Molecular cloning and characterization of the gene encoding N-acetylglucosaminyltransferase I

Abstract

One of the strategies for studying the function of N-linked complex carbohydrates during mammalian development is to manipulate glycosylation by disruption of a glycosyltransferase gene involved in their synthesis. A key gene in the generation of N-linked carbohydrates encodes the medial Golgi enzyme N-acetylglucosaminyltransferase I (GlcNAc-TI) which catalyzes the initial step in converting high mannose to complex and hybrid N-linked oligosaccharides. As the first step toward gene manipulation, the GlcNAc-TI gene was cloned and its genomic structure and expression in mouse tissues were investigated.;A unique probe derived from the coding region of the human GlcNAc-TI gene was used to isolate full length cDNA's from human and mouse cDNA libraries. The mouse gene was shown by Southern blot analyses to be present in a single copy, indicating the feasibility of gene disruption. Northern blot analyses revealed two size classes of mRNA which are differentially expressed in mouse tissues. Most tissues possess a shorter form {dollar}(\sim{dollar}2.9 kb), while brain has predominantly a longer form {dollar}(\sim{dollar}3.3 kb). The molecular basis of the size differences was investigated by an RNase H strategy, reverse transcription-PCR (RT-PCR) and 5{dollar}\sp\prime{dollar} anchored PCR. The results indicated that all the differences are restricted to differences at the 5{dollar}\sp\prime{dollar} end of GlcNAc-TI mRNA's that occur upstream of the cDNA sequence. To obtain additional 5{dollar}\sp\prime{dollar} sequence information, a mouse brain random primed cDNA library was PCR amplified using gene specific primers in combination with a primer from the vector. Two major PCR products with size differences that corresponded to that of the two GlcNAc-TI mRNA's were identified. The two cDNA's contain 451 nt and 772 nt 5{dollar}\sp\prime{dollar} of the ATG translational initiation codon respectively and both include the same open reading frame. These new 5{dollar}\sp\prime{dollar} GlcNAc-TI sequences were shown to be present in GlcNAc-TI mRNA's by RT-PCR. Northern blot analyses and primer extension experiments were performed to confirm the nature of the {dollar}\sim{dollar}2.9 kb and the {dollar}\sim{dollar}3.3 kb GlcNAc-TI transcripts. The combined data suggest that 5{dollar}\sp\prime{dollar} exons in the GlcNAc-TI gene are differentially utilized by tissue specific promoters. A tissue culture model which displays regulated expression of the GlcNAc-TI gene during differentiation to neuronal cells has been developed in P19 teratocarcinoma cells. (Abstract shortened by UMI.).