Using an anti-GD1b
monoclonal antibody, expression cloning of a
cDNA for the beta1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C, mouse
melanoma B16 transfected with polyoma
T antigen gene, and GM2/
GD2 synthase cDNA was used as a recipient cell line for the cDNA library transfection. A
cDNA clone of GD3 synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain
RNA using the pcDNAI expression vector. The isolated
cDNA clone pM1T-9 predicted a type II
membrane protein with 4
amino acids of cytoplasmic domain, 21
amino acids of transmembrane region, and a large catalytic domain with 346
amino acids. Introduction of the
cDNA clone into a mouse
melanoma line B16 previously transfected with a GM2/
GD2 synthase gene resulted in the neo-synthesis of GM1. Co-transfection of the cell line with pM1T-9 and a GD3 synthase
cDNA resulted in the expression of GD1b as well as GM1. Moreover, introduction of pM1T-9 into L cell (lacking
GM3 synthase), previously transfected with GM2/
GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that GD1b/GM1/GA1 synthases were identical, as previously suggested based on enzymological analysis. In Northern blots of the beta1, 3-galactosyltransferase gene with total
RNA from various rat tissues, a 1.6-kilobase
mRNA was strongly expressed in spleen, thymus, kidney, and testis. However, the expression level of the gene in the adult brain tissue was not especially high. On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.