| Abstract: |
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) monoclonal antibody for selection, A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of G(M2) as well as G(M3) but no G(D3) Or G(D2) and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression 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 single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl moth similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines. |
