New members of the 3 beta-hydroxysteroid dehydrogenase gene family.
Several bands of hydridization are detected when southern blots of human genomic DNA are proved with cDNA of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly, primer designed to amplify 3 beta-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 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 genomic clones were selected from two lambda genomic libraries by hybridization with exonic probes of 3 beta -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 (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 locates 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 fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from (lambda) clones to search for novel members of the gene family.
McBride
M W
MW
Institute of Genetics, Glasgow University.
Russell
A J
AJ
Vass
K
K
Forster
V
V
Burridge
S M
SM
Morrison
N
N
Boyd
E
E
Ponder
B A
BA
Sutcliffe
R G
RG
D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D015342:DNA Probes; D005091:Exons; D015698:Genomic Library; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D015342:DNA Probes; D007527:Isoenzymes; D011994:Recombinant Proteins; D015096:3-Hydroxysteroid Dehydrogenases