FIG. 4.

Deletion of 5α-reductase 2 gene in subjects with 5α-reductase deficiency. DNA isolated from two normal individuals and two related 5α-reductase deficiency subjects from a geographically isolated tribe in the Highlands of Papua New Guinea¹⁷ was screened by Southern blotting using the indicated 5α-reductase cDNA probes. The normal DNA analysed in the most left-hand lane was isolated from an individual from the same New Guinea tribe as the NG1 and NG3 subjects, whereas the normal DNA analysed in the right-hand lane was isolated from a caucasian American. The filter was screened with the 5α-reductase 2 cDNA probe first, and then stripped and reprobed with the 5α-reductase 1 cDNA probe. A deletion of all but a weakly hybridizing fragment of about 4.5 kilobases in the DNA of the affected NG1 and NG3 individuals is apparent from the autoradiogram obtained with the 5α-reductase 2 probe. All individuals have a normal 5α-reductase 1 gene.

METHODS. Genomic DNA was isolated from peripheral blood samples from the indicated individuals and prepared and analysed by high-stringency Southern blotting as described previously²⁴. Aliquots of DNA (20 μg) were digested with Hindlll before electrophoresis on agarose gels. Three single-stranded ³²P-labelled probes spanning the coding region of the 5α-reductase 2 cDNA shown in Fig. 1 were prepared as described by Church and Gilbert²⁵ and used in hybridization. After autoradiography for 5 days at −70 °C, the filter was stripped²⁰ and reprobed with a random hexanucleotide ³²P-labelled probe²⁰ corresponding to the full-length 5α-reductase 1 cDNA (ref. 4).