Study on Ganglioside GM3 Synthase and Its Gene.

神经节苷脂GM3合酶及其基因的研究。

• 批准号: 10670109
• 负责人: ISHII Atushi
• 金额: $ 2.11万
• 依托单位: National Cancer Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670109/
• 关键词: GM3 synthase; transcriptional promoter; Sp1; Genomic organization; NFY; Targetting mice; GH3合成酵素; ゲノムDNA; 抗体; ガングリオシドGM3; シアル酸転移酵素; HL-60細胞; 分化誘導療法

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.

我们最近克隆了人神经节苷脂GM3合成酶的基因。由于GM3合成酶只是α2，3-唾液酸基转移酶，能够生物合成神经节苷脂GM3，因此阐明GM3合成酶基因表达的调控机制是非常重要和有趣的。首先，我们分离了一个含有完整的人GM3合成酶基因的BAC克隆，并对其组织结构进行了分析。人类GM3合成酶基因全长约54kbps，有7个外显子和6个内含子，外显子长度在112~12242bp之间。FISH分析表明，该基因位于2号染色体上约臂着丝粒附近，该区域与2p11.2带相对应。为了鉴定该基因的启动子区域，将含有该基因5‘侧翼区和部分外显子1的约10kbp片段亚克隆到无启动子的荧光素酶表达载体中。缺失分析表明，从nT-285到nT+103是启动子活性发挥的关键区域。此外，突变分析表明，两个Sp1位点和一个NFY位点对于该基因的正转录调控是重要的。接下来，我们克隆了小鼠、大鼠和猴子的同源基因，以探讨GM3合成酶的结构与功能的关系。小鼠、大鼠和猴GM3合成酶与人的同源物在核酸水平上的同源性分别为85.0%、84.8%和97.6%。小鼠GM3合成酶基因位于小鼠基因组C区6号染色体上，全长约58kbps，有9个外显子，大小在112~1172bp之间，内含子有8个。与人类GM3合成酶基因不同，小鼠GM3合成酶基因的表达涉及选择性剪接和选择性启动子利用的机制，导致产生3种不同5‘端序列的转录变异体。我们试图以GM3合成酶的产生为靶标，分析神经节苷脂GM3和更复杂的神经节苷脂在个体中的功能。现在我们获得了嵌合小鼠。

项目成果

Ishii A., et al.: "Expression cloning and Functional Characterization of Human cDNA for Ganglioside GM3 Synthase."J.Biol. Chem.. 273. 31652-31655 (1999)

Ishii A. 等人：“神经节苷脂 GM3 合酶的人类 cDNA 的表达克隆和功能表征。”J.Biol。

Saito M.and Ishii A.: "Handbook of Glycosyl transferases"Springer-Verlag,NY.(印刷中).

Saito M. 和 Ishii A.：“糖基转移酶手册”，Springer-Verlag，纽约（印刷中）。