The cDNA cloning and transient expression of a chicken gene encoding a 3β-hydroxysteroid dehydrogenase/Δ5→4 isomerase unique to major steroidogenic tissues

Osamu Nakabayashi, Osamu Nomura, Katsuhiko Nishimori, Shigeki Mizuno

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Two cDNA clones, one containing a 2.7-kb and the other a 1.6-kb insert, were obtained from a chicken adrenal gland cDNA library by screening with a partial chicken cDNA fragment generated by PCR. Primers were used that corresponded to conserved regions among several published cDNA sequences of mammalian 3β-hydroxysteroid dehydrogenase/Δ5→4 isomerase (3β-HSD). Both clones contained the same open reading frame (ORF) encoding 377 amino acids (aa). The difference in their sizes was due to the use of different polyadenylation sites. The deduced aa sequence showed 54-57% overall identity with those of the human, macaque, bovine, mouse, rat and rainbow trout 3β-HSD. A 9-aa stretch (FYYISDDTP) is conserved among the chicken, mammalian and rainbow trout 3β-HSD, suggesting that it is essential for enzymatic activity. Northern blot hybridization demonstrated the presence of two types of mRNA, corresponding to the above two cDNA classes, in the adrenal gland, ovary and testis. Transient expression of the chicken ORF in COS-7 cells demonstrated coupled dehydrogenase and isomerase activitsies of 3β-HSD on three different substrates; pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone. Among the Km values determined for the three substrates, that for dehydroepiandrosterone was the lowest, which was different from the case for human 3β-HSD.

Original languageEnglish
Pages (from-to)261-265
Number of pages5
JournalGene
Volume162
Issue number2
DOIs
Publication statusPublished - 1995 Sep 11

Keywords

  • 0.15 M NaCl/0.015 M Na·citrate pH 7.0
  • 17-OH-Preg
  • 17-OH-Prog
  • 17α-hydroxypregnenolone
  • 17α-hydroxyprogesterone
  • 3β-HSD
  • 3β-hydroxysteroid dehydrogenase/Δ isomerase
  • AD
  • Conserved sequence region
  • DHEA
  • DNA complementary to RNA
  • ER
  • GAPDH
  • ORF
  • PCR
  • Preg
  • Prog
  • RT
  • SDS
  • SSC
  • TLC
  • UTR
  • aa
  • amino acid(s)
  • androstenedione
  • base pair(s)
  • bp
  • cDNA
  • dehydroepiandrosterone
  • endoplasmic reticulum
  • enzyme kinetics
  • gene (cDNA) encoding 3β-HSD
  • glyceraldehyde 3-phosphate dehydrogenase
  • kb
  • kilobase(s) or 1000 bp
  • nt
  • nucleotide(s)
  • open reading frame
  • polymerase chain reaction
  • pregnenolone
  • progesterone
  • reverse transcriptase
  • sodium dodecyl sulfate
  • thinlayer chromatography
  • transfection
  • untranslated region(s)

ASJC Scopus subject areas

  • Genetics

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