Date of Award
Master of Science (MS)
Daily rhythms in physiology and behavior are produced by a circadian system consisting of a central clock located in the brain and peripheral clocks located in various peripheral tissues. Circadian clocks track time of day through the presence of a molecular clock that functions as a transcriptional-translational feedback loop. In the nucleus, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate transcription of clock genes period (per) and timeless (tim). PER and TIM proteins slowly accumulate in the cytoplasm before entering the nucleus, where they inhibit CLK and CYC, and therefore repress their own transcription. Degradation of nuclear PER and TIM permits the next 24-hour cycle to begin. These molecular clocks must be coordinated across tissues, but the manner through which central and peripheral clocks communicate with one another to achieve this coordination is not well understood. We reasoned that the ability to track clock gene expression in specific tissues of living flies would facilitate an investigation into the relationship between different clock-containing tissues. Previous efforts to accomplish this in Drosophila have relied on reporter constructs in which regulatory elements of several different clock genes have been used to dictate expression of a luciferase reporter enzyme, the activity of which can be monitored using a luminometer. Although these reporter lines have been instrumental in our understanding of the circadian system, they generally lack cell specificity, making it difficult to compare molecular clock oscillations between different tissues. Here we report the generation of several novel lines of flies which allow for GAL4-inducible expression of a luciferase reporter construct for clock gene transcriptional activity. We have expressed these reporter constructs selectively in neurons, the fat body, and ubiquitously, and show that molecular clock oscillations can persist in the fat body and in all tissues for several days in the absence of environmental cues. Additionally, we confirm previous reports that bioluminescence rhythms are generally more robust under 12hr:12hr light:dark conditions compared to constant darkness.
Mather, Lilyan, "Development and Characterization of a Tissue-Specific Reporter Line for Monitoring Circadian Clock Transcriptional Activity" (2022). Master's Theses. 4418.
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