Transcriptional regulation of the human UDP-GlcNAc:alpha-6-D-mannoside beta-1-2-N-acetylglucosaminyltransferase II gene (MGAT2) which controls complex N-glycan synthesis.
UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is essential for the normal assembly of complex Asn-linked glycans. Northern analysis showed a major transcript at 2.0 kb and a minor band at approximately 2.9 kb in five different human cell lines. The gene (MGAT2) has three AATAAA polyadenylation sites at 68, 688 and 846 bp downstream of the translation stop codon. 3'-RACE (rapid amplification of cDNA ends) using RNA from the human cell line LS-180 indicated that all three sites were utilized for transcription termination. 5'-RACE and RNase protection analyses showed multiple transcription initiation sites at -440 to -489 bp relative to the ATG translation start codon (+1). The data show that the entire GnT II gene is on a single exon. The gene has a CCAAT box at -587 bp but lacks a TATA box and the 5'-untranslated region is GC-rich and contains consensus sequences suggestive of multiple binding sites for Sp1; these properties are typical for housekeeping genes. A series of chimeric constructs containing different lengths of the 5'-untranslated region fused to the chloramphenicol acetyltransferase (CAT) reporter gene were tested in transient transfection experiments using HeLa cells. The CAT activity of the construct containing the longest insert (-1076 bp relative to the ATG start codon) showed a approximately 38-fold increase as compared to that of the control. Removal of the region between -636 and -553 bp caused a dramatic decrease in CAT activity indicating this to be the main promoter region of the gene.
Chen
S H
SH
Research Institute, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Ontario, Canada.
Zhou
S
S
Tan
J
J
Schachter
H
H
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; D012260:Ribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases