ISOLATION OF DIFFERENTIALLY EXPRESSED SEQUENCE TAGS FROM STEROID-RESPONSIVE CELLS USING MESSENGER-RNA DIFFERENTIAL DISPLAY

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited nu mber of target genes. Although much is known about the structure and f unction of steroid receptors, relatively few cell-specific steroid-reg ulated genes have been isolated and characterized. In this paper we de scribe results using mRNA differential display reverse transcriptase P CR (DDPCR) to identify and isolate short cDNA sequence tags from thymo cyte and prostate cells under Various hormone conditions. Using this t echnique we have isolated several differentially expressed sequence la gs (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these D ESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homolo gue of a human expressed sequence tag isolated from activated B lympho cytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to c orrespond to the 3' end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR pro tocol, which we found can potentially decrease the frequency of isolat ing false-positive DESTs, are described and the merits of DDPCR, relat ive to other differential cDNA cloning strategies, are discussed.