Rajput B, Ma J, Vijay I K
Department of Animal Sciences, University of Maryland, College Park 20742.
J Biol Chem. 1994 Apr 1;269(13):9590-7.
The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, was isolated and characterized. Southern blot analyses demonstrated a single copy gene for GPT. The gene spans about 7.5 kilobase pairs of DNA and is divided into 9 exons by 8 introns. All the introns are found in the coding region, and most of them occur in segments separating the putative membrane-spanning domains. The exon/intron organization of the gene also correlates with the presence of several highly conserved regions of potential functional importance among yeast, leishmania, hamster, and mouse enzymes. Primer extension and reverse transcription-polymerase chain reaction analyses suggested the presence of several potential transcription start sites, with the closest one being approximately 200 base pairs upstream from the translation initiation codon. The 5'-flanking region lacks a typical TATA box, but is high in GC content and contains two putative Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 3'-end reverse transcription-polymerase chain reaction analysis indicated that the first of the two polyadenylation sites was used predominantly, in agreement with a approximately 2.0-kilobase pair GPT message seen on Northern blots of RNA from a wide variety of mouse tissues. This is the first report of cloning of a gene for an enzyme of the dolichol cycle in higher eukaryotes. A novel finding of this study is the observation of a G-->A change between the genomic sequence and nucleotide 280 in the cDNA. This could have important implications as an RNA editing mechanism for regulating the expression of the gene and therefore, protein N-glycosylation. A previous study (11) had shown that the activity of GPT was developmentally regulated in mouse mammary gland, with possible involvement by the hormone prolactin. The availability of the GPT gene with its promoter should facilitate future studies on delineating the mechanism for the hormonal regulation of GPT.
