Investigating cryptochrome as the primary photoreceptor involved in resetting the circadian clock in Chlamydomonas reinhardtii
Jonathan Howton and Sigrid Jacobshagen
Department of Biology, Western Kentucky University, 1906 College Heights Blvd. #11080, Bowling Green, KY 42101-1080, USA
The blue-light photorecepter cryptochrome is a strong candidate for the primary photoreceptor that perceives light as an external time cue to reset the circadian clock in Chlamydomonas. To test whether cryptochrome performs this task, the strain CC48 was transformed using an RNA interference construct designed to silence the production of cryptochrome. The transformants were then screened for reduced amounts of cryptochrome using western blot analysis. Several strains were found to have cryptochrome production reduced to various degrees. To test the ability of these strains to reset their circadian clocks, cultures were grown in a strict 12-hour light/12-hour dark cycle to synchronize their rhythms, and then they were treated with light pulses of varying intensities at specific times in their dark phase. This would cause a phase shift in their rhythms if the photoreceptor responsible for perceiving these time cues were present in the cells. After the light treatment, the samples were placed into a machine, which monitored their circadian rhythm of phototaxis, or swimming towards light. The data collected by the machine were visualized as graphs and analyzed using a Mathematica-based program. The data analysis made it possible to determine the phase shift of each sample compared to the control without light treatment. The phase shifts of the wild-type strain, CC48, were then compared to those of a transformed strain with reduced cryptochrome production, RNAi #16, to determine if the reduced cryptochrome production in RNAi #16 reduced its ability to reset its circadian clock. There was no apparent reduction in its ability to reset its circadian clock, but this is likely due to the light intensity of the light pulses not being optimized yet. Currently, the strains are being tested with white light pulses of lower intensity and with pulses of blue light.
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