Study on the relationship between erythroid differentiation and expression of blood group substances.

Rh血型系统的基因结构分析表明，RHD和RHCE基因均由10个外显子和9个内含子组成，其中一个8kb的间隔区连接这两个基因。通过基于逆转录聚合酶链式反应的Rh相关基因亚型的系统分析，分别获得了11个和5个截短的RhCE和RhD cDNAs。对人红系分化过程中Rh相关基因表达的研究表明，在红系成熟过程中存在可诱导的异构体。我们通过构建编码6个正常和嵌合体Rh基因的逆转录病毒基因，对Rh抗原进行了表位分析。将该基因导入KU812E细胞。结果表明，Rh表位不仅由短的多态表位环构成，而且还与其他多肽片段和其他膜组分构成。RH50和RHD或RHCE基因在非红系细胞293中的共表达研究没有发现任何Rh抗原，提示至少需要第二个共表达因子才能在质膜上表达RhD或RhCE抗原。对一例Rhnull表型先证者的基因分析证实，Rh50糖蛋白是共表达因子之一。我们发现了一种新的Duffy mRNA的第一外显子和剪接体，它是红系和毛细血管后微静脉内皮细胞的主要转录物。我们还在600bp5‘侧翼区发现了两个反向GATA基序。近端GATA位于内皮起始部下游，红系起始部上游。凝胶漂移分析表明，GATA基因近端为GATA-1的靶序列。缺失突变研究表明，该GATA基序代表Duffy基因的红系调控核心区。

Gene structure analyzes of the Rh blood group system revealed that both the RHD and RHCE genes consist of 10 exons and 9 introns that a 8kb-spacer region connects these two genes. By a systemic analysis of Rh-related cDNA isoforms based on reverse transcription-polymerase chain reaction, 11 and 5 truncated isoforms of the RhCE and RhD cDNAs, respectively. Studying on Rh-related gene expression during human erythroid differentiation exhibited the existence of isoforms inducible during erythroid maturation.We performed epitope analysis of Rh antigen by constructing retroviral gene coding 6 normal and chimera Rh cDNAs. The cDNAs were introduced into KU812E cells. The results indicated that the Rh epitopes were not constructed only with short polymorphic exofacial peptide loops but with other peptide fragments and other membrane components. Co-expression studies of RH50 and RHD or RHCE gene in non-erythroid cells, 293, did not show any Rh antigens and suggested that at least a second co-expressing factor was needed to express RhD or RhCE antigens on the plasma membrane. The gene analysis of a propositus with Rhnull phenotype confirmed the Rh50 glycoprotein as one of the co-expressing factors.We found a novel first exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium. We also identified two inverse GATA motifs in the 600 bp 5' flanking region. The proximal GATA was positionated downstream from the start position of endothelium and upstream from that of erythroid. Gel shift assay showed the proximal GATA gene is the target sequence of GATA-1. Deletion mutagenesis study revealed that this GATA motif represent the erythroid regulatory core region for Duffy gene.