DIFFERENTIAL DISPLAY RT PCR OF TOTAL RNA FROM HUMAN FORESKIN FIBROBLASTS FOR INVESTIGATION OF ANDROGEN-DEPENDENT GENE-EXPRESSION

Male sexual differentiation is a process that involves androgen action via the androgen receptor, Defects in the androgen receptor, many res ulting from point mutations in the androgen receptor gene, lead to var ying degrees of impaired masculinization in chromosomally male individ uals, To date no specific androgen regulated morphogens involved in th is process have been identified and no marker genes are known that wou ld help to predict further virilization in infants with partial androg en insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genita l skin fibroblasts cultured in the presence or absence of 100 nM testo sterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens, Most of these sequences show the charact eristics of expressed mRNAs but showed no homology to sequences in the database, However 15 clones with significant homology to previously c loned sequences were identified, Seven cDNAs appear to be induced by a ndrogen withdrawal, Of these, five are similar to ETS (expression tagg ed sequences) from unknown genes; the other two show significant homol ogy to the cDNAs of ubiquitin and human guanylate binding protein 2 (G BP-2), In addition, we have identified 8 cDNA clones which show homolo gies to other sequences in the database and appear to be upregulated i n the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences tha t appear to be upregulated in the presence of testosterone show signif icant homology to the cDNAs of L-plastin and one to the cDNA of testic an. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The result s of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression. (C) 1996 Wiley-Liss, In c.