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Genetics, Vol. 167, 119-129, May 2004, Copyright © 2004

A Genetic Selection for Circadian Output Pathway Mutations in Neurospora crassa

Michael W. Vitalinia, Louis W. Morgana, Irene J. Marcha, and Deborah Bell-Pedersena
a Center for Biological Clocks Research and Program for the Biology of Filamentous Fungi, Department of Biology, Texas A&M University, College Station, Texas 77843

Corresponding author: Deborah Bell-Pedersen, Department of Biology, Texas A&M University, College Station, TX 77843., dpedersen{at}mail.bio.tamu.edu (E-mail)

Communicating editor: M. S. SACHS


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

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.

CIRCADIAN (daily) rhythms are endogenous, self-sustaining oscillations that are regulated by a central pacemaker composed of one or more biochemical oscillators (EDMUNDS 1988 Down; YOUNG and KAY 2001 Down). These rhythms are observed in a wide variety of organisms, ranging from daily rhythms in photosynthesis in cyanobacteria and plants, to development in fungi, to activity and sleep-wake cycles in rodents and humans. The core oscillators of several organisms consist of autoregulatory feedback loops, where the positive elements of the loop activate transcription of the negative elements. The negative elements then inhibit their own transcription by blocking the activity of the positive elements (DUNLAP et al. 1999 Down; YOUNG and KAY 2001 Down; REPPERT and WEAVER 2002 Down).

The circadian clock system of Neurospora crassa is one of the best understood (LOROS and DUNLAP 2001 Down). N. crassa displays an easily viewed natural 22-hr rhythm of asexual spore development (conidiation) in constant darkness. This conidiation rhythm provided the basis for "brute force" genetic screens for mutations that affect the clock. The frequency (frq) gene was identified through such screens (FELDMAN and HOYLE 1973 Down; LOROS and FELDMAN 1986 Down) and was later shown to be a core oscillator component required for circadian rhythmicity (MCCLUNG et al. 1989 Down; ARONSON et al. 1994B Down).

The frq-oscillator consists of interconnected positive and negative feedback loops involving the products of the frq and white collar (wc-1, wc-2) genes (LOROS and DUNLAP 2001 Down). The WC-1 blue light photoreceptor (FROEHLICH et al. 2002 Down; HE et al. 2002 Down) forms a complex with WC-2 [white collar complex (WCC)]. This complex directly activates transcription of the frq gene (FROEHLICH et al. 2003 Down). Levels of FRQ protein then slowly increase. At high levels, FRQ protein binds to and inhibits the activity of the WCC (CHENG et al. 2001 Down; DENAULT et al. 2001 Down). This causes frq transcription and the accumulation of FRQ protein to stop. Subsequent phosphorylation-induced decay of FRQ leads to reactivation of frq transcription, allowing the cycle to start again (LIU et al. 2000 Down). In addition to the frq-oscillator, several lines of evidence have suggested the presence of additional oscillators in the cell (LOROS and FELDMAN 1986 Down; ARONSON et al. 1994A Down; MERROW et al. 1999 Down; LAKIN-THOMAS and BRODY 2000 Down; MORGAN et al. 2001 Down; CORREA et al. 2003 Down). However, components of the other oscillator(s) have not yet been identified.

Time of day information is signaled from the oscillator(s) to genes and gene products residing in output pathways to generate the observed daily rhythms. Genes that are rhythmically expressed under control of the clock, and reside downstream of the output pathways from the clock, are termed clock-controlled genes (ccgs; LOROS et al. 1989 Down). To date, >150 ccgs have been identified in N. crassa (LOROS et al. 1989 Down; BELL-PEDERSEN et al. 1996B Down; ZHU et al. 2001 Down; CORREA et al. 2003 Down; NOWROUSIAN et al. 2003 Down), yet few details of the output pathways from the N. crassa, or any other organism's, pacemaker are known.

The functions of the N. crassa ccg products are varied, ranging from involvement in development to stress responses and metabolic processes. The two most highly characterized ccgs are the morning-specific genes ccg-1 and eas(ccg-2) (hereafter called ccg-2 for simplicity). Clock control for both ccg-1 and ccg-2 requires a functional frq-oscillator (ARPAIA et al. 1993 Down, ARPAIA et al. 1995 Down). The abundantly expressed ccg-2 gene encodes a member of a class of low molecular weight, cysteine-rich secreted hydrophobic proteins called hydrophobins (BELL-PEDERSEN et al. 1992 Down; LAUTER et al. 1992 Down). The hydrophobins coat the outer cell wall of fungi and maintain the cell-surface hydrophobicity essential for air dispersal of mature conidiospores. Inactivation of ccg-2 results in an "easily wettable" (Eas) phenotype in which the asexual conidiospores have a darkened, wetted appearance and mix readily into water (BELL-PEDERSEN et al. 1992 Down). The function of ccg-1 is unknown; ccg-1-null strains do not display any discernible phenotype (LINDGREN 1994 Down). In addition, the CCG-1 protein sequence does not share similarity to other characterized proteins, but CCG-1 is conserved among filamentous fungi.

Both the ccg-1 and ccg-2 genes were shown by nuclear run-on assays to be controlled by the clock, at least partially, at the level of transcription (LOROS and DUNLAP 1991 Down). 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 –150 and –118 nucleotides (nt) from the start of transcription (BELL-PEDERSEN et al. 1996A Down, BELL-PEDERSEN et al. 2001 Down). Deletion of the ACE resulted in constitutive low-level expression of ccg-2 mRNA in constant darkness, suggesting that rhythmicity results from activation of transcription by the clock. A negative promoter element required for ccg-1 rhythmicity was identified between –444 and –91 nt upstream from the transcriptional start site (LINDGREN 1994 Down). Deletion of this element resulted in randomly fluctuating high levels of ccg-1 mRNA in constant darkness. These data suggested that, while ccg-1 and ccg-2 both peak in expression in the subjective early morning, they are regulated differently by the clock.

The use of genetics in the study of clocks has been restricted to brute force screening for altered overt rhythmicity or misexpression of a reporter gene to identify clock-associated mutations (MILLAR et al. 1995 Down; GOLDEN et al. 1998 Down; LOROS and DUNLAP 2001 Down). No targeted selections or suppressor screens have been described. We have utilized a genetic selection (CARATTOLI et al. 1995 Down) to identify additional components of the N. crassa circadian clock system by taking advantage of the clock-dependent regulation of the ccg-1 and ccg-2 genes.

To accomplish the selection, promoter fragments of the ccg-1 and ccg-2 genes containing the clock regulatory elements were fused to the mtr (methyl tryptophan resistance) gene. The N. crassa mtr gene encodes an amino acid permease required for uptake of neutral aliphatic and aromatic amino acids (KOO and STUART 1991 Down; STADLER et al. 1991 Down; DILLON and STADLER 1994 Down; CARATTOLI et al. 1995 Down). Loss of mtr function can be selected for on the basis of resistance to the toxic amino acid analogs p-fluorophenylalanine (FPA) or 4-methyltryptophan. Gain of mtr function can be selected for on high-arginine (Arg)/low-tryptophan (Trp) medium in a trp-2 mutant background. Arg blocks the basic amino acid transporter, making the permease encoded by mtr essential for transport of Trp. The ccg::mtr fusions were individually transformed into a mtr trp-2 frq10 strain. The frq10 mutation is a null mutation that was produced by replacement of the frq open reading frame with the hygromycin resistance gene (ARONSON et al. 1994A Down). In frq10 strains, expression of the ccg-1::mtr transgene is high and the cells are Mtr+ (grow on Trp/Arg medium, but not on FPA). Alternatively, in the frq10 strain, expression of the ccg-2::mtr transgene is low and the cells are Mtr– (grow on FPA, but not on Trp/Arg medium). To identify genes involved in regulating ccg-1 and ccg-2 expression, mutations that altered the Mtr-based growth phenotype of strains containing either the ccg-1::mtr transgene or the ccg-2::mtr transgene were selected and analyzed for expression of the endogenous ccg-1 and ccg-2 genes.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Plasmid construction:
The –481 to +57 promoter region of the ccg-1 gene was amplified by PCR using pKL119 (LINDGREN 1994 Down) as a template and 5'-CTAGGAATTCTGTTGACCATTGGTTAC-3' and 5'-CTAGAGGCCTGAGAAGAATAGAAGC-3' as primers. The –1079 to +1 promoter region of the ccg-2 gene was amplified by PCR using pLW1K (BELL-PEDERSEN et al. 1992 Down) as a template and the primers 5'-CTAGGAATTCGTCACAGACAACGGGTGA-3' and 5'-CTAGAGGCCTGGTTTGAGTGTTGTGTTG-3'. Because the basal level of ccg-2 transcription from promoter fragments containing only the ACE is low, we used a promoter fragment that drives higher levels of ccg-2 expression and contains promoter elements involved in clock (ACE) and light regulation of ccg-2 (BELL-PEDERSEN et al. 1996A Down, BELL-PEDERSEN et al. 2001 Down). The use of the larger ccg-2 promoter fragment may reduce the sensitivity of the selection and allow for the isolation of mutations that affect light regulation of ccg-2. The primer pairs introduced an EcoRI site (underlined) at the 5' end and a StuI site (underlined) at the 3' end of the amplified DNA. The promoters were cloned separately into pNC807 (KOO and STUART 1991 Down) digested with EcoRI and StuI, creating ccg promoter::mtr gene fusions. The 3.2-kb ccg-1::mtr and 3.6-kb ccg-2::mtr EcoRV fragments of the resulting plasmids were subsequently cloned into the EcoRV site of pMLSBml, generating plasmids pCCG1M and pCCG2M, respectively. pMLSBml was kindly provided by Drs. Mari Shinohara and Jay Dunlap, Dartmouth Medical School, and contains the benomyl resistance gene isolated from pSV50 (Fungal Genetics Stock Center, Kansas City, MO) inserted into the SmaI site of pBSKII+ (Stratagene, La Jolla, CA).

Strains and culture conditions:
The mtrSR33 trp-241 mat A strain was obtained from David Stadler (University of Washington). The mtr (methyl-tryptophan resistance) mutation carried by this strain is nonrevertible due to a small deletion in the coding sequence. The trp-2 gene encodes anthranilate synthetase (KEESEY et al. 1981 Down). The mtrSR33 trp-241 mat A strain was crossed to lab strain 40-1 (bd frq10 mat a) to obtain 106-20 (bd frq10 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 Down). The frq10 null mutation was generated by replacement of the frq open reading frame with the hygromycin resistance gene (ARONSON et al. 1994A Down). 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 Down). 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-µm filter (EBBOLE and SACHS 1990 Down) 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 according to standard procedures (DAVIS and DESERRES 1970 Down). Strains with the frq10 mutation were maintained on hygromycin B (200 µg/ml). Benomyl was added at 1 µg/ml to media before autoclaving to select for strains carrying pCCG1M or pCCG2M constructs. Strains carrying the trp-2 mutation were supplemented with 10 µg/ml anthranilate. Anthranilate is a metabolic precursor of tryptophan that does not require mtr for uptake. Strains carrying the mtr mutation are resistant to the toxic amino acid analog FPA at 60 µg/ml. Selection of mutant strains that negatively affect expression of the ccg::mtr transgene, resulting in low-level mtr expression, were performed on media containing FPA, anthranilate, and benomyl (FA medium). Selection of mutant strains that positively affect expression of the transgene, resulting in high-level mtr expression, was performed on 0.01 mg/ml Trp, 1 mg/ml Arg, and benomyl (TA medium). Race tube assays were carried out at 25° on glucose-arginine media (0.1% glucose, 0.5% arginine, 1x Vogel's salts, 10 µg/ml anthranilate, 2% agar). For RNA analyses, tissue was grown and synchronized by light-to-dark transfers following published procedures (LOROS et al. 1989 Down).

Mutagenesis:
Conidial suspensions (15 ml) of strains CCG1M or CCG2M (106 conidia/ml) were placed into a sterile petri dish and exposed, with continuous shaking, to a short-wave UV light (6 W) held 12 cm away. To select for mutations, dosages that corresponded to ~25 and 50% survival were plated onto the appropriately supplemented media and incubated in the dark at 30°.

Nucleic acid isolation and hybridization:
RNA isolation, Northern hybridization, and densitometric data analysis protocols have been previously described (BELL-PEDERSEN et al. 1996A Down). Radioactive riboprobes were synthesized from pKL119 (ccg-1) or pLW1K (ccg-2) using T3 polymerase in the presence of [{alpha}-32P]UTP. Radioactive mtr DNA probes were generated from pNC807 using [{alpha}-32P]dATP. Northern blot signals were normalized in each experiment to rRNA, which remains at constant levels under the growth conditions used (LOROS et al. 1989 Down).


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

A genetic selection for mutations that affect expression of the clock-controlled ccg-1 and ccg-2 genes:
Rhythmic expression of the morning-specific ccg-1 and ccg-2 genes requires frq (ARPAIA et al. 1993 Down, ARPAIA et al. 1995 Down); however, the expression levels of these genes in frq-null (frq10) strains have not been determined. The mRNA levels of ccg-1 and ccg-2 were examined by Northern assays from frq+ and frq10 strains harvested after 12 and 20 hr of growth at 25° in constant darkness, representing subjective dawn (the time of peak ccg-1 and ccg-2 mRNA levels in wild-type strains) and subjective early evening (the time of trough ccg-1 and ccg-2 mRNA levels in wild-type strains), respectively (Fig 1A). After 20 hr in the dark (DD20), ccg-1 mRNA levels were elevated ~10-fold in frq10 as compared to the frq+ strain. The high level of ccg-1 mRNA at DD20 in the frq10 strain was similar to the peak level observed at DD12 in frq+. Little difference was observed in ccg-1 mRNA levels between the two strains at DD12. Conversely, ccg-2 mRNA levels were ~30-fold lower in frq10 as compared to expression in the frq+ strain at DD12, and the levels were low in both strains at DD20. 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 et al. 2000 Down; data not shown). These data suggest that the altered levels of ccg-1 and ccg-2 mRNA in the frq10 strain may not be a direct consequence of the absence of FRQ, but rather result from the loss of a functional frq-oscillator. Together, these data support the idea that ccg-1 expression is negatively regulated and ccg-2 expression is positively regulated by a pathway involving the frq-oscillator (Fig 1B; LINDGREN 1994 Down; BELL-PEDERSEN et al. 1996A Down, BELL-PEDERSEN et al. 2001 Down; YANG et al. 2002 Down).



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  		Figure 1. The clock-controlled ccg-1 and ccg-2 genes are differentially regulated by the clock. (A) The levels of ccg-1 and ccg-2 mRNA were assayed by Northern blot analyses in the frq+ strain 87-3 and the frq10 strain 40-1. Liquid cultures of mycelia were grown in constant darkness and harvested after 12 (DD12) and 20 (DD20) hours in the dark, representing subjective morning and early evening, respectively. Equivalent loading of RNA was verified by hybridization to rRNA. (B) A simple model representing the N. crassa frq-oscillator with output pathways from the clock positively regulating rhythmic levels of ccg-2 mRNA in the morning and negatively regulating rhythmic levels of ccg-1 in the evening (see the text for details).

Electrophoretic mobility shift assays indicated that FRQ protein is not part of protein complexes that bind to the clock regulatory element (ACE) of ccg-2 (BELL-PEDERSEN et al. 2001 Down), suggesting that the FRQ-mediated regulation of ccg-2 is indirect. Thus, the gene products that link FRQ levels to expression of ccg-2 and the components that regulate ccg-1 expression are unknown. 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 25° (Table 1). Control strain 106-20 did not grow in high-Arg/low-Trp (TA) media, but did grow in FPA/anthranilate (FA) media reflecting the Mtr– 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 frq10 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 import both Trp and FPA.


 
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  		Table 1. Strains used in this study

To establish conditions for the selection of mutations, strains CCG1M and CCG2M were examined on TA and FA plating media following 2 days of growth in the dark at 30° (Fig 2). Optimal conditions were found with plating media containing Trp at 0.01 mg/ml plus 1 mg/ml Arg or 60 µg/ml FPA. Under these conditions, the CCG1M strain grew on TA but not on FA; the CCG2M strain grew on FA, but not on TA.



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  		Figure 2. Reconstruction experiments to demonstrate the selection scheme for mutations in clock output pathways. Strains CCG1M and CCG2M were spread onto plating medium containing tryptophan/arginine (TA) or p-fluorophenylalanine/anthranilate (FA) and photographed after 2 days of growth in the dark at 30°.

Isolation of mutant strains that affect expression of ccg-1 and ccg-2:
UV mutagenesis was conducted on strain CCG1M to obtain mutant strains that grew on plating medium containing FA and on strain CCG2M to obtain mutant strains that grew on TA medium. Eighty mutant strains were obtained from the CCG1M strain and 165 mutant strains from the CCG2M strain out of ~6 x 105 and 2 x 105 cells, respectively.

The mutant strains were examined for growth on minimal, FA, and TA media. Some mutant strains (5/165) selected from CCG2M that grew on TA medium were also able to grow on minimal medium. These data suggested that these strains no longer required Trp for growth and therefore were undesired trp-2 revertants or suppressors (data not shown). These strains were not studied further. In addition, most (152) of the 160 CCG2M mutants that showed some growth on TA medium grew poorly on this medium and/or were still able to grow on FA medium (data not shown). Of the remaining eight FPA-sensitive CCG2M mutant strains, we examined the levels of endogenous ccg-2 mRNA in cultures harvested after 20 hr in the dark (DD20). This time of harvest represents subjective early evening, a time of day when cycling ccg-2 levels are normally low in wild-type strains (LOROS et al. 1989 Down) and in the frq10 strain (Fig 1). We reasoned that mutations that affect 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 (Fig 3A). The eight COP2 strains developed normally; however, since they carried the frq10 mutation, the conidiation rhythm could not be observed using standard race tube assays (data not shown). Thus, other than the Mtr+ 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 Down). Because we were unable to readily cross the COP2 strains to isolate a single mutation associated with altered expression of ccg-2, these strains have not yet been further characterized.




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  		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 frq+ strain 30-7, bd frq10 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.

Twenty-two of the 80 mutant strains isolated from the 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, designated as COP1-1 through COP1-58, were examined by Northern assays for endogenous ccg-1 mRNA levels from cultures that were harvested after 20 hr of growth in the dark (DD20). This represents a time of day when cycling ccg-1 levels are normally low in wild-type strains (LOROS et al. 1989 Down) and high in frq10 strains (Fig 1). A representative Northern blot is shown in Fig 3B. The amounts of ccg-1 mRNA from some of the 58 FPA-resistant COP1 strains were reduced from <10-fold to slightly less than the amount of ccg-1 mRNA detected in the unmutagenized CCG1M strain. Eighteen FPA-resistant COP1 strains had endogenous ccg-1 mRNA levels that were equal to, slightly less than, or slightly greater than that observed in the control bd frq+ strain 30-7. We reasoned that mutations that reduce ccg-1 mRNA levels in the frq10 strain to near those of the frq+ strain at DD20, the low point in the cycle, would likely represent mutations that affect clock control of ccg-1. Thus, these 18 FPA-resistant COP1 mutant strains were chosen for further study.

Characterization of the COP1 mutant strains:
We focused on 18 COP1 strains that grew on FA, but not TA media, and exhibited ccg-1 expression levels that were comparable to the mRNA levels observed in the bd frq+ strain 30-7 at DD20. Two major groups of phenotypes were observed among homokaryotic isolates of these strains. One group (n = 13) was visually similar to the parental CCG1M strain (data not shown). A second group (n = 5) showed an Eas-like phenotype of wetted conidia on slants, and the conidia were more readily suspended in water than the unmutagenized CCG1M strain (Table 1 and Fig 4). The 5 COP1 strains with the Eas-like phenotype (COP1-2 through COP1-6) also showed the increased pigmentation phenotype associated with the eas mutation. For each of the 5 Eas-like COP1 mutant strains, the phenotype was slightly less severe than the Eas phenotype. In addition, each of the 5 Eas-like COP1 strains had an unpleasant odor (Table 1). This phenotype was heritable; progeny from crosses with 3 out of 3 of the COP1 strains (COP1-2, COP1-3, and COP1-4) also had the odor associated with the Eas-like phenotype (data not shown, see Table 1).



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  		Figure 4. Several COP1 mutant strains selected from the CCG1M strain have an Eas-like phenotype. Slant cultures for control strain CCG1M, bd frq+ strain 30-7, and 101-3 (bd, easUCLA191 mat A) along with homokaryotic isolates of three representative Eas-like mutant strains (COP1-2, COP1-3, and COP1-4) are shown. The Eas phenotype is characterized by dark, wetted asexual conidiospores.

The COP1-2 through COP1-6 strains were of special interest because the Eas phenotype is caused by loss of function of the ccg-2 gene (BELL-PEDERSEN et al. 1992 Down; LAUTER et al. 1992 Down), but the mutations were isolated from the ccg-1::mtr selection. These data suggested the possibility that a single mutation affected expression of not only ccg-1, but also other ccgs. To test this hypothesis, the five Eas-like COP1 mutant strains were first examined for endogenous ccg-1 and ccg-2 mRNA levels, along with expression of the mtr transgene from cultures grown in the dark and harvested after 20 hr (Fig 5). As previously observed, in each mutant strain, the levels of endogenous ccg-1 mRNA and mtr mRNA from the transgene were lower than those of the control unmutagenized strain. Furthermore, the levels of endogenous ccg-2 transcripts were reduced in each of the five Eas-like COP1 mutant strains from their already low levels in the CCG1M parental strain. However, there was notable variability in the levels of ccg-2 transcripts, ranging from slightly lower in COP1-5 to almost undetectable in COP1-3. These data suggest that different loci, or the severity of mutation of a single locus, resulted in different effects on ccg-1 and ccg-2 expression levels. None of the five Eas-like COP1 mutant strains displayed the characteristic wc-1 or wc-2 mutant phenotype of white hyphae resulting from reduced carotenoids (BALLARIO et al. 1998 Down), indicating that the mutations were not in either of these genes or in genes that greatly alter WC-1 or WC-2 function.



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  		Figure 5. The Eas-like COP1 mutant strains have reduced levels of ccg-2 mRNA. Densitometry of ccg-1, mtr, and ccg-2 mRNA levels from Northern blots (not shown) in the control CCG1M strain and COP1-2, COP1-3, and COP1-4 mutant strains is shown. In each experiment, the levels of mRNA 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 ccg-1 and ccg-2. In each experiment, RNA was isolated from cultures harvested following 20 hr of growth in the dark, representing subjective early evening.

We also examined ccg-2 mRNA levels in the other 13 FPA-resistant mutations from CCG1M that exhibited reduced ccg-1 expression, but did not display the Eas phenotype. One representative mutant strain, COP1-1, is shown in Fig 5. For COP1-1, the levels of ccg-2 mRNA were similar to the levels observed in the CCG1M strain, suggesting that the defect in the COP1-1 mutant strain specifically affects ccg-1 gene regulation.

To verify that the effects on ccg-1 and ccg-2 gene expression in the COP1-2 through COP1-6 strains were the result of a single mutation, each strain was crossed to the bd frq+ strain 30-7. Haploid progeny were scored for the Eas-like phenotype and for low-level endogenous ccg-1 expression at DD20. Both frq+ and frq10 progeny with low-level endogenous ccg-1 expression and the Eas-like phenotype were obtained from the COP1-2, COP1-3, and COP1-4 strains. In all first- and second-generation crosses, the Eas-like phenotype segregated 100% of the time with low-level ccg-1 mRNA (n >= 9; Fig 6 and data not shown). These data suggested that the mutant phenotypes in the COP1-2, COP1-3, and COP1-4 strains are due to mutation of a single gene. The COP1-5 and COP1-6 strains were infertile and therefore were not examined further here.



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  		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 frq+ strain 30-7 and bd frq10 strain 40-1 and four frq10-carrying progeny of COP1-4 (labeled a, b, c, and d) was hybridized to a ccg-1 probe. Ethidium bromide-stained rRNA is shown below to demonstrate equal loading of the gel. (B) Slant cultures of the COP1-4 progeny strains a–d are shown to illustrate the Eas-like phenotype.

We used forced heterokaryon analyses, in which different haploid nuclei are present in the same cell, to determine if the mutations in the five Eas-like COP1 strains were dominant or recessive to the wild-type allele (DAVIS and DESERRES 1970 Down). The mutations in the COP1-5 and COP1-6 strains showed dominance to the wild-type allele for the Eas-like phenotype, whereas the mutations in the COP1-2, COP1-3, and COP1-4 strains were recessive (data not shown).

The mutations in the COP1-1, COP1-2, and COP1-4 strains affect overt rhythmicity and circadian clock output:
To examine if the frq-oscillator is affected in the COP1-2, COP1-3, and COP1-4 mutant strains we examined progeny from crosses with the 30-7 strain (bd frq+). Race tube assays were used to examine the conidiation rhythms of frq+ COP1-2, COP1-3, and COP1-4 mutant strains (designated as COP1-2F+, COP1-3F+, and COP1-4F+ strains; Table 1), along with those of the parental frq10 mutant strains (Fig 7A). Of the frq+ progeny, the COP1-3F+ and COP1-4F+ strains displayed conidiation rhythms with periods of 20.1 ± 0.1 hr and 19.9 ± 0.4 hr, respectively. This ~20-hr rhythm observed in both mutant strains is ~2 hr shorter than that observed in the wild-type clock strain 30-7. Following transition from light to dark, the COP1-2F+ mutant strain was rhythmic for 2–4 days, but produced qualitatively low levels of conidia. After this initial period, the COP1-2F+ strain became arrhythmic and produced more conidia. These data are consistent with the mutations in the COP1-2, COP1-3, and COP1-4 strains affecting either the conidiation output pathway or the circadian oscillator itself. Interestingly, the parental COP1-2 strain (which carries the frq10 allele) displayed rhythmic conidiation in constant darkness in ~1 out of 10 experiments, whereas the COP1-3 and COP1-4 strains were always arrhythmic. Representative race tubes of the COP1-2 strain are shown in Fig 7B. Diagnostic PCR was used to confirm that these COP1-2 cells harbored the frq10 allele (data not shown). Because the COP1-2 strain is capable of rhythmcity in the frq10 background, albeit erratic, these data suggest that the mutation present in the COP1-2 strain is a partial suppressor of the frq-null phenotype.



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  		Figure 7. Conidiation rhythms are altered in the Eas-like COP1 mutant strains. (A) Race tube assays show the conidiation rhythms of control bd frq+ 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 frq+ allele are also shown (labeled F+). Two representative race tubes are shown for the COP1-2F+ strain. Race tubes were incubated in the light for 1 day at 25° and then transferred to the dark (25°). The solid lines indicate the growth front after 24 hr. The period lengths of the rhythmic strains are indicated on the left (n >= 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.

The COP1-2F+, COP1-3F+, and COP1-4F+ progeny strains were also examined by Northern assays for circadian rhythms in ccg-1 and ccg-2 mRNA accumulation (Fig 8A and Fig B). In the control 30-7 (bd frq+) strain, both ccg-1 and ccg-2 mRNA accumulated rhythmically and peaked in mRNA levels in the subjective morning (8–16 hr in the dark on day 1 and 36–40 hr in the dark on day 2). Both COP1-2F+ and COP1-3F+ strains had randomly fluctuating, but primarily low, levels of ccg-1 and ccg-2 mRNA over the course of the day. Interestingly, the COP1-4F+ strain had widely variable levels of both ccg-1 and ccg-2 mRNA. However, ccg-1 or ccg-2 mRNA levels in the COP1-4F+ strain did not display a typical circadian pattern. These data demonstrate that circadian regulation of ccg-1 and ccg-2 is defective in the COP1-2F+, COP1-3F+, and COP1-4F+ mutant strains.



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  		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 frq+ strain 30-7 and the COP1-2, COP1-3, and COP1-4 mutant strains in the frq+ background (labeled COP1-2F+, COP1-3F+, and COP1-4F+). 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 Down), 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 COP1-2F+; thin shaded lines with solid triangles represent COP1-3F+; and dashed shaded lines with solid squares represent COP1-4F+.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

The output pathway from the clock appears to involve a repressor of ccg-1 and an activator of ccg-2:
In this study, we utilized a selection for mutations (CARATTOLI et al. 1995 Down) that affect the expression of two clock-controlled genes, ccg-1 and ccg-2. The selection was based on the differential expression of the ccgs in response to the absence of the clock component FRQ. Both ccg-1 and ccg-2 are under control of the circadian clock and peak in mRNA accumulation in the subjective morning. However, in the frq10 strain, ccg-1 mRNA fluctuates at a level comparable to the peak levels in wild-type strains over the course of the day, while ccg-2 mRNA remains at levels comparable to its lowest level in wild-type strains.

Deletion of promoter elements required for rhythmic ccg-1 expression results in high-level arrhythmic expression of ccg-1 mRNA, suggesting that clock regulation of ccg-1 involves a repressor (LINDGREN 1994 Down). Conversely, deletion of the ccg-2 ACE results in low-level arrhythmic expression, indicating that an activator of transcription is involved in rhythmic transcription of ccg-2 (BELL-PEDERSEN et al. 2001 Down). One simple model to explain this regulation would be that the frq-oscillator regulates two independent pathways: one that induces a repressor of ccg-1 in the evening and another that induces an activator of ccg-2 in the morning (Fig 1B). In the absence of the frq-oscillator, the repressor of ccg-1 and the activator of ccg-2 would both have reduced activity resulting in high levels of ccg-1 mRNA and low levels of ccg-2 mRNA at all times of day. On the basis of this simple model, we anticipated identifying mutations that affected just ccg-1 or just ccg-2 expression levels. Some of the identified mutations in the frq10 background indeed caused reduced ccg-1 mRNA levels, but had no effect on the levels of ccg-2 mRNA (e.g., the mutation in the COP1-1 strain, Fig 5).

Identification of single-gene mutations that affect both ccg-1 and ccg-2 gene expression:
We identified five FPA-resistant COP1 mutant strains that had reduced ccg-1 expression and that also had reduced expression of ccg-2. These strains grew and developed normal conidiospores (with the exception of the Eas-like phenotype), suggesting that the mutations did not affect the general transcriptional machinery or primary development. Importantly, the Eas-like phenotype and low-level ccg-1 expression in the frq10 background segregated together in crosses, implying that the phenotypes are the result of a single mutation. Furthermore, previous studies have shown that the overall expression levels and rhythmicity of ccg-2 mRNA are unaffected in strains that lack ccg-1 (LINDGREN 1994 Down), and the overall expression levels and rhythmicity of ccg-1 mRNA are unaffected in strains that have low-level constitutive ccg-2 mRNA expression (BELL-PEDERSEN et al. 1996A Down). Together, these data suggest that the simple model of frq-oscillator regulation of two distinct output pathways arising directly from the oscillator, one pathway involving a ccg-1 repressor and a second pathway involving an activator of ccg-2 (Fig 1B), is not correct.

One mechanism to explain how a single mutation could cause the reduction of expression for both ccg-1 and ccg-2 mRNA in the frq10 background is that the mutation simultaneously increases the activity of a repressor of ccg-1 and decreases activity of an activator of ccg-2. In this scenario, a bifurcation in the clock output pathway from the frq-oscillator to the ccg-1 and ccg-2 genes would be predicted. In the frq+ background, the mutations present in the COP1-2F+, COP1-3F+, and COP1-4F+ strains abolish ccg-1 and ccg-2 rhythmicity. The gene(s) mutated in these strains could function in an output pathway upstream of the bifurcation or may possibly act in the frq-oscillator or in a second coupled oscillator. However, the lack of a correlation between the race tube data, in which period defects in the conidiation rhythm are observed in the COP1-3 and COP1-4 strains, and the complete lack of rhythmicity of ccg-1 and ccg-2 mRNA levels in the same strains suggest that the mutations directly affect the output pathways rather than the oscillator itself. The mutation in the COP1-1 strain, on the other hand, appears to be specific for ccg-1 regulation, and thus the gene product likely functions downstream of the branch point. Verification of these hypotheses awaits a molecular examination of oscillator components in the COP1-1, COP1-2, COP1-3, and COP1-4 mutant strains and the cloning and analyses of the genes uncovered by the mutations.

One of the most interesting mutant strains studied was COP1-2, because in the frq10 background this strain often showed unusual and inconsistent rhythms in conidial development on race tubes. The reason for the variability in observing the rhythm and of the rhythm itself is not known at this time, but may be related to the nature of the mutation and the growth conditions used to examine the rhythms. When the COP1-2 strain was crossed to isolate the mutation in the frq+ background (COP1-2F+), haploid progeny that were Eas-like also showed unusual rhythms; the strains were rhythmic for up to 4 days and then became arrhythmic. Interestingly, rhythmicity corresponded to low levels of conidiospore production, whereas arrhythmicity correlated with higher levels of conidiospores. Furthermore, the rhythm observed in the parental COP1-2 strain (carrying the frq10 allele) differs substantially from the overt conidiation rhythms that are sometimes observed in frq-null strains (LOROS and FELDMAN 1986 Down; ARONSON et al. 1994A Down). These frq-null rhythms have been attributed to a second cellular oscillator called the FRQ-less oscillator (FLO; MERROW et al. 1999 Down; LOROS and DUNLAP 2001 Down). However, with the exception of COP1-2 strains, strains carrying the frq10 allele were arrhythmic in our growth conditions. Thus, it is possible that the erratic rhythm observed in the COP1-2 strain is due to its effect on the FLO, which results in partial suppression of the frq-null phenotype.

Each of the five Eas-like COP1 mutant strains had an unpleasant odor. The volatile molecule responsible for the smell is not known. During mating, strains of each mating type secrete a mating-type-specific pheromone that gives off a strong odor similar to the odor given off by the five Eas-like Cop1 mutant strains. However, because the pheromones have not been purified, we do not know if the odor from the Eas-like Cop1 mutant strains is the pheromone. The N. crassa pheromone genes ccg-4(mfA) and mfa are regulated by the circadian clock (BOBROWICZ et al. 2002 Down). Therefore, it is possible that the effect on the output pathways in the Eas-like COP1 mutant strains also causes misexpression of the pheromone genes and that this is responsible for the odor. Assuming that high levels of pheromone have an effect on fertility, this idea is consistent with the finding that while the COP1-2, COP1-3, and COP1-4 strains were fertile in crosses, the level of fertility was reduced. The two strains with the strongest odor, COP1-5 and COP1-6, were infertile in crosses. Alternatively, the reduced fertility of the mutant strains could be related to the benomyl resistance gene that is present in the transformed strains (STABEN et al. 1989 Down).

The wild-type genes specified by the recessive mutations in the FPA-resistant COP1-2, COP1-3, and COP1-4 strains can now be cloned through complementation with existing cosmid or genomic libraries to identify clones that complement the loss of mtr function (i.e., growth on TA), but that do not themselves contain mtr. Furthermore, this selection scheme is now being used to identify mutations that affect expression of ccg-1 and ccg-2 in the frq+ background and will be used in the future to select for extragenic suppressors of existing mutations using the opposite selection.


*  ACKNOWLEDGMENTS

We thank members of our laboratory for critical discussions and Richard Gomer and Andrew Greene for comments on the manuscript. We are also grateful to Susan Andershock and Amy Kotara for technical assistance and Jay Dunlap, Jennifer Loros, and David Stadler for strains. This work was supported by the National Institutes of Health (GM58529).

Manuscript received September 27, 2003; Accepted for publication January 22, 2004.


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