Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation.
This laboratory has previously identified a human gene encoding N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) by complementation of the glycosylation defect in the Lec1 Chinese hamster ovary (CHO) cell mutant. A phage lambda library prepared from genomic DNA of a tertiary Lec1 transfectant (3 degrees T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [approximately 4.6 kilobases (kb)], isolated from an Alupositive lambda clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An approximately 1.3-kb probe generated from this DNA fragment detected unique but distinct DNA fragments in human and CHO genomic DNA. The probe also hybridized to a poly(A)+ RNA of approximately 2.7 kb in human and CHO cells and allowed the isolation of a full-length cDNA encoding human GlcNAc-TI activity. The overall features of the cDNA and deduced protein sequence (445 amino acids) are typical of other Golgi transferases that are type II transmembrane proteins. Northern blot analysis with the same probe showed that Lec1 mutant cells also possessed an approximately 2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation.
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
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R
Kumar
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J
Yang
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RD
Larsen
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P
Stanley
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D005260:Female; D005796:Genes; D005816:Genetic Complementation Test; D015698:Genomic Library; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases; D009693:Nucleic Acid Hybridization; D010053:Ovary; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D015183:Restriction Mapping; D014162:Transfection
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I