Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment.
To investigate the molecular basis and physiological regulation of exceptionally high levels of aromatase (P450arom) activity in the brain of teleost fish, a 2927 bp P450arom cDNA encoding a 510 amino acid protein was isolated from a goldfish brain cDNA library. The brain-derived cDNA had 53% and 61-62% sequence identity when compared with human placental and fish ovarian P450arom forms, respectively, and higher homologies in conserved functional domains. Goldfish brain poly(A) RNA was translatable in vitro to a 56 kDa P450arom immunoprecipitation product. Northern blot analysis using the brain cDNA revealed a major 3.0 kb transcript of high abundance in brain (FB, forebrain > M/HB, mid/hindbrain), but no signal in ovary, testis or liver. P450arom mRNA varied seasonally in brain, with a peak at the onset of gonadal regrowth (February) that preceded the annual rise in enzyme levels and was 4-fold (FB) or 50-fold (M/HB) higher than during reproductive inactivity (July-December). Known markers of neurogenesis and estrogen action in brain (28S rRNA, beta-actin and beta-tubulin transcripts) each had unique seasonal patterns which differed from P450arom mRNA. In vivo steroid treatment showed that estrogen and aromatizable androgen increase FB and M/HB levels of P450arom mRNA 8- and 4-fold, respectively. P450arom mRNA in pituitary and retina had a different regulation. Southern analysis provided no evidence for multiple genes encoding the brain derived cDNA or for brain-specific gene amplification. Results imply that high accumulated levels of P450arom mRNA are the major determinant of high measured enzyme activity in goldfish brain, and that physiological regulation of mRNA expression in the natural environment is mediated by aromatization of androgen to estrogen.
Department of Biology, Boston University, MA 02215, USA.
D
D
Gelinas
G A
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Pitoc
G V
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Callard
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D018076:DNA, Complementary; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006054:Goldfish; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010053:Ovary / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D012621:Seasons; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013737:Testis / Q000201:enzymology
D018076:DNA, Complementary; D001141:Aromatase