Quantitative detection of alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in aromatase-expressing human cells by competitive RT-PCR.
C19 steroids are converted to oestrogens in a number of tissues by a specific form of cytochrome P450, namely aromatase P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II-X. The tissue-specific expression is determined by the use of tissue-specific promoters, which give rise to P450arom transcripts with unique 5'-untranslated sequences. The majority of the transcripts present in the ovary contain promoter-II-specific sequences, while transcripts in the placenta contain exon I.1. Transcripts in adipose tissue possess exon I.3 and exon I.4. Also, the distribution of alternative transcripts in adipose stromal cells depends on the culture conditions. Therefore, a competitive RT-PCR method was designed to quantitatively detect alternatively spliced transcripts present in various tissues and cells maintained in different culture conditions. Specific synthetic transcripts with different 5' termini (exon I.3, exon I.4 and promoter-II-specific sequences) and the coding region were used as internal standards. This competitive RT-PCR method was used to quantitatively detect three 5' termini, i.e. promoter-II-specific sequence, exon I.3 and exon I.4, in transcripts in human adipose stromal cells and ovarian granulosa cells in primary culture. The quantity of total P450arom transcripts was judged by amplifying the coding region. We were also able to quantify rare transcripts which could not be detected previously by Northern analysis.
Agarwal
V R
VR
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, USA.
Bulun
S E
SE
Simpson
E R
ER
D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D001141:Aromatase / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017931:DNA Primers; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005091:Exons / Q000235:genetics; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D017370:Interleukin-11 / Q000494:pharmacology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D012680:Sensitivity and Specificity
D017931:DNA Primers; D017370:Interleukin-11; D012333:RNA, Messenger; D005576:Colforsin; D003907:Dexamethasone; D001141:Aromatase