MOLECULAR-CLONING OF A HUMAN CDNA-ENCODING BETA-1,4-GALACTOSYLTRANSFERASE WITH 37-PERCENT IDENTITY TO MAMMALIAN UDP-GAL-GLCNAC BETA-1,4-GALACTOSYLTRANSFERASE

A cDNA encoding a beta-1,4-galactosyltransferase named beta-1,4-GalT I I was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3'-rapid amplification of cDNA ends by using two nested degene rate oligonucleotide primers based on a highly conserved amino acid se quence found in the catalytic domain of mammalian beta-1,4-galactosylt ransferases and Lymnaea stagnalis beta-1,4-N-acetylglucosaminyltransfe rase (beta-1,4-Glc-NAcT), both of which utilize the same sugar accepto r, This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human beta-1,4-galactosyltransferase (referre d to as beta-1,4-GalT I) and of 28% with that of L, stagnalis beta-1,4 -GlcNAcT. Study of the properties of the beta-1,4-GalT II fused to pro tein A expressed as a soluble form in COS-7 cells revealed that it is a genuine beta-1,4-GalT but has no lactose synthetase activity in the presence of alpha-lactalbumin. Northern blot analysis of 24 human tiss ues shored that they all express the beta-1,4-GalT II transcript, alth ough the levels varied, These results indicate that human cells contai n another beta-1,4-GalT.