Identification of a new 5'-noncoding exon region and promoter activity in human N-acetylglucosaminyltransferase III gene

Abstract

In a previous paper (Kim et al., 1996a), the immediate 5′-flanking region and coding region of the human UDP-N-acetylglucosamine:-D-mannoside-1,4-N-acetylglucosaminyltransferase III (N-acetylglucosaminyltransferase-III; GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5′-noncoding region of the GnT-III mRNA by single-strand ligation to single-stranded cDNA-PCR (5′-RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells. A cDNA clone was obtained with 5′ sequences (96 bp) that diverged seven nucleotides upstream from the ATG (+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5′ to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5′-noncoding region and that the cDNA was an incompletely reverse-transcribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT-III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5′-flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5′-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.