pdk-1 loss-of-function mutation sa709 is epistatic to zfp-1(ok554) and rde-4(ne299).

(A) Schematic of the pdk-1 gene with numbered boxes for exons and lines for introns, location of the sa709 mutation and predicted effects of the mutation on mRNA and protein; exons coding for the kinase domain and the pleckstrin homology domain (PH) according to [4] are indicated. (B) Expression levels of correctly spliced pdk-1 mRNA and intron 3-containing mRNA were determined by RT-qPCR, and ratios of spliced/unspliced isoforms in the indicated mutants were calculated and presented on a graph. The forward primer used spanned the exon 1/exon 2 junction, the reverse primer for the spliced isoform spanned the exon 3/exon 4 junction and the reverse primer for the intron 3-containing isoform was intron 3-specific (see Materials and Methods). (C) Intron 3-containing pdk-1 mRNA levels were measured by real time RT-qPCR in indicated mutants (L4 stage animals) and normalized to pdk-1(sa709). Results of two biological replicas are shown; error bars represent Standard deviation. (D, E) DAF-16::GFP nuclear localization in indicated mutants was assessed as described in Materials and Methods. Representative epifluorescence images of intestinal cells (D) were taken on a Zeiss AxioImager Z1 microscope at 630x total magnification; white arrowheads point to the nuclei.