kismet and cry genetically interact.

(A) Increasing CRY signaling in flies expressing kis dsRNAs rescues the LL phenotype. Actograms show the locomotor activity of tim-GAL4/VDRC46685 (tg4/VDRC46685; homozygous for ls-tim) and tim-GAL4 UAS-myccry/VDRC46685 flies (tg4 cry/VDRC46685; s-tim/ls-tim heterozygous). The presence of one s-tim allele in flies overexpressing CRY potentially contributes to the rescue, because the S-TIM protein is more sensitive to CRY signaling [87]. This contribution is small however. Indeed, 50% of tim-GAL4/VDRC46685 flies carrying one s-tim allele are rhythmic in constant light (see Table 3). In addition, CRY overexpression also rescues arrhythmicity in a ls-tim homozygous background, although a slight increase in residual rhythms was detected during the first 3 days of LL (Figure S3). n = 15–16 flies/genotype. (B) Western blot showing TIM oscillations in whole head extracts of control (w1118), timGAL4 UAS-dcr2/+; crym/crym (tg4 dcr2/+; crym) and tim-GAL4 UAS-dcr2/VDRC46685; crym/crym (tg4 dcr2/VDRC46685; crym) flies entrained to an LD cycle. All genotypes are homozygous for ls-tim. (C) The amplitude of TIM protein rhythms is reduced in crym flies expressing kis dsRNAs. TIM protein levels were quantified by Western Blot at ZT17 (peak) and ZT5 (trough) and normalized with the Tubulin loading control. The histogram represents the ratio between the two values. Each bar represents the average of 4 independent experiments per genotypes, expect y w; crym (n = 3). Error bars represent standard deviations (*p<0.05; t-test).




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