Chlamydomonas reinhardtii isa haploid green alga with many advantages for genetic ... bridized in a solution containing 6x SSC (lx SSC = 0.15 M. NaCl/0.015 M ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 6449-6453, September 1989 Biochemistry
Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii (restriction fragment length polymorphisms/regulatory mutant/transformation)
EMILIO FERNANDEZ*, ROGENE SCHNELL, LAURA P. W. RANUM, SUSANNE C. HUSSEY, CAROLYN D. SILFLOW, AND PAUL A. LEFEBVREt Department of Genetics and Cell Biology and Plant Molecular Genetics Institute, University of Minnesota, Saint Paul, MN 55108
Communicated by David J. L. Luck, April 10, 1989
nium has been shown to play a central role in the repression of NR (3). The NR structural locus and molybdopterin cofactor loci have also been defined in higher plants, but regulatory genes have not been identified by mutation (1, 4, 8, 18, 19). In this paper we describe the isolation of the Chlamydomonas NR gene and analyze its expression in wild-type cells and in the regulatory mutant nit-2. The identity of the cloned gene was tested by examining its genetic linkage to the nit-i locus by using restriction fragment length polymorphism (RFLP) mapping and by obtaining a partial DNA sequence of the gene A The cloned NR gene was also shown to complement a nit-i mutation when introduced into cells by transformation.
ABSTRACT The nitrate reductase structural gene of Chlamydomonas reinhadt has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-l. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binig domains of tobacco and Arabidopsis thauliana nitrate reductases and human NADH cytochrome bs reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-i mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, sign tly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.
MATERIALS AND METHODS Strains and Growth Conditions. C. reinhardtii wild-type 21 gr mt+ and mutant strains 203 (nit-2 mt+), 305 (nit-i mt-), and 305d (nit-i mt+) (16, 20) were grown in liquid TAP medium (21). The RFLP mapping crosses and scoring of the mutant phenotypes are described in detail by Ranum et al. (15). The segregation of the two nit genetic markers (nit-i and nit-2) in the tetrads used for RFLP experiments was scored by using the recent observation that nit-2 mutants cannot grow on low levels (2 mM) of nitrite as sole nitrogen source, whereas wild type and nit-i mutants grow well on 2 mM potassium nitrite (E.F., unpublished observations). All tetrad progeny were tested for their ability to grow on media containing ammonium, nitrate, or nitrite as sole nitrogen
Nitrate is the major compound for nitrogen acquisition in nature. The first enzyme of the nitrate assimilatory pathway, nitrate reductase (NR), catalyzes the reduction of nitrate to nitrite and is subject to tight control at the levels of enzyme activity, synthesis, and degradation (1-3). In photosynthetic eukaryotes, NR is an enzyme complex of 220-480 kDa containing FAD, b-type heme, and molybdopterin cofactor as prosthetic groups that drive electrons from NAD(P)H to nitrate (1-3). The enzyme of higher plants and some green algae is a dimer of identical subunits (1, 3-5), although two kinds of subunits were reported for the NR of Chlamydomonas (6, 7). The isolation and characterization of nitrate reductase-deficient mutants has greatly contributed to the understanding of the structure and regulation of NR (1, 3, 8). The gene has been cloned from several higher plants and fungi (9-13), and its expression has been shown to be induced by nitrate. Chlamydomonas reinhardtii is a haploid green alga with many advantages for genetic and molecular studies, including the availability of tetrad analysis, a relatively well-marked genetic map with both molecular and genetic markers (14, 15), and a genome of low molecular complexity. Several loci are involved in the synthesis of an active NR complex in Chlamydomonas: nit-] encodes the NR apoprotein(s), nit-2 appears to be a regulatory gene, and the products of four genes (nit-3, 4, -5, and -6) are needed to synthesize the molybdopterin cofactor (16,17). In Chlamydomonas, ammo-
source. Derepression of NR. Cells were grown at 250C under continuous light in 10 mM ammonium chloride minimal medium (21) and were bubbled with air enriched with 2-5% CO2. After growth to a density of 2-5 x 106 cells per ml, the cells were collected by centrifugation (3500 x g for 10 min), washed twice with 75 mM potassium phosphate buffer (pH 7.5), and then transferred to either minimal medium with 10 mM ammonium chloride (repression conditions) or minimal medium with 4 mM potassium nitrate (derepression conditions). After 3 hr, cells were collected by centrifugation as described above and processed immediately for RNA extrac-
tion (see below). Screening of the Genomic Library. Approximately six genome equivalents (45,000 plaques) were screened from an amplified C. reinhardtii 21gr genomic DNA library conAbbreviations: NR, nitrate reductase; RFLP, restriction fragment
length polymorphism. *Permanent address: Department of Biochemistry, Faculty of Sciences, University of C6rdoba, C6rdoba-14071, Spain. tTo whom reprint requests should be addressed. Vrhe sequences reported in this paper have been deposited in the GenBank data base [accession nos. M26074 (fragment A) and M26075 (fragment B)].
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6449
6450
Biochemistry: Ferndndez et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
structed in bacteriophage A EMBL4 by J. J. Youngblom (22). Nitrocellulose plaque lifts (23) were prehybridized and hybridized in a solution containing 6x SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate), 5x Denhardt's solution (lx Denhardt's = 0.02% Ficoll 6000/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 25 mM sodium phosphate (pH 7.0), 100 ,ug of denatured calf thymus DNA per ml, and 1% SDS at 60'C. The radioactive probe (24) for screening the plaque lifts (about 1 x 109 pACi/jug of DNA; 1 Ci = 37 GBq) consisted of the insert from plasmid bNRp1O containing a partial barley NR cDNA sequence (9) (kindly provided by Howard Goodman, Department of Molecular Biology, Massachusetts General Hospital, Boston). The filters were washed three times with 0.8x SSC/0.2% SDS at 550C. RNA and DNA Isolation from C. reinhard and Hybridization Analyses. Techniques for genomic DNA isolation and Southern transfer analysis were as described by Ranum et al. (15). Techniques for isolation and gel fractionation of poly(A)+ RNA and for hybridizations to nick-translated probes were described earlier (25). RNA was transferred to reusable nylon membranes (Zeta-Probe, Bio-Rad) according to the instructions of the manufacturer. DNA Sequencing. DNA fiagments were subcloned into pUC119 (see Fig. 1), and single-stranded phage DNA was prepared (26). The DNA sequence was obtained by the dideoxynucleotide chain-termination method using Sequenase (United States Biochemical) according to the instructions of the manufacturer. Sequences of both DNA strands were obtained and analyzed by using IntelliGenetics programs (through the University of Minnesota Molecular Biology Computing Facility). Predicted amino acid sequence was compared with sequences from the GenBank database (Release 55). Transformation of Chiamydomonas. The plasmid used for transformation, pMN24, consisted of the intact nit-i gene from wild type (21gr) on a 14.5-kilobase (kb) Sal I-Bgl II genomic fragment in pUC119 (see Fig. 1). The pMN24 plasmid was introduced into a nit-la mutant strain [nit-i (allele 305) CW15 mt] by using a procedure described previously for fusing Chlamydomonas protoplasts (27). The host strain contained a cell-wall deficiency (CW15), obviating the need to prepare protoplasts (28, 29). For transformation, the host strain was grown in TAP medium to a density of 6 x 106 cells per ml. A 10-ml portion of the culture was harvested and washed once in Sager and Granick minimal medium (30) without ammonium, and the pelleted cells were gently mixed for 3 min in the presence of 0.1 ml of PEG solution [30o (wt/vol) polyethylene glycol 8000, 50 mM CaCl2, 10 mM Hepes (pH 8.0 with NaOH)] plus 8 ,ug of pMN24 DNA. The cells were immediately spread
onto minimal medium containing nitrate as sole nitrogen source (4 mM KNO3). After 10 days, a single nit' colony was isolated and analyzed further.
RESULTS Isolation of the nit-I Gene. A Chlamydomonas genomic DNA library constructed in the A bacteriophage EMBL4 was screened with the radioactively labeled barley NR cDNA insert from plasmid bNRplO. Four positive plaques were identified among approximately six genome equivalents. Three clones (B2a, B4a, and B6a) contained an identical insert of 13 kb. The fourth clone (B7a), with an insert of 15.5 kb, contained two Sma I restriction fragments in common with the first three clones. The restriction map deduced for the genomic region covered by these clones (about 24 kb) is shown in Fig. 1. Hybridization analysis of genomic DNA digested with a variety of different restriction enzymes (Pst I, Sst I, HindIII, Sma I, or Sal I) showed that the cloned region is present in the genome in only one copy and that the restriction map is colinear with the map of the cloned fragment. Only the 1.8-kb Sma I fragment (B6a-2) hybridized strongly with the barley NR probe (bNRplO) under the same conditions of stringency used for screening the library. Subsequent digestions of the B6a-2 fragment delimited the region of homology to the rightmost 300 base pairs of the fragment (Fig. 1). RFLP Mapping. That the isolated clones contained the NR structural gene was first tested by RFLP mapping using tetrad progeny from crosses between the interfertile strains C. reinhardtii and Chlamydomonas smithii. RFLPs for the parental strains were identified by using the Sma I fragment B6a-1 (Fig. 1) as a probe. Tetrad progeny were scored for segregation of the RFLP associated with the B6a-1 sequence and for segregation of nit-i as well as another marker from linkage group IX, sr-i (14). Fig. 2 shows the segregation of the polymorphic bands for the parental strains and two complete tetrads. Close linkage of nit-i and the B6a-1 sequence was indicated by cosegregation of the C. reinhardtii restriction fragment (1.0 kb) with the nit-i mutant allele and cosegregation ofthe C. smithii fragment (1.6 kb) with the nit-i I allele in 67 progeny representing 22 tetrads (10 of which were complete). No exceptions to linkage of the B6a-1 fragment to the nit-i locus were seen. Other faint bands, presumably representing sequences cross-hybridizing with the NR probe, were seen in the genomic DNA analysis, but these bands segregated independently of the nit-i marker. The segregation of the sr-i mutation, which confers streptomycin resistance, showed a clear linkage with nit-i and B6a-1, which
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to show other restriction sites, as well as the two fragments A and B, which have been sequenced in the regions indicated by the arrows. The extent of the transcribed region of the nit-i gene, as determined by RNA hybridization analysis, is shown by the arrow under the restriction map. The dashed portion of the line indicates the maximum boundaries of the RNA transcript. The direction of transcription was determined from the predicted amino acid sequence as compared to the predicted amino acid sequences of nitrate reductases from tobacco (11) and Arabidopsis (13). B, BamHI; Bg, Bgl II; E, EcoRI; H, HindIII; K, Kpn I; P, Pst I; S, Sst I; Sa, Sal I; Sm, Sma I; X, Xba I.
Biochemistry: FernAndez et aL nit-i RFLP
Proc. Nati. Acad. Sci. USA 86 (1989)
6451
FRAGMENT A:
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30 CCG GGT GGC GCC GAG TCC ATC CTG ATC ACT GCG GGC GCG GAC GCC G G A E S I L I T A G A D A P
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FIG. 3. Partial nucleotide sequence and predicted amino acid sequence of the Sma I fragment 2 (B6a-2) from the NR clone B6a. Fragments A and B were sequenced in both directions as indicated in Fig. 1. The nucleotides indicated by the boxes correspond to the predicted sequences for the 5' ends of intervening sequences in Chlamydomonas by comparison with previously sequenced genes (22, 31, 32). The arrowheads indicate the predicted splicing sites.
FIG. 2. Southern hybridization analysis of genomic DNA isolated
from tetrad progeny of a cross between C. reinhardtii and the polymorphic strain C. smithii. DNA was isolated from C. reinhardtii (lane R) and C. smithii (lane S) and from all four progeny from two tetrads (marked by the heavy lines) and was digested with Alu I. The filter was probed with the Sma I fragment B6a-1. The nit phenotype
For both fragment A and fragment B, the similarity of the predicted amino acid sequences to higher plant NR sequences ends abruptly (after amino acid 66 in Fig. 4, fragment A and after amino acid 211 in Fig. 4, fragment B). The nucleotides immediately following the region of homology (G/GTGGGT for fragment A; G/GTGAGC for fragment B; see Fig. 3) are similar to the sequence at the 5' end of introns in other Chlamydomonas genes (22, 31, 32). According to the sequence data, the direction oftranscription of the nit-i gene (5' to 3') is from left to right in the diagram shown in Fig. 1. The Cloned nit-1 Gene Complements a nit-i Mutation. To test the feasibility ofusing the cloned NR gene as a selectable marker for developing procedures for transforming Chlamydomonas, a cell wall-less mutant strain (CW15) (28) containing a nit-i mutation was transformed with plasmid pMN24, which contained the full-length wild-type gene from the nit-i locus on a 14.5-kb Sal I-Bgl II genomic fragment in pUC119. After 10 days on selective medium, a single nit' colony was isolated. DNA from this transformed strain, designated TH6, was analyzed to determine whether the nit' phenotype was due to complementation ofthe nit-i defect by the cloned gene or to reversion of the nit-i mutation. Southern hybridization analysis of DNA from the transformant indicated that a single copy of the exogenously added DNA was integrated into the genome of the transformed cells. As shown in Fig. 5a, DNA from the host strain (nit-1-305) digested with Pvu II contained four fragments that hybridized to pMN24 (Fig. 5a, lane 2). DNA from the nit' isolate (TH6) contained the same four fragments plus two new fragments (9.5 kb and 5.4 kb) (Fig. 5a, lane 4). The vector
of each of the progeny is indicated (+, growth on nitrate medium; -, no growth on nitrate medium). R, restriction fragment from C. reinhardtii; S, restriction fragment from C. smithii. Sizes of standard markers (in kb) are indicated.
supports the localization of the B6a sequence to linkage group LX. Partl Sequence Analysis of nit-i. To confirm that the B6a clone was NR, we sequenced the ends of the 1.8-kb Sma I fragment B6a-2 (labeled A and B in Fig. 1). One major open reading frame was predicted from each sequence (Fig. 3). A comparison of the predicted amino acid sequences from the B6a-2 clone with the predicted amino acid sequences of NRs from tobacco (11) and Arabidopsis (13) revealed several regions of sequence similarity. In fragment A, the Chlamydomonas sequence has the same amino acids as the two higher plant NRs at 16 of 23 positions that correspond to the heme-binding domain (11, 13) (Fig. 4, fragment A). This domain also shows clear similarity to different b5-type hemoproteins such as yeast flavocytochrome b2 (33) and horse microsomal cytochrome b5 (34) (Fig. 4, fragment A). In fiagment B, there is a high degree of conservation at the amino acid level with the sequences for the FAD/ NADH-binding domain of NR from Arabidopsis (13) (Fig. 4, fragment B). Surprisingly, although the Chlamydomonas sequence is identical to Arabidopsis NR in 16 of 31 amino acids, the same region of the Chlamydomonas NR sequence is identical to human cytochrome b5 reductase at 21 of 31 amino acids (Fig. 4, fragment B) (35). FRAGMENT A : 45
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FIG. 4. Predicted amino acid sequence of Chlamydomonas NR (CHLNR) in the hemebinding domain (fragment A) and FAD/NADH domain (fragment B). Identity with Chlamydomonas NR is shown in boxes. Fragments A and B are those described in Fig. 1. The residues for the heme-binding domain are numbered as described for horse microsomal cytochrome b5 (CBH05) (27), and those for the FAD/NADH domain are numbered as described for human cytochrome b5 reductase (RDHUB5) (28). FLAVB2, yeast flavocytochrome b2 (26); TOBNR, NR from tobacco (11); ATNR, NR from Arabidopsis thaliana (13).
Biochemistry: Fernandez et al.
6452
Proc. Natl. Acad. Sci. USA 86
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FIG. 5. DNA hybridization analysis of the TH6 transformant. (a) DNA from either the nit-1-305 mutant (lanes 1 and 2) or the transformant TH6 (lanes 3 and 4) was analyzed by Southern hybridization using the labeled plasmid pMN24 as probe. Lanes: 1 and 3, uncut DNA (5 ,ug); 2 and 4, DNA digested with Pvu 11 (5 Ag). (b) The transformant (TH6) was backcrossed to a nit-1-305 strain, and the phenotypes of the progeny were scored (+, growth on nitrate medium; -, no growth on nitrate medium). DNA was isolated from the members of one complete tetrad (lanes a-d on the left) and from seven surviving members of an octad (lanes a-g on the right) and was digested with Pvu 11 (5 ,ug per lane).
alone (pUC119) hybridized only to the 9.5-kb fragment from TH6 (data not shown). When undigested DNA was analyzed (Fig. Sa, lanes 1 and 3), pMN24 hybridized only to sequences that migrated in the very high molecular weight chromosomal DNA region. These results indicate that pMN24-derived sequences are present in TH6 in approximately one copy per cell and that they are probably integrated into the genome. To test whether these plasmid-derived sequences conferred the nit+ phenotype of TH6, the putative transformant was crossed with a nit-i mt strain. Among the progeny, the 9.5- and 5.4-kb fragments were present in each of the nit' segregants and absent from the nit- segregants (Fig. Sb), suggesting that the nit+ phenotype of TH6 is due to the integrated DNA sequences from the pMN24 plasmid. The genetic location of the integrated sequences relative to the nit-i locus was examined by crossing the transformant with strains containing both pf-13 and nit-l. Because pf-13 is tightly linked to nit-i (14), linkage of the nit+ phenotype to pf-13 would indicate linkage to nit-l. Among 79 randomly chosen progeny from 79 different tetrads, 46 showed recombination between the nit+ genetic marker and pf-13. Thus the nit+ marker is unlinked to the nit-i locus, confirming that the nit+ phenotype of the transformant was not caused by reversion of the nit-i mutation. Together these results show that the cloned nit-i gene along with a portion of the plasmid vector integrated nonhomologously into the genome of the recipient strain and that the cloned nit-i gene is able to complement a mutation in that gene. Expression of the nit-i Gene. We used the cloned Chlamydomonas NR gene to determine whether the derepression of NR enzyme activity seen after removal of ammonium from the medium is accompanied by increased levels of NR mRNA. Poly(A)+ RNA was isolated from both ammoniumgrown and nitrate-derepressed wild-type cells and was ana-
(1989)
lyzed by using RNA gel blots with the B6a-2 subclone as a probe (Fig. 6). A single 3.4-kb RNA species was observed in RNA from cells derepressed for NR expression, with no evidence of the transcript in RNA from ammonium-grown cells. The Sma I fragments 5, 3, and 2 from clone B6a and Sma I-BamHI and BamHI-EcoRI fragments within B7a-7 (Fig. 1) hybridized with the same ammonium-repressible transcript of 3.4 kb in RNA blots. Fragments B6a-1 and B6a-4 and the EcoRI-Bgl II fragment within B7a-7 did not hybridize with the putative NR transcript. Taken together, these observations indicate that the nit-i gene covers a region of 5-8 kb of the genome. Expression of the nit-i Transcript in a nit-2 Mutant. The wild type nit-2 gene product is required for the expression of NR enzyme activity and has been postulated to encode a positive regulator of the nit-i gene (37). To determine whether the nit-2 gene affects the NR transcript level, the cloned nit-i gene was used to compare the levels of the nit-i transcript in the isogenic wild-type (21gr) strain and the nit-2 (allele 203) mutant strain. The ammonium-repressible 3.4-kb transcript was barely detectable in poly(A)+ RNA from the nit-2 mutant (Fig. 6). In some experiments, a minor transcript slightly smaller than 3.4 kb (and not repressed by ammonium) was seen after a long exposure. As a control for equal loading of poly(A)+ RNA, the probe was removed and the filter was rehybridized with a cDNA probe of Chlamydomonas atubulin. The control probe hybridized with equal intensity to the a-tubulin transcripts in each lane (Fig. 6).
DISCUSSION The results presented here indicate that the C. reinhardtii nit-i gene has been isolated and is present in only one copy in the genome. Genomic DNA digested with any one of several different restriction endonucleases produced a single band when using the NR subclone B6a-1 as a probe. RFLP analysis showed that the cloned sequence cosegregated in genetic crosses with the nit-i locus. A comparison of the Chlamydomonas amino acid sequence derived from partial DNA sequence analysis with the published amino acid sequence of NR from either tobacco or WiId-type A
N
A
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A N A
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FIG. 6. RNA hybridization analysis of nit-i gene expression in wild-type (21gr) and mutant nit-2 (allele 203) cells. Equal amounts of poly(A)+ RNA (3 gg) isolated from ammonium-grown cells (A) or cells derepressed with nitrate for 3 hr (N) were analyzed by RNA hybridization analysis using the B6a-2 fragment as probe. The same filter, stripped and reprobed with the Chlamydomonas a-tubulin clone 10-2 (36) as a probe, is shown in the right two lanes of each set.
Biochemistry: FernAndez et al. Arabidopsis revealed significant similarities. Specifically, the 5' and 3' ends of the B6a-2 fragment correspond to the heme-binding and the FAD/NADH-binding domains of NR, respectively. Furthermore, the domains within the Chlamydomonas gene are positioned relative to one another as they are in the plant genes. It is notable that the similarity between the Chlamydomonas NR sequence and either of the higher plant sequences is not as great as the similarity between the higher plant sequences. For the FAD/NADH-binding domain, the Chlamydomonas sequence is even more similar to human cytochrome b5 reductase than to Arabidopsis NR. These observations illustrate the extent of divergence between the NR genes of Chlamydomonas and dicots, consistent with the observation that the Chlamydomonas nit-i gene did not hybridize with the squash NR cDNA clone pCmc-1 (ref. 10; E.F., unpublished observations). The cloned NR gene was used to demonstrate directly that derepression of NR activity when cells are washed from ammonium-containing to nitrate-containing medium is accompanied by an accumulation of the NR transcript. Cloned fragments of the NR gene hybridized to a transcript of 3.4 kb in poly(A)+ RNA isolated from nitrate-derepressed cells. This transcript was not present in RNA isolated from ammonium-grown cells. This mRNA is similar in size to those reported for NR from higher plants and fungi (3.2-3.5 kb) (9-13). Chlamydomonas nit-i mutants belong to two different complementation groups and the enzyme has been proposed to have two different protein subunits of about 45 and 67 kDa (3, 6). Since only one transcript (of 3.4 kb) has been detected, the possible existence of two smaller subunits may be accounted for by either a specific cleavage (maturation) of a native apoprotein into two functional smaller subunits or a nonspecific proteolysis of the high molecular weight subunits during the preparation of protein extracts giving rise to two moieties carrying partial functions ofthe enzyme. Our results indicate that there is a single gene for the NR apoprotein(s) and that the complementation between mutants belonging to the two complementation groups nit-l a and nit-lb represents intragenic complementation. Further studies are required to determine the size of the functional Chlamydomonas NR subunits and to explain the mechanism for the efficient in vitro and in vivo complementation (6, 37) that occurs between various nit-i alleles. The nit-2 gene of Chlamydomonas is the only biochemically and genetically characterized regulatory gene for NR expression in photosynthetic eukaryotes (reviewed in ref. 3). nit-2 mutant alleles are codominant in heterozygous diploids (37), show leaky growth on nitrate media, and have almost undetectable NR levels (3). The analysis of the expression of the NR mRNA in the nit-2 mutant showed that under conditions of nitrate derepression the NR message was present in greatly reduced amounts, indicating that the wildtype product of the nit-2 gene is involved in the control ofNR mRNA levels. The cloned NR gene was shown to be a useful selectable marker for developing transformation techniques. We have since, in collaboration with Karen Kindle of Cornell University, isolated a large number of transformants by using the particle gun technique (38) to introduce the cloned DNA, confirming the usefulness of NR as a selectable marker for transformation experiments (K. Kindle, R.S., E.F., and P.A.L., unpublished results). We would like to thank the following individuals for their help and advice: P. Savereide, J. Larkin, J. Carpenter, F. Galvan, J. Cardenas, and S. Christensen. H. Goodman and N. Crawford are gratefully acknowledged for providing
cDNA
clones. E.F. thanks the Ful-
bright-Ministry of Education and Science (Spain) program for fellowship support. This work was funded by grants from the National
Proc. Natl. Acad. Sci. USA 86 (1989)
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