Study on Ganglioside GM3 Synthase and Its Gene.

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We recently cloned a human cDNA for ganglioside GM3 synthase. Elucidation of the mechanisms controlling expression of the GM3 synthase gene is very important and interesting, since the enzyme is only α2, 3-sialyltransferase capable of the biosynthesis of ganglioside GM3. Firstly, we isolated a BAC clone containing an entire human GM3 synthase gene and analyzed its organization. Human GM3 synthase gene spans approximately 54 kbps of genomic DNA with seven exons, ranging from 112 to 1,242 bp, and six intron. FISH analysis revealed gene is located near the centromere of the about arm on chromosome 2, an area which corresponds to band 2p11.2. To identify the promoter region of the gene, about 10 kbp fragment containing 5'-flanking region and a part of exon 1 of the gene was subcloned into promoter-less luciferase plasmid. Deletion analyses indicated that the region from nt -285 to nt +103 is essential for full promoter activity. Furthermore, mutation analyses showed that two Sp1 sites and one NFY site are important for positive transcriptional regulation of the gene. Next, we cloned cDNA homologs from mice, rat and monkey to explore the structure-function relationship of GM3 synthases. Mouse, rat and monkey GM3 synthases to human homolog were 85.0%, 84.8% and 97.6% identical, respectively, at the nucleic acid level. Mouse GM3 synthase gene which was mapped to chromosome 6, region C on mouse genome, spans approximately 58 kbps of genomic DNA with nine exons, ranging from 112 to 1,172 bp, and eight intron. Different from that of the human GM3 synthase gene, the mechanisms of an alternative splicing and alternative promoter utilization were involved in the expression of mouse GM3 synthase gene, resulting the production of 3 transcript variants with different 5'-end sequences. We tried to the generation of GM3 synthase targetting mice to analyze the function of ganglioside GM3 and more complex gangliosides in individual. Now we obtained chimeric mice.