权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

Rh 血型抗原相关基因的分子克隆和变异分析。

基本信息

批准号：
10670399
负责人：
IWAMOTO Sadahiko
金额：
$ 2.05万
依托单位：
Jichi Medical School
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 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670399/
关键词：
rhesus blood group antigen cloning of RH genorne cloning of RH5O genome gene regulatory region DNaseI hypersensitive site

项目摘要

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.

Rhnull表型，缺乏所有的Rh抗原，通过分子遗传学程序进行分析，并根据遗传背景分为两组：“无定形型”的结果，RHCE和RHD基因的异常和“调节型”的结果，从RH的RH5O基因独立位点的异常。这些结果证实Rh5O糖蛋白是Rh抗原在红细胞膜上表达的一个不可缺少的因素。用编码RhD或RhCE cDNA的逆转录病毒载体转导原代表达Rh5O糖蛋白的红系细胞KU812E。转导Rh cDNA的细胞表达各自的Rh抗原。相反，不表达Rh5O糖蛋白的非红系细胞系（HEK293）不能在质膜上表达Rh抗原，尽管Rh和Rh5O的cDNA都被诱导。免疫电镜分析显示，诱导的细胞系的细胞质细胞器膜上的表达Rh5O抗原。提示非红系细胞缺乏Rh和Rh_（50）以外的红系特异性因子的表达，无法在质膜上表达Rh抗原。然后，我们打算将从骨髓中建立的人cDNA文库进一步诱导到双转染的HEK 293中，并通过细胞分选仪筛选经抗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基因的启动子区并对其进行了鉴定，发现RH50基因的5'侧翼序列具有一个与红系特异性启动子活性密切相关的反加塔基序。在进一步的上游区域中鉴定出红细胞特异性DNaseI超敏位点，其也编码反向加塔基序。这些结果进一步支持了RH5O基因的红系显性表达。