Molecular cloning of a cDNA showing alternative splicing of the 5′‐untranslated sequence of mRNA for human aromatase P‐450

Abstract

A new type of full-length cDNA clone encoding human aromatase P-450 was isolated from a human placental cDNA library. The clone, designated as pES-4, has a 3130-bp insert. The nucleotide sequences of the translated region and the 3'-untranslated region of the insert of pES-4 are exactly identical with those of the cDNA clone characterized previously. However, the sequence of the 5'-untranslated region of the insert has characteristic feature, i.e. an extra sequence of 109 bp is present at a junction between exon 1 and exon 2 on the processed human aromatase mRNA. Analysis of the genomic clones containing the region between exon 1 and exon 2 of the human aromatase P-450 gene reveals that the 109-bp genomic segment, encoding the same sequence as the extra sequence observed in pES-4, is located approximately 10-kbp downstream of exon 1 and that the nucleotide sequences of the 5'-flanking and the 3'-flanking regions of the segment conform to the GT-AG rule for RNA splicing. By means of reverse transcription and polymerase chain reaction, relative amounts of the pES-4-type mRNA are estimated to be approximately 4.8% and 2.3% of the processed aromatase P-450 mRNA in human placenta and human BeWo choriocarcinoma cells, respectively. These results indicate that the segment of 109 bp between exon 1 and exon 2 is a new exon hitherto unidentified and that heterogeneity observed in the 5'-untranslated sequence of human aromatase P-450 mRNA is, at least in part, caused by alternative splicing of this new exon.