Molecularand CellularProbes (1995) 9, 121-128 New members of the 3fl-hydroxysteroid dehydrogenase gene family Martin W. McBride, Alan J. Russell, Keith Vass,' Victoria Forster, Sandra M. Burridge, Norma Morrison, 2 Elizabeth Boyd, 2 Bruce A. J. Ponder ~ and Roger G. Sutcliffe* Institute of Genetics, Glasgow University, Church Street, Glasgow G I 1 5JS, ~CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, 2Department of Medical Genetics, Duncan Guthrie Institute, Yorkhill Hospitals, Glasgow, and 3CRC Human Cancer Genetics Research Group, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP (Received 19 August 1994, Accepted I September 1994) Several bands of hybridization are detected when southern blots of human genomic DNA are probed with cDNA of 3/3-hydroxysteroid dehydrogenase (3/~-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3/3-HSD gene family. Firstly, primers designed to amplify 3/3-HSD type I and II genes were found on occasion to amplify DNA products of appropriate length but which were resolved as distinct sequences by denaturing gradient gel electrophor'esis (DGGE). Five of these novel bands were cloned and their sequences were found to be closely related to 3/3-HSD types I and II. Secondly, 57 genomic clones were selected from two 2 genomic libraries by hybridization with exonic probes of 3/3-HSD type I. These were screened for novel members of the gene family by PCR amplification using various combinations of PCR primers to the type I and II genes, particularly those primers that previously amplified. novel PCR products from genomic DNA. Amplification products from ). clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3/~- HSD gene family were found, one of which locates to the 3/3-HSD type I and II gene cluster on lp13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from 2 clones to search for novel members of the gene family. KEYWORD$" 3/3-hydroxysteroid dehydrogenase, pseudohermaphroditism, gene family, denaturing gradient gel electrophoresis. INTRODUCTION The steroidogenic enzyme 3/3-hydroxysteroid de- hydrogenase (3/3-HSD) converts 3/~-hydroxy-As pre- cursors to 3-keto-zJ 4 products and initiates the pathways of mineralocorticoid, glucocorticoid and sex hormone biosynthesis.' Two forms of 3/3-HSD have been described to date in humans; the type I enzyme is expressed principally in placenta and skin and type II in adrenals and gonads. 2~ The type I and II genes are very similar in structure, consisting of a 5' untranslated exon and three coding exons (Fig. 1) * Author to whom correspondenceshould be addressed. 0890-8508195/020121 + 08 $08.0010 121 © 1995 Academic Press Limited