Isolation of differentially expressed sequence tags from steroid-responsive cells using mRNA differential display.
Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3' end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.
Chapman
M S
MS
Department of Biochemistry, University of Arizona, Tucson 85724, USA.
Qu
N
N
Pascoe
S
S
Chen
W X
WX
Apostol
C
C
Gordon
D
D
Miesfeld
R L
RL
D000728:Androgens / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000737:chemistry / Q000166:cytology; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000032:analysis / Q000235:genetics; D016324:Sequence Tagged Sites; D013950:Thymus Gland / Q000737:chemistry* / Q000166:cytology
D000728:Androgens; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D004247:DNA