Cloning, characterization, and expression of human ceramide galactosyltransferase cDNA

Galactosylceramide (galactocerebroside, GalC) and its sulfated derivative, sulfatide, are major lipid components of the central and peripheral nervous system myelin sheath. The enzyme UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) catalyzes the final step of galactosylceramide synthesis. In this report we describe isolation of the complete copy of human CGT cDNA. Total RNA from N-370 FG cells, a human fetal glioma cell line, was reverse-transcribed and dG-tailed. Degenerate primers synthesized based on rat CGT cDNA sequence were used in 5'- and 3'- rapid amplification of cDNA ends reaction (RACE). The obtained sequence was used to synthesize the primers for the complete coding region to be amplified and cloned into a pCR 3.1 expression vector. Following transfection of the CHOP cells with the resulting vector, the cell homogenate was assayed for the galactosyltransferase activity. Northern blot hybridization was used to determine the length of CGT mRNA and Southern blot hybridization was used to determine the number of homologous genes, Our results indicate that human CGT retains all conservative features of rat and mouse CGT. It is a single copy gene with mRNA transcript of about 4 kb. (C) 1997 Academic Press.