Audette M, Carrel S, Hayoz D, Giuffrè L, Mach J P, Kühn L C
Ludwig Institute for Cancer Research, Lausanne Branch, Switzerland.
Mol Immunol. 1989 Jun;26(6):515-22. doi: 10.1016/0161-5890(89)90002-3.
The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.
人类Me14-D12抗原是一种细胞表面糖蛋白，在神经外胚层起源的肿瘤细胞系上受γ干扰素（IFN-γ）调控。它由两个非共价连接的亚基组成，表观分子量大小分别为33,000和38,000。在此我们描述了使用基因转移方法对Me14-D12基因的基因组探针进行分子克隆。将小鼠Ltk-细胞与人基因组DNA和单纯疱疹病毒胸苷激酶（TK）基因进行稳定共转染。在HAT培养基中进行选择后，通过在荧光激活细胞分选仪（FACS）上反复分选分离出表达Me14-D12抗原的原代和二代转染子。从由阳性二代转染子细胞系构建的基因组文库中分离出一个携带14.3 kb人类DNA插入片段的重组噬菌体。源自噬菌体DNA插入片段的特异性探针能够在原代和二代L细胞转染子以及表达Me14-D12抗原的人类黑色素瘤细胞系中鉴定出3.5 kb和2.2 kb的两种mRNA。在L细胞转染子中保留了IFN-γ对Me14-D12抗原的调控，并且在蛋白质和mRNA表达水平均可检测到。
