference. All analysis give consistant quantitative results. The development of a high pressure liquid chromatographic system capable of separating fifteen modified (methylated) and unmodified nucleosides allowed the mass spectrometric quantitation of in vitro DNA methylation products. Using synthesized {('2)H(,3)-methyl} labeled methylated nucleosides as internal references, the comparison of the relative amounts of products formed when calf thymus DNA is reacted with the weak carcinogen, methyl methanesulfonate and the potent carcinogen, 1-methyl-1-nitrosourea is achieved. By this method only one product, 7-methyl-2'-deoxyguanosine, is observed when calf thymus DNA is reacted with methyl methanesulfonate, whereas when this DNA is reacted with 1-methyl-1-nitrosourea, six products (3-methyl-2'-deoxycytidine, 1-methyl-2'-deoxyadenosine, 7-methyl-2'-deoxyguanosine, 3-methylthymidine, O('4)-methylthymidine, O('6)-methyl-2'-deoxyguanosine) are detected and the relative distributions of these products quantitated. Extension of this methodology to in vivo (cell culture) systems requires the recovery of highly purified DNA from the cells for accurate total methylation determinations by ('14)C incorporation which is achieved here by enzymatic and hydroxyapatite purifications. Upon aklylation of V79 Chinese hamster cells with methyl methanesulfonate, again only one methyl product, 7-methyl-2'-deoxyguanosine is detected. This product represents 89.6% of the total methylation as determined by ('14)C incorporation. With 1-methyl-1-nitrosourea as the alkylating agent, 7-methyl-2'-deoxyguanosine is observed as the major product but two additional methylated nucleosides, O('4)-methylthymidine and O('6)-methyl-2'-deoxyguanosine, present in < 10 nmole amounts, are detected as 4.5% and 6.3% respectively of the total methyl incorporation.
Degree
Ph.D.
Subject Area
Analytical chemistry
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