Figure 1.
Schematic representation of the analysis of PLP1 gene structure with the ESEfinder, Human Splicing Finder and FAS-ESS programs.
(a) Schematic representation of PLP1 gene structure (7 exons) and the alternative splicing of the PLP1 gene into the major PLP1 transcript and a minor transcript encoding the shorter DM20 isoform, which differs only in terms of the latter half of exon 3 (3B) which is post-transcriptionally spliced out. (b) Only part of the sequence of exon 3A (sequence in black type) and exon 3B (sequence in red type) mutant PLP1 transcript is represented. The mutated nucleotide (c.436C>G) is underlined. According to the ESEfinder 3.0 program, the c.436C>G change created an exonic splicing enhancer (EES, green trapezium), whereas Human Splicing Finder version 2.4.1 and FAS-ESS web server predicted the creation of two exonic splicing silencers (ESS, purple trapezia).
Figure 2.
RT-PCR from Oli-neu cells transfected with the wild-type and mutated minigenes.
Lane U: untransfected cells; lane WT: cells transfected with the wild-type minigene construct; lane SM: cells transfected with the minigene construct harbouring the c.436C>G mutation; lane DM: cells transfected with the minigene construct harbouring the double mutation [c.436C>T plus c.437T>A]; lane C: negative (no template) control; lane M: φX174 DNA HaeIII-restricted molecular weight marker. The minigene-specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively. The asterisk denotes samples transfected with the pcDNA3.1/V5-His-TOPO/LacZ-PLP1-LacZ vector.
Figure 3.
Analysis of morpholino treatment: RT-PCR from minigene-transfected morpholino-treated Oli-neu cells.
RNA was extracted from cells transfected with the wild-type (Wt) and mutant (c.436C>G) minigene constructs (Mut, 2, 4, 6, 8, 10). Samples 2, 4, 6, 8 and 10 were treated with the morpholino oligonucleotide, whereas samples Mut and Wt were untreated. Treated cells received 10 mM morpholino oligonucleotide in 2, 4, 6, 8, 10 mM Endoporter reagent; lane C: no template control; lane M: φX174 DNA HaeIII restricted molecular weight marker. The minigene-specific primer 31GF/LACT2R-mediated RT-PCR products are 711 bp (PLP product) and 606 bp (DM20 product) in length, respectively.