A genetic analysis employing Drosophila melanogaster is elucidating the cellular and molecular mechanisms of the circadian clock. Findings obtained in Drosophila, because of its genetic amenability, are likely to shed light on the mechanisms of the human circadian clock as well. An understanding of the human circadian clock will contribute to treatments for sleep disorders, jet lag, cancer, and Seasonal Affective Disorder (SAD). Moreover, in a society which increasingly must function at all times of day and night, it is imperative to understand interactions between sleep/wake behavior and the circadian clock if we are to appreciate the long-term effects of such behavior on health and society. The work proposed in this grant application encompasses a behavioral, genetic, and molecular characterization of a novel circadian mutant of Drosophila (Double-time). The endogenous period of the circadian clock is reduced from approximately 24 hr to 18 hr in these mutant flies. The work will seek to establish the molecular basis for the effects of Double-time on circadian behavior. In particular, do the gene products affected by this new mutation affect the circadian oscillations of period and timeless RNA and protein, nuclear localization, phosphorylation state, and association? These oscillations are thought to comprise a feedback autoregulatory cycle underlying the circadian clock. It is possible that mutation of Double-time affects a protein which is also an integral component of this autoregulatory cycle, but it is also possible that it affects a component of the pathway by which light affects the rhythms, or a component of the pathways coupling clock biochemistry to behavior. The work proposed here will complete an already initiated phenotypic characterization of Double-time. What are its effects on the responses of the clock to light, to temperature, and on the molecular oscillations of the period and timeless gene products? Molecular cloning of the affected gene will allow the determination of the corresponding protein's amino acid sequence, which may suggest a function of the protein. Recombinant protein, expressed in prokaryotic or eukaryotic expression systems, will be tested for function in vitro and used to make antibodies. These antibodies and the cloned DNA will be used to determine the cellular and subcellular sites of expression for this novel RNA and protein. Does this expression show circadian regulation, and is it affected by any of the circadian mutants in Drosophila? Possible molecular mechanisms will be tested with mutant or conditionally expressed forms of the protein in transgenic flies. Finally, isolation of homologous genes for other species, or of molecules which interact with or are regulated by Double-time, will provide the materials for future work.
| Yu, Wangjie; Zheng, Hao; Price, Jeffrey L et al. (2009) DOUBLETIME plays a noncatalytic role to mediate CLOCK phosphorylation and repress CLOCK-dependent transcription within the Drosophila circadian clock. Mol Cell Biol 29:1452-8 |
| Price, J L (1997) Insights into the molecular mechanisms of temperature compensation from the Drosophila period and timeless mutants. Chronobiol Int 14:455-68 |
