岩本 禎彦

fRhesus-associated glycoprotein is a critical co-factor in the expression of rhesus blood group antigens. We identified and cloned an erythroid-specific major DNase I-hypersensitive site located about 10 kilobases upstream from the translation start site of the RHAG gene. A short core enhancer sequence of 195 base pairs that corresponded with the major hypersensitive site and possessed position- and orientation-independent enhancer activity in K562 cells. In vitro DNase I footprint analysis revealed four protected regions in the core enhancer; two GATA motifs, an Ets-like motif and an unknown motif. The GATA motifs bound GATA-1 and mutagenesis analysis revealed that the proximal one is critical for the enhancing activity. Homology plot analysis using the 5' sequence of the mouse RHAG gene revealed four homologous stretches and multiple insertions of repetitive sequences among them; four LINE/L1 and four Alu in the human and as well as one LINE/L1 and one LTR/MaLR in the mouse gene. The highly conservative enhancer region was flanked by SINE and LINE/L1 in both species. These results suggest that the 5'-flanking sequence of RHAG gene is a preferable target sequence for retroviral transposition and that the enhancer was inserted in the same manner, resulting in the acquisition of erythroid dominant expression.

Rh blood group antigens are associated with non-glycosylated human erythrocyte membrane proteins encoded by two closely related genes, RHCE and RHD, and with a glycoprotein, a critical co-er;pressing factor encoded by the RH50 gene. The sequence analysis of RHCE transcripts has revealed that RhE/e and C/c serological phenotypes are associated with a nucleotide substitution in exon 5 and six substitutions in exons 1 and 2 of RHCE gene, respectively. Smythe et al, have shown that the full length transcript of RhcE gene expressed c and E antigens and the transcript of RhD gene expressed D and G antigens, using retroviral-mediated gene transduction into K562 cells. We performed an epitope analysis of Rh antigen by constructing retroviral gene coding six RH cDNAs, which contain RhcE, ce, CE and D cDNAs, and CE-D, D-CE chimera cDNAs. The cDNAs were introduced into KU812E cells and the expressed antigens were analyzed by flow cytometry. These studies revealed that the C/c and E/e associated substitutions actually participated in respective polymorphic epitopes. However, the C antigen was not detected on the KU812E cells introduced with CE cDNA, despite E antigen being expressed. The study with the chimera gene between CE and D cDNAs also indicated that the Rh epitopes were not constructed with short polymorphic exofacial peptide loops only but also with other peptide fragments and membrane components. Go-expression studies of Rh50 and RhD or cE gene in non-erythroid cells, 293, and expression studies of Rh50 in another erythroid cell, HEL, did not show any Rh antigens on the transduced cells, despite the Northern blot study showing both transcripts in the cells. It was suggested that at least a second co-expressing factor was needed to express RhCE or D antigens on the plasma membrane. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes, RHCE and RHD. Rh50 glycoprotein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression of RH50 gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5' flanking region of RH50 gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence of RH genes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter of RH50 gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Kruppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms for RH vs RH50 genes. (C) 1998 Academic Press.