The circadian clock is an endogenous timer that allows an organism to anticipate and properly prepare for the daily changes in the environment. This preparation occurs in the form of daily rhythms in metabolism, physiology, and behavior. These approximately 24-hour rhythms are reset upon environmental time cues such as the daily light/dark and temperature cycles. The unicellular green alga Chlamydomonas reinhardtii is a useful model organism for circadian clock research. It shows several well-characterized circadian rhythms of behavior, and the monitoring of its rhythm of phototaxis, or swimming towards light, has been automated. The receptors involved in entraining the clock to the daily light/dark cycle have not yet been identified in this organism. Previous research has shown that blue, green, and red light are effective in resetting the clock in C. reinhardtii. This study focused on identifying the blue light sensor for resetting. One possibility was reception through photosynthesis. This was tested by looking for a defect in the ability to reset the clock upon blue light in cultures treated with the photosynthesis inhibitor DCMU. It was found that photosynthesis does not mediate this process. Instead, a photoreceptor must be involved, and plant-like cryptochrome was the most probable candidate, as it is known to perform this function in higher plants. To determine if plant-like cryptochrome serves this function, available transformants of C. reinhardtii with an RNA interference construct designed to knockdown plant-like cryptochrome expression were used. In this study, the transformants were screened for a reduction in cryptochrome amount using western blot analysis. The two strains with consistently the largest knockdown were tested for defects in resetting the circadian clock upon blue light pulses. Neither strain was found to be less sensitive than the parent strain to blue light induced entrainment. On the contrary, one strain was significantly more sensitive than the parent strain, which suggests a possible inhibitory role for plant-like cryptochrome in the photoentrainment of the clock to blue light in this organism.
| Identifer | oai:union.ndltd.org:WKU/oai:digitalcommons.wku.edu:theses-2197 |
| Date | 01 August 2012 |
| Creators | Howton, Jonathan |
| Publisher | TopSCHOLAR® |
| Source Sets | Western Kentucky University Theses |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses & Specialist Projects |
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