mutation of the gene, which encodes thioredoxin reductase ...encodes resistance to hygromycin b, was...

Copyright 0 1997 by the Genetics Society of America

Mutation of the 9s-9 Gene, Which Encodes Thioredoxin Reductase, Affects the Circadian Conidiation Rhythm in Neurospora crmsu

Kiyoshi Onai and Hideaki Nakashima

Department of Biology, Faculty of Science, Okayama University, Okayama 700, Japan Manuscript received September 10, 1996

Accepted for publication February 4, 1997

ABSTRACT Ten cysteine auxotrophs of Neurospora crassa were examined with regard to the period lengths of their

circadian conidiation rhythms. One of the these cysteine auxotrophs, 95-9, showed dramatic changes in the circadian conidiation rhythm. At 10 ~ L M methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. At this concentration of methionine, the period length was unstable after the fourth day and vaned widely from 1 1 to 31 hr. In contrast, other cysteine auxotrophs did not show such instability of the period length at any of the concentrations of methionine tested. Furthermore, only the qs-9mutant exhibited partial loss of the capacity for temperature compensation of the period length. With regard to cold-induced phase-shifting of the circadian conidiation rhythm, the cys-9 mutant was more sensitive than the wild-type strain to low temperature. The cy& gene was cloned and was found to encode NADPH-dependent thioredoxin reductase. These results indicate that mutation of the gene for thioredoxin reductase results in abnormal expression of the circadian conidiation rhythm in N. crassa.

C IRCADIAN rhythms have been observed in many organisms from prokaryotic cyanobacteria to hu- man beings. The filamentous fungus Neurospora crassa shows a circadian conidiation rhythm (SARGENT et al. 1966); conidiation occurs at an interval of -22 hr in continuous darkness. In several organisms, including N. c~assa, clock mutants, which have period lengths that differ from those of wild-type strains, have been isolated (DUNLAP 1993; KONDO et al. 1994; HOTZ-VITATERNA et al. 1994).

Clock mutants have generally been isolated from mutants whose growth and differentiation appeared almost normal by direct observations of circadian rhythms. However, if part of the clock mechanisms is included in or shared with reactions that are involved in cellular housekeeping mechanisms, it may not be possible to isolate such mutants due to lethality or the excessive inhibition of growth or differentiation. This possibility is supported by the observation that inhibitors of several aspects of cellular metabolism affect the progress of the circadian rhythm (FELDMAN 1975; JOHNSON 1990; SUZUKI et al. 1996).

In N. crassa, biochemical, developmental, and mor- phological mutants have been examined with regard to the period length of circadian conidiation rhythms, and most mutants have been found to have a conidiation rhythm with the same period length as that of the wild- type strain (LAKIN-THOMAS et al. 1990). Slight shortening of the period length has been reported in two cys-

Corresponding author; Hideaki Nakashima, Department of Bioloe, Faculty of Science, Okayama University, Tsushimanaka 3-1-1, Oka- yama 700, .Japan. E-mail: [email protected]:jp.

(kmrtics 146 101-1 10 (May, 1997)

teine auxotrophs, cys-4 and cy-12, but only when the growth rate of mycelia in race tubes is limited by an insufficient supply of cysteine or methionine. Similar phenomena have not been observed with mutants in the biosynthesis of methionine, which is closely related to the biosynthesis of cysteine (FELDMAN et ul. 1979). These results suggest that some aspects of sulfate reduction might interact with the circadian clock in N. crassa. Furthermore, in a photosynthetic mutant of Euglena that had lost the capacity to synchronize the circadian rhythm of cell division in continuous darkness, this capacity was restored by the addition of sulfur-containing compounds, such as cysteine or methionine, to the medium (EDMUNDS et al. 1976).

In this study, we analyzed the properties of the circadian conidiation rhythm of 10 cysteine auxotrophs, and particularly the 95-9 mutant, which exhibits major changes in the circadian conidiation rhythm. We also cloned the cys-9 gene.

MATERIALS AND METHODS

Strains of N. crassm The strains of N. crmsa used in this study are listed in Table 1. Strains 0253 (bd A) and 0254 (bd a) were used as the wild-type strain because these strains allow clear observation of conidial banding (SARGENT et al. 1966). The double-mutant cys-Z3;cys-Z4 requires cysteine, while the individual cys-13 and cys-14 mutants do not because the mu- tated genes encode sulfate permeases I1 and I, respectively ( MARZLUF 1970).

Genomic and cDNA libraries of N. crassa: A genomic DNA library of N. crassa, the cosmid pDC107 library (AKIym and NAKASHIMA 1996), was used for cloning of the cys-9t gene. The pDC107 library contains inserts of -40 kb of genomic DNA from N. crassa cys-9 strain and a benomyl-resistance

102 K. Onai and H. Nakashima

TABLE 1

N. crassa strains used in this study

Strain Genotype or description Source

0253 bd A 0254 0314 bd;cys-1 (84605) This study 0336 bd; cys-2 ( 3840 1 ) This study

Laboratory stock hd a Laboratory stock

0395 bd, rys-4 (K7) This study 0313 9.~-5 (35001);bd A This study 0'294 cyrs-9 (T156);bd A This study

0339 9s-Zl (85518);bd This study 0340 bd,cys-lO (39816) This study

0315 cys-12 (NM268);bd This study 0541 9.5-13 (Pl);cys-Il (P2),bd This study 0342 bd,cys-15 (1) This study 0378 cys-pP;bd TCS67

This study ~ys-T::hy$ rys-9 (T156);bd A This study

FGSCl094 cys-9 (T156) A FGSC FGSC4450-4487 Strains for WLP mapping FGSC

FGSC, Fungal Genetic Stock Center.

with a PRISM Dye-Primer Cycle Sequencing Kit (Perkin-Elmer Japan, Chiba, Japan) and an automated DNA sequencing system (model 3 7 3 4 Perkin-Elmer Japan) as recommended by the manufacturer. The nucleotide sequence and deduced amino acid sequence were analyzed with the INHERIT system (Perkin-Elmer Japan) and the BLAST algorithm (ALTSCHUI. et al. 1990). Other techniques, including Southern blotting and subcloning, were as described by SAMBROOK et al. (1989).

Construction of plasmid DNA: The 7.5-kb BglII fragment from cosmid 5:AlO that complemented the cys-9mutation was ligated into the BglII site of a variant of pUC18 in which the SalI site had been changed to a BglII site by PALMITER et al. (1987). This construct was called pCS901. The 3.5-kb BglII- BamHI fragment from pCS901 was subcloned into the BamHI site of pES200 (STABEN et al. 1989), which included the hy,$ gene as a dominant selectable marker for N. crussa, and the construct was called pCS909. The 2.4kb BglII-SmuI fragment from pCS901 was subcloned into pUC118 (Takara, Shiga, Japan) and the resultant construct was called pCS907. Both the 2.2-kb BglII-KfmI fragment from pCS901 and the hy,$ gene from pES200 were ligated into pBluescript I1 SK(+) (Stra- tagene, La Jolla, CA, USA) and the product was called pCS911.

contained either SA medium [ lx Vogel's salts (VOGEI. 1956), 1.2% sodium acetate, 2% agar] (SARGENT et al. 1966) or GA medium (1X Vogel's salts, 0.15% glucose and 0.25% arginine, and 1.5% agar) (SARCENT et al. 1966) and various concentrations of L-methionine. After culture in continuous light over- night, all of the samples were transferred to continuous darkness at an appropriate temperature. Circadian time (CT) 12 is defined as the time of the transfer from light to darkness. The period length and the phase of each circadian conidiation rhythm were calculated by the procedure described by DHARMANANDA and FELDMAN (1979). Phase was defined as the time at which the first conidiation band appeared in the race tubes.

Transformation of N. crassa: Preparation of spheroplasts and transformation of N. crassa were performed as described by VOLLMER and YANOFSKY (1986), except that no fructose was added to the medium. When the cys-9 mutants (strains 0294 and FGSC1094) were used for transformation experi- ments, we used INA-AGAR BA70 ( h a Foods, Nagano, Japan), which contains lower levels of sulfur-containing compounds than other commercial agars. When the hy$ gene, which encodes resistance to hygromycin B, was used as the dominant selectable marker, transformation was performed as described

Period lengths and mycelial growth rates of cysteine auxotrophs: Ten cysteine auxotrophs were examined with regard to the period lengths of their circadian conidiation rhythms and for mycelial growth rates in race tubes that contained SA medium and various concentrations of methionine at 26" (Figure 1).

Mycelial growth rates of these 10 cysteine auxotrophs were maximum at 1 or 10 mM methionine. Three cysteine auxotrophs, 0314 (bd;cys-l) , 0339 (cys-1I;bd) and 0315 (cys-I2;bd), were leaky and cells grew on solid SA medium, which does not contain methionine. The seven other cysteine auxotrophs, 0336 (bd;cys-2), 0395 (bd,cys-4), 0313 (cys-5;bd A ) , 0294 (cys-9;bd A ) , 0340 (bd,cys-lO), 0341 (cys-13;cys-14, bd), and0342 (bd,cys-15), failed to grow significantly without methionine. Myce- lial growth rates of these mutant strains were reduced by limiting the amount of methionine added to the medium. At 10 p~ methionine, mycelial growth rates of these mutant strains. but not of 0314,0339 or 0315,

by STABEN et al. (1989). were reduced to "50-7596 of the maximum rate, while Preparation and sequencing of DNA. Cosmid and plasmid

DNA were prepared with a Flexi Prep Kit (Pharmacia Biotech at the same concentration of methionine in liquid me- Japan, Tokyo, Japan), which is based on the alkaline lysis dium mycelial growth was reduced to -2°-40% Of max- method (SAMBROOK et al. 1989). Genomic DNA was prepared imum. The growth rate of mycelia in race tubes does from N. rrassa as described by METZENBERC and BAISCH not always coincide with mycelial growth in liquid me- (1981).

- The clones used for sequencing were generated by intro-

dium because the thickness of mycelial mats formed

ducing progressive deletions from one end of a cloned DNA on the surface of agar-solidified medium differs among fragment using exonuclease 111 (Pharmacia Biotech Japan), different strains. as described by HENIKOFF (1984). Sequencing was performed The period length of wild-type strain 0253 (bd A ) was

qs-9 Gene of N. crassa 103

d" Group I

/ !?-9 D-

h' I 1

P' !

Group I I

4-

0

p' 4" o 10" 10' o 10" 10' lo4 lom2

Methionine (M)

FIGURE 1 . -Period lengths of the circadian conidiation rhythms (A and B) and mycelial growth rates (C and D) of the various cysteine auxotrophs. Period lengths and growth rates were determined on SA medium that contained different concentrations of methionine at 26". Ten cysteine-requiring mutants were divided into two groups in terms of whether shortening of the period length was observed at low concentrations of methionine (A and C; group I) or not (B and D; group 11). At 10 ,UM methionine, the period length of the 9 - 9 mutant 0294, plot- ted in Figure lA, was determined from the period lengths of the first three cycles of conidial banding, since the SD of the period lengths was quite large after the fourth cycle (SD = 4.2). In Figure 1, A and C, symbols are as follows: wild-type 0253 (bd A ) ( 0 ) ; 0336 (bd;qs-2) (V); 0395 (bd,qs-4) (A); 0313

(cys-9;bd A ) (0); 0340 (bd,qs-10) ( 0 ) ; 0315

(bd,qs-15) (0). In Fig- ure 1, B and D, symbols were as follows: wild-type 0253 (bd A ) ( 0 ) ; 0314

( ~ ~ ~ - 5 ; b d A ) (8); 0294

(cys-12;bd) (A); 0342

(bd; qs-1) (0) ; 0339 ( TS- 1l;bd) 0; 0341 (TS- 13;cys-I4,bd) (H). Error bars indicate SDs of the results from six different race tubes.

-21.6 hr a t all of the concentrations of methionine tested. In contrast, the cysteine auxotrophs could be divided into two groups in terms of whether or not the period length was reduced at low concentrations of methionine. Seven cysteine auxotrophs, 0336, 0395, 0313,0294,0340,0315, and 0342, were included in the former group (group I), while three auxotrophs, 0314, 0339, and 0341, were included in the latter group (group 11). In group I, six mutants, but not the qs-9 mutant 0294, had almost the same period length as that

of the wild-type strain 0253 at 1 and 10 mM methionine. At concentrations of methionine below 1 mM, the period lengths of these six mutant strains decreased to as little as -19 hr.

The period length of the 9s-9mutant 0294 was 2 hr shorter than that of the wild-type strain 0253, even at 10 mM methionine (Figure 1A). Furthermore, at 50 /AM methionine, the period length of the qs-9mutant 0294 was shortened dramatically (to - 17 hr) , but the period length was relatively stable over entire cycles. At 10 /AM

K. Onai and H. Nakashima

10 12 14 16 18 20 22 24 26 28 30 32 10 12 14 16 18 20 22 24 26 28 30 32

Period length (hours)

FIGURE 2.-Period lengths of the circadian conidiation rhythms of the q.~9strain 0294 at 10 mh4 (A) and 10 ~ L M (B) methionine. Period lengths were examined on SA medium at 26".

methionine, the period length of the cys-9mutant 0294 was -5 hr shorter than that of the wild-type strain 0253 during the first 3 days after transfer to continuous darkness (Figure lA), but the period length became unstable after the fourth day. At this concentration of methionine, the mycelial growth rate of the cys-9mutant 0294 in race tubes was reduced to about 50% of maximum. Histograms of the period lengths of the 9s-9 mutant 0294 at 10 mM and 10 p~ methionine are shown in Figure 2. At 10 mM methionine, -75% of the cycles had period lengths that ranged between 19 and 22 hr, and the mycelial growth rate of this mutant was maximum at this concentration. In contrast, at 10 p~ methionine, the period lengths varied widely from 11 to 31 hr and the mycelial growth rate was reduced to about 50% of maximum. This instability in the period length was not observed with other cysteine auxotrophs at any of the concentrations of methionine tested (data not shown).

Effects of temperature on the period length of cysteine auxotrophs: The effects of temperatures between 20" and 28" on the period lengths of 10 cysteine auxotrophs were examined (Figure 3). GA medium, which contains 1 mM methionine, was used for this experi- ment because several cysteine auxotrophs showed slow mycelial growth and poor conidial banding on SA medium at low temperatures (below 22"). The period lengths of nine cysteine auxotrophs were compensated from 20" to 28", but the 9s-9 mutant 0294 exhibited partial loss of the capacity for temperature compensation of the period length.

Phase-shifting after brief exposure to a low temperature: We examined phaseshifting in both the cys-9 mutant 0294 and the wild-type strain 0253 after exposure to a low temperature (15") for 3 hr at various times (Figure 4A). In the wild-type strain 0253, there was a maximum phase delay of 4 hr at CT 6 and a maximum phase advance of 3 hr at CT 7. In the cys-9mutant 0294,

20 22 24 26 28

Temperature ("C) FIGURE 3.-Effects of temperature on the period length

of the circadian conidiation rhythm of the various cysteine auxotrophs. The period length was determined on GA medium, which contained 1 mw methionine, at different temperatures. The symbols are defined in the legend to Figure 1. Error bars indicate SDs of the results from six different race tubes.

there was a maximum phase delay of 8 hr at CT 6 and maximum phase advance of 8 hr at CT 9. The shapes of the phase-response curves for 9.7-9 mutant 0294 and the wild-type strain 0253 were almost the same, but the cys-9 mutant 0294 showed larger phaseshifting in phases of both the maximum delay and maximum advance than the wild-type strain 0253. This result was confirmed by exposure to a low temperature (15") for different times at CT 6 and CT 9 in the 9.7-9 mutant 0294, and at CT 6 and CT 8 in the wild-type strain 0253 (Figure 4B). The phase shifts observed in the qys- 9 mutant 0294 were larger than those in the wild-type strain 0253 at both the phase with the maximum delay and the phase with the maximum advance.

Cloning and restriction fragment length polymorphism (RFLP) mapping of the 9s-9' gene: To identify the function of the 9 s - P gene, we cloned the gene by complementation of the cysteine requirement of the cys-9 mutant FGSC1094 using the sihselection procedure (AKINS and LAMROWITZ 1985). One clone, 5:A10, among 3800 cosmid clones in the pDC107 library complemented the cys-9 mutation. The cosmid, 5:A10, was digested with several restriction enzymes and the mix- tures after digestion were tested for their ability to com- plement the cysteine requirement of the 9.7-9 mutant FGSC1094. Subclones from 5:AlO and the results of complementation assays are shown in Figure 5. The 7.5- kb BgflI fragment from cosmid 5:AlO rescued the cys-9 mutation. Subclones pCS909, pCS907 and pCS911 were

qs-9 Gene of N. mussu 105

10

5

0

-5

-1 0

A -r

0 6 12 18 24 0 60 120 180 240

Circadian time Duration of pulse (min.) FIGURE 4.-Phaseshifting by a low temperature (15") in qs -9 mutant 0294 and wild-type strain 0253. Conidia of both strains

were inoculated in race tubes that contained SA medium with 1 mM methionine at 26". (A) Phase-response curves for q s - 9 mutant 0294 (0) and wild-type strain 0253 (0 ) were determined from the growth in race tubes that had been exposed to a 3- hr pulse of low temperature (15") at 3-hr intervals from the 48th hr after the light-dark transition. (B) Six race tubes were exposed to low temperature (15") for different times at CT 6 (A) and at CT 9 ('J) in the qs-9 strain 0294, and at CT 6 (A) and at CT 8 (V) in the wild-type strain 0253. The phase of the conidiation rhythm was compared with that of a control that had not been exposed to the low temperature. Error bars indicate SDs of the results from six different race tubes.

constructed from this 7.5-kb fragment. Complementa- RFLPs. RFLP mapping was performed as described by tion tests showed that the c y s - q gene was located within METZENBERC et ul. (1984). The 7.5-kb BgflI fragment the 2.4kb BglII-SmuI segment indicated by an arrow in from pCS901 was used as a probe for Southern blotting Figure 5. of EcoRI digests of DNA that had been isolated from 38

The genetic location of the cloned 7.5-kb BglII frag- progeny (strains FGSC4450 to 4487) obtained from a ment from pCS901 was determined by mapping of cross between the Oak Ridge strain FGSC4411 and Mau-

Plasmid Complementation of cvs-9

pCS909

pcs907

pCS911

BamHl Bgl II

I + Sma I Bgl II

- 1 kb FIGURE 5.-Subclones of the q s - F gene and complementation of the cys-9 mutation. Complementation of the q s - 9 mutation

was determined by transformation of q s - 9 spheroplasts (strain FGSC1094) and subsequent examination of whether the requirement for cysteine had been overcome (indicated as +) or not (indicated as -). The orientation and approximate location of the q s - F gene are indicated by an arrow. The sequenced region is dotted. Both plasmid vectors pCS909 and pCS911 contained a h y f gene as a dominant selectable marker for N. mussu.


TABLE 2

RFLP mapping of the cloned DNA fragment

Gene RFLP Linkage group

cys-9 MMMMMOOMMMOOMMMOM"M"OOMOOOOOMMOOOOOM lys-4 MMMMOOMMMMOO"MMMMMMMMOOOOOMMOOOOMM IR un-2 MM"OOMMMMOO"MMMMMMMMOOOOOOOOMMOOOOMM IR his-2 MMMMOOMMMMOO"MMMMMMMMOOOOOOOOMMOOOOMM IR Fsr-33 M M M M O ~ O O " M M M M M M M M O O O O O M M 0 O O O M M IR

Segregation of RFLPs of the cloned cys-p gene and markers located on the right arm of linkage group I (IR) (METZENBERG and GROTEILJESCHEN 1993) in 38 progeny (strains FGSC4450 to 4487) from a cross between the Oak Ridge (FGSC4411) and Mauriceville (FGSC4416) strains is compared. M, Mauriceville parental genotype; 0, Oak Ridge parental genotype; -, not determined.

riceville strain FGSC4416. RFLP mapping showed that was interrupted by two short introns. It encoded a pro- the cloned 7.5-kb BgZII fragment was linked to other tein of 334 residues. A search for homologues of the markers located on the right arm of linkage group I, deduced product of the cys-9 gene was made in the such as the lys-4, his-2, and un-2 genes (Table 2). The DDBJ, EMBL, and GenBank databases, using the location of the cloned DNA fragment coincided with B M T algorithm. The CYS9 protein showed a high the location of the cys-9 gene that had been suggested degree of homology to both prokaryotic and eukaryotic by a recombination analysis (PERKINS et al. 1982). NADPH-dependent thioredoxin reductases (NTRs) .

Sequencing of the cys-9+ gene: The nucleotide se- The putative CYS9 protein and three eukaryotic NTRs quence of the cloned 2.4kb BgZII-SmaI fragment that are aligned in Figure 7. CYS9 was closely related to NTR complemented the cys-9 mutation was determined on of Penicillium chrysogenum (COHEN et al. 1994) with both strands. Three cys-9 cDNAs isolated from a 78% identity (89% similarity) to NTR of Saccharomyces lambda-ZAP cDNA library of the cys -9 strain were also cmevisiae (CHAE et al. 1994) with 61% identity, and to sequenced. The complete nucleotide sequence of the NTR of Arabidopsis thaliana UACQUOT et al. 1994) with cys-9 gene and the deduced amino acid sequence are 60% identity. In the deduced amino acid sequence of shown in Figure 6. The 2.4kb fragment was 2429 nucle- CYS9, the NAD(P)H-binding domain, the redox-active otides long and contained an open reading frame that site, and two FAD-binding domains (COHEN et al. 1993)

1 AGATCTGAGCACCAAATGTACTGCGTGGAATGGCCGCATCCTGATTGTCGGTTTCGCTGCCGGCAAGATCGAGAAGGTGGCCATGAACAAGGTTCTTCTC

ZOlAAGGCAAGTTCAAACCGACTGTGTTCAGCGACAAGGAGTTCGTCGGTCTCGAARAAATTCCGGATGCATTGATCGCTTTGGGGGGTCGTGAGACCTGGGG 101AAGAACATCAGCCTCGTCGGTGTTCACTGGGGCCAATACGCTGTCCATGAGAAGGAGACGGTGGTGACCGTGTGGCAGGGCATCATGAAGTTGATTGCCG

301GAAGGTGGTAGTTCAGGTCCCGCAGCAGGAAGGGAAAAGCAGGTTGTAAATTTTATGGATAGCTTCACTAGGTTCCGGTGCCGTGTCGAGTGTCGTTGAC 4OlCACAAAATCTCCGTCGGCATCTGATGGAAATTGAAATACCTTCACCGATAACCCTAACCCACATTGTGACCTTTGTTTTTTGCTTTGGGCCTCGGCTGCA SOlACCCGCGGACTGGCTGCCTCGGTGACTGCATAGAGCTGCAACCGAGGAGCCTCTAAAAGGCGGAACGACCCCGCTGGATGCAGTGGGTATTTAAGTGGGA 601ATTTTGGTGAGCACGTCAGAGGGGCATGGCCCTGGACCCTGGGCGGTCCGTTAGGGACGTTTCCTCGAATGGACGGCGTCCATCCTTCCGAGTTCGCCCC 701CAAGCATGGAAATCCAGGCCGTTTTTTGAGGCAGCCGGGGTCTAGATACGGGATTAGAAATCGCGACTCGATTTGARAAATGGCGCCGGAACAACGCGCC 8OlTGATCTGGCATCGCCTGGACTGTCTGACAAGATCGCAGATTGGGAGAGAAAG~TCTAGAGATTATCAAATCGTTACCTTCCCTTTCCCGTAGATTCTT 901ATTCTCCATCCAGTGACCATCCCCGGCTCTGTTTCCGCCACTTGTATCTTGTCTCTTGTTAGATCCGTTTCCCATTCG-CCCCACCAACCGCCAAAC

1OOlTGTTCTCCTCAACCTTTAGAAGAACTCTCCGATCCAGCGCATCTGGTCTCACAGTCGCTGCTGCCGCAACCGCCTTCAAGAAAAACACTCCGGTTCAACC 11OlTGCCGAACTTCAGGCCGTCACAAAAAGAATGCACAGCAAAGTAGTCATCATCGGCTCTGGGCCGGCCGCCCACACTGCCGCCATCTACCTGGCCCGC

1201GCCGAGCTGAAGCGTGAGTTTCAATGCCATTGCGATTGTCGACGTCAGGTTACAAGGACCAATTC~AATTCCTCGCCCTTTG=TGTACTC M H S K V V I I G S G P A A H T A A I Y L A R

A E L K P INTRON 1 V L 13OlTACGAGGGCTTCATGGCCAATGGTATCGCCGCCGGTGGCCAGCTCACCACCACGACCGAGATCGAGAACTTTCCCGGTTTCCCCGATGGCATCATGGGCC

14OlAGGAGCTGATGGACAAGATGAAGGCTCAGTCTGAGCGTTTCGGCACCCAAATTATCAGCGAGACCGTTGCCAAGGTCGACCTCTCTGCGCGCCCCTTCAA Y E G F M A N G I A A G G Q L T T T T E I E N F P G F P D G I M G Q

E L M D K M K A Q S E R F G T Q I I S E T V A K V D L S A R P F K 15OlGTACGCTACCGAGTGGTCCCCCGAGGAGTACCACACTGCCGACTCCATCATCCTGGCCACCGGCGCCTCTGCCCGCAGACTTCATCTCCCCGGAGAGGAG

16OlAAGTACTGGCAGAACGGTATCTCTGCCTGCGCCGTTTGCGATGGTGCCGTTCCTATTTTCCGCAACAAGCACCTCGTTGTCATTGGTGGTGGCGACTCCG

1701CTGCTGAGGAGGCCATGTACCTCACCAAGTATGGCAGCCACGTTACCGTGTTGGTTCGCAAGGACAAGTTGAGGGCCAGCAGCATCATGGCCCATAGGTT K Y W Q N G I S A C A V C D G A V P I F R N K H L V V I G G G D S A

Y A T E W S P E E Y H T A D S I I L A T G A S A R R L H L P G E E

A E E A M Y L T K Y G S H V T V L V R K D K L R A S S I M A H R L 1801GTTGAACCACGAGAAGGTCACTGTCAGGTTCAACACCGTCGGTGTCGAGGTCAAGGGTGACGAC~GGGTCTCATGAGCCACCTGGTCGTCAAGGATGTT

L N H E K V T V R F N T V G V E V X G D D X G L M S H L V V K D V 19OlACCACTGGCAAGGAGGAGACTCTTGAGGCCAACGGTCTTTTCTATGCCATCGGTCACGATCCTGCTACCGCCCTCGTCAAGGGCCAGCTTGAGACTGACG

ZOOlCCGATGGCTATGTTGTCACCAAGCCTGGCACTACCCTTACCAGCGTTGAGGGTGTCTTTGCTGCTGGTGATGTTCAGGATAAGAGGTACAGACAGGCTAT T T G K E E T L E A N G L F Y A I G H D P A T A L V K G Q L E T D A

D G Y V V T K P G T T L T S V E G V F A A G D V Q D K R Y R Q A I ZlllCACCAGTGCCGGTACGTAACCCATTGCCTCCCGTGAACCTGGTTTGGAAGAGTTGCCTGTTATG=TACCGGCTGCATGGCTGCCCTCGATG

T S A G INTRON 2 T G C M A A L D A ZZOlCCGAGAAGTTTTTGTCGGAGCACGAGGAAACCCCCGCCGAACACCGCGACACCAGCGCCGTACAGGGCAACCTCTAGAGTTGATTTCTTGGATAGACAGC

2301 GTTTCGCTTAAAAAGTTGGTTCTGAAAATACAGGAATGATACCCAGAGTATGGCCGGCAATTTTGAATGCTTGAAATTTTAGATGTGCCATGGAA~AA 2401GAGAGATGTCGGTTTTTGCCGCACCCGGG 2429

E K F L S E H E E T P A E H R D T S A V Q G N L

FIGURE 6,"Complete nucleotide sequence of the q s - 9 gene and the deduced amino acid sequence of CYS9. The cloned 2.4kb BgllI-SmaI fragment, which complemented the qs-9 mutation, was sequenced. Identification of the two introns (indicated as IN- TRON 1 and INTRON 2) was based on the sequences of three cDNAs. The presumed CAAT and polyadenylation sig- nal sequences (BRUCHEZ et al. 1993) are underlined. Conserved intron boundaries and putative lariat sequences ( BRU- CHEZ et al. 1993) are double-underlined. The nucleotide sequence of the cys-9t gene of N. crassa has been deposited in the DDBJ, EMBL, and GenBank DNA databases under the acces- sion number D45049.

9s-9 Gene of N. crassn 107

FAD I

I 1 S G ~ A H T A A I Y L D R A E L P V L Y E G A N G O A A G G 0 L 46 of CYSI) and three eukaly- I I ~ S G P A A ' H T A A I Y L A R A E L K P V L Y E G A N G I A A G G o L 45 FICUKE 7.--Alignment I Y L A R A E & ' P I L Y E G l G s G P A A H T m A l I ~ ~ s G P A A H T A A I Y m A R A E L K P ~ E G otic NADPH-dependent

thioredoxin reductases. The amino acids of o(S9

Penicdlium Saccharomyces 47 T T T T E I E N F P G F P D G L T E L M D R M R E S a F G T E I I T E T V S K V D L S S K P F 97 [ium chlysoRaum (COHEN

47 T T T T ~ E N F P G F P ~ I A E L M D N M R A Q S E R F G T E I I T E T I S K ~ D L S R P F 97 andofNTRsfromPaici/- Arahidopsis 52 N O P P R - E N F P G F P E I L V E L T D K F R K S E R F G T T I F T E T V T K V D F S K P F 101

cvsq 46 T T T T E I E N F P G F P D G I M E L M D K K A Q S E R F G T I 1 E T V A K V D L S A R P F 96

-lKJJ =Gu w.Fl et al. 1994), Saccharomvces 09 9- a rn0WDW

cvsq 97 K Y A T E P E E - - - - Y H T A D I I L A T G A S A R R L H L P G E E K - - - - Y W Q N G l S A 1 8 cmmiriut (CHAE el al.

Pcniclllium 811 K M T E N D D B S E P V R T A D A V I O A T G A O A R R L N L P G E E T - - - - Y W O N G I S A 14 1994)andArbi~~~ris1ha'- Saccharomyces 811 K L W A E F N E D - - A E P V - A T D A I I L A T G A S A K R M H L P G E E T - - - - Y W O K I S A 141 i~n~(JACQUOTe~tl.1994) Arahidopsis 102 K L F T D S K A I - - - - - - - L A D A V l L A l A V A K L S F V G E V L G G L N R I S A 1 6 werealignedtomaximize

similarity using the CLUS CYS9 140 A V C D G A V P I F R N K H L V V I G G G D S A A E E A M Y L T K Y G S H V T V L V R K D K L R A S 190 T&Valgorithm(L~p~~~ Saccharomyces 142 A V C D G A V P I F R N K P L A V I G G G D S A E E A F L T K Y G S K C L C L E K T I C V L L 19L andPEARsoN 1985)'Resi- PCnlCllliUm I S A V C D G A V P I F R N K P L Y V I G G G D S A A E E A M F L B K Y G S V T V L V R K D K L R A S 195

Arahidopsls 146 A V C D G A A P I F R N K P L A V I G G G D S A M E E A N F L T K Y G S K ~ Y I I D R R D A F ~ S I S duesidenticaltothosein CXS9 are boxed and dot-

CYS9 191 s I M A H R L L N H E K V T V R F N T V V E V K G D D K - - L M H L V V K D V T ~ K E E T L E r ~ 9 ted,andgaps(introduced

Arahidopsis 197 K ~ Q R A ~ N P K I D ~ I o F q 9 E l N S S V V E A Y D G E R D - V L G G L K V K N O V ~ D V S D L K Q5l57 p3 2 6 areindicatedbyhyphens (-). Two FAD-binding do- Saccharomyces 193 P L C K K R A E K N E K I E I L Y N T V A L E A K G D K - - - L L N A L R I K N T K K N E E T D L P 243 Penicillium 196 N I M A D R L L A H P K K V R F N T V A T E V I E N K P N L M T H L R V K D V L S N A E E V V E 243 to maximize homology) cvsq 240 A N G L F Y A I G H D P A T A L V K G O L E T D A D G Y V V T K P G T T L T S V E G V F A A G D V Q D 290 mains(indicatedasFm1 Penicillium 247 A N G L F Y A m H D P A G L V K G O V E L D D E Y I I T K P G T F T N V E G V F A D D V Q D 297 and FAD II ) , an N m ( P ) Saccharomyces 241 V S L F Y A I G H T P A T K I V A O V D T D E A Y O T O P G S L T S V P O F A A G D V O D 291 H-binding domain [in& Arahidopsis 247 5 L Z P l Q a a V S L F F A I G H E P A T K F L D V E L D S D G Y V V T K P G T T T S V P V F A A G D V O D 297 cated as NAD(P)H], and

Y R Q A I T S A G T G C M A A L D A E E H E E T P A E R D T S A V G N L Y R Q A I T S A G C O A A L O A E L ; I T i T - - - - B E A K P I $ - -lJ Y R O A I T S A G B C M A A L D A E S L E - - - - - - - - - - - - - - - - Y R Q A I T m A G T G C M A A L D A E a l G S Q O G K S D - - - - - - - - -

a redox-active site (indi- 33.( cated as Redox) (COHEN 319 et nl. 1993) are boxed. 334

352

were conserved. These results indicate that CYS9 is the NTR of N. crmsa.

Analysis of transformants and disruption of the qs- 9' gene by RIP: To determine whether or not the shortening and instability of the period length observed in the cys-9mutant 0294 were due to the cys-9mutation itself, we examined the period length of cys-P transformants and that of a strain with a disrupted cys-9gene.

Spheroplasts of the cys-9 mutant 0294 were transformed with pCS909, which contained both the 9 s - P and hy$ genes. The period lengths of the conidiation rhythms of 102 heterokaryotic qs-P transformants that were resistant to hygromycin B and whose cysteine requirement had been complemented were determined on SA medium at 26" (Figure 8). It was impossible to determine the period length of the cys-9 mutant 0294 on SA medium, which does not contain methionine or cysteine, because this mutant failed to grow significantly. In contrast, the period length of the wild-type strain 0253 on SA medium was 21.7 2 0.3 hr (average from 18 tubes). Nine of the 102 transformants had almost the same period length as the wild-type strain 0253. Most of the other transformants had shorter period lengths than the wild-type strain 0253. The period length of one of the q s - 9 transformants, TCS67 (cys- 9+::hy$ cys-9;bd), which had almost the same period length and rate of mycelial growth as the wild-type strain 0253, was determined on SA medium that contained different concentrations of methionine at 26" (Figure 9). The period length of strain TCS67 was unaffected by the concentration of methionine, and the mycelial

growth rate was also the same as that of the wild-type strain 0253.

The 9 s - 9 gene was disrupted by RIP. RIP results in heavy methylation of cytosine residues and many transi- tions from G C pairs to A-T pairs in duplicated DNA sequences (SELKER 1990). One RIP strain at the cys-9 locus, 0378 (cys-p";bd), was examined for the effects of methionine on period length and mycelial growth rate on SA medium at 26" (Figure 9). The period length and mycelial growth rate of cys-$" strain 0378 were the same as those of the cys-9 mutant 0294 at all of the concentrations of methionine tested. Furthermore, the instability of the period length observed in the 9.5-9 mutant 0294 at 10 PM methionine was also observed with cys-p" strain 0378.

DISCUSSION

Cysteine auxotrophs of N. crmsa were divided into two groups based on whether or not shortening of the period length was observed (Figure 1). The sites at which defects in the individual cysteine auxotrophs occur on the pathway for the assimilation of sulfate have been described previously (PERKINS et al. 1982). Muta- tions in strains in group I resulted in modification of reactions between the phosphorylation of adenosine-5'- phosphosulfate (APS) and the synthesis of thiosulfate. Shortening of the period length of mutants in group I was not due to starvation of cysteine or methionine itself because the same phenomena did not occur in methionine auxotrophs under the same conditions


(FELDMAN et al. 1979). This conclusion is supported by the result that ethionine, which is an analogue of methionine, does not affect the period length of the wild-type strain, even though mycelial growth is inhib ited by ethionine (data not shown).

Only the cys-9 mutant in group I showed a dramatic change in the circadian conidiation rhythm: (1) this strain had a period length 2 hr shorter than the wild- type strain, even when mycelial growth was not limited by an insufficient supply of methionine (Figure 1); (2) this strain showed considerable shortening of the period length during growth on medium that contained a low concentration of methionine (Figure 1); (3) at 10 methionine, instability of the period length was observed in this strain after the fourth day (Figure 2); and (4) this strain partially lost the capacity for temperature compensation of the period length (Figure 3). Fur- thermore, the 9s-9 mutant was more sensitive to a low temperature (15") than the wild-type strain, and this increased sensitivity resulted in larger phase-shifting (Figure 4). However, the phase-response curve for a light pulse had almost the same features as that in the wild-type strain (data not shown). Instability of the period length has been reported infrqmutants (ARONSON et nl. 1994), but not in other mutants, including other clock mutants.

The results of the analyses of cys-9+ transformants and of the strain with a disrupted 95-9 gene (Figures 8 and 9) indicated that shortening and instability of the

v)

S c,

30

20

10

0 - 14 15 16 17 18 19 20 21 22 23 24 25

Period length (hours) FIGURE 8.-Relationship between the number of 9s-9+

transformants and the period lengths of the circadian conidiation rhythm. The period lengths of 102 heterokaryotic transformants that had been resistant to hygromycin B and had been able to grow on minimal medium were determined on SA medium at 26". The period length of the wild-type strain 0253 was 21.7 hr and is indicated by an arrow.

23 - 22 E = 21 5

0 r - 20 m 19 c

18 'CI .g 17

I

5 e 16 15

50

n )r 45 (II 2 40 v 35 Q)

30

E CI

c z 25 0 2o e

15

T

0 1 o4 1 o4 10" 1 o 2

Methionine (M) FIGURE 9.-Effeca of methionine on the period length (A)

and mycelial growth rate (B) of the q s - T transformant TCS67 and of the qs-Misrupted strain 0378. Experimental proce- dures were the same as those described in the legend to Figure 1. At 10 ,UM methionine, the period lengths of the qs-9mutant 0294 and qs-$'"strain 0378 were determined from the period lengths of the first three cycles of conidial banding because the SDs of the period lengths were quite large after the fourth cycle (0294, SD = 4.2; 0378, SD = 4.5). Symbols were as follows: wild-type 0253 (bd A) (0 ) ; 0294 (9s-9;bd A ) (0); TCS67 (qs-9 t : :hyf cy-9; bd A ) (0); 0378 (qs-p"';bd) ( 0 ). Error bars indicate SDs of the results from six different race tubes.

period length were due to mutation of the cys-9 gene itself, and did not depend on the site(s) of mutation in this gene. Period lengths of 102 heterokaryotic cys- T transformants were examined, but only 9% of these transformants were completely rescued. Since most pri- mary transformants of N. cTassa are heterokaryotic, the nuclear ratio of transformed to untransformed nucleus may be different in each transformant. Furthermore,

cys-9 Gene of N. crassa 109

as transforming DNAs were integrated into ectopic sites of the N. crussu genome (PANDIT and RUSSO 1992), the expression level of the integrated gene may be different in each transformed nucleus, possibly resulting in the low proportion of apparent complementation by the exogenous cys-9 gene.

CYS9 was identified as an NADPH-dependent thioredoxin reductase (NTR) of N. crussu, which reduces thioredoxin (TRX) (Figure 7). TRX reduced by NTR is used to reduce 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to sulfite in the pathway for assimilation of sulfate (RUSSEL et ul. 1990). Therefore, the cys-9 mutant must require cysteine for mycelial growth because of a defect in the capacity for the reduction of PAPS. This conclusion is supported by the result that the 95-9 mutant can use sulfite for mycelial growth (MURRAY 1965).

Responses of the period lengths of strains in group I to limited sulfur were similar (Figure 1). One of the mutants in group I, cys-5, was previously reported as a strain that lacks PAPSreduction activity (PERKINS et al. 1982). The period length of the double-mutant cys-5,cys- 9 was almost the same as that of the cys-9 mutant, and instability of the period length was also observed in this double-mutant strain (data not shown). This result suggests that the shortening of the period length observed in mutants in group I might be due to the same defect as that caused by the cys-9 mutation. However, the cys-9 mutant exhibited major changes in the circadian rhythm that were not observed in other cysteine auxotrophs, as previously stated. The cause of these differences in the circadian rhythm between the c y - 9 mutant and other mutants in group I remains un- known. However, the evidence presented in this paper suggests that loss of NTR activity and, at the same time, a defect in the pathway for assimilation of sulfate between APS and thiosulfate may result in major changes in the circadian conidiation rhythm, such as instability of the period length.

Recent models of the circadian clock include several fundamental aspects of metabolism, such as the phosphorylation (EDEKY et al. 1994) and degradation (HONGKUI et al. 1996) of protein. The thioredoxin system, which involves NTR and TRX, functions to intro- duce specific disulfide bonds in targeted proteins and plays an important role in posttranslational control of the activities of various proteins, including regulation of transcription factors and protein kinases (HOLMGKEN and BJOKNSTEDT 1995). In N. crussa, there may exist clock-related reaction (s) that require the thioredoxin system and support from sulfur metabolism.

The authors thank Mr. KENJ TAKAYANAGI for his technical assis- tance.

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Communicating editor: J. J. LOROS

mutation of the gene, which encodes thioredoxin reductase ...encodes resistance to hygromycin b, was...

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