A Genetic Selection for Circadian Output Pathway Mutations in Neurospora Crassa

A Genetic Selection for Circadian Output Pathway Mutations in Neurospora crassa

Michael W. Vitalini, Louis W. Morgan, Irene J. March and Deborah Bell-Pedersen1 Center for Biological Clocks Research and Program for the Biology of Filamentous Fungi, Department of Biology, Texas A&M University, College Station, Texas 77843 Manuscript received September 27, 2003 Accepted for publication January 22, 2004

ABSTRACT In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq- oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.

IRCADIAN (daily) rhythms are endogenous, self- and negative feedback loops involving the products of C sustaining oscillations that are regulated by a cen- the frq and white collar (wc-1, wc-2) genes (Loros and tral pacemaker composed of one or more biochemical Dunlap 2001). The WC-1 blue light photoreceptor oscillators (Edmunds 1988; Young and Kay 2001). These (Froehlich et al. 2002; He et al. 2002) forms a complex rhythms are observed in a wide variety of organisms, with WC-2 [white collar complex (WCC)]. This complex ranging from daily rhythms in photosynthesis in cyano- directly activates transcription of the frq gene (Froeh- bacteria and plants, to development in fungi, to activity lich et al. 2003). Levels of FRQ protein then slowly and sleep-wake cycles in rodents and humans. The core increase. At high levels, FRQ protein binds to and inhib- oscillators of several organisms consist of autoregulatory its the activity of the WCC (Cheng et al. 2001; Denault feedback loops, where the positive elements of the loop et al. 2001). This causes frq transcription and the accu- activate transcription of the negative elements. The neg- mulation of FRQ protein to stop. Subsequent phosphor- ative elements then inhibit their own transcription by ylation-induced decay of FRQ leads to reactivation of blocking the activity of the positive elements (Dunlap frq transcription, allowing the cycle to start again (Liu et al. 1999; Young and Kay 2001; Reppert and Weaver et al. 2000). In addition to the frq-oscillator, several lines 2002). of evidence have suggested the presence of additional The circadian clock system of Neurospora crassa is one oscillators in the cell (Loros and Feldman 1986; Aron- of the best understood (Loros and Dunlap 2001). son et al. 1994a; Merrow et al. 1999; Lakin-Thomas N. crassa displays an easily viewed natural 22-hr rhythm and Brody 2000; Morgan et al. 2001; Correa et al. of asexual spore development (conidiation) in constant 2003). However, components of the other oscillator(s) darkness. This conidiation rhythm provided the basis have not yet been identified. for “brute force” genetic screens for mutations that af- Time of day information is signaled from the oscilla- fect the clock. The frequency (frq) gene was identified tor(s) to genes and gene products residing in output through such screens (Feldman and Hoyle 1973; Loros pathways to generate the observed daily rhythms. Genes and Feldman 1986) and was later shown to be a core that are rhythmically expressed under control of the clock, oscillator component required for circadian rhythmicity and reside downstream of the output pathways from the (McClung et al. 1989; Aronson et al. 1994b). clock, are termed clock-controlled genes (ccgs; Loros et al. The frq-oscillator consists of interconnected positive 1989). To date, Ͼ150 ccgs have been identified in N. crassa (Loros et al. 1989; Bell-Pedersen et al. 1996b; Zhu et al. 2001; Correa et al. 2003; Nowrousian et al. 2003), yet few details of the output pathways from the N. crassa, 1Corresponding author: 3258 TAMU, Department of Biology, Texas A&M University, College Station, TX 77843. or any other organism’s, pacemaker are known. E-mail: [email protected] The functions of the N. crassa ccg products are varied,

Genetics 167: 119–129 (May 2004) 120 M. W. Vitalini et al. ranging from involvement in development to stress re- ler et al. 1991; Dillon and Stadler 1994; Carattoli sponses and metabolic processes. The two most highly et al. 1995). Loss of mtr function can be selected for on characterized ccgs are the morning-specific genes ccg-1 and the basis of resistance to the toxic amino acid analogs eas(ccg-2) (hereafter called ccg-2 for simplicity). Clock con- p-fluorophenylalanine (FPA) or 4-methyltryptophan. trol for both ccg-1 and ccg-2 requires a functional frq- Gain of mtr function can be selected for on high-argi- oscillator (Arpaia et al. 1993, 1995). The abundantly nine (Arg)/low-tryptophan (Trp) medium in a trp-2 expressed ccg-2 gene encodes a member of a class of low mutant background. Arg blocks the basic amino acid molecular weight, cysteine-rich secreted hydrophobic transporter, making the permease encoded by mtr essen- proteins called hydrophobins (Bell-Pedersen et al. tial for transport of Trp. The ccg::mtr fusions were indi- 1992; Lauter et al. 1992). The hydrophobins coat the vidually transformed into a mtr trp-2 frq 10 strain. The frq 10 outer cell wall of fungi and maintain the cell-surface mutation is a null mutation that was produced by re- hydrophobicity essential for air dispersal of mature co- placement of the frq open reading frame with the hygro- nidiospores. Inactivation of ccg-2 results in an “easily mycin resistance gene (Aronson et al. 1994a). In frq 10 wettable” (Eas) phenotype in which the asexual conidio- strains, expression of the ccg-1::mtr transgene is high spores have a darkened, wetted appearance and mix and the cells are Mtrϩ (grow on Trp/Arg medium, readily into water (Bell-Pedersen et al. 1992). The but not on FPA). Alternatively, in the frq 10 strain, expres- function of ccg-1 is unknown; ccg-1-null strains do not sion of the ccg-2::mtr transgene is low and the cells are display any discernible phenotype (Lindgren 1994). In MtrϪ (grow on FPA, but not on Trp/Arg medium). addition, the CCG-1 protein sequence does not share To identify genes involved in regulating ccg-1 and ccg-2 similarity to other characterized proteins, but CCG-1 is expression, mutations that altered the Mtr-based growth conserved among filamentous fungi. phenotype of strains containing either the ccg-1::mtr Both the ccg-1 and ccg-2 genes were shown by nuclear transgene or the ccg-2::mtr transgene were selected and run-on assays to be controlled by the clock, at least par- analyzed for expression of the endogenous ccg-1 and tially, at the level of transcription (Loros and Dunlap ccg-2 genes. 1991). Analysis of the ccg-2 promoter localized a positive activating clock element (ACE) required for ccg-2 mRNA rhythmicity to within a 68-bp sequence located between MATERIALS AND METHODS Ϫ150 and Ϫ118 nucleotides (nt) from the start of tran- Plasmid construction: The Ϫ481 to ϩ57 promoter region of scription (Bell-Pedersen et al. 1996a, 2001). Deletion the ccg-1 gene was amplified by PCR using pKL119 (Lindgren of the ACE resulted in constitutive low-level expression 1994) as a template and 5Ј-CTAGGAATTCTGTTGACCATTG of ccg-2 mRNA in constant darkness, suggesting that GTTAC-3Ј and 5Ј-CTAGAGGCCTGAGAAGAATAGAAGC-3Ј Ϫ ϩ rhythmicity results from activation of transcription by as primers. The 1079 to 1 promoter region of the ccg-2 gene was amplified by PCR using pLW1K (Bell-Pedersen et al. the clock. A negative promoter element required for Ј Ϫ Ϫ 1992) as a template and the primers 5 -CTAGGAATTCGTCA ccg-1 rhythmicity was identified between 444 and 91 CAGACAACGGGTGA-3Ј and 5Ј-CTAGAGGCCTGGTTTGA nt upstream from the transcriptional start site (Lind- GTGTTGTGTTG-3Ј. Because the basal level of ccg-2 transcrip- gren 1994). Deletion of this element resulted in ran- tion from promoter fragments containing only the ACE is low, domly fluctuating high levels of ccg-1 mRNA in constant we used a promoter fragment that drives higher levels of darkness. These data suggested that, while ccg-1 and ccg-2 expression and contains promoter elements involved in clock (ACE) and light regulation of ccg-2 (Bell-Pedersen et al. ccg-2 both peak in expression in the subjective early 1996a, 2001). The use of the larger ccg-2 promoter fragment morning, they are regulated differently by the clock. may reduce the sensitivity of the selection and allow for the The use of genetics in the study of clocks has been isolation of mutations that affect light regulation of ccg-2. The restricted to brute force screening for altered overt primer pairs introduced an EcoRI site (underlined) at the 5Ј Ј rhythmicity or misexpression of a reporter gene to iden- end and a StuI site (underlined) at the 3 end of the amplified DNA. The promoters were cloned separately into pNC807 tify clock-associated mutations (Millar et al. 1995; (Koo and Stuart 1991) digested with EcoRI and StuI, creating Golden et al. 1998; Loros and Dunlap 2001). No tar- ccg promoter::mtr gene fusions. The 3.2-kb ccg-1::mtr and 3.6- geted selections or suppressor screens have been de- kb ccg-2::mtr EcoRV fragments of the resulting plasmids were scribed. We have utilized a genetic selection (Carat- subsequently cloned into the EcoRV site of pMLSBml, generat- toli et al. 1995) to identify additional components of ing plasmids pCCG1M and pCCG2M, respectively. pMLSBml the N. crassa circadian clock system by taking advantage was kindly provided by Drs. Mari Shinohara and Jay Dunlap, Dartmouth Medical School, and contains the benomyl resis- of the clock-dependent regulation of the ccg-1 and ccg-2 tance gene isolated from pSV50 (Fungal Genetics Stock Cen- genes. ter, Kansas City, MO) inserted into the SmaI site of pBSKIIϩ To accomplish the selection, promoter fragments of (Stratagene, La Jolla, CA). the ccg-1 and ccg-2 genes containing the clock regulatory Strains and culture conditions: The mtr SR33 trp-2 41 mat A strain elements were fused to the mtr (methyl tryptophan resis- was obtained from David Stadler (University of Washington). The mtr (methyl-tryptophan resistance) mutation carried by this tance) gene. The N. crassa mtr gene encodes an amino strain is nonrevertible due to a small deletion in the coding acid permease required for uptake of neutral aliphatic sequence. The trp-2 gene encodes anthranilate synthetase (Kee- and aromatic amino acids (Koo and Stuart 1991; Stad- sey et al. 1981). The mtr SR33 trp-2 41 mat A strain was crossed to Genetic Selection for Circadian Clock Mutations 121

lab strain 40-1 (bd frq 10 mat a) to obtain 106-20 (bd frq 10 mtr trp-2 mat a). The bd (band) mutation allows the circadian rhythm of conidiation to be easily observed on race tubes (Sargent et al. 1966). The frq 10 null mutation was generated by replace- ment of the frq open reading frame with the hygromycin resistance gene (Aronson et al. 1994a). The mating types of the strains are indicated (mat A or mat a). The 106-20 strain was transformed with the pCCG1M or pCCG2M plasmid by electroporation and transformants were selected on benomyl- containing media using standard techniques (Chakraborty et al. 1991). The resulting CCG1M and CCG2M strains are homokaryotic isolates of the pCCG1M and pCCG2M transformants, respectively. Homokaryons were obtained from a microconidial suspension passed through a 5-␮m filter (Ebbole and Sachs 1990) and germinated on media containing benomyl. Genomic DNA isolated from the transformants was analyzed by Southern blotting to verify integration of a single plasmid into the chromosome (data not shown). All strains used in this study were maintained on Vogel’s minimal media with the appropriate supplements and handled ac- cording to standard procedures (Davis and deSerres 1970). Strains with the frq 10 mutation were maintained on hygromycin B (200 ␮g/ml). Benomyl was added at 1 ␮g/ml to media before autoclaving to select for strains carrying pCCG1M or pCCG2M constructs. Strains carrying the trp-2 mutation were Figure 1.—The clock-controlled ccg-1 and ccg-2 genes are ␮ supplemented with 10 g/ml anthranilate. Anthranilate is a differentially regulated by the clock. (A) The levels of ccg-1 metabolic precursor of tryptophan that does not require mtr and ccg-2 mRNA were assayed by Northern blot analyses in for uptake. Strains carrying the mtr mutation are resistant to the frq ϩ strain 87-3 and the frq 10 strain 40-1. Liquid cultures ␮ the toxic amino acid analog FPA at 60 g/ml. Selection of of mycelia were grown in constant darkness and harvested after mutant strains that negatively affect expression of the ccg::mtr 12 (DD12) and 20 (DD20) hours in the dark, representing transgene, resulting in low-level mtr expression, were per- subjective morning and early evening, respectively. Equivalent formed on media containing FPA, anthranilate, and benomyl loading of RNA was verified by hybridization to rRNA. (B) A (FA medium). Selection of mutant strains that positively affect simple model representing the N. crassa frq-oscillator with out- expression of the transgene, resulting in high-level mtr expres- put pathways from the clock positively regulating rhythmic sion, was performed on 0.01 mg/ml Trp, 1 mg/ml Arg, and levels of ccg-2 mRNA in the morning and negatively regulating benomyl (TA medium). Race tube assays were carried out at rhythmic levels of ccg-1 in the evening (see the text for details). 25Њ on glucose-arginine media (0.1% glucose, 0.5% arginine, 1ϫ Vogel’s salts, 10 ␮g/ml anthranilate, 2% agar). For RNA analyses, tissue was grown and synchronized by light-to-dark transfers following published procedures (Loros et al. 1989). ccg-1 and ccg-2 were examined by Northern assays from ϩ 10 Mutagenesis: Conidial suspensions (15 ml) of strains frq and frq strains harvested after 12 and 20 hr of growth CCG1M or CCG2M (106 conidia/ml) were placed into a sterile at 25Њ in constant darkness, representing subjective dawn petri dish and exposed, with continuous shaking, to a short- (the time of peak ccg-1 and ccg-2 mRNA levels in wild- wave UV light (6 W) held 12 cm away. To select for mutations, type strains) and subjective early evening (the time of and 50% survival were 25ف dosages that corresponded to plated onto the appropriately supplemented media and incu- trough ccg-1 and ccg-2 mRNA levels in wild-type strains), bated in the dark at 30Њ. respectively (Figure 1A). After 20 hr in the dark (DD20), -fold in frq 10 as com-10ف Nucleic acid isolation and hybridization: RNA isolation, ccg-1 mRNA levels were elevated Northern hybridization, and densitometric data analysis proto- pared to the frqϩ strain. The high level of ccg-1 mRNA cols have been previously described (Bell-Pedersen et al. 1996a). at DD20 in the frq 10 strain was similar to the peak level Radioactive riboprobes were synthesized from pKL119 (ccg-1) ϩ or pLW1K (ccg-2) using T3 polymerase in the presence of [␣-32P] observed at DD12 in frq . Little difference was observed UTP. Radioactive mtr DNA probes were generated from in ccg-1 mRNA levels between the two strains at DD12. fold lower in-30ف pNC807 using [␣-32P]dATP. Northern blot signals were nor- Conversely, ccg-2 mRNA levels were malized in each experiment to rRNA, which remains at con- frq10 as compared to expression in the frqϩ strain at stant levels under the growth conditions used (Loros et al. DD12, and the levels were low in both strains at DD20. 1989). Similar results were observed in comparisons of ccg-1 and ccg-2 mRNA levels between wild type and a strain that produces a truncated WC-1 protein (wc-1ER53; Lee RESULTS et al. 2000; data not shown). These data suggest that the A genetic selection for mutations that affect expres- altered levels of ccg-1 and ccg-2 mRNA in the frq10 strain sion of the clock-controlled ccg-1 and ccg-2 genes: Rhyth- may not be a direct consequence of the absence of mic expression of the morning-specific ccg-1 and ccg-2 FRQ, but rather result from the loss of a functional frq- genes requires frq (Arpaia et al. 1993, 1995); however, oscillator. Together, these data support the idea that the expression levels of these genes in frq-null (frq 10) ccg-1 expression is negatively regulated and ccg-2 expres- strains have not been determined. The mRNA levels of sion is positively regulated by a pathway involving the 122 M. W. Vitalini et al.

TABLE 1 Strains used in this study

Straina Genotypeb TA FA Phenotype Other comments 30-7 bd mat A ϩϪwt 22.6-hr period 87-3 bd mat a ϩϪwt 40-1 bd frq 10 mat a ϩϪPale orange conidia 181 mtr SR33 trp-2 41 mat a Ϫϩwt 96-11 mtr bd trp-2 mat A Ϫϩwt 106-20 mtr bd trp-2 frq10 mat a ϪϩPale orange conidia CCG1M 106-20 ccg-1::mtr(EC) ϩϪPale orange conidia CCG2M 106-20 ccg-2::mtr(EC) ϪϩPale orange conidia COP1-1 106-20 ccg-1::mtr (EC) cop1-1 ϪϩPale orange conidia COP1-2 106-20 ccg-1::mtr(EC) cop1-2 ϪϩEas-like, odor Recessive COP1-3 106-20 ccg-1::mtr(EC) cop1-3 ϪϩEas-like, odor Recessive COP1-4 106-20 ccg-1::mtr(EC) cop1-4 ϪϩEas-like, odor Recessive COP1-5 106-20 ccg-1::mtr(EC) cop1-5 ϪϩEas-like, strong odor, sterile Dominant COP1-6 106-20 ccg-1::mtr(EC) cop1-6 ϪϩEas-like, strong odor, sterile Dominant COP1-2Fϩ bd cop1-2 ϩϪEas-like, odor Arrhythmic COP1-3Fϩ bd cop1-3 ϩϪEas-like, odor 20.1-hr period COP1-4Fϩ bd cop1-4 ϩϪEas-like, odor 19.9-hr period wt, wild type. a Fϩ refers to the strains carrying the wild-type frq gene. b EC, ectopic integration of plasmid. frq-oscillator (Figure 1B; Lindgren 1994; Bell-Peder- Pedersen et al. 2001), suggesting that the FRQ-mediated sen et al. 1996a, 2001; Yang et al. 2002). regulation of ccg-2 is indirect. Thus, the gene products Electrophoretic mobility shift assays indicated that that link FRQ levels to expression of ccg-2 and the com- FRQ protein is not part of protein complexes that bind ponents that regulate ccg-1 expression are unknown. to the clock regulatory element (ACE) of ccg-2 (Bell- We reasoned that mutations in genes in the circadian output pathways could be identified by finding mutant strains that express low levels of ccg-1 mRNA or high levels of ccg-2 mRNA in the frq10 background. Promoter regions containing sequences required for circadian expression of ccg-1 and ccg-2 were independently fused to the coding sequence of the mtr gene to produce pCCG1M and pCCG2M, respectively (materials and methods). The benomyl-resistant pCCG1M and pCCG2M plasmids were transformed into 106-20 (bd mtr trp-2 frq10) to produce the CCG1M and CCG2M strains, respectively. These transformants were chosen on the basis of their growth phenotypes in liquid culture at 25Њ (Table 1). Con- trol strain 106-20 did not grow in high-Arg/low-Trp (TA) media, but did grow in FPA/anthranilate (FA) media re- flecting the MtrϪ phenotype and inability of this strain to uptake Trp. Strain CCG1M grew in TA, but not in FA media. This growth pattern suggested high expression of the ccg-1::mtr fusion in the frq 10 strain and the ability of this strain to import both Trp and FPA. Strain CCG2M grew well in FA and had very little growth in TA media. This growth pattern suggested a low level of expression of the ccg-2::mtr fusion and decreased ability of this strain to im- Figure 2.—Reconstruction experiments to demonstrate the port both Trp and FPA. selection scheme for mutations in clock output pathways. To establish conditions for the selection of mutations, Strains CCG1M and CCG2M were spread onto plating medium containing tryptophan/arginine (TA) or p-fluorophenyl- strains CCG1M and CCG2M were examined on TA and alanine/anthranilate (FA) and photographed after 2 days of FA plating media following 2 days of growth in the dark growth in the dark at 30Њ. at 30Њ (Figure 2). Optimal conditions were found with Genetic Selection for Circadian Clock Mutations 123

Figure 3.—Endogenous ccg-1 and ccg-2 mRNA levels in selected mutant strains. (A) Densitometry of ccg-2 mRNA levels in control strains (bd frqϩ strain 30-7, bd frq 10 strain 40-1, and the CCG2M strain) and COP2-1 through COP2-8 mutant strains. The strains are indicated on the x-axis and the relative levels of ccg-2 mRNA are indicated on the y-axis. (B) A representative Northern blot showing ccg-1 mRNA levels from control (30-7, 40-1, and CCG1M) and COP1 mutant strains. rRNA is shown as a loading control. (C) Densitometry of ccg-1 mRNA levels in control (30-7, 40-1, and CCG1M) and COP1 mutant strains derived from CCG1M. The strains are indicated on the x-axis and the relative levels of ccg-1 mRNA are indicated on the y-axis. In each experiment, RNA was isolated from cultures harvested following 20 hr of growth in the dark, representing subjective early evening.

plating media containing Trp at 0.01 mg/ml plus 1 mg/ml also able to grow on minimal medium. These data sug- Arg or 60 ␮g/ml FPA. Under these conditions, the gested that these strains no longer required Trp for CCG1M strain grew on TA but not on FA; the CCG2M growth and therefore were undesired trp-2 revertants strain grew on FA, but not on TA. or suppressors (data not shown). These strains were Isolation of mutant strains that affect expression of not studied further. In addition, most (152) of the 160 ccg-1 and ccg-2: UV mutagenesis was conducted on strain CCG2M mutants that showed some growth on TA me- CCG1M to obtain mutant strains that grew on plating dium grew poorly on this medium and/or were still medium containing FA and on strain CCG2M to obtain able to grow on FA medium (data not shown). Of the mutant strains that grew on TA medium. Eighty mutant remaining eight FPA-sensitive CCG2M mutant strains, strains were obtained from the CCG1M strain and 165 we examined the levels of endogenous ccg-2 mRNA in ϫ 105 cultures harvested after 20 hr in the dark (DD20). This 6ف mutant strains from the CCG2M strain out of and 2 ϫ 105 cells, respectively. time of harvest represents subjective early evening, a The mutant strains were examined for growth on mini- time of day when cycling ccg-2 levels are normally low mal, FA, and TA media. Some mutant strains (5/165) in wild-type strains (Loros et al. 1989) and in the frq 10 selected from CCG2M that grew on TA medium were strain (Figure 1). We reasoned that mutations that affect 124 M. W. Vitalini et al. clock regulation of ccg-2 should result in increased levels of ccg-2 mRNA in the mutant strains as compared to the CCG2M strain. Indeed, the eight FPA-sensitive mutant strains, designated as COP2-1 through COP2-8, showed increased levels of endogenous ccg-2 mRNA as compared to the CCG2M strain (Figure 3A). The eight COP2 strains developed normally; however, since they carried the frq 10 mutation, the conidiation rhythm could not be observed using standard race tube assays (data not shown). Thus, other than the Mtrϩ phenotype, these strains lacked any readily observable phenotype that could be followed in standard genetic crosses. More importantly, the eight COP2 strains were sterile in crosses (data not shown). This phenotype might suggest that the mutations affect several ccgs, including those involved in mating (Bobrowicz et al. 2002). Because Figure 4.—Several COP1 mutant strains selected from the we were unable to readily cross the COP2 strains to CCG1M strain have an Eas-like phenotype. Slant cultures for ϩ isolate a single mutation associated with altered expres- control strain CCG1M, bd frq strain 30-7, and 101-3 (bd, ea- UCLA191 sion of ccg-2, these strains have not yet been further s mat A) along with homokaryotic isolates of three representative Eas-like mutant strains (COP1-2, COP1-3, and COP1- characterized. 4) are shown. The Eas phenotype is characterized by dark, Twenty-two of the 80 mutant strains isolated from the wetted asexual conidiospores. CCG1M strain grew poorly on selective FA medium or also grew on TA medium. The other 58 FPA-resistant mutant strains derived from the CCG1M strain, desig- showed the increased pigmentation phenotype associ- nated as COP1-1 through COP1-58, were examined by ated with the eas mutation. For each of the 5 Eas-like Northern assays for endogenous ccg-1 mRNA levels from COP1 mutant strains, the phenotype was slightly less cultures that were harvested after 20 hr of growth in severe than the Eas phenotype. In addition, each of the the dark (DD20). This represents a time of day when 5 Eas-like COP1 strains had an unpleasant odor (Table cycling ccg-1 levels are normally low in wild-type strains 1). This phenotype was heritable; progeny from crosses (Loros et al. 1989) and high in frq 10 strains (Figure 1). with 3 out of 3 of the COP1 strains (COP1-2, COP1-3, A representative Northern blot is shown in Figure 3B. and COP1-4) also had the odor associated with the Eas- The amounts of ccg-1 mRNA from some of the 58 FPA- like phenotype (data not shown, see Table 1). resistant COP1 strains were reduced from Ͻ10-fold to The COP1-2 through COP1-6 strains were of special slightly less than the amount of ccg-1 mRNA detected interest because the Eas phenotype is caused by loss of in the unmutagenized CCG1M strain. Eighteen FPA- function of the ccg-2 gene (Bell-Pedersen et al. 1992; resistant COP1 strains had endogenous ccg-1 mRNA lev- Lauter et al. 1992), but the mutations were isolated els that were equal to, slightly less than, or slightly from the ccg-1::mtr selection. These data suggested the greater than that observed in the control bd frqϩ strain possibility that a single mutation affected expression of 30-7. We reasoned that mutations that reduce ccg-1 not only ccg-1, but also other ccgs. To test this hypothesis, mRNA levels in the frq 10 strain to near those of the frqϩ the five Eas-like COP1 mutant strains were first exam- strain at DD20, the low point in the cycle, would likely ined for endogenous ccg-1 and ccg-2 mRNA levels, along represent mutations that affect clock control of ccg-1. with expression of the mtr transgene from cultures Thus, these 18 FPA-resistant COP1 mutant strains were grown in the dark and harvested after 20 hr (Figure 5). chosen for further study. As previously observed, in each mutant strain, the levels Characterization of the COP1 mutant strains: We fo- of endogenous ccg-1 mRNA and mtr mRNA from the cused on 18 COP1 strains that grew on FA, but not TA transgene were lower than those of the control unmuta- media, and exhibited ccg-1 expression levels that were genized strain. Furthermore, the levels of endogenous comparable to the mRNA levels observed in the bd frqϩ ccg-2 transcripts were reduced in each of the five Eas- strain 30-7 at DD20. Two major groups of phenotypes like COP1 mutant strains from their already low levels in were observed among homokaryotic isolates of these the CCG1M parental strain. However, there was notable strains. One group (n ϭ 13) was visually similar to the variability in the levels of ccg-2 transcripts, ranging from parental CCG1M strain (data not shown). A second slightly lower in COP1-5 to almost undetectable in group (n ϭ 5) showed an Eas-like phenotype of wetted COP1-3. These data suggest that different loci, or the conidia on slants, and the conidia were more readily severity of mutation of a single locus, resulted in differ- suspended in water than the unmutagenized CCG1M ent effects on ccg-1 and ccg-2 expression levels. None strain (Table 1 and Figure 4). The 5 COP1 strains with of the five Eas-like COP1 mutant strains displayed the the Eas-like phenotype (COP1-2 through COP1-6) also characteristic wc-1 or wc-2 mutant phenotype of white Genetic Selection for Circadian Clock Mutations 125

Figure 6.—The mutant phenotypes in the COP1-4 strain are due to mutation of a single gene. (A) Progeny from a second-generation cross of the COP1-4 strain were examined for ccg-1 mRNA levels after 20 hr of growth in the dark (DD20). RNA isolated from duplicate cultures of control bd frqϩ strain 30-7 and bd frq 10 strain 40-1 and four frq 10-carrying progeny of Figure 5.—The Eas-like COP1 mutant strains have reduced COP1-4 (labeled a, b, c, and d) was hybridized to a ccg-1 levels of ccg-2 mRNA. Densitometry of ccg-1, mtr, and ccg-2 probe. Ethidium bromide-stained rRNA is shown below to mRNA levels from Northern blots (not shown) in the control demonstrate equal loading of the gel. (B) Slant cultures of CCG1M strain and COP1-2, COP1-3, and COP1-4 mutant the COP1-4 progeny strains a–d are shown to illustrate the strains is shown. In each experiment, the levels of mRNA Eas-like phenotype. from the CCG1M strain were normalized to 1. The strains are indicated on the x-axis and the relative levels of mRNA are indicated on the y-axis. Four independent experiments were used to determine the standard deviation of RNA levels for like phenotype were obtained from the COP1-2, COP1-3, ccg-1 and ccg-2. In each experiment, RNA was isolated from and COP1-4 strains. In all first- and second-generation cultures harvested following 20 hr of growth in the dark, crosses, the Eas-like phenotype segregated 100% of the representing subjective early evening. time with low-level ccg-1 mRNA (n Ն 9; Figure 6 and data not shown). These data suggested that the mutant phenotypes in the COP1-2, COP1-3, and COP1-4 strains hyphae resulting from reduced carotenoids (Ballario are due to mutation of a single gene. The COP1-5 and et al. 1998), indicating that the mutations were not in COP1-6 strains were infertile and therefore were not either of these genes or in genes that greatly alter WC-1 examined further here. or WC-2 function. We used forced heterokaryon analyses, in which dif- We also examined ccg-2 mRNA levels in the other 13 ferent haploid nuclei are present in the same cell, to FPA-resistant mutations from CCG1M that exhibited determine if the mutations in the five Eas-like COP1 reduced ccg-1 expression, but did not display the Eas strains were dominant or recessive to the wild-type allele phenotype. One representative mutant strain, COP1-1, (Davis and deSerres 1970). The mutations in the is shown in Figure 5. For COP1-1, the levels of ccg-2 COP1-5 and COP1-6 strains showed dominance to the mRNA were similar to the levels observed in the CCG1M wild-type allele for the Eas-like phenotype, whereas the strain, suggesting that the defect in the COP1-1 mutant mutations in the COP1-2, COP1-3, and COP1-4 strains strain specifically affects ccg-1 gene regulation. were recessive (data not shown). To verify that the effects on ccg-1 and ccg-2 gene ex- The mutations in the COP1-1, COP1-2, and COP1-4 pression in the COP1-2 through COP1-6 strains were strains affect overt rhythmicity and circadian clock out- the result of a single mutation, each strain was crossed put: To examine if the frq-oscillator is affected in the to the bd frqϩ strain 30-7. Haploid progeny were scored COP1-2, COP1-3, and COP1-4 mutant strains we exam- for the Eas-like phenotype and for low-level endogenous ined progeny from crosses with the 30-7 strain (bd frqϩ). ccg-1 expression at DD20. Both frqϩ and frq 10 progeny Race tube assays were used to examine the conidiation with low-level endogenous ccg-1 expression and the Eas- rhythms of frqϩ COP1-2, COP1-3, and COP1-4 mutant 126 M. W. Vitalini et al.

Figure 7.—Conidiation rhythms are altered in the Eas-like COP1 mutant strains. (A) Race tube assays show the conidiation rhythms of control bd frqϩ strain 30-7 and CCG1M strains, along with mutant strains COP1-2, COP1-3, and COP1-4. Progeny from each of these strains that carry the frqϩ allele are also shown (labeled Fϩ). Two representative race tubes are shown for the COP1-2Fϩ strain. Race tubes were incubated in the light for 1 day at 25Њ and then transferred to the dark (25Њ). The solid lines indicate the growth front after 24 hr. The period lengths of the rhythmic strains are indicated on the left (n Ն 18; standard deviations are indicated). (B) Race tube assay of the COP1-2 strains in the frq10 background. Several independent experiments are shown to depict the unusual rhythm in conidiation for COP1-2 strains that lack frq. strains (designated as COP1-2Fϩ, COP1-3Fϩ, and albeit erratic, these data suggest that the mutation pres- COP1-4Fϩ strains; Table 1), along with those of the ent in the COP1-2 strain is a partial suppressor of the parental frq 10 mutant strains (Figure 7A). Of the frqϩ frq-null phenotype. progeny, the COP1-3Fϩ and COP1-4Fϩ strains dis- The COP1-2Fϩ, COP1-3Fϩ, and COP1-4Fϩ progeny played conidiation rhythms with periods of 20.1 Ϯ 0.1 hr strains were also examined by Northern assays for circa- hr rhythm dian rhythms in ccg-1 and ccg-2 mRNA accumulation-20ف and 19.9 Ϯ 0.4 hr, respectively. This ,hr shorter than (Figure 8, A and B). In the control 30-7 (bd frqϩ) strain 2ف observed in both mutant strains is that observed in the wild-type clock strain 30-7. Follow- both ccg-1 and ccg-2 mRNA accumulated rhythmically ing transition from light to dark, the COP1-2Fϩ mutant and peaked in mRNA levels in the subjective morning strain was rhythmic for 2–4 days, but produced qualita- (8–16 hr in the dark on day 1 and 36–40 hr in the dark tively low levels of conidia. After this initial period, the on day 2). Both COP1-2Fϩ and COP1-3Fϩ strains had COP1-2Fϩ strain became arrhythmic and produced randomly ﬂuctuating, but primarily low, levels of ccg-1 more conidia. These data are consistent with the muta- and ccg-2 mRNA over the course of the day. Interestingly, tions in the COP1-2, COP1-3, and COP1-4 strains affect- the COP1-4Fϩ strain had widely variable levels of both ing either the conidiation output pathway or the circa- ccg-1 and ccg-2 mRNA. However, ccg-1 or ccg-2 mRNA dian oscillator itself. Interestingly, the parental COP1-2 levels in the COP1-4Fϩ strain did not display a typical strain (which carries the frq 10 allele) displayed rhythmic circadian pattern. These data demonstrate that circa- out of 10 experi- dian regulation of ccg-1 and ccg-2 is defective in the 1ف conidiation in constant darkness in ments, whereas the COP1-3 and COP1-4 strains were COP1-2Fϩ, COP1-3Fϩ, and COP1-4Fϩ mutant strains. always arrhythmic. Representative race tubes of the COP1-2 strain are shown in Figure 7B. Diagnostic PCR DISCUSSION was used to conﬁrm that these COP1-2 cells harbored the frq 10 allele (data not shown). Because the COP1-2 The output pathway from the clock appears to involve strain is capable of rhythmcity in the frq 10 background, a repressor of ccg-1 and an activator of ccg-2: In this Genetic Selection for Circadian Clock Mutations 127

Figure 8.—The ccg-1 and ccg-2 mRNA levels are arrhythmic in the Eas-like COP1 mutant strains. The levels of ccg-1 and ccg-2 mRNA were assayed by Northern blots in control bd frqϩ strain 30-7 and the COP1-2, COP1-3, and COP1-4 mutant strains in the frqϩ background (labeled COP1-2Fϩ, COP1-3Fϩ, and COP1-4Fϩ). Liquid cultures of mycelia were grown in constant darkness and harvested after the indicated times in the dark (DD). The RNA was hybridized with either ccg-1 (A) or ccg-2 (B) probes and rRNA probes for normalization. Following autoradiography, mRNA levels were normalized to rRNA, which remains at constant levels under these conditions (Loros et al. 1989), and plotted as relative band intensity vs. time in the dark. The lowest point for each individual blot was set to 1. For both A and B plots, solid lines with solid diamonds represent mRNA from 30-7; thick shaded lines with solid circles represent COP1-2Fϩ; thin shaded lines with solid triangles represent COP1-3Fϩ; and dashed shaded lines with solid squares represent COP1-4Fϩ.

study, we utilized a selection for mutations (Carattoli independent pathways: one that induces a repressor of et al. 1995) that affect the expression of two clock-con- ccg-1 in the evening and another that induces an activa- trolled genes, ccg-1 and ccg-2. The selection was based tor of ccg-2 in the morning (Figure 1B). In the absence on the differential expression of the ccgs in response to of the frq-oscillator, the repressor of ccg-1 and the activa- the absence of the clock component FRQ. Both ccg-1 tor of ccg-2 would both have reduced activity resulting and ccg-2 are under control of the circadian clock and in high levels of ccg-1 mRNA and low levels of ccg-2 peak in mRNA accumulation in the subjective morning. mRNA at all times of day. On the basis of this simple However, in the frq 10 strain, ccg-1 mRNA fluctuates at a model, we anticipated identifying mutations that af- level comparable to the peak levels in wild-type strains fected just ccg-1 or just ccg-2 expression levels. Some of over the course of the day, while ccg-2 mRNA remains at the identified mutations in the frq 10 background indeed levels comparable to its lowest level in wild-type strains. caused reduced ccg-1 mRNA levels, but had no effect Deletion of promoter elements required for rhythmic on the levels of ccg-2 mRNA (e.g., the mutation in the ccg-1 expression results in high-level arrhythmic expres- COP1-1 strain, Figure 5). sion of ccg-1 mRNA, suggesting that clock regulation of Identification of single-gene mutations that affect both ccg-1 involves a repressor (Lindgren 1994). Conversely, ccg-1 and ccg-2 gene expression: We identified five FPA- deletion of the ccg-2 ACE results in low-level arrhythmic resistant COP1 mutant strains that had reduced ccg-1 expression, indicating that an activator of transcription expression and that also had reduced expression of is involved in rhythmic transcription of ccg-2 (Bell-Ped- ccg-2. These strains grew and developed normal conidi- ersen et al. 2001). One simple model to explain this ospores (with the exception of the Eas-like phenotype), regulation would be that the frq-oscillator regulates two suggesting that the mutations did not affect the general 128 M. W. Vitalini et al. transcriptional machinery or primary development. Im- rhythm observed in the parental COP1-2 strain (car- portantly, the Eas-like phenotype and low-level ccg-1 ex- rying the frq 10 allele) differs substantially from the overt pression in the frq 10 background segregated together in conidiation rhythms that are sometimes observed in frq- crosses, implying that the phenotypes are the result of null strains (Loros and Feldman 1986; Aronson et al. a single mutation. Furthermore, previous studies have 1994a). These frq-null rhythms have been attributed to shown that the overall expression levels and rhythmicity a second cellular oscillator called the FRQ-less oscillator of ccg-2 mRNA are unaffected in strains that lack ccg-1 (FLO; Merrow et al. 1999; Loros and Dunlap 2001). (Lindgren 1994), and the overall expression levels and However, with the exception of COP1-2 strains, strains rhythmicity of ccg-1 mRNA are unaffected in strains that carrying the frq10 allele were arrhythmic in our growth have low-level constitutive ccg-2 mRNA expression (Bell- conditions. Thus, it is possible that the erratic rhythm Pedersen et al. 1996a). Together, these data suggest observed in the COP1-2 strain is due to its effect on the that the simple model of frq-oscillator regulation of two FLO, which results in partial suppression of the frq-null distinct output pathways arising directly from the oscilla- phenotype. tor, one pathway involving a ccg-1 repressor and a second Each of the five Eas-like COP1 mutant strains had an pathway involving an activator of ccg-2 (Figure 1B), is unpleasant odor. The volatile molecule responsible for not correct. the smell is not known. During mating, strains of each One mechanism to explain how a single mutation could mating type secrete a mating-type-specific pheromone cause the reduction of expression for both ccg-1 and that gives off a strong odor similar to the odor given ccg-2 mRNA in the frq 10 background is that the mutation off by the five Eas-like Cop1 mutant strains. However, simultaneously increases the activity of a repressor of because the pheromones have not been purified, we do ccg-1 and decreases activity of an activator of ccg-2. In not know if the odor from the Eas-like Cop1 mutant strains this scenario, a bifurcation in the clock output pathway is the pheromone. The N. crassa pheromone genes ccg-4 from the frq-oscillator to the ccg-1 and ccg-2 genes would (mfA) and mfa are regulated by the circadian clock be predicted. In the frqϩ background, the mutations (Bobrowicz et al. 2002). Therefore, it is possible that present in the COP1-2Fϩ, COP1-3Fϩ, and COP1-4Fϩ the effect on the output pathways in the Eas-like COP1 strains abolish ccg-1 and ccg-2 rhythmicity. The gene(s) mutant strains also causes misexpression of the phero- mutated in these strains could function in an output mone genes and that this is responsible for the odor. pathway upstream of the bifurcation or may possibly act Assuming that high levels of pheromone have an effect in the frq-oscillator or in a second coupled oscillator. on fertility, this idea is consistent with the finding that However, the lack of a correlation between the race tube while the COP1-2, COP1-3, and COP1-4 strains were data, in which period defects in the conidiation rhythm fertile in crosses, the level of fertility was reduced. The are observed in the COP1-3 and COP1-4 strains, and two strains with the strongest odor, COP1-5 and COP1-6, the complete lack of rhythmicity of ccg-1 and ccg-2 mRNA were infertile in crosses. Alternatively, the reduced fertil- levels in the same strains suggest that the mutations ity of the mutant strains could be related to the benomyl directly affect the output pathways rather than the oscil- resistance gene that is present in the transformed strains lator itself. The mutation in the COP1-1 strain, on the (Staben et al. 1989). other hand, appears to be specific for ccg-1 regulation, The wild-type genes specified by the recessive muta- and thus the gene product likely functions downstream tions in the FPA-resistant COP1-2, COP1-3, and COP1-4 of the branch point. Verification of these hypotheses strains can now be cloned through complementation awaits a molecular examination of oscillator compo- with existing cosmid or genomic libraries to identify clones nents in the COP1-1, COP1-2, COP1-3, and COP1-4 that complement the loss of mtr function (i.e., growth mutant strains and the cloning and analyses of the genes on TA), but that do not themselves contain mtr. Further- uncovered by the mutations. more, this selection scheme is now being used to identify One of the most interesting mutant strains studied mutations that affect expression of ccg-1 and ccg-2 in the was COP1-2, because in the frq 10 background this strain frqϩ background and will be used in the future to select often showed unusual and inconsistent rhythms in co- for extragenic suppressors of existing mutations using nidial development on race tubes. The reason for the the opposite selection. variability in observing the rhythm and of the rhythm We thank members of our laboratory for critical discussions and itself is not known at this time, but may be related to Richard Gomer and Andrew Greene for comments on the manuscript. the nature of the mutation and the growth conditions We are also grateful to Susan Andershock and Amy Kotara for techni- used to examine the rhythms. When the COP1-2 strain cal assistance and Jay Dunlap, Jennifer Loros, and David Stadler for was crossed to isolate the mutation in the frqϩ back- strains. This work was supported by the National Institutes of Health ground (COP1-2Fϩ), haploid progeny that were Eas-like (GM58529). also showed unusual rhythms; the strains were rhythmic for up to 4 days and then became arrhythmic. Interest- ingly, rhythmicity corresponded to low levels of conidi- LITERATURE CITED ospore production, whereas arrhythmicity correlated Aronson, B. D., K. A. Johnson and J. C. Dunlap, 1994a Circadian with higher levels of conidiospores. Furthermore, the clock locus frequency: protein encoded by a single open reading Genetic Selection for Circadian Clock Mutations 129

frame defines period length and temperature compensation. terial circadian system: a clock apart. Curr. Opin. Microbiol. 1: Proc. Natl. Acad. Sci. USA 91: 7683–7687. 669–673. Aronson, B. D., K. A. Johnson, J. J. Loros and J. C. Dunlap, 1994b He, Q., P. Cheng, Y. Yang, L. Wang, K. H. Gardner et al., 2002 Negative feedback defining a circadian clock: autoregulation of WHITE COLLAR-1, a DNA binding transcription factor and a the clock gene frequency. Science 263: 1578–1584. light sensor. Science 297: 840–843. Arpaia, G., J. J. Loros, J. C. Dunlap, G. Morelli and G. Macino, Keesey, J., J. Paukert and J. Demoss, 1981 Subunit structure of 1993 The interplay of light and the circadian clock. Indepen- anthranilate synthase from Neurospora crassa: preparation and dent dual regulation of clock-controlled gene ccg-2(eas). Plant Physiol. characterization of a protease-free form. Arch. Biochem. Biophys. 102: 1299–1305. 207: 103–109. Arpaia, G., J. J. Loros, J. C. Dunlap, G. Morelli and G. Macino, Koo, K., and W. D. Stuart, 1991 Sequence and structure of mtr, 1995 Light induction of the clock-controlled gene ccg-1 is not trans- an amino acid transport gene of Neurospora crassa. Genome 34: duced through the circadian clock in Neurospora crassa. Mol. Gen. 644–651. Genet. 247: 157–163. Lakin-Thomas, P. L., and S. Brody, 2000 Circadian rhythms in Ballario, P., C. Talora, D. Galli, H. Linden and G. Macino, 1998 Neurospora crassa: lipid deficiencies restore robust rhythmicity to Roles in dimerization and blue light photoresponse of the PAS null frequency and white-collar mutants. Proc. Natl. Acad. Sci. USA and LOV domains of Neurospora crassa WHITE COLLAR proteins. 97: 256–261. Mol. Microbiol. 29: 719–729. Lauter, F. R., V. E. Russo and C. Yanofsky, 1992 Developmental Bell-Pedersen, D., J. C. Dunlap and J. J. Loros, 1992 The Neuro- and light regulation of eas, the structural gene for the rodlet spora circadian clock-controlled gene, ccg-2, is allelic to eas and en- protein of Neurospora. Genes Dev. 6: 2373–2381. codes a fungal hydrophobin required for formation of the conid- Lee, K., J. J. Loros and J. C. Dunlap, 2000 Interconnected feedback ial rodlet layer. Genes Dev. 6: 2382–2394. loops in the Neurospora circadian system. Science 289: 107–110. Bell-Pedersen, D., J. C. Dunlap and J. J. Loros, 1996a Distinct cis- Lindgren, K. M., 1994 Characterization of ccg-1,aclock-controlled gene acting elements mediate clock, light, and developmental regula- of Neurospora crassa. Thesis, Dartmouth Medical School, Hanover, tion of the Neurospora crassa eas (ccg-2) gene. Mol. Cell. Biol. 16: NH. 513–521. Liu, Y., J. Loros and J. C. Dunlap, 2000 Phosphorylation of the Bell-Pedersen, D., M. L. Shinohara, J. J. Loros and J. C. Dunlap, Neurospora clock protein FREQUENCY determines its degrada- 1996b Circadian clock-controlled genes isolated from Neurospora tion rate and strongly influences the period length of the circa- crassa are late night- to early morning-specific. Proc. Natl. Acad. dian clock. Proc. Natl. Acad. Sci. USA 97: 234–239. Sci. USA 93: 13096–13101. Loros, J. J., and J. C. Dunlap, 1991 Neurospora crassa clock-controlled Bell-Pedersen, D., Z. A. Lewis, J. J. Loros and J. C. Dunlap, 2001 genes are regulated at the level of transcription. Mol. Cell. Biol. The Neurospora circadian clock regulates a transcription factor 11: 558–563. that controls rhythmic expression of the output eas(ccg-2) gene. Loros, J. J., and J. C. Dunlap, 2001 Genetic and molecular analysis of Mol. Microbiol. 41: 897–909. circadian rhythms in Neurospora. Annu. Rev. Physiol. 63: 757–794. Bobrowicz, P., R. Pawlak, A. Correa, D. Bell-Pedersen and D. J. Loros, J. J., and J. F. Feldman, 1986 Loss of temperature compensa- Ebbole, 2002 The Neurospora crassa pheromone precursor tion of circadian period length in the frq-9 mutant of Neurospora genes are regulated by the mating type locus and the circadian crassa. J. Biol. Rhythms 1: 187–198. clock. Mol. Microbiol. 45: 795–804. Loros, J. J., S. A. Denome and J. C. Dunlap, 1989 Molecular cloning Carattoli, A., E. Kato, M. Rodriguez-Franco, W. D. Stuart and G. of genes under control of the circadian clock in Neurospora. Macino, 1995 A chimeric light-regulated amino acid transport Science 243: 385–388. system allows the isolation of blue light regulator (blr) mutants McClung, C. R., B. A. Fox and J. C. Dunlap, 1989 The Neurospora of Neurospora crassa. Proc. Natl. Acad. Sci. USA 92: 6612–6616. clock gene frequency shares a sequence element with the Drosoph- Chakraborty, B. N., N. A. Patterson and M. Kapoor, 1991 An ila clock gene period. Nature 339: 558–562. electroporation-based system for high-efficiency transformation Merrow, M., M. Brunner and T. Roenneberg, 1999 Assignment of germinated conidia of filamentous fungi. Can. J. Microbiol. of circadian function for the Neurospora clock gene frequency. 37: 858–863. Nature 399: 584–586. Cheng, P., Y. Yang and Y. Liu, 2001 Interlocked feedback loops Millar, A. J., I. A. Carre, C. A. Strayer, N. H. Chua and S. A. contribute to the robustness of the Neurospora circadian clock. Kay, 1995 Circadian clock mutants in Arabidopsis identified by Proc. Natl. Acad. Sci. USA 98: 7408–7413. luciferase imaging. Science 24: 1161–1163. Correa, A., Z. A. Lewis,A.V.Greene,I.J.March,R.H.Gomer et al., Morgan, L. W., J. F. Feldman and D. Bell-Pedersen, 2001 Genetic 2003 Multiple oscillators regulate circadian gene expression in interactions between clock mutations in Neurospora crassa: can Neurospora. Proc. Natl. Acad. Sci. USA 100: 13597–13602. they help us to understand complexity? Philos. Trans. R. Soc. Davis, R. L., and D. deSerres, 1970 Genetic and microbial research Lond. B Biol. Sci. 356: 1717–1724. techniques for Neurospora crassa. Methods Enzymol. 27A: 79–143. Nowrousian, M., G. E. Duffield, J. J. Loros and J. C. Dunlap, Denault, D. L., J. J. Loros and J. C. Dunlap, 2001 WC-2 mediates 2003 The frequency gene is required for temperature-dependent WC-1-FRQ interaction within the PAS protein-linked circadian regulation of many clock-controlled genes in Neurospora crassa. feedback loop of Neurospora. EMBO J. 20: 109–117. Genetics 164: 923–933. Dillon, D., and D. Stadler, 1994 Spontaneous mutation at the mtr Reppert, S. M., and D. R. Weaver, 2002 Coordination of circadian locus in Neurospora: the molecular spectrum in wild-type and a timing in mammals. Nature 418: 935–941. mutator strain. Genetics 138: 61–74. Sargent, M. L., W. R. Briggs and D. O. Woodward, 1966 Circadian Dunlap, J. C., J. J. Loros, Y. Liu and S. K. Crosthwaite, 1999 nature of a rhythm expressed by an invertaseless strain of Neuro- Eukaryotic circadian systems: cycles in common. Genes Cells 4: spora crassa. Plant Physiol. 41: 1343–1349. 1–10. Staben, C., B. Jensen, M. Singer, J. Pollock, M. Schechtman et al., 1989 Use of bacterial hygromycin B resistance gene as a domi- Ebbole, D., and M. Sachs, 1990 A rapid and simple method for nant selectable marker in Neurospora crassa transformation. Fun- isolation of Neurospora crassa homokaryons using microconidia. gal Genet. Newsl. 36: 79–81. Fungal Genet. Newsl. 37: 16–17. Stadler, D., H. Macleod and D. Dillon, 1991 Spontaneous muta- Edmunds, L. N., 1988 Cellular and Molecular Bases of Biological Clocks. tion at the mtr locus of Neurospora: the spectrum of mutant Springer-Verlag, New York. types. Genetics 129: 39–45. Feldman, J. F., and M. N. Hoyle, 1973 Isolation of circadian clock Yang, Y., P. Cheng and Y. Liu, 2002 Regulation of the Neurospora mutants of Neurospora crassa. Genetics 75: 605–613. circadian clock by casein kinase II. Genes Dev. 16: 994–1006. Froehlich, A. C., Y. Liu, J. J. Loros and J. C. Dunlap, 2002 WHITE Young, M. W., and S. A. Kay, 2001 Time zones: a comparative COLLAR-1, a circadian blue light photoreceptor, binding to the genetics of circadian clocks. Nat. Rev. Genet. 2: 702–715. frequency promoter. Science 297: 815–819. Zhu, H., M. Nowrousian, D. Kupfer, H. V. Colot, G. Berrocal- Froehlich, A. C., J. J. Loros and J. C. Dunlap, 2003 Rhythmic Tito et al., 2001 Analysis of expressed sequence tags from two binding of a WHITE COLLAR-containing complex to the fre- starvation, time-of-day-specific libraries of Neurospora crassa reveals quency promoter is inhibited by FREQUENCY. Proc. Natl. Acad. novel clock-controlled genes. Genetics 157: 1057–1065. Sci. USA 100: 5914–5919. Golden, S. S., C. H. Johnson and T. Kondo, 1998 The cyanobac- Communicating editor: M. S. Sachs