Fig. 4

Alternative splicing out of the entire EK in the WT Ler genome. a Three alternative variants (SV1, SV2 and SV3) of splicing out of the entire EK revealed by direct sequencing of the PCR product amplified by primers flanking the EK insertion site (P6 + P7). Vertical broken line marked the border with exon 14. The sequences of the three spliced variants and of the proximal exon 14 are shown. b Analysis of the proportion of spliced variants. An example of PCR analysis of spliced variants (SV1 and SV2 + SV3) cloned into pJET1.2 (lanes 1–15). Randomly isolated colonies were subjected to PCR followed by separation on 2.5% agarose gel. The position and expected sizes of the various spliced variants are shown on the right. M indicates the DNA size markers. c CMT1-EK spliced variant 1 (SV1). The proportion (58%) of SV1 in WT Ler is indicated. The DNA sequence at the EK insertion site is shown with EK marked in blue and intron 13 in red. The fragment spliced out from the chimeric transcript is indicated by a horizontal bracket. The splicing donor (GT) and acceptor (AG) sites are boxed yellow. DNA sequences related to exons 13 and 14 are indicated. The chromatogram and sequence of pJET1.2-SV1 as well as amino acid sequence and premature stop codon (underlined) are shown. d CMT1-EK spliced variant 2 (SV2) yielding the correct CMT1 reading frame. The proportion (25%) of SV2 in WT Ler is indicated. The DNA sequence at the EK insertion site is shown with EK marked in blue and intron 13 in red. Horizontal brackets indicate the fragments spliced out from the chimeric transcript. The splicing donor (GC, non-canonical) and acceptor (AG) sites for the EK and the GT-AG canonical splicing sites for intron 13 are boxed yellow. DNA sequences related to exons 13 and 14 are indicated. The chromatogram and sequence of pJET1.2-SV2 and the amino acid sequence are shown. e CMT1-EK spliced variant 3 (SV3). The proportion (17%) of SV3 in WT Ler is indicated. The DNA sequence at the EK insertion site is shown with EK marked in blue and intron 13 in red. Horizontal brackets indicate the fragments spliced out from the chimeric transcript. The canonical GT-AG splicing sites are boxed yellow. DNA sequences related to exons 13 and 14 are indicated. The chromatogram and sequence of pJET1.2-SV3 as well as amino acid sequence and premature stop codon (underlined) are shown. The green, red, blue and black peaks in all chromatograms represent the bases ‘A’, ‘T’, ‘C’ and ‘G,’ respectively