HGPRT-Positive and HGPRT-Negative Erythrocytes in Heterozygotes for HGPRT Deficiency

Abstract

A method for autoradiography of tritium labelled mononucleotides in red blood cells has been developed and used to examine heterozygotes for HGPRT deficiency. Although HGPRT-positive and HGPRT-negative cell populations have been demonstrated by autoradiography in cultured fibroblasts from heterozygotes for the severe HGPRT deficiency found in the Lesch-Nyhan syndrome, erythrocyte lysates have always shown normal HGPRT activity. In heterozygotes for the partial HGPRT deficiency, on the other hand, erythrocyte lysates have shown a range of activities between 20% of normal and completely normal values. Although erythrocytes do not synthesize nucleic acids, they are capable of accumulating significant quantities of labelled mononucleotides when incubated with labelled purines. However, these soluble mononucleotides are lost when the selective permeability of the cell membrane is destroyed by fixing or drying. Thus the problem of autoradiographically demonstrating tritiated hypoxanthine uptake by red cells involves fixing the labelled nucleotide. This has been achieved by precipitating the mononucleotides with lanthanum simultaneously with fixation of the red cells with osmium tetroxide.