BIO411H5 Lecture Notes - Lecture 1: Freerunning, Rna Interference, Molecular Clock
Document Summary
The circadian clock speed in eukaryotes could be determined by phosphorylation that drives the levels of period. It was hypothesized slow phosphorylation at phospho-clusters caused by time-delay phospho-circuits could be a key aspect of the timer in eukaryotic circadian clock. In order to study this in general eukaryotes, Drosophila per (dper) was mutated at several ser/thr sites of phosphorylation and then expressed in cultured s2 cells which had recombinant of dbt and dper. These sites and specifically pershort (pers) allele which changed a ser amino acid at position. 589 to asn (s589n) were found to have faster degradation rate mediated by dbt. To identify the slimb-binding site on dper, tev/tag versions of dper and of different per-short domain mutants in the first 100 aa were generated. Here, there was a trend of increased dbt-mediated phosphorylation kinetics. Also, there was a higher phospho-occupancy of ser47 (in slimb) for the s596a, s589a, and s585a mutants compared to the control.
