The Neurospora crassa clock-controlled gene (ccg-2) is transcriptionally activated by the circadian clock in a time-of-day-specific manner. Transcript and sequence analyses of ccg-2 reveal that the predicted Ccg-2 polypeptide bears significant similarity to a class of low-molecular-weight, cysteine-rich, hydrophobic proteins (hydrophobins), first identified in Schizophyllum, and including the product of the developmentally regulated Aspergillus gene, rodletless, required for spore surface rodlets. Allelism between ccg-2 and easily wettable (eas) (one of the first developmental genetic loci identified in Neurospora) was predicted on the basis of this similarity, their close genetic linkage, and previous findings demonstrating that eas mutants lack rodlets. In this study allelism is confirmed experimentally by showing that (1) transformation of an eas mutant strain with ccg-2 DNA results in phenotypic complementation, including restoration of surface rodlets, (2) inactivation of the ccg-2 gene, by RIP, results in an eas phenotype including loss of rodlet fascicles, and (3) the original eas strain has dramatically reduced levels of ccg-2 mRNA. Thus, the clock-controlled ccg-2 gene encodes an integral component of fungal asexual spores important for spore dispersal. The dramatic reduction of ccg-2 expression in the eas mutant has no apparent effect on the normal operation of the circadian clock, confirming that there is no feedback of this clock output on the oscillator itself. These data, in conjunction with the previous observation that ccg-2 is light induced, serve to focus attention on the dual interacting role of light and the circadian clock in the regulation of fungal spore development.[Key Words: Clock-controlled gene; ccg-2; easily wettable., hydrophobins; rodlets; circadian clock] Received August 13, 1992; revised version accepted September 15, 1992.Circadian rhythms are biological rhythms that are present in most, if not all, eukaryotes (Dunlap 1990). Daily fluctuations in cellular and organismal activities are controlled by an endogenous oscillator, commonly referred to as the biological clock. The clock exerts its effects on a wide variety of processes ranging from asexual spore formation in the ascomycete Neurospora crassa and the control of cell division in the marine protist Gonyaulax to photosynthesis in plants and cognitive functions in people (for review, see Edmunds 1988). However, at all phylogenetic levels, clocks appear to be assembled at the level of intracellular regulation and not intercellular communication. Thus, simple eukaryotes provide appropriate and powerful systems for molecular genetic analysis of this phenomenon.Of the eukaryotes known to possess a well-defined circadian clock, the filamentous fungus N. crassa provides an ideal model system for clock analysis {for review, see Feldman and Dunlap 1983;Dunlap 1990; Lakin-Thomas tCorresponding authors. et al. 1990). Advantages include an easily measurable circadian rhythm in developmental potential (rhythmic formation of aerial hy...