ISOLATION OF G(D3) SYNTHASE GENE BY EXPRESSION CLONING OF G(M3) ALPHA-2,8-SIALYLTRANSFERASE CDNA USING ANTI-G(D2) MONOCLONAL-ANTIBODY

For the isolation of ganglioside G(D3) synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-G(D2) mono clonal antibody for selection, A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high le vels of G(M2) as well as G(M3) but no G(D3) Or G(D2) and was construct ed from mouse B16 melanoma cells transfected with the polyoma large tu mor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylg alactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfecti on, monoclonal antibody 3F8 panning, and Hirt extraction resulted in t he isolation of two cDNA clones, transfection of which directed the ex pression of G(D3) in KF3027 and B16 melanoma cells and G(D3) and G(D2) in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a singl e open reading frame. The deduced amino acid predicted a type II membr ane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the prese nce of a sialyl moth similar to that found in the other sialyltransfer ases cloned so far. As expected, mRNA of this gene (2.6 kb) was strong ly expressed in human melanoma lines.