Molecular cloning and variant analysis of the genes associated with Rh blood group antigens.

采用分子遗传学方法对缺乏Rh抗原的Rhnull表型进行了分析，并根据遗传背景将其分为两组：无性型由RHCE和RHD基因共同异常引起，调节型由RH50O基因非依赖于RH的基因座异常引起。这些结果证实了Rh5O糖蛋白是红细胞膜上表达Rh抗原不可缺少的因子。用编码RhD或RhCE基因的逆转录病毒载体转导原表达Rh50O糖蛋白的红系细胞系KU812E。转导Rh基因的细胞表达相应的Rh抗原。相反，不表达Rh50O糖蛋白的非红系细胞株(HEK293)在Rh和Rh50cDNA的诱导下，不能表达质膜上的Rh抗原。免疫电子显微镜分析表明，诱导后的细胞系在细胞质-细胞器膜上表达Rh50抗原。提示非红系细胞不表达Rh和Rh50以外的红系特异因子来表达质膜上的Rh抗原。在此基础上，进一步将从骨髓中构建的人c DNA文库导入HEK293细胞，并通过细胞分选仪筛选抗Rh抗体染色的诱导细胞。然而，诱导Rh抗原在非红系细胞质膜上表达的cDNA克隆仍在寻找中。我们还分离了RH50基因的启动子区域并对其进行了鉴定。RH50O基因5‘侧翼序列有一个反向GATA基序，该基序与红系特异性启动子活性密切相关。在更远的上游区域发现了一个红系特异的DNaseI超敏位点，它也编码了一个反向GATA基序。这些结果进一步支持RH50O基因在红系中的显性表达。

Rhnull phenotypes, which lack all Rh antigens, were analyzed by molecular-genetic procedures and categorized into two groups by the genetic backgrounds; 'amorph type' results from abnormality of both RHCE and RHD genes and 'regulator type' results from abnormality of RH5O gene independent locus from RH. These results confirmed that Rh5O glycoprotein is an indispensable factor for the expression of Rh antigens on erythrocyte membrane. A erythroid cell line (KU812E) originally expressing Rh5O glycoprotein was transduced by retroviral vector encoding RhD or RhCE cDNAs. The cells transduced Rh cDNAs expressed the respective Rh antigens. On the contrary, non-erythroid cell line (HEK293) not expressing Rh5O glycoprotein failed to express Rh antigens on the plasma membrane despite the induction of both cDNAs of Rh and Rh5O. Immuno-electro-microscopic analysis of the induced cell lines revealed that the cell lines expressed Rh5O antigen on the cytoplasmic-organella membranes. It was suggested that non-erythroid cell lack the expression of erythroid specific factors other than Rh and Rh5O to express Rh antigens on the plasma membrane. Then, we have intended to induce further a human cDNA library established from bone marrow into the double transfected HEK293 and screen the induced cell stained by anti-Rh antibodies through cell-sorter. However, the cDNA clone which induce the expression of Rh antigens on the plasma membrane of non-erythroid is still under hunting. We also isolated the promoter region of RH50 gene and characterized it. Just 5' flanking sequence of RH5O gene has an inverse GATA motif which is critically involved in the erythroid specific promoter activity. An erythroid specific DNaseI hypersensitive site was identified in the further up-stream region, which also encoded an inverse GATA motif. These results support further the erythroid dominant expression of the RH5O gene.