Specific detection of kinetoplast DNA in cytological preparations of trypanosomes by hybridization with complementary RNA

Fibroblasts from patients with Xeroderma pigmentosum (X.P.) were used together with normal fibroblasts, in order to test (1) whether complementation takes place in heterokaryons formed by these cells; (2) whether the ‘factor’ defective in X.P. limits the rate of DNA repair synthesis in normal fibroblasts. Proximity to normal fibroblasts as well as treatment with their extract does not significantly affect the DNA repair synthesis of the abnormal cells, as measured by autoradiography. By contrast, in heterodikaryons a corrective substance rapidly diffuses into the abnormal nuclei which then perform a normal amount of DNA repair synthesis. Such complementation does not require de novo protein synthesis, since it occurs in the presence of daunomycin or cycloheximide. Furthermore, the dilution of normal ‘factor’, which follows diffusion, does not prevent each nucleus in these hybrids from showing a normal amount of DNA repair synthesis even after UV doses capable of saturating the DNA repair system of the normal parental cells. Thus it seems that in normal fibroblasts the factor defective in X.P. is not rate limiting.Nevertheless, a comparison of heteropolykaryons with a high (3:1) and a low (1:1.25) average ratio of X.P. to normal nuclei shows that, in the former, DNA repair synthesis is reduced. This effect, which seems rather long lasting, indicates that the carrier state for X.P. could be detected using the dosimetric help of heteropolykaryons.