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

Abstract

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.

Key Words:

• Neurospora crassa
• frq gene
• prd-4 gene
• Genetic analysis