Identification of a mutation in the Clock1 gene affecting zebrafish circadian rhythms

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1(dg3), revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.