FIG. 5.

RT-PCR analysis of trans splicing of mutant leaders. (A) Lanes 1 to 3, trans splicing of SL1 or mutant leaders to the myo-3 message in wild-type (N2) elongated embryos; lanes 4 to 6, homozygous wDf1 embryos rescued with the wild-type SL1 RNA transcribed from the U2-3 promoter (WT-SL1); lanes 7 to 9, homozygous wDf1 embryos (wDf1); lanes 10 to 12, wDf1 embryos carrying the 11–20 shuffle transgene. Three independent RNA samples were prepared and analyzed for each strain. Primers specific for the wild-type SL1 leader or for the 11–20 shuffle leader, together with a downstream primer specific for the myo-3 coding region, were used in the RT-PCRs (see Materials and Methods). The myo-3 message is embryonically transcribed (9), and both the message and protein are expressed robustly in wDf1 mutant embryos (12), although no trans splicing to SL1 is detected (therefore, these reactions serve as negative controls for contamination in the RT-PCR). There are no detectable bands in lanes 7 to 12, as described above, even in overexposed autoradiograms. The bands present in lanes 4 and 6, which appear faint in this exposure, are clearly evident in overexposed autoradiograms (data not shown). (B) RT-PCR analysis of the myo-3 message in wDf1 embryos carrying the 11–20 shuffle (lanes 1 to 3) transgene, using primers specific for an internal portion of the myo-3 message (see Materials and Methods). The positions of the molecular size markers (in base pairs) are shown at the left.