| Abstract: | By crossing two strains of Saccharomyces cerevisiae deficient for each of the two methionine adenosyltransferase isoenzymes (ATP: L-methionine S-adenosyltransferase EC 2.5.1.6) respectively, we have constructed a strain strictly auxotrophic for S-adenosylmethionine and used it as a source of undermethylated mRNA suitable for in vitro transmethylation studies. RNA has been phenol-extracted from yeast cells shifted down to S-adenosylmethionine-free medium for 90 min and poly(A)-rich RNA has been prepared by oligo(dT)-cellulose chromatography. Upon incubation in vitro in the presence of methyl-labeled S-adenosylmethionine and mRNA (guanine-7-)-methyltransferase purified from wheat germ or yeast, undermethylated poly(A)-rich RNA became significantly labeled as compared to non-starved cells from the same strain, or from a wild-type control. Cap structures were resolved by paper chromatography afer T2 and P1 RNase digestion, and shown to be a mixture of m7G5'ppp5'G and m7G5'ppp5'A, irrespective of the enzyme source, in agreement with earlier in vivo studies in yeast mRNA capping and methylation. |
