Figure 2 Analysis of restriction sites that define the 5′ and 3′ boundaries of the region of identity between RHD and the Ce allele. TaqI (a) and HinfI (b) sites bracket the 5′ boundary, and PstI (c) defines the 3′ boundary (see Fig. 1). In (a) TaqI fragments of 306, 203 and 97 bp are only generated when RHD is present. In the case of HinfI (b) two sites are common to all genes, hence the presence of 211 and 172 bp fragments in all lanes. The residual 1060 bp fragment is resistant to further digestion when it originates from a c allele. Similarly, in the case of PstI (c); all products are cut twice yielding fragments of322 and 107 bp, and a residual product which is 749 bp when it originates from C alleles, 640 bp from RHD and c alleles. The c allele product is further cut to 390 and 250 bp. Sizes of restriction fragments (in base pairs) are shown. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Table 2 RH gene extended haplotypes From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Table 1 Serologically defined Rh haplotypes and their population frequencies. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Figure 1 The relationship between the RHD gene and alleles of RHCE in the neighbourhood of exon 2. A total of 5501 bp was sequenced around exon 2 of RHD and the Ce and ce alleles of RHCE (the limits of the region sequenced are denoted by vertical arrows). Restriction sites relevant to the present study only are shown. The starred AflIII site in the RHD sequence is polymorphic (see text). These sequences have been deposited in GenBank, accession numbers U66340 and U66341. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Figure 3 New polymorphisms used for haplotype construction Figure 3 New polymorphisms used for haplotype construction. (a) Intron 8 simple sequence repeat polymorphism. A single, extended, pedigree segregating CDe, cDE and cde RH haplotypes and alleles 2, 3 and 4 of the repeat is shown. PCR products were resolved by polyacrylamide gel electrophoresis followed by silver staining. (b) RHD AflIII RFLP. The uncut product is 1295 bp from both RHD and RHCE. That from the C alleles is cut twice by AflIII to 627, 531 and 137 bp. The product from the c alleles is reduced to 764 and 531 bp. The RHD product is either cut twice, like C, or once, yielding fragments of 1158 and 137 bp (see Fig. 1). Fragment sizes (in base pairs) are shown. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Figure 4 5′ and 3′ end analysis of an RH YAC clone Figure 4 5′ and 3′ end analysis of an RH YAC clone. (Left) DNA from the 450 kb RH YAC clone was amplified using PCR primers that flank a HindIII site 1670 bp 5′ of intron 1 in RHCE only. The 1.97 kb product was incubated with HindIII overnight at 37°C. The presence of the 150 bp HindIII fragment is diagnostic of RHCE; the residual 1.82 kb fragment is not resolved from undigested PCR product on the gel shown here. (Right) YAC DNA was amplified with RHCE- and RHD-specific exon 10 primers in a duplex reaction; the RHCE-specific product is 133 bp, the RHD-specific product is 291 bp. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press

Figure 5 Evolution of the human RH genes Figure 5 Evolution of the human RH genes. (a) Recombination between the more common haplotypes generates the less common ones. In cdE (r″), haplotype data cannot distinguish between the alternative sites of exchange shown. The rare haplotype CdE (r^y) requires recombination between two uncommon recombinant haplotypes. (b) Pathway showing the evolutionary relationship between all RH haplotypes. From: Evolution of the Human RH (Rhesus) Blood Group Genes: A 50 Year Old Prediction (Partially) Fulfilled Hum Mol Genet. 1997;6(6):843-850. doi:10.1093/hmg/6.6.843 Hum Mol Genet | © 1997 Oxford University Press