Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals.DOI: http://dx.doi.org/10.7554/eLife.00426.001

Glucokinase (GK) is activated by glucose binding to the substrate site, is inhibited by GK regulatory protein (GKRP) but stimulated by GK activator drugs (GKAs). To further explore the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady state kinetics of the enzyme and tryptophan fluorescence (TF). TF studies of the normal binary GK/glucose complex corroborate recent crystallography showing that it exists in a closed conformation greatly different from the open conformation of the ligand free structure but indistinguishable from the ternary GK/glucose/GKA complex. GKAs did activate and GKRP did inhibit normal GK while its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: 1) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (kcat, glucose S0.5, nH and ATP Km) but usually strong GKRP inhibition (13/31); and 2) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S0.5 but with reduced or no GKRP inhibition in cases (14/31). These and pertinent literature data are best explained by envisioning a common allosteric regulator region with spatially non overlapping GKRP and GKA binding sites.

Random mutagenesis combined with phenotypic screening using carefully crafted functional tests has successfully led to the discovery of genes that are essential for a number of functions. This approach does not require prior knowledge of the identity of the genes that are involved and is a way to ascribe function to the nearly 6000 genes for which knowledge of the DNA sequence has been inadequate to determine the function of the gene product. In an effort to identify genes involved in the visual system via this approach, we have tested over 9000 first and third generation offspring of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for visual defects, as evidenced by abnormalities in the electroretinogram and appearance of the fundus. We identified 61 putative mutations with this procedure and outline the steps needed to identify the affected genes.

Background: ENU mutagenesis was used to generate new animal models of diabetes. Results: We identified two novel mutations in glucokinase, with glucose Ͼ400 mg/dl in homozygotes, and differential responsiveness to glucokinase activators. Conclusion: Increased GCK thermolability is a major cause of hyperglycemia in Gck mutant mice. Significance: Chemical genetics creates new models to study glucose homeostasis and diabetes drugs.

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