Circadian clock genes and seasonal behaviour

Circadian and photoperiodic phenomena serve to organize the temporal pattern of various biological processes. While the former generates endogenous daily rhythms, the latter is related to seasonality. In Drosophila melanogaster, the gene timeless (tim) encodes a cardinal component of the circadian clock and also contributes to photoperiodism, which is observed as an adult reproductive diapause. In this work, natural tim variants were examined for diapause across different temperatures and photoperiods. The newly derived allele, ls-tim, exhibited consistently higher diapause levels than the ancestral one, s-tim, implicating a putative adaptive advantage in the seasonal European environment and providing a perfect substrate for the recently proposed scenario of directional selection. To investigate further genetic links between circadian and photoperiodic mechanisms, classical clock mutations and transgenes were placed on a natural congenic background and assayed for locomotor activity behaviour and diapause response. Surprisingly, the results not only highlighted the importance of tim, and its natural alleles, but also revealed the participation of other clock components in diapause, suggesting that both daily and seasonal timers might have molecularly coevolved. The phenotypic effects promoted by ls-tim arise from the protein isoform LTIM, which expresses an additional N-terminal fragment. To study the adaptive significance of the N-terminal residues, including putative phosphorylation sites, a number of mutagenized TIM constructs were generated and functionally analysed. At the molecular level, it was demonstrated that both the N-terminus length and the order of its residues are important variables modulating the interaction dynamics between TIM and CRYPTOCHROME (CRY). At the behaviour level, the overall amino acid composition, rather than a particular order, appeared to be more critical for the phaseshift responses. Interestingly, despite the functional importance of the N-terminus, a deletion mapping analysis revealed that CRY directly binds to a protein sequence located at TIM C-terminus.