Isolation and characterization of the cDNA encoding the channel catfish (Ictalurus punctatus) form of cytochrome P450arom.
Cytochrome P450arom (aromatase) is responsible for the conversion of androgens to estrogens. A cDNA library was constructed from poly(A)-enriched mRNA isolated from channel catfish (Ictalurus punctatus) ovary and ligated into EcoRI-cut lambda arms. The amplified library was screened using a human aromatase DNA probe. The longest clone isolated (2.1 kb) contained 20 bp of the 5'-untranslated region, a 1572-bp open reading frame, and a 509-bp 3'-untranslated region. Northern blot analysis indicated a single 3.4-kb transcript. A putative polyadenylation signal (AATAAA) is 13 bp from the 3'-end of the clone. The deduced amino acid sequence of the catfish form of aromatase is 65% identical to the rainbow trout form. The catfish form is 50 to 53% identical to mammalian (rat, human, mouse, bovine) and chicken forms. There are large regions of extremely high identity shared among all the forms. The deduced catfish aromatase protein is 524, 517, or 502 residues in length, depending on the translation initiation site. Steroidogenic activity of gonadal tissue is correlated with mRNA and protein content. Nonsteroidogenic COS cells, transfected with the catfish aromatase cDNA, converted androstenedione to estrone, thus verifying its identify. The catfish P450arom was modified to facilitate production in transformed Escherichia coli and its subsequent purification by a single column technique. This purified protein was used to raise antisera for Western blot analysis. Western blot analysis indicates that the catfish form is similar in size (57 kDa) to other vertebrate forms.
Trant
J M
JM
Department of Zoology and Physiology, Louisiana State University, Baton Rouge 70803.
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007059:Ictaluridae / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012098:Reproduction / Q000502:physiology; D012621:Seasons; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D001141:Aromatase