Figure 1.
Expanded GAA- and TTC-Triplet Repeats Did Not Affect the Levels of Nascent Transcription
(A) Schematic representation of pEDA minigenes. Gray and white boxes correspond to α-globin and fibronectin exonic sequences, respectively; lines indicate intronic sequences. The position of the GAA and TTC repeats is indicated. The position of the u1, d1, and d2 nascent-transcript amplification products is indicated. The length and position of the M13 antisense Nuclear Run On probes relative to the minigenes are shown.
(B) Typical NRO analysis of HeLa cells. Cells were transfected with one of the indicated minigenes together with the cotransfection control VA construct. M13 corresponds to the empty M13 vector. Antisense single-strand M13 probes (A to F and VA) are indicated at the top.
(C) The graph shows the VA-normalized quantitation of three independent transfections relative to the values obtained with the pEDA (black columns) constructs over the corresponding probes. Data are expressed as means + SD. Light-gray and white columns show transcription levels obtained with pEDA NcoGAA and pEDA NcoTTC minigenes. All signals are corrected for the background hybridization shown in the M13-negative control.
(D) Analysis of pre-mRNA transcript abundance. The indicated minigenes were transfected in COS cells, and the resulting RNAs were coamplified with the u1-d1 (top) or u1-d2 (bottom) primer set and resolved on agarose gels. The pEDA without repeats is indicated with Ø. Controls without reverse transcriptase (RT) and untransfected cells (C) are indicated. M is the molecular 1 Kb marker.