Molecular ananysis of the fly circadian clock
thesisposted on 15.12.2014, 10:38 by Karen. Garner
I have investigated the co-evolutionary divergence of two central circadian clock genes; period (per) and timeless (tim). The molecular components of the clock were derived from a common ancestor in higher eukaryotes, so that closely related species possess homologous genes and mechanisms. By substituting a clock gene from one species into another closely related one, it is probable that the clock will still be able to function to some degree because it maintains its interactions with other clock proteins. Previous behavioural analysis of Drosophila melanogaster per01 transfromants carrying a single copy of the, Musca domestica per gene, revealed a successful rescue of rhymicity, plus Musca - specific circadian phenotypes were transferred to the transformants. I have generated a series of M. domestica / D. melanogaster chimeric per transgenes and transformed them into a per-null background, in an attempt to define the regions of the per gene which give species-specific patterns of behaviour.;To further understand how PER and TIM interact via the PAS domain of PER, I have used an Evolutionary Trace procedure to predict functionally important residues of the PASA region. I have generated a number of PAS mutations based on the results of this bioinformatics analysis and studied the effect of these mutations on PER-TIM interactions using both the yeast two-hybrid system and for nuclear transport and retention in Schneider 2 cells.;Finally I have focused on TIM and its interaction with Drosophila Axin (dAxin), a protein that was originally discovered in the wingless pathway. dAxin is known to facilitate the phosphorylation and subsequent degradation of ARM (protein product of the armadillo gene). dAxin does this by acting as a scaffold between ARM and the serine-threonine kinase SGG (protein product of the shaggy gene). SGG has been shown to phosphorylate TIM, promoting nuclear localization.