Molecular Analysis of the Neurospora Clock: Cloning and Characterization of the frequency and period-4 Genes

Genetic analysis of Neurospora crassa has identified many mutants that affect the biological clock. In this article we review the cloning of two of these genes, frq and prd-4. Both genes were isolated using a chromosome walk technique. Subcloning experiments and subsequent Northern analysis of frq implicate the importance of two transcripts that emanate from this locus. In preliminary data, no protein-coding region is evident in the smaller transcript; the larger transcript contains a 962-amino acid open reading frame. The open reading frame shows limited homology to per, a clock gene identified in Drosophila. Sequence analysis of all existing. frq alleles suggests that the defect in each case lies within the open reading frame. Successful cloning of the prd-4 gene required walking a distance of >40 kb. A physical map of this region has been constructed using restriction analysis. The dominance-recessive relationship of prd-4 and prd-4+ was established by examining the period lengths of strains harboring a wide range of prd-4 / prd-4+ nuclear ratios.