Figure 5.

Transcription-induced instability of the (GAA•TTC)_n sequence is related to transcript stability. pT115H, which showed transcription-induced expansions and contractions, was modified to prevent splicing by introducing base substitutions in the splice donor and acceptor sites to generate pT115HM. Both constructs, transfected in COS Tet-On cells in the absence or presence of doxycycline-induced transcription, were analyzed for splicing efficiency, transcript stability, and mutational spectrum of the (GAA•TTC)₁₁₅ sequence. (A) RT-PCR of RNA extracted from transfected COS Tet-On cells in the presence of doxycycline showed the predicted spliced product (115 bp) in pT115H but not in pT115HM (which only showed the unspliced product of 246 bp), indicating that the splice site mutations prevented splicing. Note that splicing of the synthetic intron in pT115H was inefficient. (B) Transcript stability was measured by estimating the amount of residual transcript upon withdrawal of doxycycline via real-time quantitative RT-PCR of RNA extracted at hourly intervals. pT115HM-derived transcript was significantly unstable compared with pT115H, showing a precipitous fall in transcript level to ∼10% within 2 h. The pT115H transcript was comparatively stable, remaining at ∼70% through 4 h. Error bars (SEM) are from triplicate RT-PCR assays of two separate experiments. (C and D) Expansions and contractions were analyzed by comparing the background instability prior to transfection (white bars) to instability following replication in the absence (gray bars) or presence (black bars) of doxycycline-induced transcription. pT115HM did not show the transcription-induced expansions seen with pT115H, but the contractions were seen with both constructs. The same result, i.e. increased contractions but abolition of expansions was also seen with an independent mutant of pT115H, made by removal of the synthetic intron (pT115HR). P-values are derived using Chi-square test (*P < 0.05 and **P < 0.01).