NEW MEMBERS OF THE 3-BETA-HYDROXYSTEROID DEHYDROGENASE GENE FAMILY

Several bands of hybridization are detected when southern blots of hum an genomic DNA are probed with cDNA of 3 beta-hydroxysteroid dehydroge nase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly primers desig ned to amplify 3 beta-HSD type I and II genes were found on occasion t o amplify DNA products of appropriate length but which were resolved a s distinct sequences by denaturing gradient gel electrophoresis (DGGE) . Five of these novel bands were cloned and their sequences were found to be closely related to 3 beta-HSD types I and II. Secondly, 57 geno mic clones were selected from two lambda genomic libraries by hybridiz ation with exonic probes of 3 beta-HSD type I. These were screened for novel members of the gene family by PCR amplification using various c ombinations of PCR primers to the type I and II genes, particularly th ose primers that previously amplified novel PCR products from genomic DNA. Amplification products from lambda clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3 beta-HSD gene family were found, one of which locate s to the 3 beta-HSD type I and II gene cluster on 1p13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fr agments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from l ambda clones to search for novel members of the gene family.