Chromosomal organization of the DNA regions located 5′ to the RHD and RHCE genes.

(A) The proposed structure of the RHCE and RHD 5′ flanking regions is depicted. A total of 4941 bp immediately 5′ of the ATG start codons are homologous between the RHCE andRHD genes (vertically hatched bars). No homology is present beyond this homology region (diagonally hatched bars). We used 2 genomic clones, dJ469D22 and dJ465N24, for primer design. DJ469D22 comprises the full length of the depicted RHCE region, whereas dJ465N24 extends only 466 bp into the homology region. The positions of several PCR primers are indicated: a, rey14a; b, rend32; c, rey15a; d, re014; and e, re011d. (B) This proposed structure is supported by several PCR reactions. Forward priming was done with primer a (RHCE specific, lane 1-3), primer b (RHD specific, lane 4-6), and primer c (RHCE and RHD homology region, lane 7-9). Amplicons were lacking for primer a with RHD-specific reverse primer e (lane 2) and for primer b withRHD⁻ DNA (lane 6). The other 7 PCR reactions yielded amplicons of the predicted sizes in accordance with the genomic structure shown in panel A.