Cloning and expression of the murine gene and chromosomal location of the human gene encoding N-acetylglucosaminyltransferase I

Ravindra Kumar, Jing Yang, Roger L. Eddy, Mary G. Byers, Thomas B. Shows, Pamela Stanley

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

A mouse cDNA clone previously isolated from an F9 teratocarcinoma cell library and shown to confer N-acetylglucosaminyltransferase I (GlcNAc-TI) activity on Lec1 Chinese hamster ovary (CHO) cell transfectants [Kumar, R., Yang,J., Larsen,R.D. and Stanley,P. (1990) Proc. Nail. Acad Sci. USA, 87, 9948-9952] has been sequenced. The nucleotide and deduced amino acid sequences are highly homologous to previously described human and rabbit GlcNAc-TI cDNAs. A 1250 bp portion of the mouse cDNA encoding all but the first 34 amino acids of the deduced protein sequence was inducibly expressed in Escherichia coli andgave rise to a prominent fusion protein of mol. wt ̃45 kDa whose presence correlated with high levels of GIcNAc TI activity in cell lysates. Probes generated from the cDNA were used to show that the GlcNAc-TI gene is present in a single copy in mammals and that a homologous gene was not detectable (under low-stringency hybridization conditions) in DNA from yeast, sea urchin, Drosophila or Chaenorhabditis elegans. Genomic DNA clones that hybridized to probes generated from the GlcNAc-TI cDNA were isolated from a mouse liver library. Restriction analyses, Southern hybridization and DNA sequence analyses of subcloned genomic DNA fragments and a polymerase chain reaction (PCR) product provided evidence that the coding and 3' untranslated regions of the cDNA reside in a single exon. However, the mouse GlcNAc-TI gene (Mgat-1) includes at least one additional exon 5' of the coding region. Southern analyses of DNA from mouse-human somatic cell hybrids and in situ hybridization were used to locate the human GlcNAc-TI gene (MGAT-1) between positions q31.2 and q31.3 on chromosome 5, a region of chromosome 5 that is syntenic with a region of mouse chromosome 11. Northern analyses of adult mouse tissues revealed two GlcNAc-TI gene transcripts that are differentially expressed in different tissues.

Original languageEnglish (US)
Pages (from-to)383-393
Number of pages11
JournalGlycobiology
Volume2
Issue number4
DOIs
StatePublished - Aug 1 1992

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Keywords

  • Bacterial expression
  • Chromosomal mapping
  • Cloned glycosyltransferase
  • Mouse gene expression

ASJC Scopus subject areas

  • Biochemistry

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