Novel Strategies for the Identification of Clock Genes Neurospora With Insertional Mutagenesis

As the molecular mechanism of circadian clocks has reached high complexity, the fungal model system, Neurospora crassa , is increasingly important for clock research. It offers the possibility of extensive biochemical experimentation and thorough description of circadian properties. Realization of the full potential is dependent on efficient, high-throughput methods. We have combined several protocols to develop abundant and inexpensive production of mutants, and subsequent identification of the affected gene. We applied a novel screening protocol and, after screening several hundred mutants, identified a known clock gene, frequency . Furthermore, the methods described here can easily be adapted to various insertional constructs (e.g., those with alternative selection markers or that facilitate overexpression) or combined with strains carrying clock-regulated reporter genes.