Identification of nt 836 G→A (Gly₂₇₉→Glu) missense mutation in the Rh50 transcript. The strategy for synthesis and amplification of Rh50 cDNA is shown. The Rh50 mRNA was reverse-transcribed with the 3′-UTa primer and then amplified with two pairs of upstream primers. (A) The resultant cDNA products, designated by the primers used, were electrophoresed on 1.8% agarose gel. Size markers ofHaeIII-cleaved◊X174 DNA are shown at left. Lanes are designated as in Fig 1. No difference in size is seen between normal subjects and Rh_null(YT). (B) DNA sequencing profiles for the nt 836 G→A missense change. The G residue is seen in normal subjects, whereas the A residue is seen in Rh_null(YT) only. (C) Immunoblot analysis of RhD and Rh50 in red blood cell membranes. Lanes are the same as in (A). The antibodies used are indicated. Note that no band is seen in Rh_null (YT), even though a normal RhD and a missense Rh50 transcript were expressed. Note also that RhD-negative control did not react with LOR-15C9.21