Molecular cloning of cDNA for rat ovarian 20 alpha-hydroxysteroid dehydrogenase (HSD1).
20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD, EC 1.1.1.149) catalyses the conversion of progesterone into 20 alpha-dihydroprogesterone (20 alpha-OHP). Previously, we purified the enzyme (37 kDa) from rat ovary and determined its N-terminal amino acid sequence. In the present study we succeeded in cloning a full-length 20 alpha-HSD cDNA. mRNA was extracted from immature rat ovaries after successive treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). A cDNA library was constructed in lambda ZAP. For screening, a 576 bp probe was amplified by the PCR using mixed primers based on the N-terminal sequence of 20 alpha-HSD, and labelled with [32P]dCTP. Eight positive clones were isolated from 1.2 x 10(4) recombinants. Analysis of the nucleotide sequence revealed that one clone of 1.2 kbp cDNA (pHSD12-07) contained a polyadenylation site and an open reading frame encoding 323 amino acids with the N-terminal sequence of 20 alpha-HSD. The fusion protein of pHSD12-07 produced by Escherichia coli reacted with a specific polyclonal antibody generated against rat ovarian 20 alpha-HSD. In addition, the in vitro transcription-translation product produced by Xenopus oocytes showed 20 alpha-HSD activity and Northern-blotting analysis revealed that the ovaries from normal adult rats contained a 1.2 kb mRNA. Thus we succeeded in isolating a clone encoding the full length of rat ovarian 20 alpha-HSD. The sequence showed high similarity with those of rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), bovine lung prostaglandin F synthase (PGFS), human liver chlordecone reductase (CDR), frog lens rho-crystallin and aldose reductases, indicating that 20 alpha-HSD belongs to the aldo-keto reductase family.
Miura
R
R
Laboratory of Cellular Biochemistry, University of Tokyo, Japan.
Shiota
K
K
Noda
K
K
Yagi
S
S
Ogawa
T
T
Takahashi
M
M
D015089:20-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D009865:Oocytes / Q000378:metabolism; D010053:Ovary / Q000187:drug effects / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014982:Xenopus laevis
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase