ISOLATION OF G(D3) SYNTHASE GENE BY EXPRESSION CLONING OF G(M3) ALPHA-2,8-SIALYLTRANSFERASE CDNA USING ANTI-G(D2) MONOCLONAL-ANTIBODY
ISOLATION OF G(D3) SYNTHASE GENE BY EXPRESSION CLONING OF G(M3) ALPHA-2,8-SIALYLTRANSFERASE CDNA USING ANTI-G(D2) MONOCLONAL-ANTIBODY
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DOI：
10.1073/pnas.91.22.10455
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发表时间：
1994-10-25
影响因子：
11.1
通讯作者：
FURUKAWA, K
中科院分区：
文献类型：
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作者：
HARAGUCHI, M;YAMASHIRO, S;FURUKAWA, K
为了分离神经节苷脂G（D3）合酶（EC 2.4.99.8）cDNA，我们开发了使用抗G（D2）单克隆抗体进行选择的表达克隆方法。还利用了我们命名为KF 3027-Hyg 5的宿主受体细胞系。该细胞系表达高水平的G（M2）和G（M3），但不表达G（D3）或G（D2），并且由用多瘤大肿瘤抗原基因（KF 3027）和先前克隆的β-1，4-N-乙酰氨基半乳糖转移酶（EC 2.4.1.92）cDNA转染的小鼠B16黑素瘤细胞构建。通过4轮转染、单克隆抗体3F 8淘选和Hirt提取，分离出两个cDNA克隆，其转染指导了G（D3）在KF 3027和B16黑色素瘤细胞中的表达以及G（D3）和G（D2）在KF 3027-Hyg 5细胞中的表达。该cDNA含有一个1650 bp的插入片段和一个开放阅读框。推导的氨基酸预测的II型膜拓扑结构组成的细胞质（14个氨基酸），跨膜（18个氨基酸），和催化（309个氨基酸）域。该序列还预测了一种唾液酸蛾的存在，类似于迄今为止克隆的其他唾液酸转移酶。正如预期的那样，该基因的mRNA（2.6 kb）在人黑色素瘤细胞系中强烈表达。
For the isolation of ganglioside G(D3) synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-G(D2) monoclonal antibody for selection, A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of G(M2) as well as G(M3) but no G(D3) Or G(D2) and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of G(D3) in KF3027 and B16 melanoma cells and G(D3) and G(D2) in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl moth similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.