Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.
Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.
Bobes
Raúl José
RJ
Dpto. de Fisiología, Biofisica y NC, CINVESTAV-IPN, Apdo. Postal 14-740, 07000 México D.F., Mexico.
Miranda
Carolina
C
Pérez-Martinez
Mario
M
Luu-The
Van
V
Romano
Marta C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry* / Q000302:isolation & purification*; D005260:Female; D006041:Goats / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D017385:Sequence Homology
D018076:DNA, Complementary; D012333:RNA, Messenger; D001141:Aromatase