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

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. Natl. 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 and gave rise to a prominent fusion protein of mol. wt approximately 45 kDa whose presence correlated with high levels of GlcNAc-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 Chaenorhaditis 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.