frq9 mutant

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Figure 11.6 Diagram of a race tube and the frq mutants of N. crassa.

Figure 11.6 Diagram of a race tube and the frq mutants of N. crassa.

(circadian), which persists ("free runs") in the laboratory under constant illumination or darkness for several days. The rhythmic formation of conidia is a manifestation of an endogenous time-keeping system, a biological clock. This feature of Neurospora makes it an attractive model for investigating how living organisms keep time. In nature the rhythm is synchronized (entrained) to a 24 hour light-dark cycle. How this happens is not understood, although the light-dark cycle is undoubtedly the major factor that continuously resets the internal time-keeping mechanism.

11.2.1 A Clock Gene

Several mutants of Neurospora show altered period lengths of 16 to 29 hours or are arrhythmic, suggesting that genes affect the operation of circadian clock. For example, one mutant has a period of about 19 hours, another has a period of about 22 hours and another is arrhythmic. These mutants (Figure 11.6), frq2, frq7, and frq8, respectively, are alleles of the frequency (frq) gene; their distinct phenotypes are the results of different mutations in this gene. By transformation experiments, it was found that a 7.7 kb DNA fragment from a wild strain could restore circadian rhythm in an arrhythmic mutant, allowing the cloning of a frq gene (McClung et al., 1989). Using the frq probe, Northern and Western blot analyses were made to monitor frq transcription and translation. The relative levels of the frq mRNA and FRQ protein levels cycle with a 22-hour period in the wild-type strain grown in constant darkness. frq is therefore the oscillator determining the conidiation rhythm. The FRQ protein has helix-turn helix DNA-binding domain and nuclear-localization motifs, which suggest that it is a transcription regulator (Bell-Pedersen, 2000).

The circadian day, about 22 hours in length, is divided into 24 equal parts: the circadian hours. A molecular model for the Neurospora clock (Figure 11.7) views the day in a linear fashion. By convention, CT0 corresponds to subjective dawn and CT12 to subjective dusk. The cultures maintain their rhythmicity in liquid media, making it possible to monitor changes in mRNA and protein product FRQ at different time intervals. At dawn of circadian time (CT0), both frq mRNA and FRQ proteins are low but frq transcript starts to rise at CT4. The amount of frq mRNA and FRQ protein cycle in circadian manner, suggesting they control a cascade of clock-controlled output genes and thereby rhythmicity of the organism. The stability of FRQ is a major determining factor for the period length of the clock. Light causes a rapid increase in the levels of frq transcript and resets the clock by resetting the rhythm of frq transcription. The FRQ protein feeds back negatively to regulate the amount of frq transcript. This autoregulatory loop involving the expression of genes to proteins, which in turn inhibit their own expression, is thought essential for the self-sustained circadian rhythmicity. FRQ can be considered to have a function similar to oscillator clock proteins Period (Per) and Timeless (Tim) of Drsophila melanogaster and CRY1 (crypto chrome) and CRY2 of mammals. The N. crassa clock is a generally relevant model for clock studies.

11.2.2 Regulatory Genes

In Neurospora, mutants called the white collar (wc) produce pigmented conidia on a collar of white mycelium and exhibit arrhythmic conidiation. They are "blind," being blocked in light-induced pigmentation (carotenogenesis) in mycelium. The finding of frq mRNA in a wc-2 mutant suggested that light signaling acted primarily through wc-1. Cloning of wc-1 and wc-2 revealed that their sequences are quite similar. Both genes encode zinc-finger

Figure 11.7 Oscillations in Neurospora clock components. The relative amounts of frq mRNA and FRQ protein are plotted over 24 hours, one circadian cycle. (From Bell-Pedersen et al. (1996). With permission of Indian Academy of Science.)

transcription factors and have a "PAS" domain in common that serves as protein-protein dimerization domain found in many transcription factors and signaling components. Purified WC-1 and WC-2 form a white collar complex—a protein complex. (In the Neurospora nomenclature for proteins, the protein is given the same name as the gene but with letters in upper case without italicization.) The association of a flavin chromophore with WC-1 suggests that complex is a light-signaling molecule. Its nucleotide sequence suggests that the complex of WC-1 and WC-2 also act as a transcription factor—they bind to the frequency (frq) gene and induce its expression (Crosthwaite et al., 1997; Merrow et al., 2001). Null mutants of either gene are arrhythmic. A model of regulation of frq by white collar genes proposed by Dunlap and co-workers (Liu et al., 2000) is shown in Figure 11.8. The model envisages that FRQ plays a dual role—acting to depress its own synthesis by allowing transcription only when the concentration falls below a certain threshold and also to activate wc-1 and wc-2 genes that encode the DNA-binding proteins and act as transcriptional factors (positive element) in the feedback loop for the photoinduction of frq transcript. Phosphory-lation of FRQ triggers its turnover and is a major determinant of period length in the clock. The FRQ proteins inhibit frq activation, making a negative autoregulatory driving feedback loop rhythm in frq RNA levels that is the basis of circadian rhythm in Neurospora and, likely, in other organisms too. The rate of degradation of FRQ is most likely the major determining factor for the period length of the clock.



Figure 11.8 Genes and regulation of Neurospora clock. Adapted from Merrow and Roenneberg (2001).

11.2.3 Clock-Controlled Genes

Many genes have a role in conidiation (Chapter 7). In Neurospora, screening for clock-regulated genes was carried out using a subtractive hybridization of subjective morning vs. subjective evening mRNAs (Loros et al., 1989). In general, the transcripts of these genes accumulate in late night to early morning. The best-characterized clock-controlled gene is easily wettable (eas), which encodes a hydrophobic component of conidial wall and is, therefore, important for spore dissemination (Lauter et al., 1992). Conidia of eas are easily wettable because hydrophobin is missing. Genes associated with carotenoid biosynthesis and conidiation are also found to display a daily rhythm in mRNA accumulation (Lauter, 1996). Experiments using transcriptional profiling with DNA microarrays will provide a means to determine the full extent of clock-regulation of genes.

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