小鼠3种α1,2岩藻糖转移酶基因的比较  

Comparison of the Three Murine GDP-fucose: β-galactoside α1,2-fucosytransferase MFUT-I, MFUT-II and MFUT-III

作　　者：林蓓[1] 齐藤真木子[2] 岩森正男[3] 

机构地区：[1]中国医科大学第二临床学院妇产科,沈阳110004 [2]东京大学医学部小儿科 [3]近畿大学理工学部化学科

出　　处：《细胞生物学杂志》2005年第4期464-468,共5页Chinese Journal of Cell Biology

摘　　要：利用RT-PCR方法克隆小鼠3种GDP岩藻糖:β-半乳糖苷α1,2-岩藻糖转移酶(α1,2-fucosyltansferase,α1,2-FT)基因编码区MFUT-I、MFUT-II、MFUT-III,序列分析结果表明MFUT-I、MFUT-II和MFUT-III间具有相当的同源性,且分别与人类H基因(78.4%)、Se基因(79.0%)和Sec1基因(74.9%)具有序列同源性。将3种基因编码区分别插入表达载体pcDNA3.1的多克隆位点,并将其分别转染于COS-7细胞,结果显示MFUT-I和MFUT-II基因转染后的COS-7细胞具有α1,2-FT活性,但在MFUT-III基因转染后的COS-7细胞中检测不到这种活性。应用Northern印迹杂交法研究基因在小鼠组织中的表达情况。证实MFUT-II可在多种组织中产生3.5kb大小的mRNA转录产物,然而MFUT-I和MFUT-III分别只在附睾和睾丸中显著表达。Southern印迹杂交分析结果显示:基因MFUT-II仅为一个拷贝,而MFUT-I和MFUT-III可能存在2个拷贝。因此,MFUT-I、MFUT-II和MFUT-III分别为鼠的H基因、Se基因和Sec1基因。Three members of a GDP-fucose： β-galactoside α1,2-fucosyltransferase （α1,2-fucosyltransferase, α1,2-FT）, MFUT-Ⅰ, MFUT-Ⅱ, and MFUT-Ⅲ, from a cDNA of murine small intestine, were cloned by RT-PCR. MFUT-Ⅰ, MFUT-Ⅱ, and MFUT-Ⅲ exhibited sequence homology together and with the human H （78.4%）, Se （79.0%）, and Secl （74.9%） gene products, respectively. The open reading frames were ligated into mammalian expression vector pcDNA 3.1（pcDNA3.1-MFUT-Ⅰ, pcDNA3.1-MFUT-Ⅱ, pcDNA3.1-MFUT-Ⅲ,） and then transiently transfected into COS-7 cells using a Cellphect transfection kit and the cells was used for determination of α1,2-FT. COS-7 cells transfected with MFUT-Ⅰ and MFUT-Ⅱ exhibited α1,2-FT activity, but no activity was detected in COS-7 cells with MFUT-Ⅲ. The expression of three gene in murine tissues were analyzed by Northern blotting. MFUT-Ⅱ yielded a 3.5 kb mRNA transcript in several tissues, whereas MFUT-Ⅰ and MFUT-Ⅲ were predominantly expressed in epididymis and testis, respectively. Southern blot analysis showed that MFUT-Ⅱ was present in the mouse genome as a single-copy gene but MFUT-Ⅰ and MFUT-Ⅲ were present as tow-copy gene. Our results suggest that MFUT-Ⅰ, MFUT-Ⅱ and MFUT-Ⅲ correspond to the human H, Se and Secl genes, respectively.

关 键 词：小鼠 α1 2-岩藻糖转移酶 基因 克隆 表达 酶基因 岩藻糖 COS-7细胞 RT-PCR方法 PCDNA3.1 

分 类 号：Q78[生物学—分子生物学]