Figure 1. Positional cloning of Past-time (Psttm) and identification of the Fbxl21 mutation.

A. Histogram showing circadian period distribution of ENU mutagenized generation 3 (G3) mutant mice in a recessive screen. The shaded area represents the average period for WT C57BL/6J mice ± 3 standard deviations. The Past-time founder mouse is indicated by the arrow.

B. Representative actogram of a WT C57BL/6J mouse (upper). The actogram is double plotted where each horizontal line represents 48 hrs of activity. The mice were kept on an LD12:12 cycle (represented in the bar above) for the first 7 days and then released into constant darkness for 21 days (indicated by the arrowhead on the right). Actogram of the Past-time founder G3 mouse (period = 22.91 hours) (lower).

C. Period distribution of F2 intercross mice used for genetic mapping. The 3 panels from top to bottom represent WT, Psttm/+ and Psttm/Psttm mice, respectively. ANOVA of the period was performed on the 3 populations of F2 mice (grouped by genotype) (DF = 2; F= 78.80; p = 3.983 ×10⁻²⁹).

D. Psttm was initially mapped to a 40 Mb region on chromosome 13 between rs13481788 and rs367922 (left). The chromosome 13 schematic lists the markers used to map Psttm to a smaller genetic interval. Haplotypes of the 78 Psttm/Psttm F2 intercross progeny (156 meioses) are shown on the right. Black boxes represent C57BL/6J WT alleles, and white boxes represent C3H/HeJ alleles. The number of recombinants per total meioses is indicated to the right of the haplotype map. Sequencing of Fbxl21 where a single-base change from G to A is indicated by the asterisk (right).

E. Structural modeling of the FBXL21 LRR3 motif based on the Skp2 structure in WT and mutant proteins. See also Figure S1.