Project 4: Structure/activity analysis of mammalian clock components by saturation mutagenesis
Dr. Carla Green and Dr. John Hogenesch, Project Leaders
Although many components of the central clock mechanism
have been identified, very little is known about the biochemical
details of their action. In order to further refine the
molecular oscillator model and to better understand how
the circadian clock controls rhythmicity, we propose to
perform saturation mutagenesis to generate allelic series
of the central clock components and to analyze their functions
in biochemical and cell-based assays.
Random mutagenesis will be used to create libraries of
random mutations in Bmal1, Clock, Cryptochrome 1, Cryptochrome
2, Period 1, and Period 2. The mutant proteins
will be analyzed in high-throughput cell-based assays for
alterations in their activity. The mutants of interest will
be analyzed further, using a host of biochemical and cell
assays to study protein-protein interactions, DNA-binding,
post-translational modifications and intracellular localization.
Information about the nature of the mutations and their
corresponding activities will be used to identify structural
features or domains that are important for specific aspects
of their functions. The mutants of interest will be analyzed
for effects on circadian properties (period, amplitude of
rhythmicity, etc) in cycling cell assays containing per2::luciferase
reporters. The definition of these functional domains will
aid in the biochemical analysis of the response to small
molecule perturbations and will allow better interpretations
of the functions of the human genetic variants identified.
Finally, these mutant forms could provide genetic tools
for future manipulation of the mammalian circadian system.