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.