Analysis of the sequences of human β-1,4-galactosyltransferase cDNA clones

Abstract

UDP-galactose:β-1,4 N-acetyl glucosamine galactosyltransferase (4βGT) is a promising tumor marker for ovarian cancer. To study the role of 4βGT in malignant transformation at the molecular level human 4βGT cDNA and genomic clones were isolated and analyzed. For the isolation of 4βGT cDNA and genomic clones, a human fetal liver cDNA library in λgt11 and a human genomic library in EMBL-3B vectors respectively were screened using a 4βGT cDNA insert as the probe. Complete sequence of the cDNA clones were determined by subcloning in plasmid vectors, and compared with the published sequence of human liver 4βGT. Presence of various 4βGT exons in the genomic clones were determined by Southern blot analysis using specific oligodeoxynucleotide probes. Among the 5 cDNA clones isolated, 2 clones GTN 6 and GTN 17 were sibling clones and had a nucleotide sequence identical to the published 4βGT cDNA sequence, except at the 3′-end, where these clones had 7 unique nucleotide sequences. One cDNA clone, GTN2 also had a nucleotide sequence identical to that of 4βGT, except for 3 G residues at the 5′-end. One cDNA clone, GTN 1, had a unique sequence at the 5′-end comprising of 74 nucleotides. Another clone, GTN 20, was unrelated to 4βGT. Analysis of genomic clones showed that 4βGT exons 3, 4, 5 and 6 were present in a 14 kb genomic clone, EMGT-4. Exon 1 was present in a separate 16 kb clone, EMGT-6. No clone was detected for exon 2. Southern blot analysis and partial restriction mapping of EMGT-6 showed that the 74 nucleotide unique sequence of GTN1 was present in this genomic clone. But this DNA fragment has separated from exon 1 by a distance of about 10 kb. This study showed that human 4βGT cDNA had divergent sequences, both at the 5′- and 3′-ends. Among them, a 74 nucleotide unique sequence at the 5′-end of GTN1 is present along with exon 1 in a genomic clone. It is proposed that the divergent cDNA clones represent various isoenzymes of 4βGT found in normal and malignant cells.