Novel strategies for identification of clock genes in 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.
Sveric
Kruno
K
Institute for Medical Psychology, University of Munich, Germany.
Mason
Moyra
M
Roenneberg
Till
T
Merrow
Martha
M
D001483:Base Sequence; D002940:Circadian Rhythm / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D017931:DNA Primers; C087405:FRQ protein, Neurospora crassa; D005656:Fungal Proteins