Fig. 3

Linkage and sequence analysis of Aey69 mutation. a) Haplotype analysis defines the critical interval between the markers D3Mit188 and D3Mit11 at mouse chromosome 3. The analysis was performed in two steps separated by the black line; the markers D3Mit188 and D3Mit76 were used only in the 9 mice with a recombination between D3Mit141 and D3Mit11. The numbers of mice for each haplotype are given; 7 mice of the F2 panel had the B6 allele of all markers tested, but carried the Aey69 mutation. Black squares are heterozygotes, and empty squares represent homozygotes for the C57Bl/6 J allele. The red arrows mark the critical interval for the underlying mutation; the genetic distances (given in cM) and the exact physical positions of the markers (given in Mb) are from the MGI database (http://www.informatics.jax.org/; Dec. 2018). b) Sanger sequencing confirmed the exome sequencing data (c. 358 A-> C; red circles). c) The change in the amino acid sequence (Ile120Leu) is given below and boxed in yellow with a red surrounding line; the underlined DNA sequence (CCTC) demonstrates the new MnlI restriction site in the mutants. Schematic drawing of the mouse Hist2h3c1 gene (ENSEMBL) is given below the nucleotide sequence; the red arrow points to the site of the mutation at the C-terminal end of this single-exon gene. d) The novel MnlI restriction site is present in all homozygous mutant mice tested. It is absent in 5 tested wild-type strains indicating that it is a mutation and no widespread polymorphism. The schema above the gels explains the digestion pattern of the fragment, and the size of the critical bands is given in red or green. The red arrows point to these critical bands and their sizes are indicated; +, with MnlI restriction enzyme; -, without restriction enzyme.