Organization of the human beta-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis.
UDP-GlcNAc: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-T I) is a medial-Golgi enzyme which catalyses the first step in the conversion of oligomannose-type to N-acetyl-lactosamine- and hybrid-type N-glycans and is essential for normal embryogenesis in the mouse. Previous work indicated the presence of at least two exons in the human GlcNAc-T I gene MGAT1, exon 2 containing part of the 5' untranslated region and the complete coding and 3' untranslated regions, and exon 1 with the remainder of the 5' untranslated region. We now report the cloning and sequencing of a human genomic DNA fragment containing exon 1, which is between 5.6 and 15 kb upstream of exon 2. Transient transfection, ribonuclease protection and reverse transcriptase-mediated PCR indicated the absence of transcription start sites in intron 1 between exons 1 and 2. Northern analysis, ribonuclease protection, primer extension analysis and rapid amplification of 5'-cDNA ends showed that there are multiple transcription start sites for exon 1 compatible with the expression by several human cell lines and tissues of two transcripts, a broad band ranging in size from 2.7 to 3.0 kb and a sharper band at 3.1 kb. The 5' flanking region of exon 1 has a GC content of 81% and has no canonical TATA or CCAAT boxes but contains potential binding sites for transcription factors Sp1, GC-binding factor and epidermal growth factor receptor-specific transcription factor. Chloramphenicol acetyltransferase (CAT) expression was observed on transient transfection into HeLa cells of a fusion construct containing the gene for CAT and a genomic DNA fragment from the 5' flanking region of exon 1. It is concluded that MGAT1 is a typical housekeeping gene although there is, in addition, tissue-specific expression of the larger 3.1 kb transcript.
Yip
B
B
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Chen
S H
SH
Mulder
H
H
Höppener
J W
JW
Schachter
H
H
D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017931:DNA Primers; D005091:Exons; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000502:physiology*; D011134:Polysaccharides / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D011134:Polysaccharides; D015500:Chloramphenicol O-Acetyltransferase; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D012260:Ribonucleases