Novel strategies for the identification of clock genes in Neurospora crassa with insertional mutagenesis

Circadian clocks are endogenous cellular mechanisms that control daily rhythms of physiology and behaviour. The adjustment of the circadian clock to the 24 h period of a day is commonly accomplished by several environmental cues, e.g. temperature, light and nutrition. For one light input pathway the mechanism that synchronises or entrains Neurospora’s clock is supposed to be known. Nevertheless, there are plenty more environmental cues that have an obvious impact on the circadian clock, e.g. temperature. The environmental cue “temperature” was underrepresented in studies about Neurospora’s circadian clock, while several clock studies focused on stationary conditions rather than changing ones. As a result, the functionality and adaptation of circadian clocks were underestimated, and thus new clock components could be overlooked due to screening on constant darkness. It was therefore important to develop a novel strategy in screening mutants that challenged the circadian clock of Neurospora crassa entirely on temperature alternations. A temperature cycle of low amplitude (22° C cold and 27° C warm) and of short period (8 h cold and 8 h warm) applied as Zeitgeber stimulus. A mutant library was created with insertional mutagenesis via electroporation in order to transform a BASTA resistance gene into conidial nuclei. As a consequence, a novel method of rescuing the mutations was established, which combined the process of mapping, partial cloning and PCR and was called Size Selected Fragment Plasmid Rescue or SSFPR in short. During the screening of several hundred mutants among the novel protocol, a known clock gene, frequency, was identified and characterised. The identification of several mutants with altered clock phenotypes has on one hand confirmed the general approach of this study and on the other proved that the greater sensitivity for the temperature screen can bee used to detect mutant phenotypes