Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation

Ravindra Kumar, Jing Yang, Robert D. Larsen, Pamela Stanley

Research output: Contribution to journalArticle

131 Citations (Scopus)

Abstract

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 A library prepared from genomic DNA of a tertiary Lec1 transfectant (3°T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [≈4.6 kilobases (kb)], isolated from an Alu-positive λ clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An ≈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 ≈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 ≈2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation. (.

Original languageEnglish (US)
Pages (from-to)9948-9952
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number24
StatePublished - 1990

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Transferases
Organism Cloning
Carbohydrates
Cricetulus
Ovary
DNA
Complementary DNA
Clone Cells
Messenger RNA
Genomic Library
Cell Separation
DNA Probes
Glycosylation
Point Mutation
Northern Blotting
Bacteriophages
Transfection
Amino Acid Sequence
Proteins
alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I

Keywords

  • Cloning Golgi glycosyltransferase
  • Gene rescue
  • Genomic DNA transfection

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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abstract = "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 A library prepared from genomic DNA of a tertiary Lec1 transfectant (3°T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [≈4.6 kilobases (kb)], isolated from an Alu-positive λ clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An ≈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 ≈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 ≈2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation. (.",
keywords = "Cloning Golgi glycosyltransferase, Gene rescue, Genomic DNA transfection",
author = "Ravindra Kumar and Jing Yang and Larsen, {Robert D.} and Pamela Stanley",
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T1 - Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation

AU - Kumar, Ravindra

AU - Yang, Jing

AU - Larsen, Robert D.

AU - Stanley, Pamela

PY - 1990

Y1 - 1990

N2 - 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 A library prepared from genomic DNA of a tertiary Lec1 transfectant (3°T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [≈4.6 kilobases (kb)], isolated from an Alu-positive λ clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An ≈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 ≈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 ≈2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation. (.

AB - 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 A library prepared from genomic DNA of a tertiary Lec1 transfectant (3°T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [≈4.6 kilobases (kb)], isolated from an Alu-positive λ clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An ≈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 ≈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 ≈2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation. (.

KW - Cloning Golgi glycosyltransferase

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KW - Genomic DNA transfection

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