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 resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen 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 genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.
Nitsche
E M
EM
W.A. Jones Cell Science Center, Lake Placid, New York, USA.
Moquin
A
A
Adams
P S
PS
Guenette
R S
RS
Lakins
J N
JN
Sinnecker
G H
GH
Kruse
K
K
Tenniswood
M P
MP
D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007231:Infant, Newborn; D007256:Information Systems; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D016324:Sequence Tagged Sites; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D013739:Testosterone / Q000494:pharmacology*; D014452:Ubiquitins / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011944:Receptors, Androgen; D014452:Ubiquitins; D013739:Testosterone; D019204:GTP-Binding Proteins