Figure 1.

Mutations in Dnaaf1 and Lrrc48. (A) A chromatogram from Sanger sequencing showing a T > C mutation (asterisk) at the junction of exon 4 and intron. (B) RT-PCR result showing amplification of a fragment corresponding to exon 3–6 of Dnaaf1 (lanes 1 and 2). Multiple bands with incorrect sizes were detected in Dnaaf1^m4Bei, indicating a splicing defect. Cloned RT-PCR products revealed abnormal transcripts with exon 4 skipping (lane 4, insert size 279 bp) or intron 4–5 insertion (lane 5, insert size 617 bp). The largest band (nonspecific) was not a Dnaaf1 transcript. (C) A chromatogram from Sanger sequencing showing a T > C mutation (asterisk) causing an amino acid change (Leu89Pro). (D) Multiple protein sequence alignment adapted from NCBI HomoloGene search result, including conserved leucine-rich repeat domains near the mutation. Leucine residues in the domain are highlighted in yellow. The leucine residue substituted in Lrrc48^m6Bei mutants is shown in red (asterisk). Other conserved residues are marked in blue. (E) RT-PCR results using three different primer sets spanning the Lrrc48 transcript. All amplified weaker bands from the mutant. Actb (Beta-actin) was used as a control. NCBI, National Center for Biotechnology Information; NTC, no template control; RT-PCR, reverse transcription polymerase chain reaction.