Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary.
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, approximately 14:1), the ratios are reversed in retina (B/A, approximately 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.
Tchoudakova
A
A
Department of Biology, Boston University, Massachusetts 02215, USA.
Callard
G V
GV
D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005260:Female; D006031:Glycosylation; D006054:Goldfish / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010053:Ovary / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D001141:Aromatase