Isolation and Characterization of a Temperature - Sensitive Circadian Clock Mutant of Neurospora c ~ clssu

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period6 (pd -6 ) , has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21") the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21") the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperaturesensitive aspect of the phenotype of the p d 6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal pd-P protein is required for low temperature to "rescue" the p d 6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock. G ENETIC analyses of circadian clock mechanisms have resulted in the isolation of mutants in several organisms that exhibit alterations in one or more clock properties (reviewed in DUNLAP 1993). Of these, only the frequency (frq) gene of Neurospora and the period and timekss genes of Drosophila have been cloned and studied at a molecular level. Most previous work in Neurospora has focused on the frq gene for which five mutant allelic phenotypes are known. A molecular model for the Neurospora clock has been proposed, involving periodic transcription and translation of frq, that is controlled by negative regulation of frq transcription by FRQ protein (ARONSON et al. 1994; CROSTHWAITE et ul. 1995). It seems likely, however, that a full understanding of the mechanism of the Neurospora clock and the role of each clock gene in the clock mechanism will require characterization and analysis of other clock components. In Drosophila, preliminary models of the clock involving the period gene were greatly enhanced with the discovery and characterization of timeless (SEHGAL et al. 1995; HUNTER-ENSOR et al. 1996). In N. crussa there are seven known genes that, when mutated, alter the 21.5-hr period length of the circadian rhythm in asexual spore formation (DUNW 1993). Among these genes are both short period (frq', f r q , prd-4) and long period ( f ig , f r4 , prd-1, prd-2, prd-3) mutants. Efforts to establish functional relationships between these genes have failed to reveal any synergistic versity of' California, Santa Cruz, CA 95064. E-mail: [email protected] Corresponding author: Jerry F. Feldman, Department of Biology, UniGenetics 146 525-530 (June, 1997) interactions since all double and triple mutant combinations previously tested show additive (or multiplicative) behavior (FELDMAN et al. 1979; GARDNER and FELDMAN 1980). We report here the characterization of a new clock mutant strain in N. crussu, pm'od-6 (prd-h), which has a temperature-sensitive shortening of circadian period length and is thus the first known temperature-sensitive clock mutation. In addition, the prd-6 mutation shows an unusual epistasis to the previously isolated clock mutation period-2 (prd-2) that suggests a direct physical interaction between the products of the two genes. MATERIALS AND METHODS Strains and culture conditions: All strains used in this study were isolated in this lab or were obtained from the Fungal Genetics Stock Center (FGSC). Unless otherwise indicated in the text, all strains contain the band ( 6 4 mutation that allows clear expression of the circadian rhythm of conidiation without affecting the period length (SARGENT and WOODWARD 1969). The strains used in this study are shown in Table 1. Strains were handled according to standard procedures (DAVIS and DESERRES 1970) and maintained on minimal media with required supplements (see PERKINS et al. 1982). Racetube assay of circadian rhythm: Racetube assays were performed on glucose-arginine media as described ( SARGENT et al. 1966; SARGENT and KALTENBORN 1972) with supplements as required. Circadian period was calculated as previously described (FELDMAN and HOYLE 1973). Unless otherwise indicated all racetube assays were done at 25". Genetic mapping: Genetic crossing and linkage analyses were conducted using random ascospores as described (DAVIS and DESERRES 1970). Auxotrophic markers were scored for growth on Vogel's minimal media with and without supple526 L. W. Morgan and J. F. Feldman