Screening for New Circadian Clock Components in Drosophila

The endogenous circadian clock adjusts the physiology and behaviour of an organism to advantageous periods of the day, and represents an adaptation to daily environmental cycles, such as light and temperature. Locomotor activity in Drosophila melanogaster represents a robust behavioural rhythm used to study the clock. This clock is located in the lateral and dorsal neurons of the fly and in the suprachiasmatic nuclei (SCN) of the hypothalamus in the mammal. The molecular bases of underlying circadian timing mechanisms in insects and mammals are conserved. Although we have a basic knowledge of the Drosophila molecular clock circuits functioning, many questions regarding the nature of the protein complexes that subserve circadian pacemakers, the connections between the oscillator and the overt rhythms and the entrainment signals to the clock remain unanswered. To identify new D. melanogaster circadian components I used three different approaches. The first is based on immunoprecipitation of protein complexes using tagged CYC, a dedicated clock protein, to pull down its partners. The second employs a comparative approach with the mammalian circadian SCN proteome and the third uses a tap-tagging design which is used to screen the proteome. Expression studies of candidate proteins, and behavioural analyses using mutants and transgenes to disrupt and silence some of these factors, have revealed a number of candidate genes that may affect aspects of clock function. Two novel genes involved in glutamate metabolism are particularly compelling, and appear to contribute to the circadian mechanism by mediating the neurons that are important for light input. A further synaptic gene may be involved in setting the clock pacemaker.