Human ϵ-globin and β-globin mRNAs display different stabilities in cultured cells. (A) Structures of doxycycline-conditional genes encoding human ϵ- and β-globin mRNAs. TRE-hϵ and TRE-hβ were constructed by inserting the full-length human ϵ-globin gene (gray) and β-globin gene (black) into plasmid pTRE-2, immediately downstream of the TRE transcriptional control element (diagonal shading). Important structural features of both genes are indicated. (B) Representative transcriptional chase analysis of globin mRNA stability in erythroid cells. tTA-expressing MEL cells were cotransfected with TRE-hϵ and TRE-hβ and total RNA prepared at defined intervals following doxycycline (dox) exposure. Levels of hϵ- and hβ-globin mRNAs were determined by RPA using [³²P]-labeled mRNA-specific probes. Aliquots containing 2- and 4-fold excess of the T = 0 sample were assessed in parallel to ensure assay linearity (lanes 2X and 4X). The interval after doxycycline exposure (top) and the positions of the protected hϵ- and hβ-probe fragments are indicated. (C) Human ϵ- and β-globin mRNAs are differentially stable in erythroid MEL cells. The study described in panel B was performed in triplicate. The hϵ/hβ band intensities at defined intervals after doxycycline exposure were determined by PhosphorImager densitometry and average values plotted. A gray line emphasizes the temporal hϵ/hβ ratio that would be observed if the 2 mRNAs were equally stable. Error bars indicate 1 SD. (D) Representative transcriptional chase analysis of globin mRNA stability in nonerythroid cells. Total RNA from tTA-expressing HeLa cells that had been cotransfected with TRE-hϵ and TRE-hβ was analyzed as described in panel B. Linearity controls have been cropped to preserve image clarity. (E) Human ϵ- and β-globin mRNAs are differentially stable in nonerythroid HeLa cells. The study described in panel D was performed in triplicate; average hϵ/hβ band intensities are plotted. Error bars indicate 1 SD.