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Cloning and Expression of Drosophila melanogaster UDP-GlcNAc:?-3-D-Mannoside ? 1,2-N-Acetylglucosaminyltransferase I
A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDPNacetylglucosamine: ?-3-Dmannoside ?-1,2-Nacetylglucosaminyltransferase I (GnT I, EC 188.8.131.52) as probe yielded a clone (GM01211) with 56% identity over 36 carboxyterminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in ?ZAP II and two cDNAs lacking a start ATG codon were obtained. 5Rapid amplification of cDNA ends (5RACE) yielded a 2828 bp cDNA containing a fulllength 1368 bp open reading frame encoding a 456 amino acid protein with putative Nterminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the Nterminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an Nterminal (His) tag. Protein purified by adsorption to and elution from nickel beads converted Man?1-6(Man?1-3)Man?octyl (M3-octyl) to Man?1-6(GlcNAc?1-2Man?1-3)Man?octyl. The K values (0.7 and 0.03 mM for M3-octyl and UDPGlc NAc respectively), temperature optimum (37 C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GTAG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.
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