Copyright 0 1993 by the Society of America

Isolation of the Chlamydomonas Regulatory GeneNIT2 by Transposon Tagging

Rogene A. Schnell and Paul A. Lefebvre

Department of Genetics and Cell Biology and Plant Molecular Genetics Institute, University of Minnesota, St. Paul, Minnesota 55108 Manuscript received September 22, 1992 Accepted for publication March 8, 1993

ABSTRACT Genetic evidence suggests that the NIT2 of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOCI, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA re- arrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrateassimilation pathway in Chlamydomonas.

HE assimilatory reduction of nitrate to ammo- trate permease, and nitrite uptake activities are un- T nium by plants, algae and some fungi and bac- detectable or greatly reduced compared to their wild- teria is the predominant point of entry of inorganic type levels (FERN~NDEZ MATAGNE and 1984; GALVAN, nitrogen into the biological realm (GUERRERO, VEGA CARDENASand FERN~NDEZ 1992;NICHOLS, SHEHATA and LOSADA1981). In this process, nitrate is trans- and SYRETT 1978; E. FERN~NDEZ,personal commu- ported into the cell and is reduced to nitrite and to nication). Comparison of NITl transcript levelsin ammonium by the consecutive action of nitrate and wild-type and nit2 mutant strains confirmed that the nitrite reductases. In the unicellular green alga Chlam- wild-type NIT2 gene is required for accumulation of ydomonas reinhardtii, mutations in seven genes (NITI- the NITl transcript (FERNANDEZet al. 1989). To our NIT7)have been described that block the assimilation knowledge, NIT2 is the first regulatory gene involved of nitrate (AGUILARet al. 1992; FERNANDEZand MA- in nitrate assimilation to be identified in a photosyn- TAGNE 1984; NICHOLSand SYRETT 1978;SOSA, OR- theticeukaryote. Progress toward identifying the TEGA and BAREA 1978). These mutations were iso- product of the NIT2 gene and understanding its reg- lated because they confer resistance to chlorate, an ulatory role is contingent upon isolation of the gene. analog of nitrate. It has been suggested that chlorate Transposable elements have provided an invaluable toxicity in wild-type cells is mediated by nitrate reduc- means for isolating genes whose productsare un- t!se, which convertschlorate to a toxic compound known (BINGHAM,LEVIS and RUBIN198 1 ; MOERMAN, (ABERG 1947), althoughother mechanisms of chlorate BENIANand WATERSTON1986; OKKEMA andKIMBLE toxicity have been proposed (BAEUERLEand HUTTNER 1991; SCHMIDT,BURR and BURR1987; TOMLINSON, 1986; COVE 1976; PRIETOand FERNANDEZ1993). KIMMELand RUBIN1988). Cloning a geneby transpo- The NITI gene encodes the nitrate reductase apoen- son tagging requires an active zyme and NIT3 through NIT7 are required for syn- for which a molecular probe is available, and a readily thesis of a molybdopterin cofactor that is essential for identifiable mutation caused by insertion of the trans- nitrate reductase activity (AGUILARet al. 1992; FER- posable element into or near the gene of interest. In NANDEZ and AGUILAR1987; FERNANDEZand MA- Chlamydomonas, two transposableelements have TAGNE 1984; FERNANDEZet al. 1989). been identified. The TOCI element has an unusual The NIT2 gene is proposed to encode a positive long terminal direct repeat structure at its ends and regulator of the nitrateassimilation pathway (FERN~N- resembles RNA-mediated transposons (DAY and Ro- DE2 and MATAGNE 1986; FERNANDEZet al. 1989). In CHAIX 1991a,b; DAY et al. 1988). The Gulliver ele- nit2 mutants, nitrate reductase, nitrite reductase, ni- ment has a short inverted repeat at its ends and is

Genetics 134: 737-747 (July, 1993) 738 R. A. Schnell and P. A. Lefebvre flanked by an 8-bp target site duplication, hallmarks Where appropriate,NH4NOJ (5 mM) was replaced by of DNA-mediated transposons (FERRIS1989). KNOJ(4 mM), KN02 (2 mM), or urea (2 mM). Filter- sterilized solutions of KNOn, urea and potassium chlorate members thetransposable element families If of were added to media after autoclaving. Solid media con- described in Chlamydomonastranspose during mi- tained 1%agar thathad been washed extensively with toticgrowth, and if integrationoccurs randomly distilled water. Chlamydomonas strains were grown at 25" throughout the genome, then it shouldbe possible to under continuouslight, except that strains containing recover mutations causedby insertion of a transposon supcsl-2 were grown at 33" and maintained at 25". Standard methods were used for tetrad analysis (LEVINE into a particular is well-suited gene. The NIT2 gene and EBERSOLD1960). For complementation testing, Nit- for testingthe feasibility of transposontagging in mutant strains were mated with nit2 and nit1 tester strains Chlamydomonas because loss-of-function nit2 muta- (B20, B2 1, B14, B 15) andstable diploids were selected using tions can be selected on medium containing chlorate complementing mutations (EBERSOLD1967). For this selec- and wild-type revertants can be selected on medium tion, cold-sensitive supcsl-2 strains were mated with acetate- requiring ac17 strains and the mating mixtures were plated containing nitrate as the sole nitrogen source. onto solid medium without acetate (M) and incubated at the In thisstudy a transposon-induced nit2 mutation restrictive temperature for supcsl-2 (15 ") for 10 days. To was identified by analyzing spontaneous nit2 mutant assay complementation, at least three colony-purified dip- strains for the presence of additional or rearranged loid isolates were spotted onto medium containing nitrate copies of transposable elements. A Gulliver element as the sole nitrogen source. Growth was scored after 3 days at 25". Noncomplementing mutations should result in dip- associated with a nit2 mutation was isolated, and se- loid strains that are unable to grow on nitrate. quences contiguous to the transposon were used to Chlamydomonas cells were transformed withplasmid clone the wild-typeNIT2 gene. Analysis of transcript DNA using a previously described method (KINDLE 1990). abundance revealed that expressionof the NIT2 gene Briefly, cells containing nit2-203 and m15 were grown in was repressed in medium containing ammonium. In SGII medium and cells (2-5 X 10') were vigorously mixed with supercoiled plasmid DNA (5-10 pg), glass beads (1- light of these observations, working models for the mm diameter), and polyethylene glycol (M, 8,000) using a role of NIT2 in the regulation of nitrate assimilation Vortex mixer (Scientific Products) at top speed for 45 sec. are discussed. Plasmids used for transformation were obtained by subclon- ing restriction fragments from X47-7 (Fig. 5) into pUCl18 or pUCll9 (VIERA and MESSING 1987): pMN60 contains MATERIALS AND METHODS the 6.2-kb HindIII fragment, pMN68 contains the 12-kb Strains: Chlamydomonas strains usedin this study are SstI fragment and pMN72 contains the 10.5-kb Sal1 frag- listed in Table 1. Mutant alleles nitl-305 and nit2-203 were ment. obtained from strains 305 and 203,which were provided by Spontaneous, chlorate-resistant mutants were obtained E. FERN~NDEZ(University of Cordoba,Cordoba, Spain; by plating cells from 16 independent cultures on solid R SOSA,ORTECA and BAREA 1978). Mutant alleles nitl-137 medium containing urea as the sole nitrogen source and and nit2-137 arose spontaneously in the Ebersold-Levine either 2.5 or 5 mM potassium chlorate. Chlorate-resistant line 137c (FERN~NDEZand MATAGNE 1984).Strains A54 colonies arose at frequencies of 0.8 X to 5.6 X and A55 were progeny from a cross between strain 305 per cell. Mutants were named according to the strain and (nitl-305,mt-) and strain C6+(ac17, srl, mt'). The supcsl-2 culture from which they were isolated. For example, mutant mutation confers a cold-sensitive growth and was A55-c9 is the ninth mutant obtained from culture "c" of originally isolated as an extragenic suppressor of oryl-1 strain A55. (Rcs2) (JAMES et al. 1989).Strains C6+and T897-B2 (supcsl- Revertibility of Nit- mutations was assayed qualitatively 2 mt-) were provided by S. JAMES (Gettysburg College, by plating approximately loficells (100 p1 of a 200-pl cul- Gettysburg, Pennsylvania). Strains D66 and D67 were ob- ture), grown nonselectively from a single colony, onto me- tained from the fifth consecutive cross inwhich meiotic dium containing nitrate as the sole nitrogen source. Rever- progeny containing the cell wall mutation cw15 (DAVIESand sion frequencies from 1O-' to 1 O-fi revertants per cell were PLASKITT197 1; HYAMS andDAVIES 1972), were successively detected by this method. The rates of reversion of nit2-1 backcrossed to strains A33, A53, 305, B27, B26 and A35. and nit2-2 were measured using the method of the median Culture conditions, media and genetic analysis: TAP (JAMES et al. 1989; LEAand COULSON1948). For this assay, medium (GORMAN andLEVINE 1965) contained 20 mM Tris cultures were grown from individual whole colonies in TAP (Sigma7-9), 7.5 mM NH,CI, 0.01% Mg~S04*7H20,0.005% medium at 25" under constant light. In strain A55-c9 (nit2- CaC12.2H20, 0.62 mM K2HP04,0.41 mM KHPPO4, 1 ml 1)and A55-a5 (nit2-2),Nit+ revertants arose at arate of 4.4 Hutner trace elements (SURZYCKI1971), and 1 ml glacial X lo-' and 5.0 X reversions per cell division, respec- acetic acid per liter. TAP-N was identical to TAP except tively. that NH,CIwas omitted. M medium (minimal medium I) To determine whether nit2 mutantscontained TOCl (SAGERand GRANICK 1953) was prepared accordingto dimorphisms, genomic DNA (3.5 pg) from each mutant was HARRIS (1989)except that the trace metal concentration digested with SstI or with PvuII, fractionated through 0.8% was reduced fourfold and the phosphate composition was agarose, transferred to a nylon membrane, and hybridized changed to 0.53 mM KzHP04 and 0.51 mM KHzP04. R with the radiolabeled TOCl . 1 R 1 plasmid (DAYet al. 1988). medium was the same as M except that it also contained 13 To test mutants for Gulliver dimorphisms, genomic DNA mM sodium acetate and the phosphate concentration was from each mutant was digested with HindIII, transferred to increased to 1.5 mM KzHP04 and 1.5 mM KHzP04. SGII a nylon membrane and hybridized with the AB3 clone medium (SAGERand GRANICK1953) was the same as M (FERRIS 1989). except that it also contained 14.7 mM sodium acetate and Isolation of nucleic acids from Chlamydomonas: the phosphate was changed to 6.6 mM K2HP04 and 26.6 Chlamydomonas DNA was purified using a procedure mM NaH2P04. adapted from WEEKS,BEERMAN and GRIFFITH(1986). Sta- Chlamydomonas NIT2 Gene 739 tionary phase cells grown in 250 ml of TAP medium bub- TABLE 1 bled with filtered air, were pelleted by centrifugation and resuspended in water to a volume of 2 ml. Cells were lysed Chlamydomonas reinhardtii strains by the addition of 0.35 ml 20% SDS, followed immediately by the addition of 0.5 ml Extraction Buffer (0.138 M EDTA, Strain Genotype Aliasa 1.38 M NaCI, and 0.345M Tris-HCI, pH 8.0), and incubated A35 nit2-203 mt+ for 30-60 min at 42”. Thecell lysate was mixed with solid A54 acl7 srl mt’ CsCl (3.1 g), loaded into a quick-seal tube (Beckman No. A55 acl7 srl mt- 358980) containing 0.1 5 ml ethidium bromide (10 mg/ml), A55-c9 nit2-1 ac17 srl mt- (CC-2841) and centrifuged at 90,000 rpm for at least 4 hr (Beckman A55-a5 nit2-2 ac17 srl mt- (CC-2842) TLNlOO rotor). The banded DNA was collected using a A55-d8 nit2-3 ac17 srl mt- syringe adapted with an 18-gauge needle and ethidium A54-a2 nit2-4 ac17 srl mt+ bromide removed by extraction using isopropanol saturated B14 supcsl-2 nitl-137 mt+ with water and sodium chloride. The DNA was precipitated B15 supcsl-2 nitl-I37 mt- by addition of one volume of water and one volume of B20 supcsl-2 nit2-137 mt+ isopropanol. The precipitated DNA was washed once with B2 1 supcsl-2 nit2-I37 mt- 70% ethanol, dried and resuspended in TE for at least 2 B26 srl mt+ days at 4’. B27 srl mt- Chlamydomonas RNA was isolated from cells grown to a B52 nit2-2 srl mt+ density of 1.5-3.5 X lo6 cells per mlin 1.5 liters TAP D66 nit2-203 nu15 mt- medium. Cultures were bubbled continuously with air sup- D67 nit2-203 nu15 mt+ plemented with 2-5% Con. Forderepression, cells were S1-D2 mt- (CC-2290) washed three times with TAP-N (without ammonium), re- suspended in 1 liter TAP-N and bubbled with air for 45 a Strains available from the Chlamydomonas Genetics Center. min. Approximately 90 min elapsed between the first wash with TAP-N and the harvesting of the cells following de- DH5a (Bethesda Research Laboratories). Plasmids contain- repression. Total RNA was isolated by phenol-chloroform ing Gulliver sequences were identified by colony hybridiza- extraction and centrifugation through cesium chloride as tion (MANIATIS,FRITSCH and SAMBROOK1982). described previously (SCHLOSS,SILFLOW and ROSENBAUM 1984). Poly(A)+RNA was selected by batchwise adsorption RESULTS tooligo(dT) cellulose (Collaborative Research) and size- fractionated through agarose in the presence of formalde- Isolation of mutants: Spontaneous, chlorate-resist- hyde (MANIATIS,FRITSCH and SAMBROOK1982). ant mutants were selected from Nit+ strains A54 and Nucleic acid hybridizations: DNA fragments to be used A55 (Table 1) and assayed for theirability to grow on as hybridization probes were purifiedfrom low-melting- temperature agarose (Seaplaque, FMC) by phenol extrac- nitrate as a sole nitrogen source. Most of the chlorate- tion and ethanol precipitation (MANIATIS,FRITSCH and SAM- resistant mutants (335 outof 384) were unableto BROOK 1982). Probes were radiolabeled by nick-translation utilize nitrate (Nit-). To identify putative nit2 alleles, (RIGBYet al. 1977). Conditions for transfer of nucleic acids 122 Nit- mutants were analyzed for their ability to from agarose gels to Nytran membranes and for hybridiza- complement a known nit2 mutation in diploids (see tion were as described by the manufacturer (Schleicher & Schuell, 1987). MATERIALS AND METHODS). When mated with a nit2 Library construction and screening: To generate a wild- strain (B20 or B21), approximately one-third of the type Chlamydomonas DNA library, DNA from strain 21gr mutants (42 out of 122) gave rise to Nit-diploids. mt+ was isolated as described above except that, following When mated with a nitl strain (B14 or B15), all but gradient centrifugation, CsClwas removed by dialysis two of these 42 mutants gave rise to Nit+ diploids. against TEN (5mM Tris-HCI, 2.5 mM EDTA, 20 mM NaCI, pH 7.6) rather than by precipitation in ethanol. The result- Genetic mapping confirmed that one of the mutants ing DNA was partially digested with MboI, and the vector that failed to complementboth a nitl and a nit2 DNA (XFIXII; Stratagene) was digested with XhoI, and both mutation (A54-f24) contained mutationsin both genes were partially filled in using the Klenow fragment of Esche- (data not shown). Only mutations that failed to com- richia coli DNA polymerase I to generate compatible over- plement the nit2 mutation, and fully complemented hangs andto prevent the formation of multiple inserts during ligation. The ligated mixture was packaged in vitro the nit1 mutation were retained for further analysis. (GigapakIIXL; Stratagene)and plated on E. coli strain FERNANDEZet al. (1989) observed that a strain con- P2392. A library containing 0.5 X lo6 recombinant phage taining a nit2 mutation grew poorly on nitrite. To was amplified by plating on E. coli strain LE392. Using a determinethe generality of this phenotypeamong single-copy sequence as a probe (probe2, Figure 4) positively nit2 alleles, we tested the ability of each of the Nit- hybridizing phage were detected ata frequency of 2 X 1 per phage and the insert lengths averaged 18 kb (Figure 5). mutants to grow on medium containing nitrite as the Standard methods were used for plating phage andfor sole nitrogen source. Each of the 40 mutants assigned screening by hybridization (MANIATIS,FRITSCH and SAM- to the nit2 complementation group grew slowly on BROOK 1982) andfor isolation of X DNA (CHISHOLM1989). nitrite compared to wild-type strains. Thus, the “ni- To isolate the Gulliver element associated with the nit2-1 trite minus” phenotype would bea useful test for mutation, genomic DNA from strain A55-c9 was digested withHind111 and size-fractionated by electrophoresis identifying potential nit2 mutants among large num- through a low-melting-temperature agarosegel. DNA from bers of chlorate-resistant strains. the fractioncontaining the desiredfragment was ligated Mutability, or phenotypic instability has been ob- intothe plasmid vector pUCl19and transformed into served in association with transposable element-in- 740 R. A. Schnell and P. A. Lefebvre

1 23 4 56 709 position of a Gulliver element into the NIT2 locus. Alternatively, the new band might have arisen by a transposition or recombination event independent of the nit2 mutation. To distinguish between these alter- natives, we tested whether the new Gulliver element in each of these strainswas genetically linked with the 14.7 - nit2 mutation. Eachof the mutant strains (A55-c9, A55-a5 and A55-d8) was mated with a wild-type strain (B26) and meiotic progeny were isolated. In more than 20 tetrads from each cross, the Nit- phenotype segregated as singlea nuclear mutationand was linked to the ac17 locus on linkage group 111 (less than 15 4.7 - cM), as predictedfor mutations atthe NIT2 locus " "W"" (HARRIS 1989).In all three tetrads analyzed by hy-

3.7 - I "*"- >-:- 'f bridization from the first cross, the new Gulliver band in A55-c9 cosegregated with the Nit- phenotype (Fig- ure 2A). For thesecond cross, a nit2-2 dstrain (B52), obtained by crossing A55-a5 and a wild-type strain (B26), was mated with a wild-type field isolate (S 1-D2) which is devoid of Gulliver elements. (FERRIS 1989; GROSS,RANUM andLEFEBVRE 1988).In all four te- 2.0 - trads analyzed from this cross, the new Gulliver ele- FIGURE1 .-Hybridization analysis of Gulliver elements in nit2 ment cosegregated with the Nit- phenotype (Figure mutant strains. DNA from wild type (lane 1) and isogenic nit2 2B). In contrast, in the third cross, between mutant mutant strains (lanes 2-9) was digested with HindIII, fractionated through 0.8% agarose, transferred to a nylon membrane and hy- A55-d8 and a wild-type strain (B26), thenew Gulliver bridized to a fragment corresponding to one end of the Gulliver band segregated independently from the nit2 muta- transposon (Figure 4A, probe 1). Lanes contain DNA (3.5 pg) from tion (1 PD and 2 NPD tetrads; data notshown). These strain A55 (lane 1). A55-c9 (lane 2), A55-a5 (lane 3), A55t8 (lane results indicated that the Gulliver dimorphisms in the 4). A55-el2 (lane 5). A55-h1 (lane 6). A55-h9 (lane 7), A55-d8 first two strains (A55-c9 and A55-a5) were linked to (lane 8) and A55-al4 (lane 9). DNA size markers are indicated in kilobase pairs (kb) throughout. the nit2 mutations in those strains and that the di- morphism in the third strain (A55-d8)was not associ- duced mutations in a number of organisms (SHAPIRO ated with the nit2 mutation in that strain. 1983). To determine whether the nit2 mutations iso- To determine whether thenit2 mutations in strains lated in this study were unstable, multiple cultures of A55-a5 and A55-c9 were caused by the insertion of each mutant strain were grown from single cells with- Gulliver intothe NIT2 locus, or whetherthe new out selection and spotted onto nitrate medium. More Gulliver elements in these strains were simply linked than half of the nit2 mutant strains (24 out of 40) to the nit2 mutations, Nit+ revertants were isolated. gave rise to Nit+ colonies. Thirteen of these unstable For each of the nit2 mutants, genomic DNA from mutants and one nonreverting mutant (A55-d8) were each of three independent revertant strains was ex- selected for further analysis. amined for the presence of the Gulliver dimorphism Gulliver elements associated with nit2 mutations: (Figure 3).In each case, loss of the dimorphic Gulliver To determine whether a Gulliver element was associ- band accompanied reversion to the wild-type pheno- ated with any of the nit2 mutations, DNA from 14 type. These results confirmed that the nit2 alleles in different or independently isolated nit2 mutant strains strains A55-c9 and A55-a5 were caused by the inser- was analyzed for the presence of a Gulliver dimorph- tion of a Gulliver element intoor near theNIT2 gene. ism. For this experiment, genomic DNA was cleaved Cloning of a nit2-associated transposon and the with HindIII, fractionated through agarose, blotted, wild-type NIT2 locus: To isolate sequences flanking and hybridized to a radiolabeled DNA fragment con- a nit2-associated transposable element, genomic DNA taining one endof a Gulliver element. Each fragment from mutant strainA55-c9 was digested with HindIII detected by the Gulliver end-specific probecorre- and fractionated on an agarose gel. DNA fragments sponds to a single Gulliver element(FERRIS 1989). from the region of the gel containing the 8-kb di- Compared with the isogenic wild-type strain (A55), morphic Gulliver fragment were purified, ligated into three of the nit2 mutantstrains contained a new a plasmid vector, and transformed into E. coli. Among Gulliver band (A55-c9, A55-a5 and A55-d8) and in 6,000 colonies that were screened, nine hybridized to one of thesestrains (A55-c9) aband was also lost the Gulliver probe. To identify plasmids that con- (Figure 1, lanes 2, 3 and8). The appearance of a new tained inserts of the appropriate size, plasmid DNA Gulliver band in these three strains might reflect trans- from each of the positive bacterial clones and genomic Chlamydomonas NIT2 Gene 74 1 A B

1 2 3 2 3 4 abcd abcd abcd abcd abcd abcd NIT2 +-+- - +-+ +- +- NIT2 - + - - ++ +- +- - ++' ++- -

4- 14.7 -

6.9 - . n - ..G .I, vm m N 4.7 -

FIGURE2.-Hybridi~ation analysis of tetrad progeny. (A) Cosegregation of a Gulliuer element with nit2-1. Progeny from tetrads (labeled 1-3) were obtained by crossing a nit2-l mt- strain (A55-c9) with a wild-type mt+ strain (B26). DNA samples (3.5 pg) were digested with HindllI, fractionated through 0.8% agarose, and hybridized with XB3, a clone containing one end of a Gulliuer element (FERRIS1989). The NIT2 phenotype, listed above each lane, was determined by testing growth on medium containing nitrate as the sole nitrogen source. The arrows show the position of the relevant Culliuer band. (B) Cosegregation of a Culliuer element with nit2-2. Tetrads were obtained by crossing a nit2-2 mt* strain (B52) with a wild-type,. mt-strain (SI-D2). The progeny were analyzed as in panel A except that a purified fragment was used as the Gulliver probe (probe 1, Figure 4).

DNA from strain A55-c9 were digested with HindIII, The cloned sequence encoded a functional NZT2 size-fractionated and hybridized with the Gulliver gene product: To test whether the cloned fragments probe. Inserts from six of the plasmids comigrated were able to complement a nit2 mutation, restriction with the 8-kb dimorphic band in A55-c9. A repre- fragments from the phage inserts were subcloned into sentative plasmid from this set, designated pMN47, a plasmid vector and introduced intoChlamydomonas was characterized further (Figure 4). cells by transformation. For this experiment,three To determine the relative position of Gulliver and plasmids were introduced into anit2-203, nu15 strain flanking sequences within the pMN47insert, the (D66 or D67) by vortexing cells and plasmid DNA in cloned region was subdivided into smaller fragments the presence of glass beads and polyethylene glycol which were used as probes for genomic hybridization (KINDLE 1990). The treated cells were spread onto analysis. The rightwardprobe (probe 1,Figure 4) medium containing nitrate as the sole nitrogen source hybridized exclusively to Gulliver elements (Figure l), and the number of Nit+ colonies was scored after 10 whereas the leftward probe (probe 2, Figure 4) hy- days (Figure 6A). In four control transformations, in bridized to a 6.2-kb HindIII fragment in the wild-type which plasmid DNAwas omitted, only one Nit+ colony strain(A55) andan 8-kbHindIII fragment in the was observed. When plasmid pMN6O or pMN68 was nit2-I strain. In tetrads from across between the two strains, the 6.2-kb HindIIIfragment cosegregated added, the total number of colonies increased to 37 with the Nit+ phenotype (Figure 4B). These results and 105, respectively. Addition ofplasmid pMN72 suggested that the leftward fragment from pMN47 resulted in five colonies. hybridized to the NIT2 locus. To isolate the complete Since nucleartransformation in Chlamydomonas NIT2 gene, the leftward fragment was used to screen occurs almost exclusively by integration of the trans- a phage X library containing DNA from a wild-type forming DNA into the genome by nonhomologous Chlamydomonas strain. Restriction analysis of DNA recombination(DIENER et al. 1990; KINDLE et al. from eight of the positively hybridizing phage indi- 1989; MITCHELL and KANG 1991),a transformant cated that the inserts were overlapping and spanned should contain at least one extra copy of the intro- a 27-kb region (Figure 5). duced gene. Putative transformants were tested for 742 R. A. Schnell and P. A. Lefebvre

" ASS 12345678 A

0 - Probe 2 Probe 1 8.6 - H 6.9 -

4.7 - B

2 3.7 - 1 3 v) a~mo) abcd abcdabcd NIT2 + - +- +- - +-+ +-+-

FIGUREJ.-Hybridi7ation analysis of Nit+ revertant strains. Ge- 8kb - nomic DNA from nit2 mutants and revertants was digested with Hind111 and hybridized to a Gulliver probe as described in the 6.2 kb - legend for Figure 1. Lanes contain DNA from nit2 strains A55-a5 (lane 1) and A55-c9 (lane 5) and from independently isolated Nit+ revertantsobtained from A55-a5 (lanes 2-4) and from A55-c9 FIGURE4.-The 8-kb transposon-tagged Hind111 fragment from (lanes 6-8). The arrows indicate the positions of the polymorphic A55-c9. (A) Restriction map of the pMN47 insert. The shaded Hindlll fragments associated with the Nit- phenotype of the nit2 areas indicate fragments that were used for hybridization probes mutant strains. (probe 1 and 2). The unshaded rectangle represents the portion of the presence of extra gene copies by digesting ge- the insert corresponding to the transposon. (B) The filter shown in Figure 2A was stripped and rehybridized with the 1.5-kb Hindlll- nomic DNAwith SstI and using afragment from Sal1 fragment from pMN47 (panel A, probe 2). within the transforming DNA as a probe. Transform- ants obtained with pMN68 or pMN6O contained one or more new bands comparedwith the untransformed parentstrain (Figure 6B, lanes 1-12). Incontrast, putative transformants obtained using pMN72 con- 141-1 I tained a single band and were indistinguishable from k 41-2 I the untransformed strain, suggesting that these colo- 147-3 I A41-4 , I nies arose by reversion of the nit2-203 mutation rather 141-5 than by transformation with pMN72 (Figure 6B,lanes 147-6 147-1 1 13-1 5). A 47-8 4 If the introduced gene rescues the nit2 mutation, n then the Nit+ phenotype should segregate with the FIGURE5.-Restriction map of wild-type NIT2 genomic clones. extra gene copy during meiosis. To test this predic- Lines beneath the map represent lambda inserts. The rectangle corresponds to probe 2 in Figure 4. The scale bar represents 1 kb. tion, a pMN68-transformed strain (Figure 6B,lane 2) was mated with a nit2 mutant strain(A35). In all four cloned NIT2 gene (probe B, Figure 7A). In the wild- tetrads examined,the extra genecopy segregated with type strain and in four of the mutant strains, the probe the Nit+ phenotype (Figure 6C). Together, these re- hybridized to a 6.2-kb fragment. However, in 10 of sults indicate that plasmids pMN68 and pMN60, but the mutant strains, the probehybridized to fragments not pMN72, were able to rescue the nit2-203 muta- of various lengths (Figure 7B and data not shown). tion. Therefore, 10 of the 14 mutant strains contained a Physicalmapping of nit2 mutations: Transfor- restriction fragment length polymorphism within the mation experiments showed that a 6.2-kb Hind111 6.2-kb HindIII region. The location of each of the 10 fragment was sufficient to complement a nit2 muta- polymorphisms was determined more precisely by di- tion. A reciprocal approach, to identify functionally gesting genomic DNA with both HindIII and SaZI, or important sequences, is to map mutations that disrupt with HindIII and KpnI, and hybridizing filters with a gene's function. For this experiment, DNA from 14 subfragmentsfrom the cloned region (probes A spontaneous nit2 mutant s rains was digested with through E were used to detect polymorphisms within HindIII and hybridized kith a fragment from the regions athrough e, respectively; Figure 7A). A Chlamydomonas NIT2 Gene 743

pMN6O 37

pMN68 105

pMN72 5 H no DNA 1

B

Lc F m pMN68 pMN6O pMN72 C

1 2 3 4 5 6 7 8 9 10 11 121314 15 1 2 3 4 I -+-++--++-+-++--I I I I

8.6 - 6.9 -

4.7 -

3.7 -

2.0 -

FIGURE&-The cloned wild-type NIT2 sequence rescues a nit2 mutation. (A) Mutant rescue by genetic transformation. Regions contained in plasmid subclones are indicated by lines beneath the restriction map. The scale bar represents 1 kb. The number of Nit+ colonies refers to the total number obtained in four trials. The line above the restriction map shows the 0.9-kb Sall-Pstl fragment used as a probe in panels B and C. (B) Hybridization analysis of transformants. Genomic DNA (2 pg) from the untransformed strain D66 (lane 1) or from putative transformants obtained with plasmid pMN68 (lanes 2-9). pMN6O (lanes 10-12) or pMN72 (lanes 13-1 5). was digested with SstI. fractionated through 0.8% agarose. transferred to a nylon membrane and hybridized with the probe indicated in panel A. (C) Cosegregation of a transgene and the Nit+ phenotype. Tetrads were obtained by crossing the pMN68-transformant shown in panel B, lane 2, and a nit2-203 strain (A35). The progeny from four tetrads were analyzed as described for panel B. Segregation of the NIT2 phenotype is indicated above each lane. change in the hybridization patternrelative to the ulation by ammonium: When ammonium, the prod- wild-type control indicated a mutation within the re- uct of nitrate assimilation, is available, each compo- gion detected by that probe. The results of this analy- nent of the nitrate assimilation pathway is repressed sis are summarized in Table 2. For example, DNA (FLORENCIO and VEGA 1983; GALVANet al. 199 1; insertions associated with nit2-4, nit2-2 and nit2-1 HERRERAet al. 1972). In the case of NITl, the only resided within region b, c and d, respectively. No component of the pathway whose expression has been restriction fragment length polymorphisms were ob- examined at the RNA level, ammonium stringently served within regions a or e in any of the mutants. represses accumulation of the transcript (FERN~NDEZ These results are consistent with the suggestion from et al. 1989). In addition, transcripts fromat least four transformation experiments that the NIT2 gene re- genes that are tightly linked to NITl, but whose sides within the 6.2-kb Hind111 fragment, although functions are unknown, are repressed by ammonium sequences important for the regulation of the NIT2 (QUESADA 1992). To ask whether ammonium also gene may extend beyond this region. affects expression of the NIT2 gene, we compared the Identification of the NIT2 transcript and its reg- abundance of the NIT2 transcript in cells grown in 744 R. A. Schnell and P. A. Lefebvre A TABLE 2 Summary of new nit2 mutations AB c DE Probe: H - IsolateaAllele ~ype mutation'LocationC - of nit2-4 A54-a2 Insertion (TI) b nit2-12 A5442 ND nit2-6 A54-flO lnsertion (Tl) C nit2-7 A54-g18 d (T2)lnsertion nit2-13 A54-g21 ND ab c d e nit,?-I1 A54-g24 b Rearrangement nit2-2 A55-a5 Insertion (G) C nit2-14 A55-al4 ND nit2-I0 A55c8 b Rearrangement nit2-1 A55c9 Insertion (G) d nit2-3 A5548 ND nit2-5 A55-12 Insertion (Tl) b nit,?-8 A55-h 1 Insertion (T2) C B nit2-9 A55-h9 (T3)lnsertion C 1 23456789 These 14 different nif2 mutants were isolated from eight independent cultures, designated by the parental strain name (A54 or A55) and the culture name (a, b, etc.). ' Polymorphismscaused by DNA insertions are classified accord- ing to the identity of the inserted sequence: G denotes a Culliuer element and TI, T2, and T3 denote three unrelated sequences. 14.7 - The abbreviation ND indicates that no DNA polymorphisms were W detected. 8.6 - Regions that contain DNA polymorphisms (b, c, and d) are a- defined in Figure 7A. 6.9 - W 0 wild-type cells, and in derepressed nit2-Z mutant cells,

4.7 - all three transcripts were undetectable(Figure 8A, lanes 1 and 3). As a control for equal loading, the

3.7 - filter was rehybridized with an a-tubulin probe (Fig- ure 8B). These results confirm that ammonium re- pressed the expression of NITl and of two neighbor- ing genes, that transcripts from all three genes were detectable within 1 hr after removal of ammonium, and that accumulation of these transcripts required 2.0 - the wild-type NIT2 gene. FIGURE7.-Localization of nit2 mutations. (A) Genomic restric- To determine whether theNIT2 gene was regulated tion map of the wild-type NIT2 locus. DNA regions that were tested by ammonium,the filter was rehybridized using a for thepresence of restriction length polymorphisms are designated fragment from the cloned NIT2 gene as aprobe by small letters below the restriction map (a through e). Fragments (probe C, Figure 7A). Thisprobe detected a 6-kb used as probes are designated by capital letters above the restriction RNA in derepressed wild-typecells and ashorter, map (A through E). Polymorphisms in four mutants were located 2.45-kb RNA in mutant cells (Figure 8C, lanes within region b (A54-a2, A54-g24, A55c8 and A55-el2);four were nit2-1 in region c (A55-a5, A54-fl0, A55-hl and A55-h9); and two resided 2 and 3). The altered length of the RNA in the nit2 in region d (A55c9 and A54-g18). (B) Hybridization analysis of mutant confirmed thatthe 6-kb RNA detected in wild- Hindlll-digested DNA from strain A55 (lane 1) and from nit2 type cells is the NIT2 transcript. In the presence of mutant strains A55c9 (lane 2), A55-a5 (lane 3), A55c8 (lane 4). ammonium, the level of the NIT2 transcript was dra- A55-el2 (lane 5). A55-hl (lane 6). A55-h9 (lane 7). A55-d8 (lane 1). 8) and A55-aI4 (lane 9). For this experiment, the filter described matically reduced(Figure 8B, lane Therefore, in the legend to Figure 1 was rehybridized with probe B, shown in accumulation of the NIT2 transcript was repressed by panel A of this figure. ammonium. the presence of ammonium and in cells transferred to DISCUSSION nitrogen-free medium for one hour prior to harvest- ing. To test the effectiveness of the repression and Isolation of the NIT2 gene from C. reinhardtii dem- derepression regimens, a fragment that encompasses onstrates the first use of transposon tagging in this the NITl locus was used as a probe for RNA hybrid- organism. In principle, transposon tagging provides a ization analysis. In derepressed wild-type cells, the means for isolating any locus for which a transposon control probe detected the NITl transcript (4 kb) plus insertion allele can be identified. However, if Gulliver two shorter transcripts from genes that flank NITl integrates preferentially into particular sequences or (Figure 8A, lane 2). Whereas in ammonium-grown if the rate of transposition is influenced by environ- Chlamydomonas NIT2 Gene 745 A C

NIT2 nit2 NIT2 nit2 NIT2 nit2 +N -N -N +N -N -N +N -N -N 12 3 12 3 12 3

FIGURE&--RNA hybridilation analysis of the NIT2 9.49 - transcript. RNA from wild-type NIT2 strain A55 (lanes 7.46 - 1 and 2) or the isogenic nit2-I strain A55-c9 (lane 3). was isolated from cells grown in the presence of ammonium 4.40 - (+N) or from cells transferred to ammonium-free me- dium for one hour beforeharvesting (-N). In each lane, 10 rg poly(A)+ RNA were fractionated through a 1.2%

2.37 agarose gel containing formaldehyde, transferred to a - nylon membrane and hybridized with (A) the 13.5-kb SphI fragment that contains the NITZ gene from plasmid pMN24 (KINDLEet al. 1989); (B) an a,-tubulin cDNA 1.35 - (1x10-2) (SILFLOWet al. 1985); (C) the 1-kb Pstl-KpnI fragment corresponding to probe C in Figure 7A.

N IT1 Tubulin N IT2

mental or genetic factors, then this transposon may tritereductase andnitrite uptake levels (GALVAN, not be useful for tagging certaingenes. One approach CARDENAS and FERNANDEZ1992; NICHOLS,SHEHATA forovercoming this limitation would beto isolate and SYRETT 1978;E. FERNANDEZ,personal commu- probes for additional transposable element families, nication). In the case of nitrate reductase, regulation thereby increasing the numberof transposon-induced by NIT2 occurs at the RNA level, since accumulation mutations that can be detected. Strikingly, most of of the nitrate reductase transcript requires the pres- the spontaneous nit2 mutations isolated in this study ence of a wild-type NIT2 gene. Whether NIT2 acti- contained insertions or rearrangements within the vates other components of the pathway at the RNA NIT2 gene. Six of the mutations were caused by the level is not known since those genes have not been insertion of sequences that share no homology with identified. However, at least four genes of unknown Gulliver or TOCI.Characterization of these sequences function, noted by their proximity to the NITl gene, has so far led to the identification of two new trans- require NIT2 for their expression (QUESADA1992). posable element familes, TcrI and Tcr2 (our unpub- The 6-kb NIT2 transcript could be unequivocally lished results). Future studies will resolve whether identified because in a mutant strain that contained a transposon tagging can be used routinely for isolating transposon inserted within the NIT2 locus, this tran- genes in Chlamydomonas. script was replaced by a shorter RNA. Furthermore, Most significantly, isolation of the NIT2 gene pro- the accumulation of this mutant nit2 transcript sug- vides a means of determining the productof this gene. gests that theNIT2 gene does not require the product The properties of nit2 mutants suggest that theNIT2 of its own gene to be expressed. Comparison of the gene encodes apositive regulator of the nitrate assim- abundance of the NIT2 transcript in cells under am- ilation pathway. Specifically, nit2 mutations are reces- monium-repressed and derepressed conditions re- sive and prevent the utilization of both nitrate and vealed that the NIT2 gene was repressed by ammo- nitrite. The role of NIT2 appears to be specific for nium. the nitrate assimilation pathway, since nit2 mutants Previous studies have shown that every gene or grew as well as wild-type strains in the presence of biochemical activity that has been found to be acti- other nitrogen sources, including acetamide, adenine, vated by NIT2 is also repressed by ammonium. The ammonium, arginine, asparagine, glutamine, guanine, observation thatthe NIT2 gene itself is subject to ornithine and urea (data not shown). Consistent with metabolite repression suggests that ammonium might their inability to utilize either nitrate or nitrite, nit2 repress the expression of NITl and other NIT2-de- mutants contain little if any nitrate reductase or ni- pendent genesby controlling the amount of the NIT2 trate permease activity and significantly reduced ni- gene product. Assuming that regulation occurs at the 746 R. A. Schnell and P. A. Lefebvre

level of RNA synthesis rather than RNA stability, it nitrate reductase-deficient mutants in Chlamydomonas reinhar- should be possible to test this model by introducing a dii. Curr. Genet. 8: 635-640. NIT2 gene placed under the control of a constitutive FERNANDEZ, E.,and R. F. MATAGNE,1986 In vivo complementa- tion analysis of nitrate reductase-deficient mutants in Chlam- promoter. The model predicts that expression of the ydomonas reinhardtii. Curr. Genet. 10 397-403. NIT2 gene in the presence of ammonium would lead FERNANDEZ,E., R. SCHNELL, L.P. W. RANUM,S. C. HUSSEY,C. D. to constitutive expression of NIT2-dependent genes, SILFLOWand P. A. LEFEBVRE,1989 Isolation and character- including NITl. Alternatively, if ammonium represses ization of the nitrate reductase structural gene of Chlamydo- both NIT2 and NIT2-dependent genes directly, then monas reinhardtii. Proc. Natl. Acad. Sci. USA 86: 644945453, FERRIS,P. J., 1989 Characterizationof a Chlamydomonas tran- constitutive expression of the NIT2 gene would not sposon, Gulliver, resembling those in higher plants. Genetics influence the ability of ammonium to repress NITl 122: 363-377. and otherNIT2-dependent genes. FI~ORENCIO,F. J., and J. M. VEGA, 1983 Utilizationof nitrate, nitriteand ammonium by Chlamydomonasreinhardii. Photo- We thank PATRICK FERRIS and URSULA GOODENOUGH for pro- production of ammonium. Planta158: 288-293. viding the Gulliver clone, STEVEJAMES and EMILIOFERN~NDEZ for GALV~N, A,, J. C~RDENAS andE. FERN~NDEZ, 1992 Nitratere- Chlamydomonasstrains and CAROLYN SILFLOWfor the tubulin ductase regulates expression of nitrite uptake and nitrite re- cDNAplasmid. We also thank EMILIO FERN~NDEZ,LESLIE ductase activities in Chlamydomonas reinhardtii. Plant Physiol. MOUNKES, ROBIN WRIGHT and DONGHONGZHANG for critical read- 98: 422-426. ing of the manuscript. This workwas supported by grants toP.A.L. GALVAN,A., F. CORDOBA,J. 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