FIG. 7.

Effect of the FR-α sequence from nt 234 to 293 on mRNA stability of FR-β. Cells were cotransfected with the Tet-Off plasmid and the expression plasmid pTRE, which contained either FR-β cDNA or chimeric FR in which the FR-α cDNA sequence from nt 234 to 293 was substituted in FR-β. At 48 h posttransfection, the cells were treated with 1 μg of Dox/ml to selectively turn off expression of the transfected FR for the indicated periods of time before extraction of total RNA or nuclear RNA. (A) mRNA stability in the nucleus. Nuclear RNA was purified from isolated nuclei, and mRNA for FR-β or the chimera was quantitated by RT-PCR. For HeLa cells, input x represents 0.25 μg of nuclear RNA; for MG63 cells, input x represents 0.5 μg of nuclear RNA. Before the reverse transcription reaction was initiated, 1 pg of FR-β cRNA with an internal deletion from nt 642 to 822 (FR β del) was added to each reaction tube to serve as an internal standard. The reaction products were electrophoresed on a 6% polyacrylamide gel, and the band intensities were quantified by phosphorimage analysis. Negative controls, which did not generate PCR products (data not shown), included reactions without either RNA or reverse transcriptase. The PCR was carried out for 20 cycles, and the amplification was shown to be in the linear range by using different amounts of input RNA (complete data not shown). The numbers at the bottom of each gel represent band intensities normalized against those of the corresponding cRNA standard; the error from triplicate data sets was ≤15%. (B) Total mRNA stability. For HeLa cells, input x represents 0.25 μg of total RNA; for MG63 cells, input x represents 0.5 μg of total RNA. All other procedures were carried out in the same way as for the nuclear RNA. The numbers at the bottom of each gel represent band intensities normalized against those of the corresponding cRNA standard; the error from triplicate data sets was ≤15%.