The conversion of
androgens to
estrogens is catalyzed by a complex of
enzymes that includes a specific
cytochrome P-450 aromatase (P-450arom). In this paper we describe the high level expression of
aromatase activity in the rat
Leydig cell tumor line, R2C. We also report the isolation of
cDNA clones encoding the rat
aromatase P-450arom from a cDNA library prepared from this cell line. Analysis of these
cDNA clones predicts a
protein sequence with a high degree of sequence conservation when compared to the chicken and human P-450arom
enzymes. Notably, four of the
cDNA clones were found to lack the last coding exon that contains the
heme-binding domain, a structural feature essential for
aromatase activity. These clones were found to contain instead a segment of genomic
DNA derived from an unspliced intron. Northern analysis using a fragment of the coding region of the rat P-450arom
cDNA as probe revealed that three species of P-450arom
mRNa are expressed in rat ovary that are similar to those identified in
RNA samples prepared from the rat R2C cell line. Analysis of the same samples of
RNA using a probe derived from the 3' terminal intron segment of the rat
aromatase cDNA clones that lack the
heme-binding domain indicates that two of the species of
aromatase mRNA transcripts present in both rat ovary and R2C cell lack the
heme-binding domain and thus must encode a nonfunctional
aromatase protein. These findings have important implications for the measurement of
aromatase mRNA and appear to explain why three sizes of rat P-450arom
mRNA exist on Northern analysis and why previous studies failed to demonstrate a clear relationship between
aromatase mRNA,
protein, and enzymatic activity in the rat ovary.