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Department of Biology, Boston University, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Gloria V. Callard, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: gvc{at}bio.bu.edu
Evidence to date indicates that the gene encoding cytochrome P450
aromatase (P450arom) in humans is a single member of the CYPl9 family,
but multiple CYPl9 loci and isoforms have been identified in pigs. Here
we report the cloning and characterization of a second member of the
CYP19 family in goldfish. A search for P450arom variants was prompted
by studies showing that a full-length P450arom complementary DNA (cDNA)
isolated from a goldfish brain cDNA library hybridizes with a high
abundance 3 kb transcript in brain RNA but fails to detect a message in
ovarian RNA. A stepwise PCR cloning strategy led to isolation of a
1.9-kb cDNA, which encodes a protein of 518 amino acids and has a
predicted mol wt of 58.7K. The ovary-derived P450arom (-A) shares
68–72% sequence identity with ovarian aromatases of other fish
species, but only 62% identity with the homologous brain-derived
P450arom (-B). Amino acid differences are distributed throughout the
two goldfish P450arom forms, but presumptive functional domains are
highly conserved. Both P450aromA and -B are able to aromatize
[3H]androgen to [3H]estrogen when expressed
in nonsteroidogenic COS cells. Southern analysis and PCR-restriction
analysis of genomic DNA using discriminating probes and primers
indicates that a single locus encodes the brain-derived P450aromB
(CYPl9B), whereas one or two different loci encode the ovarian form
(CYPl9A). Northern blot analysis revealed two P450aromA messenger RNAs
(1.9 >> 3.0 kb) in ovary. Simultaneous PCR amplification with A- and
B-specific primer pairs confirms that P450aromA is the only form
expressed in ovaries, but shows overlapping expression of the two genes
in neural tissues. Whereas P450aromB messenger RNA predominates in
brain (B/A, 
14:1), the ratios are reversed in retina (B/A, 1:25).
Further studies are required to resolve the evolutionary and functional
implications of multiple CYPl9 genes and P450arom isozymes in goldfish,
their differential expression in brain and ovary, and whether
observations can be generalized to other vertebrates.
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