Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1)

R. Miura, K. Shiota, K. Noda, S. Yagi, T. Ogawa, M. Takahashi

Research output: Contribution to journalArticle

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Abstract

20α-Hydroxysteroid dehydrogenase (20α-HSD, EC 1.1.1.149) catalyses the conversion of progesterone into 20α-dihydroprogesterone (20α-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 20α-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 λZAP. For screening, a 576 bp probe was amplified by the PCR using mixed primers based on the N-terminal sequence of 20α-HSD, and labelled with [32P]dCTP. Eight positive clones were isolated from 1.2 x 104 recombinants. Analysis of the nucleotide sequence revealed that one clone of 1.2 kbp cDNA (pHSD12-07) contained a poly-adenylation site and an open reading frame encoding 323 amino acids with the N-terminal sequence of 20α-HSD. The fusion protein of pHSD12-07 produced by Escherichia coli reacted with a specific polyclonal antibody generated against rat ovarian 20α-HSD. In addition, the in vitro transcription-translation product produced by Xenopus oocytes showed 20α-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 20α-HSD. The sequence showed high similarity with those of rat liver 3α-hydroxysteroid dehydrogenase (3α-HSD), bovine lung prostaglandin F synthase (PGFS), human liver chlordecone reductase (CDR), frog lens p-crystallin and aldose reductases, indicating that 20α-HSD belongs to the aldo-keto reductase family.

Original languageEnglish
Pages (from-to)561-567
Number of pages7
JournalBiochemical Journal
Volume299
Issue number2
Publication statusPublished - 1994
Externally publishedYes

Fingerprint

20-Hydroxysteroid Dehydrogenases
Cloning
Molecular Cloning
Rats
Complementary DNA
Ovary
prostaglandin-F synthase
Clone Cells
Chorionic Gonadotropin
Liver
3-Hydroxysteroid Dehydrogenases
20-alpha-Dihydroprogesterone
Equine Gonadotropins
Amino Acids
Aldehyde Reductase
Messenger RNA
Crystallins
Transcription
Xenopus
Gene Library

ASJC Scopus subject areas

  • Biochemistry

Cite this

Miura, R., Shiota, K., Noda, K., Yagi, S., Ogawa, T., & Takahashi, M. (1994). Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1). Biochemical Journal, 299(2), 561-567.

Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1). / Miura, R.; Shiota, K.; Noda, K.; Yagi, S.; Ogawa, T.; Takahashi, M.

In: Biochemical Journal, Vol. 299, No. 2, 1994, p. 561-567.

Research output: Contribution to journalArticle

Miura, R, Shiota, K, Noda, K, Yagi, S, Ogawa, T & Takahashi, M 1994, 'Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1)', Biochemical Journal, vol. 299, no. 2, pp. 561-567.
Miura R, Shiota K, Noda K, Yagi S, Ogawa T, Takahashi M. Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1). Biochemical Journal. 1994;299(2):561-567.
Miura, R. ; Shiota, K. ; Noda, K. ; Yagi, S. ; Ogawa, T. ; Takahashi, M. / Molecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehydrogenase (HSD1). In: Biochemical Journal. 1994 ; Vol. 299, No. 2. pp. 561-567.
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