A clever genetic selection
In order to identify players involved in output pathways in Neurospora, the authors focused on two well-characterized ccgs: ccg-1 and ccg-2. These genes depend on a functional FRQ-based oscillator to cycle and both peak on the subjective dawn. Clock-controlled gene-2 encodes a hydrophobin, a protein that coats the outer layer of asexual spores (conidiospores) and helps maintain the cell-surface hydrophobicity essential for their air dispersal. Loss of ccg-2 leads to a phenotype in which conidiospores “have a darkened, wetted appearance and mix readily with water” (the so-called Eas phenotype). On the other hand, the function of ccg-1 is unknown and no discernible phenotype is evident in the null strain.
The authors observed, just as it had already been reported, that on a WT strain, both ccg-1 and ccg-2 mRNA levels peak on the subjective morning and have their lowest levels in the subjective early evening. The authors noticed that in a strain carrying a frq null mutation (frq10), this pattern was altered, but in a different way for each gene. While ccg-1 mRNA levels appear to be high throughout the circadian day in the frq-less strain, ccg-2 mRNA levels were low at both time points evaluated (Figure 1).
OK, but what does this have to do with trying to identify actors involved in output pathways?
Identifying output pathway players
Among these strains, two phenotypic groups could be observed. One group was similar to the parental CCG1M strain and the other one had a phenotype resembling the ccg-2 loss-of-function phenotype (an Eas-like phenotype). The latter is remarkable, considering that it was obtained through a screen aimed at identifying strains with a dysregulated ccg-1 expression pattern. The strains in this group indeed had low endogenous levels of ccg-2 mRNA, suggesting that a single mutation could affect the expression of not only ccg-1 but other ccgs. Indeed, the authors showed that in these strains, the effects on ccg-1 and ccg-2 expression appeared to be the result of a single mutation: the Eas-like phenotype always segregated with the low-level ccg-1 mRNA one.
Vitalini MW, Morgan LW, March IJ, & Bell-Pedersen D (2004). A genetic selection for circadian output pathway mutations in Neurospora crassa. Genetics, 167 (1), 119-29 PMID: 15166141