Fml, a Negative Regulatory Required for the Repression the ...of nitrate assimilation in fungi...

Copvrighr 0 1997 by the Genetira Society ufhler ica

Fml, a Negative Regulatory Locus Required for the Repression of the Nitrate Reductase Gene in Chlamydomonas reinhardtii

Donghong Zhang' and Paul A. Lefebvre Department of Genetics and Cell Biology, and Plant Molecular Genetics Institute, University of Minnesota, St. Paul, Minnesota 55108

Manuscript received July 26, 1996 Accepted for publication January 27, 1997

ABSTRACT In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding

nitrate reductase ( N I T l ) , are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of N I T l gene expression. ( 1 ) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NZTl:PF14 chimeric construct produced a transformant (T10-1OB) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the N I T l promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia yepression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. the furl-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.

I N fungi, algae and higher plants, the reduction of nitrate to nitrite and then to ammonia is catalyzed sequentially by two enzymes, nitrate reductase (NR) and nitrite reductase (NiR) . This highly regulated pro- cess is the main pathway through which inorganic nitrogen is incorporated into living organisms (GUERRERO et al. 1981). In higher plants the expression of the NR gene is regulated by many factors, such as nitrate (reviewed by CRAWFORD 1995), light (reviewed by LILLO 1994), C o n (KAISER and FORSTER 1989), circadian rhythms (GALANGAU et al. 1988), and many others. In fungi, nitrate induces the expression of the NR gene, while ammonia ion and glutamine, the immediate metabolite of ammonia ion, repress NR gene expression (reviewed by MARZLUF 1993). Induction and repression of nitrate assimilation in fungi apparently involve two independent pathways. Low levels of NR activity are detectable when both ammonium and nitrate are present.

In the green alga Chlamydomonas reinhardtii, repression by ammonium plays a prominent role in the regulation of NIT1, the gene encoding nitrate reductase. The NI771 gene is not expressed in ammonium-grown cells. Following the removal of ammonium from the medium, the NIT1 transcript accumulates (FERNANDEZ et nl. 1989), and achieves maximal levels if nitrate is added

Cm~sponding author: Paul A. Lefebvre, Department of Genetics and Cell Biology, 250 Biological Sciences Center, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108-1095. E-mail: [email protected]

'Prrsent address: Department of Biology, Washington University, Campus Box 1229, 1 Brookings Dr., St. Louis, MO 63130.

Ck~rlrtirs 146 12 I - 193 ( May, 1997)

(QUESADA and FERNANDEZ 1994). In the presence of both nitrate and ammonium, the Chlamydomonas NIT1 gene is repressed. The dominant effect of ammonium on the regulation of NITl expression in Chlamy- domonas is an important part of the complex regulatory circuit for nitrogen metabolism, but the mechanism of this repression is not known.

Extensive genetic and molecular studies on the regulation of NR gene expression have been conducted in the filamentous fungi Aspergillus nidulans and Neurospora crassa (recently reviewed by CRAWFORD and ST 1993). Both positive and negative regulatory genes have been identified and characterized at the molecular level. In A. nidulans, nitrate induction is mediated by the pathway-specific, positively acting gene NIRA (RAND and ARST 1978; BURGER et al. 1991). Nitrogen metabolite repression is mediated by the positively-acting gene AREA ( CADDICK et al. 1990; KUDU et al. 1990). The loss- of-function phenotype of AREA is an inability to utilize nitrogen sources other than ammonium and glutamine ST and COVE 1973). Some areA mutant alleles cause derepression of NR and other enzymes normally under the control of AREA (reviewed by CRAWFORD and ARST 1993). In N. crassa, the genes involved in multiple nitrogen metabolic pathways, including those in the nitrate reduction pathway, are regulated by the positively acting NZTZ gene (Fu and MARZLUF 1987, 1990) and the negatively acting NMR gene (Fu et al. 1988; YOUNG et al. 1990). In the nmrmutant, the repression of nitrogen catabolic enzymes is defective, such that NR activity is expressed constitutively (PREMAKUMAR et al. 1980; DUNN-COLEMAN et al. 1981). NIT4, a pathway-specific

122 D. Zhang and P. A. Lefebvre

regulatory gene, is also required for nitrate induction of the synthesis of NR and NiR (SORGER and GILES 1965; HURIBURT and GARRETT 1988; FU et al. 1989; YUAN et al. 1991).

C. reinhardtii has a single gene for NR, the NITl locus, which has been cloned and sequenced (FERNANDEZ et al. 1989; D. ZHANG, M. LAVOIE, R. A. SCHNELL, S. CHRIS TENSON and P. A. LEFEBVRE, unpublished data). Chla- mydomonas NR is similar to NRs from other eukaryotic species in terms of its partial enzymatic activities (reviewed by FERNANDEZ and CARDENAS 1989) and primary amino acid sequence (D. ZHANG, M. LAVOIE, S. CHRIS TENSON and P. A. LEFEBVRE, unpublished data). NITl is located in a cluster of five genes (the NAR genes) that are proposed to be involved in nitrate assimilation (QUESADA et al. 1993). The expression of the five genes in the cluster is coregulated, being repressed by ammonium and induced by nitrate ( QUESA~A et al. 1993). The N172 gene, unlinked to the NIT1 cluster, is a positive regulator of the nitrate assimilation pathway (FERNAN- DEZ and MATAGNE 1986). In nit2 mutants, the transcripts of NITl and the other NAR genes are not detected upon derepression (SCHNELL and LEFEBVRE 1993; QUESADA et al. 1993). The NIT2 gene of Chlamy- domonas is the only known regulatory gene for nitrogen metabolism in photosynthetic eukaryotes.

For Chlamydomonas, as well as for other organisms, chlorate has been used extensively in the genetic selec- tion of mutants that are defective in nitrate reduction. Chlorate is thought to be reduced to the more toxic chlorite through catalysis by NR ERG 1947). Wild- type cells are sensitive to chlorate only under conditions in which NR is expressed, such as when cells are grown on media containing nitrate or urea as the sole nitrogen source. However, NR-deficient mutants are resistant to chlorate under the nonrepressing conditions. A mechanism for chlorate toxicity was proposed by PRIETO and FERNANDEZ (1993). They suggested that the nitrate transporter was necessary for chlorate uptake, and therefore that ammonium prevented chlorate toxicity by inhibiting the accumulation of the nitrate transporter.

Eight nit loci have been identified (NICHOLS and SYR- ETT 1978; SOSA et al. 1978; SCHNELL and LEFEBVRE 1993) in Chlamydomonas. Besides the NR structural gene (N17'1) and the positively acting regulatory gene (NIT2), five loci (NIT3, NIT4, NIT5, NIT6, and NI777) are involved in the synthesis of the molybdopterin cofactor that is required for nitrate reductase activity (FER- NANDEZ and MATAGNE 1984; FERNANDEZ and AGUILAR 1987; ACUILAR et al. 1992). NIT8, located only 1.5 kb upstream of NITl in the five-gene cluster, encodes a putative membrane protein required for growth on nitrate and nitrite (S-C. WANG, R. A. SCHNELL and P. A. LEFEBVRE, unpublished data).

In the present study, we sought to identify more regulatory gene (s) for the nitrate-reduction pathway by iso-

lating mutants with constitutive expression of NITI. A new reporter gene system was developed to facilitate mutant isolation. A chimeric construct, in which a gene required for flagellar motility (PF14) was placed under the control of the NIT1 promoter, was introduced into Chlamydomonas pf14 mutant cells by transformation. A transformant with appropriate expression of the transgene was motile in nitrate-containing medium but was paralyzed in ammonium. Mutations in genes required for the repression of the NITl promoter were sought by isolating swimming cells from the surface of cultures with ammonium as the sole nitrogen source. Most of these mutants were shown to have constitutive NR activity. The mutations in these strains affect at least two loci required for the repression of NITl gene expression in the presence of ammonium. One mutant (Jarl-1) was generated by insertional mutagenesis, and a fragment of genomic DNA flanking the site of insertion was isolated.

MATERIALS AND METHODS

Chlamydomonas strains. Most of the C. reinhardtii strains used in this study are listed in Table 1. The strains used for the recombination tests are listed in Table 2 . In addition, mutants with a constitutive swimming phenotype (sw), derived from strain TlO-lOB, are listed in Figure 3.

Culture conditions: Chlamydomonas cells were grown under continuous light at 24". The cold-sensitive mutant supcsl- 2was grown at 33" and its phenotype was scored at 16". Liquid cultures larger than 50 ml were bubbled with filtered air. For solid media, 1% agar U R H Bioscience, Lenexa, KS), washed extensively with distilled water, was added. Mating plates were prepared with 2% washed agar.

Minimal medium was used in most genetic experiments (HARRIS 1989). The acetate-requiring strains were grown on R medium, which is minimal medium supplemented with acetate and increased phosphate (SCHNELI. and LEFEBVRE 1993). When appropriate, either 3 mM KNOs or 3 mM NH&l was used in place of NH4N01 as the nitrogen source. Cells used for transformation were grown in SGII medium (SAGER and GRANICK 1953). Gametogenesis was induced in nitrogen- free minimal medium supplemented with 10 mM HEPES (pH 7.0). For selective media, 10 mM KCQ, 100 pg/ml emetine dihydrochloride, or 100 pg/ml erythromycin were added (all from Sigma Chemical Co., St. Louis, MO).

Plasmids: pMN56 is a subclone of pMN24 ( F E R N ~ D E Z et nl. 1989), in which a 8.9-kb XbuI-EcolU fragment containing the entire NZTl gene was cloned in the vector pUC118 (SAM- BROOK et nl. 1989). pJN4 carries the dominant selectable marker CRYl-1, which is fused to the promoter of the RBCS2 gene (NELSON et al. 1994). A plasmid (pDZ101) containing the upstream region of the NZTl gene was constructed by cloning a 3.6kb SalI-Hind111 fragment from pMN24, extending from within the adjacent gene (NZT8) to 42 bp up stream of the initiation codon of NZTl, into pUCl19. To make the chimeric construct pDZl03 (Figure lA), a 2.fikb NheI fragment containing the coding region of the PF14 gene was isolated from plasmid pEKrsp3.epitope (kindly provided by Dr. K. A. JOHNSON, Haverford College, Haverford, PA). The fragment was inserted into the Hind111 site in pDZ101, so that the expression of PF14 was placed under the control of the NIT1 promoter. Before ligation, the fragments were partially filled in with dCTP and dTTP using the Klenow fragment of

Chlamydomonas Regulatory Locus Far1

TABLE 1

C. reinhurdtii strains

Strain Genotype Source

123

21gr

A35 A54e 18 B26 B27 CG1032 CC-1709

2-G4

C1-1OH C2-8G C2-9E C3-1 C42Bd C49Bd C33-3F C33-10H C345B C37D-3A C39-1E C39-3E C39-6D C39-6F C39-6H C62-4H c744A c75-3c C78-3F C79-2A C80-7G C82-8A C83-2A C847E C87-3F D42 E39 G65 H11 JN142 JN143 T10-1OB

mt+ wild typ& mt+ farl-1 mt+ nit2-203 mt+ nitl-lac17 srl mt+ srl mt- srl mt+ pf14 nitl-137 nit2-137 mt- ~ $ 2 2 q 3 nitl-137 nit2-137 mt- arg7pf14 nitl-I37 nit2-137 mt arg7 pf l4 mt- arg7 pf14 mt+ nitl-l pf14 mt- farl-1 mt+ farl-1 mt- nitl-1 pf14 mt- pf l4 (pMN56 pDZl03) mt+ farl- l pf14 mt+ farl-I pf14 (FMN56 pDZlO3) mt- farl-1 acl7 mt+ farl-l acl7 mt+ farl-1 a d 7 mt+ ac17 mt+ farl-1 ac17 mt- arg7 farl-1 mt- supcsl-2 mt+ y3-2 mt- farl-2 supcsl-2 mt- farl-3 supcsl-2 mt- farl-4 supcsl-2 mt- farl-5 supcsl-2 mt- farl-6 supcsl-2 mt+ farl-7 supcsl-2 mt- pp.2 9 3 mt- arg7 nitl-137 nit2137 mt- lf3-2 nitl-137 nit.!?-137 mt- supcsl-2 nitl-137 nit2-137 mt- arg7 mt- a d 7 mt+ acl7 mt+ nitl-l pf14 (fiIWV56 pDZlO3)

SAGER (1954) * 21gr transformed with pJN4 SOSA et al. (1978) R. A. SCHNELL, unpublished data‘ SCHNELL and LEFEBVRE (1993) SCHNELL and LEFEBVRE (1993) EBERSOLD et al. ( 1962)b MYERS et al. (1984) CC-1032 X D42 B26 X C1-1OH B26 X C1-1OH A54e18 X C2-8G 2-G4 X B27 2-G4 X B27 T10-1OB X C2-9E T10-1OB X C2-9E C49B X C2-9E C345B X C3310H C49B X JN142 C49B X JN142 C49B X JN142 C49B X JN142 C49B X JN142 H11 X C42B 21gr X G65 B26 X E39 sw4 x c74-4A sw7 x c74-4A S W ~ X C74-4A sw18 X C74-4A sw19 x c74-4A ~ ~ 3 6 X C74-4A B26 X CG1709 Lux and DUTCHER (1991) BARSEL et al. (1988) JAMES et al. (1988) 21gr X D42 J. A. E. NELSON, unpublished data J. A. E. NELSON, unpublished data C3-1 cotransformed with pMN56 and pDZlO3

” -

“All strains are Nit+ unless indicated otherwise.

‘ The nitl-1 allele contains a 5-kb deletion in the NITl gene. 21gr, CG1032 and CC-1709 were obtained from E. HARRIS (Chlamydomonas Genetics Center).

C4B2 and C4B9 are available from The Chlamydomonas Genetics Center at Duke University as CG3351 and CG3352. The alleles farl-2 to farl-7 are CC-3353 through CC-3358, respectively.

DNA polymerase I. pDZlOl was digested with Hind111 and gation were used for transformation. Both pMN56 and pJN4 the ends were partially filled in with dATP and dGTP. The were linearized by digestion with EcoRI before transformation, orientation of inserts was determined by restriction enzyme while pDZlO3 was linearized by digestion with SalI. analysis. A construct containing the PF14 coding sequence in Isolation of the sw mutants: The plasmid pDZlO3 con- the sense orientation (pDZlO3) was used for transformation taining the NITI:PF14 chimeric construct was introduced into (Figure 1A). ap f l4 , nitl-1 double mutant (strain (23-1) by cotransformation

Transformation of Chlamydomonas: Chlamydomonas with pMN56. The Nit’ transformants were grown in liquid cells were transformed using the glass bead-PEG procedure nitrate medium and screened for expression of the PF14 gene (KINDLE 1990). Cotransformation using NITl as selectable by examining motility using a dissecting microscope. Cultures marker (pMN56) and the chimeric construct (pDZ103) was containing swimming cells were then single-colony isolated performed following “method 2” described by NELSON et al. on solid medium containing ammonium as the sole nitrogen (1994). Transformation using CRYI-1 as selectable marker source. Motility was tested by transferring cells into 200 p1 of (pJN4) was also performed as described (NELSON et al. 1994). liquid media containing either ammonium or nitrate in 96- Plasmids (1 -2 pg) purified using CsCl gradient ultracentrifu- well microtiter dishes. Strains that were immotile in ammo-


nium-containing medium but motile in nitrate-containing medium after 8 hr were retained for further analysis. Genomic DNA was isolated from a total of 24 cotransformants and analyzed by gel blot hybridization analysis to determine the copy number of the transforming plasmids.

The cotransformant T10-1OB was chosen as the parent for isolation of constitutively swimming mutants. To obtain independent mutants, 32 single colonies of T10-1OB were used to inoculate separate culture tubes containing 12 ml minimal medium with ammonium as the sole nitrogen source. Initially all cells accumulated at the bottom of each tube. Two weeks later, swimming cells were observed in one of the tubes. A small volume (-500 pl) of the culture was removed from the top 1 cm of the culture and added to 10 ml of fresh medium for 3-5 days to enrich for the swimming cells. After two such transfers, a small volume of medium was removed from the top of the tubes and plated on agar medium to isolate single colonies. Swimming cells arose at different times (2-8 weeks) in different tubes. During the period of 8 weeks, swimming cells were recovered from 28 of the 32 tubes. Each mutant was single-colony isolated and retested for its ability to swim in the presence of ammonium.

NR activity assay: Chlamydomonas cells were grown in minimal medium in 24well culture dishes to a density of 1-3 X lo6 cells/ml. The cells (0.4 ml of the culture) were permeabilized by adding 8 p1 toluene and vortexing for 1 min (FI.ORENCIO and VEC:A 1982). The terminal nitrate reductase activity was assayed using reduced benzyl viologen as the elec- tron donor (FLORENCIO and VEGA 1982). The nitrite produced was measured using the method of SNELL and SNELL (1949). Chlorophyll was extracted with acetone and quanti- tated by measuring absorbance at 652 nm according to BRU- I N S ~ (1961). The NR activity is expressed as nmole nitrite min" mg" chlorophyll.

Genetic tests: Gametogenesis, mating, and tetrad dissec- tion were performed as described (LEVINE and EBERSOLD 1960). Stable diploid strains were constructed to test dominance/recessiveness of the new mutations, and to test for genetic complementation between pairs of mutants. Three recessive markers, ne17 (ERERSOLD et al. 1962), arg7 (LUX and DUTCHER 1991) and supcsl-2 (JAMES et al. 1989), were used for construction of the diploids. Vegetative diploid cells were selected under conditions in which neither parent strain could grow. Diploidy was confirmed by assessing a number of characteristics including cell size, frequent appearance of cells with three flagella, and minus mating type.

To establish the dominance or recessiveness of the sw mutations, a mutation that prevents growth at 16" (supcsl-2) was introduced by genetic crosses into each of the six sw mutant strains that showed no recombination with Juri-1, namely sw4, sw7, sw8, sw18, sw19, and sw36. Each sw supcsl-2 strain was then mated to an acl 7strain (C39-1E or C39-6F) that is unable to grow in the absence of acetate. Diploids were selected on minimal medium at 16". The recessiveness of the Juri-1 mutation was established by testing diploids isolated by mating C62- 4H (juri-I urg7) and JN143 (ucl7) . Genetic complementation between furl-1 and the six sw mutations was tested in diploids made by mating a furl-l ac17 strain (C39-6D or C39-6H) to each of the six s w supcsl-2 strains. At least a dozen independent diploid strains were examined for each mutant pair.

DNA hybridization analysis: Chlamydomonas genomic DNA was purified as described by SCHNEIJ. and LEFEBVRE (1993), which is a procedure adapted from WEEm et al. (1986). The DNA was digested with restriction enzymes, frac- tionated on 1% agarose gels, and transferred to Magna NT membrane (Micron Separations Inc., Westborough, MA) (SOUTHERN 1973). The DNA fragments to be used as hybridization probes were isolated from gels made from SeaPlaque

low-melting agarose (FMC, Rockland, ME), then labeled with "PdCTP by random priming (FEINBERG and VOGEISTEIN 1983), and purified by passage through a Sephadex G50 col- umn. Prehybridization and hybridization were carried out in aqueous conditions (SAMBROOK et al. 1989). After overnight hybridization, the filters were washed three times (15 min each) with 0.2X SSC/O.l% SDS at 65".

RESULTS

Construction of a Chlamydomonas strain carrying a PF14 reporter gene under control of the NZTl promoter: The strategy used to isolate mutants insensitive to ammonium repression of the NITl promoter took advantage of the flagellar motility of Chlamydomonas. Wild-type Chlamydomonas cells are motile in liquid culture, propelled by a pair of anterior flagella. The pf14 mutation disrupts the assembly of the radial spokes of the flagellar axoneme, causing paralyzed flagella (EBER- SOLD 1962; HUANG et ul. 1981). The wild-type PF14gene, which encodes radial spoke protein RSP3, has been cloned and sequenced (WILLIAMS et al. 1989). In this study, a chimeric construct (pDZlO3) was made by ligat- ing 3.6kb of the NZl1 upstream region, extending from the SalI site in the middle of the adjacent NZ7'8 gene to the Hind111 site 42 bp upstream of the ATG initiation codon of NITl, onto the coding region of PF14 (Figure 1A). Thus the expression of the PF14 gene was placed under the control of the NITl promoter. pDZ103 was linearized at the SalI site and introduced into a pf14 nit1 strain (C3-1) by cotransformation using NZl1 (pMN56) as a selectable marker. W'e reasoned that if the NIT1 upstream region is sufficient to confer negative regulation to the reporter gene, then the rescue of the paralyzed phenotype should become conditional, controlled by the nitrogen source in the medium (Fig- ure 1B). Specifically, in media containing ammonium the cells should remain paralyzed due to the repression of transcription from the NIT1 promoter. However, in the absence of ammonium, motility should be restored due to expression of the PF14 reporter gene.

More than 2000 Niti transformants were obtained upon cotransformation of strain C3-1 (pf14 nitl-1) with pDZ103 and pMN56. To enrich for the cotransformants with a functional PF14 gene product, only those transformants that formed loose, spread-out colonies (192 colonies) were picked into nitrate medium in 96-well microtiter dishes and were examined for motility. Eighty-four strains that were motile in nitrate medium were then single-colony isolated on solid medium containing ammonium as the sole nitrogen source. Motility was tested again by transferring cells into 200 ~1 of liquid media containing either ammonium or nitrate in 96well microtiter dishes. For 81 of the cotransformants, flagellar motility was affected by the nitrogen source. After the cells grown on solid ammonium medium were transferred to liquid ammonium medium, almost all of the cells were paralyzed or nearly paralyzed. The cells

Chlamydomonas Regulatory Locus Furl 125

pDZ103

FIGURE 1.-Strategy for isolating mutants using PF14 as a reporter gene. (A) Structure of pDZ1OS. The 2.6-kb Nhd fragment containing the entire coding region of PF14 from the plasmid pEKrspS.epitope was ligated to the 3.6- kb Sun- Hind111 fragment containing the Nl771 promoter region is* lated from the plasmid pMN24. The two sites labeled as (N/ H) resulted from ligation after modifying the NheI ends and the Hind111 ends. The translation initiation codon ATG in PF14 and the polpdenylation signal TGTAA are indicated. Probe A is the 1.7-kb SphI-XbuI fragment that is present in pDZlOS but absent in pMN56. Probe B is the entire 2.6-kb NheI fragment of PF14. (B) The conditional motility of the pf14cells transformed with pDZlO3. In the presence ofNH,+, the PFZ4 gene product (flagellar protein RSPS) is not synthesized, thus cells remain paralyzed. When NH,+ is absent from the medium, the PF14 gene product accumulates and the cells become motile. (C) If a mutation blocks the action of a tramacting regulatory factor for transcription, the NITl promotor is derepressed in the presence of NH,+, and the mutant swims constitutively.

of these 81 strains gained motility several hours after being transferred to liquid medium containing nitrate as the sole nitrogen source. The observed conditional motility in the cotransformants suggested that the NITl upstream sequences are sufficient to mediate the repression of NITl expression by ammonium. The different levels of ammonium repression of motility among the transformants might be influenced by factors such as the copy number of the integrated plasmid pDZ103 and the sites of integration of the pDZ103. Three transformants were motile under both conditions, possibly due to loss or rearrangement of the NITl promoter region during integration. Deletion or rearrangement of the incoming DNA is a common occurrence during transformation of the nuclear genome of Chlamydomo- nas (KINDLE et al. 1989; TAM and LEFEBVRE 1993).

Integration of the NITI:PF14 transgene in the cotransformants was confirmed by DNA hybridization analysis using hybridization probes derived from the NITl upstream region and thePFI4 coding region

4.25 - FIGURE 2.-Cosegregation of the plasmid insertion with

the constitutive motility phenotype in backcross TlO-1OB X CS33F (pf14 nitZ-I mt). DNA from 10 randomly picked progeny was digested with SmuI, gel-frationated and blotted. The 2.6-kb Nhd fragment (probe B in Figure 1A) was used as the probe for hybridization. The growth in nitrate and conditional swimming phenotypes ( s , swimming in nitrate; p, paralyzed) are indicated under the strain names.

(probes A and B in Figure 1A). Most transformants contained multiple copies of the pDZlO3 plasmid (data not shown). In 13 of the 24 transformants examined, at least one copy of the NITl promoter from pDZlO3 remained intact.

Isolation of spontaneous, constitutively swimming mutant strains: Starting with these conditionally swimming strains, it became possible to isolate mutants with defective repression of the expression from the NZTI promoter by ammonium, If ammonium is present in the medium, cells with normal regulation of NR are paralyzed and stay at the bottom of a liquid culture tube. Mutants with defective repression of the NITl promoter should therefore swim even in the presence of ammonium (Figure 1C) and should be isolated easily by recovering swimming cells from the meniscus of the culture.

The cotransformant T10-10B was chosen as the parent strain for selecting mutants. This strain contains at least two copies of pDZlO3 (data not shown). All of the cells had paralyzed flagella in ammonium but showed normal motility within 8 hr after cells were transferred from ammonium to nitrate. When TlO-1OB was back- crossed to a pf14 nitl-1 strain (C33-3F), the conditional swimming and unconditional paralysis phenotypes segregated in a 2:2 ratio among 24 tetrads. DNA hybridization analysis was performed with DNA from the 10 randomly chosen progeny using the PF14 coding region as the probe (probe B in Figure 1A). The smaller hybridizing fragment represents the endogenous gene that is present in both parents. The two larger fragments, which are characteristic of T10-10B, cosegregated with the conditional swimming phenotype (Figure 2). These results demonstrated that the transgene NITI:PF14 be-


haved as a single Mendelian locus. Furthermore, the swimming phenotype cosegregated with the Nit+ phenotype, indicating that the inserted pMN56 is tightly linked to the NITl:PF14 transgene. Genetic linkage of separate plasmids cotransformed into the Chlamydo- monas genome has been observed previously (DIENER et al. 1990).

To isolate spontaneous mutants that were motile in medium containing ammonium as the sole nitrogen source, 36 independent cultures were inoculated with single colonies of strain T10-10B. After 2-8 weeks of growth, swimming cells were observed in 28 of the tubes (see MATERIALS AND METHODS). The mutant strains were designated as swX, X being the number of the tube from which the mutant was isolated.

The majority of the sw mutants expressed NR activity in the presence of ammonium: In addition to the de- sired class of mutants with defects in the regulation of NITl expression by ammonium, other classes of mutations might bring about the constitutive swimming phenotype. For example, since pf14 is a point mutation that changes a CAA to the nonsense codon UAA (ochre) (WILLIAMS et al. 1989), constitutive swimming could result from either reversion of the pf14 mutation or the occurrence of an extragenic suppressor mutation (Hu- ANG et al. 1982). Such mutations should have no effect on the repression of NITl by ammonium. In contrast, if a mutation affected a transacting regulatory factor that interacts with the NITl promoter, then the transgene NITl:PF14 as well as the NIT1 gene itself should be derepressed. Because the parent strain (T10- 10B) used for mutant isolation contained a functional NITl gene (contributed by the pMN56 plasmid), it was possible to test the expression of the NIT1 gene in sw mutants in the presence of ammonium.

To assay for NR activity in the presence of ammonium repression, the 28 constitutively swimming strains, along with the nitl-1 pf14 strain C3-1, the parent strain T10-10B and the wild type 21gr, were grown in medium containing NH4N03, and NR activities were assayed. In the control strains, (T10-10B and 21gr), NR activity was very low, confirming that NR was repressed under these conditions. Of the 28 constitutively swimming strains, 19 displayed elevated NR activity in the presence of ammonium compared to the wild-type control strains (Figure 3). Therefore, these 19 sw strains were retained as candidate mutants with potential defects in trans acting regulatory genes for NITl expression.

The expression of NI?'l gene can also be assessed indirectly by testing the ability of cells to grow in the presence of chlorate. Wild-type cells are resistant to chlorate in the presence of ammonium, presumably because the genes involved in nitrate assimilation are repressed by ammonium. If a mutant lost the repression of these genes by ammonium, it should be sensitive to killing by chlorate regardless of the nitrogen source in the medium.

s t r a i n s

FIGURE 3.-NR activity assay. The NR activity is expressed in units of nmol NOB- min" mg" chlorophyll. s and r indicate sensitivity or resistance to 10 mM KCIOR on solid medium containing 3 mM NH4NO:

Chlamydomonas Regulatory Locus Far1 127

dominant selectable marker to transform the wild-type strain (21gr). The wild-type CRYI gene encodes the ribosomal protein S14. The mutation in CRYI-1 changes the last amino acid residue of the S14 protein from leucine to proline, causing dominant resistance to the antibiotics cryptopleurine and emetine. The construct pJN4, which carries the coding region of CRYI- I ligated to the RRCS2 promoter (NELSON et ul. 1994), was introduced into 21gr cells by transformation and emetine-resistant transformants were selected. The de- sired mutants were then identified by screening the transformants for sensitivity to chlorate in the presence of ammonium. A total of 1824 emetine-resistant transformants were spotted on agar plates containing 10 mM KC103 in medium containing ammonium as the sole nitrogen source. One chlorate-sensitive mutant, 2-G4, was identified using this screen.

When grown in the presence of ammonium, the NR activity in the mutant 2-G4 was 29-fold higher than in wild-type cells, confirming that the endogenous NITI gene in the 2-G4 mutant was derepressed in the presence of ammonium. When the 2-G4 mutant was back- crossed to wild type, the chlorate-sensitivity and chlorate-resistance phenotypes segregated 2:2, and the chlorate-sensitivity phenotype cosegregated with emetine resistance (46 tetrads). The 2-G4 mutant therefore contains a single Mendelian mutation, hereafter designated furl-I (free from ammonium repression), that is tightly linked to the CRYI-1 transgene. Moreover, when the transgene NITl:PF14from T10-1OB was introduced into the furl-I pf14 background by mating C345B X C33 10H, the progeny containing both the furl-I mutation and the NITI:PF14 transgene (e.g., C37D-3A) were found to swim in the presence of ammonium (data not shown). The constitutive expression of the transgene NITI:PF14 in the furl-I mutant suggests that it is deficient in the repression of the NITI promoter in the presence of ammonium, as observed for the sw mutants.

To examine the structure of the inserted plasmid DNA in the furl-I mutant, genomic DNA from eight progeny of a cross between the furl-I mutant strain (C4 9B) and a wild-type strain (B27) was digested with KpnI, which cuts the CRYl-I plasmid (pJN4) only once. The genomic blot was hybridized with a 1.7-kb fragment (KpnI-B') from the 5' half of the CRYl coding region (NELSON et ul. 1994). A fragment of 5.5 kb representing the endogenous S14 gene is present in both wild-type cells (21gr, B27) and the furl-I mutant cells (2" and C49B) (Figure 4). Each extra hybridizing fragment in the mutant represents at least one copy of pJN4, and at least four copies of plasmid pJN4 were present in the genome of 2-G4. All four copies of the integrated plasmids cosegregated with the emetine-resistance and the chlorate-sensitivity phenotypes among the progeny, suggesting that the integrated copies of pJN4 were linked to each other and to the furl-I mutation.

Recombmation tests betweenfud-1 and the spontane-

emetine - - + + + + - - + + + + C10i,NH4+ + + - - - - + + - - - -

7.74 - 6.22 - 4.25 - 3.47 - 2.69 -

FIGURE 4.-DNA hybridization analysis of the firl-1 mutant 2 4 2 and the progeny of backcrosses. Genomic DNA samples were digested with KpnI. The hybridization probe was prepared from the CRYl - I region of pJN4 ( 1.7-kb KpnI-Bfl fragment). 21gr is the wild-type parent strain from which farl-1 was isolated. 2-G4 is the original firl-1 isolate. B27 is the wild- type mt- parent for the two backcrosses. C49B is a progeny strain from the first backcross and the parent of the second backcross. C11-2E, C11-2F, C11-2G, and C11-2H are the four progeny of an NPD tetrad from the backcross C4-9B X B27. C11-3B, C11-6B, C11-8G and C11-11C are four randomly c h e sen chlorate-sensitive progeny from the same backcross. + and - under the strain names represent growth under indicated conditions.

ous sw mutants furl-I and the sw mutants exhibit similar phenotypes: constitutive NR activity, chlorate sensitivity in ammonium, and constitutive expression of the chimeric gene NITI:PFI4. To determine whether the spontaneous sw mutations are allelic to furl-I, and to determine whether more than one gene is represented in the sw mutant collection, we carried out recombination tests involving furl-I and the 21 chlorate-sensitive sw mutants. In the cross of a furl-I strain to an sw strain, both parents are sensitive to chlorate in the presence of ammonium. The production of progeny that are chlorate resistant in ammonium ( i e . , wild type) would indicate recombination between furl-I and the sw mutation, suggesting that the sw mutation may identify a gene unlinked to furl.

To facilitate scoring of the chlorate-sensitivity phenotype among progeny from recombination tests with furl- I, 12 of the 21 sw mutations were first crossed into the NITl background (strain C2-9E). The resulting NITl sw mutant strains (listed in Table 2) were then crossed to a furl-I NITl strain (C42B or C49B). The progeny were spotted on medium containing chlorate and ammonium to test whether chlorate-resistant recombi- nants were produced. Among the 12 sw mutants shown in Table 2, five (sw4, sw7, sw8, sw18, and sw19) did not


TABLE 2

Recombination between 12 sw mutations and furl-1

Recombination Strain Mutations PD:NPD:T observed

C12-6H (mt') sw 1 3:5:0 Yes

C148C (mt') sw4 22:o:o No

C16-5G (mt') sw7 23:O:O No

C13-1OC (mt') S W ~ 10:11:2 Yes

C15-12H ( V I . - ) S W ~ 12:12:O Yes

C17-1C (mt') sw8 24:O:O N o C19-9H (mt-) sw13 13:9:0 Yes C81-2G ( ~ 6 ) sw14 18:25:11 Yes C23-8D (mt- ) sw18 24:O:O No C24-llC (mt- ) sw19 17:O:O No C25-9F (mt') sw20 6:16:2 Yes C26-5H (mt- ) sw2 1 11:8:5 Yes

The furl-1 parents were C42B (mt- ) or C49B (mt') . The original sw mutants were crossed to C2-9E, and the NIT+, Chl", pf progeny were chosen for the recombination tests. All but one of the sw strains were in the NITl , pf14 background. For sw14, the strain C81-2G (mt-swl4 supcsl) was crossed to C39-3E (mt+ farl-l acl7) .

recombine with furl-I and were considered potential furl alleles. The other seven sw mutations were unlinked tofurl-I, because parental ditype (PD) and non- parental ditype (NPD) tetrads were recovered in ap- proximately equal numbers in the cross to furl-I. These seven sw mutants therefore represent mutations in at least one other locus besides FARl.

A different strategy was used to test linkage of the remaining nine sw mutants with furl. The original constitutively swimming isolates (genotype swX nitl-l pf14 pDZlO3 pMN56) were crossed to the furl-l NIT1 strain (C42B). Some progeny (25%) from these crosses would inherit only the mutant nitl-l allele and thus would not be useful in the chlorate-sensitivity test for furl, because all of the nitl-1 progeny would be resistant to chlorate. Nevertheless, it was possible to score for chlorate sensitivity among the Nit+ segregants. If no recombination occurred between an sw mutation and furl-I, each of the Nitf progeny should be chlorate sensitive. The appearance of a chlorate-resistant phenotype among the Nit' progeny would indicate that recombination between furl-1 and the sw mutation had occurred. The remaining nine mutants, s w l l , sw15, sw17, sw22, sw26, sw27, sw28, sw29 and sw36, were crossed to furl-I, and tetrads were tested on nitrate plates and plates containing ammonium plus chlorate. As expected, all Nit- progeny were resistant to chlorate. At least 61 Nit' progeny were obtained from each cross. Only one of the nine sw mutations (sw36) tested in this way failed to recombine with furl-1. Overall, six of the 21 chlorate-sensitive sw mutations were shown to be tightly linked to each other and to furl-I.

Complementation tests of furl-1 and the six tightly l i e d sw mutants: To determine whether furl-I and

the six linked sw mutations define a single complementation group, vegetative diploids were constructed by mating two different mutant strains carrylng comple- menting selectable markers (see MATERIALS AND METH- ODS). If the mutations fail to complement, the diploid strain should be sensitive to chlorate in the presence of ammonium. If the mutations define different complementation groups, the diploid cells should be resistant to chlorate in the presence of ammonium. First we tested whether furl-l and the six sw mutations, namely sw4, sw7, sw8, sw18, sw19, and sw36, were recessive to wild type in diploids. The heterozygous diploids constructed with wild type and each of the seven mutants displayed the wild-type phenotype, i.e., the diploids were resistant to chlorate in the presence of ammonium, indicating that all seven mutations are recessive. Each of the six sw mutants was next mated to furl- I cells and stable diploids were isolated. The diploids constructed with jurl-I and each of the six sw mutants were sensitive to chlorate in the presence of ammonium. Thus those sw mutations and furl-1 belong to the same complementation group, confirming thatfkrl- I and the six mutations are allelic. The mutations in sw4, sw7, sw8, sw18, sw19, and sw36 are designated as jurl-2, ,f(~rl-3, jicrl-4, furl-5, furl-6, and furl-7, respectively.

Genetic localization of FARl: The FARl gene was placed on the Chlamydomonas genetic map based on the results of crosses to a number of genetically marked strains. When furl-I was crossed to the tightly centromere-linked mutation ucl7 (C49B X JN142), the ratio of PD:NPD:T was 27:28:10, corresponding to a distance of 7.7 cM between furl-1 and its centromere (WHITEHOUSE 1956). Linkage was detected between furl-I and urg7 on Linkage Group I (C42B X H11) based on an absence of NPD tetrads, although this cross exhibited poor survival of progeny (data not shown). A three-point cross, j&rl-l PF22 ERY3 (C49B) X FAR1 pf22 9 3 (C87-3F) was performed. The order of the three loci was determined to be pj22-farl-ery3, based on the observation that a single tetrad (of 51 tested), presumably resulting from a double crossover event, contained two pf22 furl q 3 and two PF22 FARl ERY3 progeny. furl was mapped to the right arm of Linkage Group I, between the centromere and cry? (Figure 5).

Genetic interaction between far l and nit2 FARl and NZTZ are both regulatory genes whose products affect the expression of NZT1, but in opposite ways. ,furl mutations result in constitutive NZTl expression in the presence of ammonium, whereas nit2 mutations abolish NZl1 expression. Thus far1 and nit2 mutants exhibit opposite phenotypes when tested for growth on nitrate medium and on medium with ammonium plus chlorate (Table 3 ) . When multiple genes act in a regulatory pathway, the mutations in downstream genes are epistatic to mutations affecting genes acting upstream (AVF.KY and WASSEWN 1992). By examining the phenotype of

Chlamydomonas Regulatory Locus Far1 129

PP2 farl I arg7 I ev3 I - I 1 I

,7.7,,7.7, 18.6 23.5 *

t 4

4"

35.6 10 4"""""- 19.4

4"""""""" 27.2 .) 4"""""""""""""""" 40.4 *

FIGURE 5.-Genetic localization of furl . - --, the published centromere distance of pf22 (HUANG et al. 1979) and the distances from arg7 to pj22 and to my3 (MYERS et al. 1984). -, the results obtained in this study. The centromere distance was determined from crosses to the centromere marker ac l7 (PD:NPD:T = 27:28:10) (WHITEHOUSE 1956). The f a r l - arg7distance was determined from the RFLP mapping relative to the cloned ARG7 gene (DEBUCHY et al. 1989) using 58 random progeny from a cross C. rrinhardtii X C. srnithii (Ri- NUM et al. 1988, C. D. SILFL.OM' and p. KATHIR, unpublished result). The results of the three-point cross furl X pf22 e93 (51 tetrads) were calculated according to PERKINS (1949). The p122-Jirl distances was 18.6 cM (PD:NPD:T = 37:1:13), the f u r l - 9 3 distance was 23.5 cM (PD:NPD:T = 32:1:18). The pj22-93 distance was 35.6 cM according to this cross.

a furl-I nit2 double mutant, it is possible to determine the epistatic relationship between the two loci, and therefore infer the order of the two genes in the regulatory pathway, assuming that they function in a single pathway. A furl-1 strain (C42B) was crossed to a nit2 strain (A35) to construct the double mutant. The furl nit2 recombinant was identified among NPD tetrads, which have two wild-type and two double mutant progeny. The phenotype of the furl-l nit2 double mutant resembled that of the nit2 parent, i.e., no growth on nitrate and resistance to chlorate in the presence of ammonium (Table 3). Thus nit2 is epistatic to furl, suggesting that if FARl and N17'2 act in the same pathway regulating N I l 1 expression, then FAR1 functions upstream of NITz'2.

Cloning of the FARl locus: Because jurl-I appeared to be generated by the insertion of the transforming pJN4 plasmid, we isolated DNA fragments flanking the site of the insertion to obtain a probe for cloning the wild-type FAR1 gene. DNA hybridization analysis indicated that in at least one of the four pJN4 inserts, the integrated plasmid vector (pUC118) was largely intact (data not shown), suggesting it might be possible to clone the DNA fragments flanking the insertion site by plasmid rescue (TAM and LEFEBVRE 1993).

Genomic DNA of thefurl-l mutant was digested with BclI, a restriction enzyme that does not cut plasmid pJN4. The resulting DNA fragments were circularized by ligation and transformed into Eschm'chia coli. Six am- picillin-resistant transformants were isolated, each carrying a 20-kb plasmid (pDZ105) that presumably contains the integrated pJN4 and some flanking sequences. By comparing the restriction maps of pDZ105 and

TABLE 3

Phenotypes of farl-I and nit2 mutants and the farl-I nit2 double mutant

Growth on Growth on medium Genotype NO$ medium with NH: and C10,

Wild type + + f a r l + nit2 + far1 nit2 - +

-

-

The cells were spotted on solid minimal medium. f, growth; -, no growth.

pJN4, and by DNA hybridization analysis of pDZ105 using pJN4 as a probe, we determined that a 1.35-kb HindIII-SmaI fragment was unique to pDZ105.

As described above, the furl-1 mutation was tightly linked to the integrated pJN4 plasmids. If the 1.35-kb fragment cloned in pDZ105 is derived from sequences flanking the site of pJN4 integration in the furl-1 mutant, then using this fragment as a hybridization probe, restriction fragment length polymorphisms (RFLPs) should be detected upon comparison of DNAs from wild-type and the furl-l mutant cells. Moreover, the RFLPs should cosegregate with the wild-type and the mutant phenotypes in a genetic cross. The 1.35-kb fragment was labeled with y2P and hybridized to a Southern blot of wild-type and furl-1 DNA digested with the restriction enzymes SmaI and XbaI. The 1.35-kb probe hybridized to fragments of different sizes in DNA from wild-type and furl-I cells (data not shown). When the same 1.35-kb probe was hybridized to genomic DNA of the progeny of the backcross furl-I X rut digested with KpnI, the polymorphic bands cosegregated with the mutant phenotypes among the 12 progeny of the backcross (data not shown). These results showed that the 1.35- kb fragment isolated from pDZ105 corresponds to a genomic region that is adjacent to the site of the pJN4 insertion and is closely linked to the fir1 mutation genetically.

To clone the wild-type FARl gene, the 1.35-kb fragment was used as a hybridization probe for screening a genomic DNA library of Chlamydomonas in WixZZ (SCHNELI. and LEFEBVRE 1993). Sixteen overlapping clones were obtained, spanning a 22-kb region (Figure 6A). To determine whether the spontaneous furl mutants contain DNA rearrangements in the cloned region, the 1.35-kb fragment from pDZ105 was used as a hybridization probe to analyze the genomic DNA from the six spontaneous furl mutants. Two of the spontaneous furl alleles, furl-3 and furl-4, showed major alter- ations relative to wild type in the size of the DNA fragment hybridizing to the 1.35-kb probe. The 2.8-kb SstI fragment in wild type (fragment C in Figure 6A) changed to 7.7 kb in furl-3, 7.0 kb in furl-4, and 5 kb in furl-1. In addition, furl-2 and furl-4 also showed an altered hybridization pattern when the 1.8-kb SstI frag-


A. A 1 kb - H I B I I I c I I D 1

w g 7.74 -

. . :

3.47 -

FIGURE 6.- (A) The restriction map of the cloned genomic region linked to the FARl locus. The location of the 1.35 kb fragment from pDZlO5 used as a hybridization probe is indicated (region A). The vertical bars indicate SstI sites. Frag- ment B is the 3.5kb SstI fragment used as a hybridization probe for RFLP mapping of the FARl locus. (B) Restriction fragment length polymorphisms between the far1 mutant alleles. The genomic DNA from the wild-type and the farl mutant strains was digested with SsA, separated by electrophoresis and transferred to nylon membrane. The 1.35-kb fragment (region A) was used as the probe for hybridization. The 2.8- kb S s d fragment (fragment C) is altered in size in DNA from farl-I, farl-3, and furl-4 cells relative to wild-type cells. The 1.8-kb SstI fragment (region D) is altered in size in DNA from farl-2 and farl-4 mutants relative to wild type (data not shown).

ment (fragment D in Figure 6A) was used as a probe (data not shown). The RFLPs observed among the furl mutants strongly support the conclusion that the DNA sequences within the region containing the SstI fragments C and D are essential for FARl gene function.

The location of the cloned DNA on the Chlamydo- monas genetic map was determined by RFLP mapping (RANUM et ul. 1988). A 3.5-kb SstI fragment from the left region (fragment B in Figure 6A) was used as a hybridization probe. The fragment was mapped to Link- age Group I, 7.7 cM from the ARG7gene (DEBUCHY et al. 1989), proximal to the centromere (see Figure 5). This map location is consistent with the genetic m a p ping of the FARI locus.

DISCUSSION

FARl is a negative regulator of the expression of NZTl: A new Chlamydomonas regulatory gene I3R1,

which is an important link in the nitrogen regulatory circuit, has been identified genetically by two independent mutant screens. FARI was mapped to the right arm of Linkage Group I by crosses using conventional markers as well as by RFLP mapping using a cloned DNA fragment from the FAR1 locus. We obtained a stable mutant allele by insertional mutagenesis, along with six spontaneous alleles, at least three of which are probably caused by transposon insertions. These furl mutants are most likely loss-of-function mutations, par- ticularly furl-I, the allele generated by insertional mutagenesis. The expression of NIT1 gene became constitutive in these mutants, implying that the function of the wild-type FARI gene involves inactivation of the NITI gene in the presence of ammonium. Therefore, we pro- pose that FARI is a negative regulator for the expression of NITI. To determine whether FAR1 is a pathway-specific regulator or a more general regulator, it will be of interest to test whether other enzymes that are normally repressed by ammonium, e+, urate oxidase (PINEDA et ul. 1984) and xanthine dehydrogenase (FERNANDEZ and CARDENAS 1981), are derepressed in the farl mutants. Recently PRIETO et ul. (1996) screened for chlorate sensitivity in the presence of ammonium and identified two unlinked mutations that confer a similar phenotype to that observed in furl mutants. The relationship of these mutations to furl has not yet been established.

It is possible that repression by ammonium occurs at several levels, through a global repressor as well as through factors specific for certain pathways. From the results of the recombination tests between farl-1 and the 21 sw mutants, it is evident that at least two fur loci are represented in the sw mutant collection. We intend to characterize the remaining 14 chlorate-sensitive sw mutants genetically and eventually to clone the relevant gene(s) by transposon tagging to identify all of the gene products required for ammonium regulation of gene expression in Chlamydomonas.

The farl mutants are not deficient in ammonium u p take: One possible reason for the constitutive expression of NIT1 in furl could be that the mutation pro- duces a defective ammonium transporter. In Chla- mydomonas, the mal mutant, conferring resistance to the ammonium analogue methylammonium, was shown to be defective in the transport of ammonium into cells (FRANCO et al. 1987). In mal mutants, NR activity was detected in the presence of both nitrate and ammonium, probably because the transport defect caused low intracellular levels of ammonium (FRANCO et al. 1987). To test the possibility that some or all of the furl mutations affect ammonium transport, ammonium uptake was assayed for furl-I and the 21 chlorate-sensitive mutants. No significant difference in the rates of ammonium uptake was found between wild type and the mutants (data not shown). We conclude that the furl-I mutants are not defective in the uptake of ammonium.


PF14 as a reporter gene: The PF14 gene encodes radial spoke protein 3 of the flagellar axoneme, which is essential for the motility of Chlamydomonas cells. Using the PF14 gene as a reporter gene under the control of the NITl promoter, we were able to isolate extragenic mutations that restored motility due to relieved ammonium repression of expression from the NITl promoter. The results demonstrate the feasibility of using flagellar genes as reporter genes for the isolation of mutants with defects in the regulation of metabolic pathways. Using this screen it was possible to eliminate the need to replica-plate thousands of colonies to isolate chlorate-sensitive mutants. Instead, the mutants of interest swam to the surface of medium in a culture tube.

Spontaneous mutations of FARl may have been caused by the insertion of transposable elements: In C. reinhardtii, a number of transposable elements have been characterized (DAY et al. 1988; FERRIS 1989; DAY and ROCHAIX 1991a,b; R. A. SCHNELL, SC. WANG and P. A. LEFEBVRE, unpublished data). SCHNELL and LEFEB VRE (1993) demonstrated that a large proportion (eight out of 14) of spontaneous nit2 mutants were caused by the movement of transposable elements, and that it was feasible to clone new Chlamydomonas genes by transposon tagging. We sought mutants that arose spontaneously using the swimming screen, hoping to obtain mutants caused by transposon insertions and to clone the affected genes by transposon tagging. Several lines of evidence indicated that at least some of the sw mutations were caused by the movement of transposons: (1) the sw mutations all occurred spontaneously; (2) most of the mutants were unstable, and the rate of spontaneous reversion varied among different mutants; (3) when the six spontaneous furl alleles were analyzed by DNA hybridization using cloned FARl gene fragments as probes, RFLPs were observed in three mutants, indicating that DNA insertion or rearrangements occurred at the FARl locus.

&acting element(s) for the repression of NITl by ammonium: Complete inactivation of the reporter gene in the presence of ammonium is crucial for the success of the swimming screen. For the transacting factors to function properly, it was essential that all of the ciselements needed for the regulation of the NITl promoter be present in the chimeric gene. During transformation of Chlamydomonas, the incoming DNA often suffers deletion or rearrangement when integrated into the genome (KINDLE et al. 1989; TAM and LEFEBVRE 1993). To ensure that all of the ciselements of NITl were included, the 3.6-kb Sua-Hind111 fragment, which begins in the middle of the upstream gene NIT8 and ends 42 bp before the ATG initiation codon of NITl, was used in construct pDZ103 (FERNANDEZ et al. 1989; D. ZHANG, SC. WANG, M. PRIGGE, M. P. LAVOIE and P. A. LEFEBVRE, unpublished data). For transformant T10-lOB, swimming was shown to be under

strict control by ammonium, and the NITl promoter region remained largely intact in at least one of the two copies of the chimeric construct integrated into the genome. Our results suggest that the 1014bp XbaI-Hin- dIII fragment of the NITl gene present in pMN56 was sufficient for both repression of NITl expression by ammonium and positive regulation by NIT2, because the ectopic NITl gene in TlO-1OB cells was regulated in the same way as the endogenous NITl gene in wild- type cells (Figure 3).

The regulatory pathway €or nitrate assimilation: The assimilation of nitrate is regulated in response to nu- merous physiological and environmental signals. The Chlamydomonas NIT2 gene is a pathway-specific positive regulator for the genes involved in nitrate reduction ( F E R N ~ D E Z and MATAGNE 1986). In a nit2 mutant, the transcripts of NITl and the other four genes in the cluster, NIT8, NARl, NAIU, and NAR4, cannot be expressed upon derepression ( F E R N ~ D E Z et al. 1989; QUESADA et al. 1993). In addition, NIT2 itself is repressed by ammonium. The NIT2 transcript levels are significantly lower in the presence of ammonium than in the absence of ammonium (SCHNELL and LEFEBVRE 1993).

The epistasis of nit2 relative to farl is consistent with the hypothesis that FARl represses NIT2 when ammonium is present. The simplest explanation would be that the two genes act in a linear pathway in which the FARl gene product represses NIT2 expression in response to ammonium. NIT2 would directly activate the expression of NITl and other target genes. By this model, N f l 2 activity would be constitutive in the furl mutants, and NITl and the genes in the cluster would be expressed as a result. Alternatively, it is possible that the repression involves a direct interaction of the FARl and NIT2 gene products. Such an interaction has been described for the regulatory proteins NMR and NIT2 of Neurospora. The Neurospora NIT2 gene encodes a protein with a putative zinc finger DNA-binding domain (Fu and MARZLUF 1990) that binds to upstream activa- tion sites of the structural genes and activates their transcription. The predicted amino acid sequence for the negatively acting NMR gene does not have an obvious DNA binding domain (YOUNG et al. 1990). XIAO et ul. (1995) showed evidence that the NMR protein inhibits the expression of the NR gene by binding to the NIT2 protein. If the Chlamydomonas NIT2 protein interacts with the FARl protein in a similar manner, then the nit2 mutation should still be epistatic to furl.

We are very grateful to PUSHPA KATHIR and Dr. CAKOI.YN D. SII, FLOW for RFLP mapping of the cloned DNA and comments on the manuscript. We thank Drs. JULIE A. E. NELSON and LA WA TAM for sharing plasmids and Chlamydomonas strains and for helpful discus- sions, and Dr. KARL A. JOHNSON for providing the HA-tagged PF24 construct. The Chlamydomonas Genetics Center at Duke University provided several mutant strains. A special thanks is given to Dr. Ro- GENE A. SCHNELL for sharing many experimental materials and unpublished data and for critical reading of the manuscript. This re-


search was supported by grants to P.A.L. from the Department of Agriculture (NRICGP/USDA 9403648) and the National Institutes of Health (NIGMS GM-34437).

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Communicating editor: V. G. FINNERTY

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