Isolation and cell-free translation of messenger ribonucleic acids specifying thymus-leukemia antigens

Messenger ribonucleic acid (mRNA) for thymus-leukemia antigens, membrane-associated glycoproteins of murine leukemia cells, was obtained from polysomes of murine leukemia cells forming thymus-leukemia antigens. Polysomes forming thymus-leukemia antigens were recovered by immunoprecipitation using alloantibodies specific for the 1,2,3 determinants of the thymus-leukemia antigen complex before they were extracted with phenol-detergent. Poly(adenylic acid)-containing RNA [poly(A)-RNA] was fractionated by oligo[deoxythymidylate] [oligo(dT)]-cellulose chromatography. The mRNA obtained had a sedimentation coefficient of approximately 17 S in agreement with the predicted size necessary for forming proteins specifying thymus-leukemia antigens. In a wheat germ system, the polypeptides formed upon addition of mRNA for thymus-leukemia antigens consisted of a major product with a molecular weight of 42,000. It was larger than the nonglycosylated heavy chain molecule of 40,000 daltons formed by the cells themselves. On the cell surface thymus-leukemia antigens exist as glycosylated molecules of 47,000 daltons associated with a light-chain equivalent to beta 2-microglobulin. Molecules of 40,000 daltons were isolated from the cells cultured in the presence of tunicamycin, an inhibitor of de novo glycosylation, and by treating thymus-leukemia heavy chains with endo-beta-N-acetylglucosaminidase H.