Figure 5

Demonstration of G1754T (Gly583Cys) mutation in SHR, SHRSP and WKY rats. Panel A shows a representative sequence electropherogram from SD, Wistar, WKA, GH and BN rats (top electropherogram) and from SHR, SHRSP and WKY rats (bottom electropherogram) for exon 12 of Abcg5. A homozygous change (G1754T, Gly583Cys) is present in the sitosterolemic rats and this mutation abolishes the recognition site for restriction enzyme Hae III. Panel B shows the restriction fragment analyses of DNA amplified from these different rat strains, further confirming the presence of the mutational change. In the absence of digestion a 252 bp PCR product is present, which, after Hae III digestion (wt) results in two products of 180 bp and 72 bp (indicated by the arrows). The sitosterolemic strains WKY, SHR and SHRSP (lanes 6–8) are all resistant to digestion by Hae III, but SD and Wistar remain sensitive (lanes 3 and 4). Lanes 1 and 10 shows molecular weight markers, and lanes 2 and 9 show undigested PCR products (252 bp) from SD rat and SHRSP respectively. Panel C shows the alignment of amino acids 565 to 592 of sterolin-1 from human, mouse, rat and Fugu. An arrowhead indicates the position of Gly583Cys mutation.