Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis of the kinetics and metal specificity of its copper-responsive expression.

We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt c6 transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262, 9062-9067) revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intron/exon boundaries. Primer extension and S1 nuclease protection analyses identified the 5' border of the Cyt c6 mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt c6 mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55, 705-717), it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo.


Isolation and Structural Characterization of the Chlamydomonas reinhardtii Gene for Cytochrome c6
ANALYSIS OF THE KINETICS AND METAL SPECIFICITY OF ITS COPPER-RESPONSIVE EXPRESSION* (Received for publication, November 21, 1990) Kent L. Hill,Hong Hua Lis,Jennifer Singer,and Sabeeha Merchant5 From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90024 We have isolated a 5-kilobase pair fragment of genomic DNA containing the entire coding region for the Chlamydomonas reinhardtii gene encoding the copper-repressible Cyt c6. A region comprising 2.6 kilobase pairs contains the entire transcribed region plus 852 nucleotides upstream of the Cyt CS transcription start site and 495 nucleotides downstream of the conserved C. reinhardtii polyadenylation signal. Comparison of the genomic sequence with the cDNA sequence (Merchant, S., and Bogorad, L. (1987) J. Biol. Chem. 262,[9062][9063][9064][9065][9066][9067] revealed that the coding region is interrupted by two introns, each of which is flanked by C. reinhardtii consensus intronlexon boundaries.
Primer extension and S 1 nuclease protection analyses identified the 5' border of the Cyt CS mRNA at approximately 79 base pairs upstream from the initiator methionine. Analysis of the 5' upstream region reveals no significant similarity to sequences found in upstream regions of other copper-regulated genes. Time-course studies indicate that 1) the mature Cyt cg mRNA has a half-life of approximately 45-60 min and is completely lost within 4 h, and 2) the primary, unspliced transcript has a half-life of approximately 10 min and is completely lost within 30 min after the addition of copper ions to copper-depleted cells. These results indicate that the response to copper occurs very rapidly upon elevation of extracellular copper levels. Although this gene is unresponsive to silver ions in vivo, in contrast to the yeast copper-responsive CUP1 gene (Furst, P., Hu, S., Hackett, R., and Hamer, D. (1988) Cell 55,[705][706][707][708][709][710][711][712][713][714][715][716][717], it does respond to mercury ions, albeit with less sensitivity. Mercury ions cannot, however, substitute for copper in allowing the accumulation of plastocyanin in vivo. The K. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequenceis) reported in thispaper has been submitted

M67448.
to the GenBankTM/EMBL Data Bank with accession number(s) $ Graduate student in the Ph.D. program of the Dept. of Biology,

UCLA.
To whom all correspondence should be addressed Dept. of Chemistry and Biochemistry, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-1569. Tel.: 213-825-8300;Fax: 213-206-4038. referred to as Cyt cS),' located in the intrathylakoid space of chloroplasts and cyanobacteria, catalyze electron transfer between the Cyt bs/f complex and the P-700 reaction center of photosystem I (Ho et al., 1979;Mathews, 1985). Whereas vascular plants utilize only the "type I" copper-protein plastocyanin (Boulter et al., 1977) for the reduction of P-700, several algae (and some cyanobacteria) can use plastocyanin and cytochrome cS interchangeably in this capacity (Wood, 1978;Sandmann and Boger, 1980;Sandmann et al., 1983;Ho and Krogmann, 1984). In the green algae (e.g. Chlamydomonas reinhardtii and Scenedesmus acutus) and cyanobacteria (e.g. Anabaena uariabilis) in which the functional replacement of plastocyanin by the heme-containing cytochrome has been studied, the accumulation of plastocyanin and Cyt cS is regulated by the availability of copper in the extracellular medium (Wood, 1978;Ho et al., 1979;Sandmann and Boger, 1980;Merchant and Bogorad, 1986a). In these organisms plastocyanin accumulates when sufficient copper is available for synthesis of the holoprotein whereas under conditions of copper deficiency only the cytochrome accumulates (Wood, 1978;Ho et al., 1979;Sandmann and Boger, 1980;Merchant and Bogorad, 1986a).
The mechanisms responsible for the coordinated and reciprocal regulation of plastocyanin and Cyt cfi expression appear to differ among the organisms studied. In A. uariabilis (van der Plas et al., 1989) and S. acutu.s,2 copper-dependent regulation of plastocyanin content occurs at the level of mRNA accumulation. By contrast, in C. reinhardtii the absence of plastocyanin in copper-deficient cells is the result of a posttranslational, copper-dependent step: uiz. rapid degradation of apoplastocyanin (Merchant and Bogorad, 1986b). Thus, although the synthesis (transcription plus translation) and intracellular and intraorganellar transport of apoplastocyanin are unaffected by copper, accumulation of plastocyanin is copper-dependent because of the instability of the apoprotein relative to the holoprotein (Merchant and Bogorad, 198613). With the exception of our own work in the C. reinhardtii system in which we have shown that Cyt c6 accumulation in C. reinhardtii is tightly regulated at the transcriptional level (Merchant et al., 1991), copper-responsive expression of Cyt cS has not been investigated. We anticipate that the mechanisms underlying the copper-regulated transcription of the algal gene for Cyt cs are similar to those that function in the The general term cytochrome c6 is used here to refer to the algal plastidic, soluble C-type cytochromes that participate in the Z-scheme of electron transfer. In previous work (from this laboratory and others) these cytochromes have been named for the absorbtion maximum of the (Y band of the reduced cytochrome, CW, c5m, c5b4, and so on. The use of the general name, Cyt cg, stresses its functional role in photosynthesis as opposed to its species specific physical properties. H. Li and S. Merchant, unpublished results. Copper-responsive Expression of the c. reinhardtii Gene for Cyt c6 15061 regulation of transcription in response to various environmental stimuli, uiz. that control is mediated uia the interaction of DNA-binding proteins with specific regions of target genes (for reviews see Johnson and McKnight, 1989;Struhl, 1989;Gruissem, 1990). We have invoked the existence of a coppertitrating factor that either directly or indirectly controls transcription of the Cyt c6 gene in response to occupancy of its metal binding site (Merchant et al., 1991). The study of Cyt c6 regulation in ac-208, a plastocyanin-deficient mutant, indicated that this putative copper-titrating factor is distinct from plastocyanin and that accumulation of plastocyanin is not a prerequisite for copper-induced repression of the gene encoding Cyt c6 (Merchant and Bogorad, 1987b). Plastocyanin does, however, play a role in governing the sensory threshhold of the copper-dependent transcriptional response since it appears that the two proteins compete for intracellular copper (Merchant et al., 1991). Our long term goal is to identify the metal-dependent components of this regulatory circuit. Toward this end we have isolated and characterized the C. reinhardtii gene for Cyt c6 and its upstream untranscribed sequences.
We have also extended our analysis of the physiology, specificity, and kinetics of copper-induced repression of the gene encoding Cyt c6 in C. reinhardtii. Such analyses could provide insight into the expression and biological properties of the cellular factors that participate in the regulatory circuit particularly in light of the proposed competition between plastocyanin and the regulatory factor for intracellular copper ions (Merchant et al., 1991). We find that the response seems to be quite specific for copper since other transition metals tested (cobalt, manganese, nickel, and zinc) do not repress Cyt c6 expression. Even silver, which activates the copperresponsive yeast metallothionein gene in uiuo as well as in vitro (Furst et al., 1988;Buchman et al., 1989;Evans et al., 1990), is not effective in specifically repressing Cyt c6 expression in C. reinhardtii (Merchant et al., 1991;and this work). These results are in line with studies of the stability of various metals at the type I ("blue") copper center in azurin, which suggests that Cu(I1) is preferred over Ni(I1) and Zn(I1). However, the type I copper center of azurin is believed to have a higher affinity for Hg(I1) than for Cu(I1) (Engeseth and McMillin, 1986). Furthermore, mercury ions are capable of competitively displacing copper ions from the type I binding site of plastocyanin (Kimimura and Katoh, 1972;Colman et al., 1978;Church et al., 1986). In fact, early evidence for the functional position of plastocyanin within the photosynthetic electron transfer chain came from studies in which mercuric salts were used to inhibit plastocyanin-dependent redox activity (Kimimura and Katoh, 1972). The recently determined three-dimensional structure of mercury-substituted plastocyanin crystals revealed only minor changes in the geometry of the metal site and few changes (none thought to be significant) elsewhere in the protein (Church et al., 1986). These observations prompted us to investigate the capacity of mercury ions to repress transcription of the gene encoding c y t c6. We report in this work that mercury ions (in contrast to other metals tested Ag(I), Co(II), Mn(II), Ni(II), and Zn(I1)) are indeed capable of specifically repressing Cyt c6 mRNA accumulation.

EXPERIMENTAL PROCEDURES
Isolation of a Cyt c6 Genomic Clone-A C. reinhardtii X-EMBL 3 genomic DNA library, obtained from Michel Goldschmidt-Clermont as five independent sublibraries (Goldschmidt-Clermont, 1986), was replicated in Escherichia coli NM 539 and screened by plaque hybridization (Maniatis et al., 1982) to a nick-translated (Genescreen Instruction Manual), "P-labeled (2 X IO9 cpm/pg DNA), gel-purified Cyt cs cDNA insert (Merchant and Bogorad, 1987a). Several positive plaques were identified from each subpool, and phage from these were further purified by rescreening.
DNA from each of seven phage isolates was prepared as described by Maniatis et al. (1982). Southern hybridization analysis (Church and Gilbert, 1984) of restriction endonuclease-digested DNA, prepared from one of these phage isolates, with 32P-labeled fragments from the cloned Cyt ce cDNA generated restriction fragment patterns identical to those obtained by similar hybridizations of C. reinhardtii genomic DNA, indicating that this isolate contained the entire coding sequence for the Cyt ce gene. Digestion of this DNA with SstI yielded a single fragment, approximately 5 kilobase pairs in length, that hybridized to the Cyt cg cDNA insert. This SstI fragment was subcloned, in both orientations, into the SstI site of pTZ19R to generate pTZlSRCrCGS1.2 and 6. Two DNA fragments (a 1,778-base pair HinfI fragment containing the entire Cyt c6 coding region and an overlapping 1,062-base pair SsA/ BstEII fragment, encompassing the promoter region; Fig. 1) were subcloned further into the SmaI sites of KSII+/-and pTZlSU, respectively. The resulting clones, KSII+/-:CrCGFl.l and pTZlSU:CrCGSBl, in two orientations (C/D), were used to generate nested deletions for DNA sequence determination.
Sequencing-Unidirectional deletions of KSII+/-:CrCGFl.l and pTZISU:CrCGSBlC/D were prepared with the help of the Exo/Mung DNA sequencing system (Stratagene) essentially as described by the manufacturer. The nucleotide sequence of each deleted clone was determined, using the appropriate primer sites in the vector, at the automated DNA sequencing core facility a t UCLA. 100% of the nucleotide sequence of both strands of the HinfI and SstIIBstEII fragments was determined. The DNA sequences were analyzed by use of the Sequence Analysis software package, version 6.1, from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (Madison, WI).
Isolation of Total RNA-Total RNA was prepared from vegetative C. reinhardtii cells by a method described previously (Schmidt et al., 1984;Merchant and Bogorad, 1986a) with the following modifications. Middle to late log phase cells (25-100 ml) were collected by centrifugation at 4,000 X g for 2 min a t 4 "C. The cells were resuspended in 1.5 ml of H20 and then lysed by slow stirring in 3 ml of "lysis buffer" (Schmidt et al., 1984) for 20 min a t room temperature (22 "C). RNA was isolated from the lysed cells after four extractions with an equal volume of phenol/chloroform/isoamyl alcohol (20:19:1) followed by two extractions of the resultant aqueous phase with an equal volume of chloroform/isoamyl alcohol (19:l). RNA was precipitated by the addition of 2.5 volumes of 100% ethanol and left to stand overnight at -20 "C. The precipitate was collected by centrifugation (7,500 X g, 30 min) and washed with 70% ethanol. After removal of the final traces of ethanol the pellet was resuspended in Northern Hybridization Analysis-Total RNA (3-7 pg/lane) was analyzed by Northern hybridization, essentially as described by Mer-200-500 p1 of H20. The presence of all indicated restriction endonuclease recognition sites has been confirmed by restriction and Southern mapping. Each arrow represents an independent determination of sequence (5' to 3') as described under "Experimental Procedures." The entire sequence was therefore determined on both strands. chant and Bogorad (1987a). 32P-Labeled (Feinberg and Vogelstein, 1984) DNA fragments, corresponding to cDNA inserts for Cyt c6 (Merchant and Bogorad, 1987a), plastocyanin (Merchant et al., 1990), or the small subunit of ribulose-bisphosphate carboxylase/oxygenase (Goldschmidt-Clermont and Rahire, 1986) were used as probes in these analyses. Hybridizing messages were visualized by exposure of Kodak XAR-5 or XRP-1 diagnostic x-ray film a t -80 "C with two enhancing screens.
Preparation of an Zntron-specific Probe-A 136-base pair DNA fragment, generated by digestion of the cloned Cyt c6 genomic DNA with PuuII and corresponding to nucleotides 633-768 of the genomic sequence ( Fig. 2), was subcloned into plasmid KSII+ using standard techniques (Maniatis et al., 1982). This 136-base pair intron-specific PuuII insert was used to prepare hybridization probes for Northern analysis as described above.
SI Nuclease Protection Analysis-The 1,062-base pair fragment generated by digestion of the cloned genomic sequence with SstI and BstEII ( Fig. 1) was separated by gel electrophoresis and purified on an NACS column according to instructions provided by the manufacturer (Bethesda Research Laboratories). The phosphoryl groups at the 5' termini were removed by digestion with calf intestinal alkaline phosphatase. The fragment was radiolabeled by phosphorylation of the free 5"OH groups with [-y-"'P]ATP and T4 polynucleotide kinase. The DNA (0.1 pg) was denatured by heating the sample to 90 "C for 10 min a 30-pl solution containing 40 mM Na/PIPES," pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% redistilled formamide. Transcripts (100 pg of total RNA) were annealed to the denatured DNA in the same solution during a 14-h incubation a t 59 "C. The annealed product was digested with S1 nuclease for 30 min at 20 "C by the addition of 300 p1 of digestion buffer (30 mM NaOAc, pH 5.0, 0.25 M NaCl, 1 mM ZnSOI, 0.5% glycerol) containing 330 units of S1 nuclease. Nucleic acids were precipitated from this reaction mixture by the addition of 2.5 volumes of ethanol. After removal of the last traces of ethanol by rotary evaporation the precipitated nucleic acids were resuspended in 10 pl of a solution containing 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyano1 FF, and 0.1 M NaOH and were analyzed after separation (50 watts, 4 h) on an 8% polyacrylamide gel (50 X 17 cm) in 0.089 M Tris, 0.089 M boric acid, 20 mM Na2-EDTA. After electrophoresis, gels were dried on to Whatman 3MM paper, and the radiolabeled fragments were detected by exposure to Kodak XAR-5 film.
Primer Extension Analysis-A synthetic oligonucleotide, GTTCGCCAACTGAAGCAT (Genetic Design, Inc., Houston, TX), that is complementary to the first six codons of the Cyt ce mRNA, was radiolabeled by phosphorylation of its free 5'-OH with [T-~'P] ATP and T4 polynucleotide kinase (Maniatis et al., 1982) for use as a primer. The primer was annealed to the Cyt cg mRNA (in a 10-pl reaction containing 50 pg of total RNA, 10 mM Tris-C1, p H 8.0, 1 mM EDTA, 250 mM KCl) by heating the reaction mixture to 80 "C for 10 min and then allowing it to cool to 49 "C over a period of 30 min. The primer was extended in a 35-p1 reaction mixture containing 15 mM Tris-HC1, pH 8.3, 70 mM MgC12, 4 mM dithiothreitol, 0.2 mM each of dNTPs, and 25 units of reverse transcriptase for 30 min a t 42 "C. The extension reaction was terminated by the addition of 2.5 volumes of ethanol to precipitate the nucleic acids. The pelleted nucleic acids were analyzed as described above.
Preparation of Soluble Protein Extracts-Cells were collected by centrifugation (3,000 X g for 2 min) and washed once in a solution containing 10 mM phosphate, pH 7.0. Pelleted cells were resuspended in 0.1 ml of the same solution. Soluble components of the cells were released quantitatively by subjecting the cell suspensions to two freeze (-80 "C)/thaw cycles.The insoluble cell debris was removed by two sequential centrifugations (12,000 X g for 15 min) at 4 "C, and supernatant fractions were used for Western blot analysis.
Cell Culture, Growth Conditions, and Miscellaneous Methods-Cultures of C. reinhardtii wild-type strain 2137 from L. Mets, University of Chicago, were grown in copper-free "TAP" medium (Merchant and Bogorad, 1986a;Harris, 1989)   Conserved cysteine residues that form thioether linkages with heme are marked with asterisks. A putative TATA box (AATAA) is underlined, and a conserved polyadenylation signal (TGTAA) is double underlined. A thick line is drawn over the region most similar to the 12-base pair ACE1 binding site consensus sequence; 8 matching base pairs (dots) are indicated. The end points of the PuuII fragment that serves as a probe for the detection of precursor mRNAs (Fig. 4) are indicated by filled arrowheads.
(125 pE/m*/s). Where indicated, cultures were supplemented with Cu(II), Hg(II), or Ag(1) salts from stock solutions. Cell densities were determined by counting (average of two determinations) in a hemocytometer after immobilization as described (Harris, 1989). The chlorophyll content of whole cells was determined spectrophotometrically after extraction into 8020 acetone/methanol using the extinction coefficients calculated by Arnon (1949).
Reagents-Plasmid vectors, KSII+/and pTZ19R/U, were purchased from Stratagene Cloning Systems, San Diego, CA, and U. S. Biochemical Corp., respectively. Restriction, DNA-, and RNA-modifying enzymes were purchased from the following manufacturers: Bethesda Research Laboratories; Promega Biotec, Madison, WI; and Stratagene and were used according to the manufacturers' instructions. [r-:"P]ATP and [n-:"P]dCTP were purchased from Du Pont-New England Nuclear Research Products or Amersham Corp. Actinomycin D was purchased from Sigma. Sources for other reagents are specified in the previous sections or have been listed elsewhere (Merchant and Bogorad, 1986a, 1986b, 1987a. 1987b.

Isolation of a Genomic Clone Encoding Cyt cG-A Cyt cs
cDNA was used to screen a X-EMBL3 library of C. reinhurdtii genomic DNA (Goldschmidt-Clermont, 1986), as described under "Experimental Procedures." Southern analysis of DNA prepared from phage yielding positive plaque hybridization signals identified an approximately 5-kilobase pair SstI fragment that contained the entire C. reinhardtii Cyt ca coding region. This DNA fragment was subcloned into plasmid pTZ19R. A 1,062-base pair SstIIBstEII fragment and an overlapping 1,778-base pair HinfI fragment were subcloned further from the resulting plasmid (pTZ19RCrCGSl) into appropriate vectors for sequence determination. The sequencing strategy employed for elucidating the nucleotide sequence of these two DNA fragments and a partial resriction map of the region encompassing the gene for Cyt c6 are shown in Fig.   1).

Determination of the 5'
Border of the mRNA for Cyt c6-Based on the cDNA sequence for Cyt c6, a synthetic oligonucleotide, complementary to the first six codons of the mRNA for Cyt c6, was used for primer extension analysis of Cyt cs transcripts. Four major products, resulting from termination at 63, 66, 69, and 79 nucleotides upstream of the initiator methionine codon, were generated (Fig. 3A, lune on extreme left). The longest product places the 5' border a t 5'ATTGCAG.. .3' (79 nucleotides upstream from the initiation codon); the shorter products probably result from premature termination caused by stable RNA secondary structures. The 5' border was also mapped by an S1 nuclease protection assay (Fig. 3B). The protected length of the 5' end-labeled probe encompassed approximately 211 nucleotides from the BstEII site ( Figs. 1 and 2). This region includes 79 nucleotides of 5"untranslated sequence, thus confirming our analysis of the results of primer extension. In addition, the presence of a single protected fragment, of the expected size, indicates that the untranscribed region is uninterrupted.
Nucleotide Sequence of the Gene Encoding Cyt cs-The nucleotide sequence of a Cyt cs cDNA from C. reinhardtii has been reported previously (Merchant and Bogorad, 1987a). The nucleotide sequence of the cloned fragment of genomic DNA (Fig. 2) reported here is in agreement with the cDNA sequence, with the exception of a thymidine at position 534, which was reported as a guanosine in the cDNA sequence (numbering is as for the genomic sequence; Fig. 2). Thymidine at this position generates an isoleucine codon, compared with a serine codon at the equivalent position in the cDNA sequence. Amino acid sequence data for Cyt ca are in agreement with the genomic nucleotide sequence (Merchant and Bogorad, 1987a). Thus, we believe that the genomic sequence is correct and that the guanosine in the cDNA sequence results from the approximately 0.1% error rate of RNA-dependent DNA polymerase (Maniatis et al., 1982).
Southern blot analysis of genomic DNA suggested that the Cyt cs gene was interrupted by one or more intervening Primer extension and S1 nuclease protection analyses of the 5' end of the Cyt c6 mRNA. A, total RNA from copperdeficient C. reinhardtii cells was annealed to a synthetic oligonucleotide (18-mer) that is complementary to the first 6 codons of the Cyt c6 mRNA, then processed as described under "Experimental Procedures" (lane on extreme left). The same 18-mer was used to prime dideoxy chain termination reactions which employed, as template, the cloned 1,062 base-pair SstIIRstEII fragment of the Cyt CR gene. The reaction products (A, T, C, and G ) were analyzed with the primer extension products to allow direct determination of the transcription start site. B, total RNA from copper-deficient C. reinhardtii cells was used in an S1 nuclease protection assay as described under "Experimental Procedures." The products of dideoxy chain termination reactions (A, T, C, and G; described above) were analyzed with the S1 nuclease digestion products (lane on extreme right) to estimate the size of the protected DNA fragment. Arrows indicate the positions of A, the longest primer extension product, and R, the single 211base pair DNA fragment (labeled at the BstEII site) that was protected from nuclease digestion. RNA from copper-supplemented cells is unable to support primer extension or to protect the labeled DNA fragment from S1 digestion (not shown).
sequences (Merchant and Bogorad, 1987a). The nucleotide sequence of the gene (Fig. 2), which contains two introns, confirms this prediction. Comparison of the genomic sequence with that of the cDNA allows prediction of splice site locations for both introns. It is intriguing that the highly conserved heme binding site (CXXCH; reviewed by Mathews, 1985) is exactly split by the first intron. The significance, if any, of this observation is unknown. The exact location of the 3' border of the second intron is ambiguous because of the presence of a 5-base pair repeat at the splice sites of this intron. However, the position of conserved splice site sequences (exon-GU.. .intron.. . AG-exon;(Breathnach and Chambon, 1981)), which are also found at the borders of other C. reinhardtii introns (Zimmer et al., 1988), suggests that the Copper-responsive Expression of the C.

reinhurdtii
Gene for Cyt c6 second intervening sequence ends at nucleotide 931 as shown. Both introns 1 and 2 (135 and 387 base pairs in length, respectively) are flanked by consensus intronlexon boundaries (Zimmer et al., 1988). Each intron also contains a consensus sequence of nucleotides, 25-55 base pair upstream from the 3' splice site, which is believed to be involved in the formation of the splicing branch site in type I1 introns (Ruskin et al., 1984;Silflow et al., 1985;Zimmer et al., 1988). Goodall andFilipowicz (1989) have suggested that AU-rich tracts present in plant introns are required for efficient splicing. Such AU-rich regions are not found in the introns of the C. reinhardtii gene encoding Cyt c6. In fact, the AT content of introns 1 and 2 (43 and 40%, respectively) is considerably lower than that of the least AT-rich dicotyledonous intron (Arabidopsis thaliana: 60.5% AT) analyzed by Goodall and Filipowicz. The GC content of the Cyt cG exons (67, 62, and 64%, respectively) are only slightly higher than that found in introns 1 and 2 (57 and 60%, respectively) and reflect the high GC nature of the C. reinhardtii nuclear genome (Chiang and Sueoka, 1967). The GC content of 5'-and 3"untranslated regions (51 and 47%, respectively) of the cyt c6 transcription unit is slightly lower than for translated sequences. The 5'nontranscribed region is moderately GC-rich (57% GC).
Computer-aided sequence analysis of the Cyt c6 promoter region revealed no striking features. There is a region approximately 20 base pair upstream from the transcription start site (underlined in Fig. 2) which roughly resembles the eukaryotic "TATA box" (Breathnach and Chambon, 1981). Similar regions have been identified in other C. reinhardtii genes (Brunke et al., 1984;Goldschmidt-Clermont and Rahire, 1986;Mayfield et al., 1987;Zimmer et al., 1988;de Hostos et al., 1989;Woessner and Goodenough, 1989). The GC-rich region described by Brunke et al. (1984), which follows such TATAlike sequences in many C. reinhardtii genes (Brunke et al., 1984;Goldschmidt-Clermont and Rahire, 1986;Imbault et al., 1988;de Hostos et al., 1989;Schloss, 1990), is not present in the gene encoding cyt Cg. Comparison of sequences upstream from the cyt Cg coding region with upstream activating sequences of the copper-regulated yeast metallothionein gene (Thiele and Hamer, 1986;Evans et al., 1990) revealed no strong similarities; the best match identified on either strand (8 of 12 base pairs) is indicated (Fig. 2).

Identification of a Precursor to the mRNA Encoding Cyt
cG-Northern analysis of Cyt c6-specific RNA molecules in total RNA preparations identified two hybridizing transcripts (Fig. 4). The smaller of these, approximately 760 nucleotides in length, has been identified previously as the Cyt Cg mature transcript (Merchant and Bogorad, 1987a). Upon the addition of copper ions, loss of the larger RNA molecule precedes the loss of the mature transcript (Fig. 5). This observation, together with the fact that the gene encoding Cyt c6 contains two intervening sequences, prompted us to examine the possibility that the larger RNA molecule was in fact an unspliced precursor to the mature transcript. As shown in Fig. 4, the larger RNA molecule hybridizes to both the Cyt cG cDNA and a Cyt Cg intron-specific probe. Hence, this molecule likely corresponds to an incompletely processed Cyt c g mRNA precursor that still contains a t least part of intron 2. Analysis of the levels of the pre-mRNA, then, is likely to provide a more sensitive gauge of the transcriptional activity of the cyt c6 gene as compared with analysis of the levels of the mature message. other lanes contain RNA from cells supplemented with 10 pM CuC12 for the indicated times (60, 100, 120, 160, or 180 min). Preparation of RNA and Northern hybridization were performed as described under "Experimental Procedures." The cDNA fragment encoding Cyt cG was used as a probe. Equivalent loading of RNA in all lanes was verified based on visualization of rRNAs. The estimate of the halflife of the Cyt cg message in copper-supplemented cells is based on the observation that the rate of its synthesis in these cells is negligible (Merchant et al., 1991). In eight independent time course experiments representing 20 time points (20-180 min) the estimates ranged from 45 to 60 min. Messages that are not known to be copper regulated (including those for @tubulin, a small subunit of ribulose-bisphosphate carboxylase, and plastocyanin) were used as internal controls for standardization of Cyt ce message levels in different populations of cells. The amount of Cyt ce-specific transcripts was quantified by differential exposures of the same blot or by densitometric scanning. The levels of Cyt cg-specific transcripts in the samples shown in this figure were quantified by densitometric scanning of several exposures of a single set of samples relative to plastocyanin transcripts. The half-life of the Cyt ce message in this experiment is thus calculated to be approximately 60 min. B, time course of the loss of mRNA for Cyt c6 in actinomycin D-treated cells. Total RNA was isolated from copper-deficient cells (open triangks) or cells supplemented with copper at 0 min (closed triangles). All samples were treated with actinomycin D (40 pg/ml) a t 0 min. RNA was isolated at the indicated times and analyzed by Northern hybridization. Cyt cG-encoding, radiolabeled fragments were used as a probe. Transcript levels were quantified by video densitometric scanning of the autoradiogram and are plotted as a percent of the level of transcripts present at the first time point (0 min). The persistence of plastocyanin-encoding transcripts in actinomycin D-treated cells shows the same pattern as that seen above (i.e. no difference between copper-supplemented uersus copper-deficient cells). tion of copper salts to copper-deficient cells results in the loss of greater than 95% of Cyt c6-specific RNA transcripts within 3 h after the addition of copper (Fig. 5). After 4 h in the presence of copper ions Cyt cfi transcripts are not detectable (Fig. 6). From a number of similar time course experiments we estimate that Cyt c6 mRNA levels decay with a half-life of approximately 45-60 min in copper-supplemented cells. This tu is significantly shorter than the tLI2 of the "average" C. reinhardtii message (150 min; Baker et al., 1984), suggesting that the Cyt cfi message is relatively unstable. However, a role for differential mRNA stability in copper-supplemented cells is not supported since the decay of mature messages in actinomycin D-treated cells is identical in copper-supplemented versus copper-deficient cells (Fig. 5B). The loss of the mature mRNA for Cyt c6, over a period of 3-4 h, is preceded by a much more rapid loss of the pre-mRNA (Fig. 5). Based on results of kinetic studies similar to the one illustrated here we conclude that this mRNA precursor is completely lost within 30 min, after the provision of copper ions, with an estimated half-life of less than 10 min (not shown).

Repression of Cyt cfi-specific m R N A Accumulation by Mer-
cury-We have provided evidence that plastocyanin and a putative copper-binding cyt c6 transcription factor compete for intracellular copper ions in C. reinhardtii cells (Merchant et al., 1991). Since mercury ions are able to substitute for copper ions at the type I copper-binding site of plastocyanin in vitro (Kimimura and Katoh, 1972;Colman et al., 1978;Church et al., 1986) we wished to examine the effect of mercury ions on Cyt c6 and plastocyanin expression in vivo.
As illustrated in Fig. 6, mercury ions are effective at repressing Cyt cc-specific mRNA accumulation. Other metal ions, Ag(I), Co(II), Mn(II), Ni(II), and Zn(II), tested a t PM concentrations, failed to repress expression of the gene encoding cyt c6 (Fig. 7).4 The effect of mercury ions is specific for cyt c6 transcripts since the levels of transcripts for plastocyanin, the small subunit of ribulose-bisphosphate carboxylase, and ptubulin (not shown) are not altered significantly in mercurysupplemented cells (Fig. 6).
To characterize further the effect of mercury ions on Cyt cs expression we determined what level of HgCl,, in the growth medium, is required to repress the gene encoding Cyt cfi (Fig.  8). A concentration in excess of 5 p~ HgCl, is required to bring about a level of repression comparable to that produced by 500 nM CuS04 (compare levels of pre-mRNA and mRNA in Figs. 6 and 8). Thus, this response is a t least 10-20 times more sensitive to extracellular copper ions than to extracellular mercury ions. Silver ions are unable to substitute for copper ions in specifically reducing Cyt cfi mRNA levels, even when tested a t concentrations 20-fold higher than that sufficient for Cyt c6 repression by copper ions (Fig. 7). We know that silver ions are indeed accessible to the organism since the cells do synthesize phytochelatin-like peptides in response to these levels of AgN03." A slight reduction in the levels of messages for both Cyt ce and plastocyanin is observed in cells supplemented with 10 PM AgNOs. However, this nonspecific effect is probably a result of the toxic effects of silver ions since cells supplemented with 10 PM AgNOs no longer divide (not shown).

Metal Specificity of Holoplustocyanin Accumulation-Mer-
cury can displace copper from the metal binding site of plastocyanin when presented either in vitro to the purified protein, or in organello to intact chloroplasts (Kimimura and Katoh, 1972;Colman et al., 1978;Church et dl., 1986). As Soluble proteins were extracted from copper-deficient cells prior to (-) and 5 h after the addition of 5 ~L M CuS04 (+Cu) or 5 PM HgCI, (+Hg) to the growth medium. The amount of plastocyanin in the extracts was visualized by Western blot analysis as described under "Experimental Procedures." hardtii cells are supplemented with copper ions, the steadystate level of plastocyanin increases because of synthesis and hence stabilization of holoplastocyanin relative to the apoprotein (Merchant and Bogorad, 1986b). Mercury ions, however, are unable to bring about a similar increase in plastocyanin steady-state levels (Fig. 9). Since mercury ions are available intracellularly (as evidenced by repression of Cyt cs mRNA accumulation (Fig. 8) and stimulation of glutathione synthesis," the inability of mercury ions to support holoplastocyanin accumulation cannot be accounted for by cellular exclusion of mercury ions. Neither is it caused by the toxicity of HgC12 since the algal cells continue to divide (at this concentration of supplemented HgC12) during the course of the experiment (not shown). Rather, we must conclude that the cellular pathway for holoplastocyanin formation and accumulation is highly specific for copper ions, perhaps suggesting that this process depends on metal-specific catalytic events in. vivo, or alternatively, that mercury-plastocyanin does not accumulate because of comparable instability of apoplastocyanin (Merchant and Bogorad, 1986b) and mercury-plastocyanin.

DISCUSSION
We have determined the nucleotide sequence of the C.
reinhardtii gene encoding Cyt cs and found that its coding region is interrupted by two introns. The transcript produced from this gene begins 79 nucleotides upstream from the initiator methionine codon. We have examined the copperresponsive regulation of accumulation of mRNA for Cyt cn and demonstrated that mature transcripts are lost with an approximate half-life of 45 min in cells supplemented with copper ions and are completely absent within 4 h after the addition of copper ions to copper-deficient cells. On the other hand, decay of the steady-state levels of an unspliced pre-mRNA occurs within minutes in parallel with a decreased ability of isolated nuclei to elongate Cyt c6 transcripts in vitro6 after the addition of copper ions to copper-deficient cells, thus demonstrating the rapidity with which algal cells respond to changes in copper availability and supporting the finding that expression of the gene encoding Cyt cfi is regulated primarily at the transcriptional level with changes in mRNA stability playing a negligible role. We expect that this swift transcriptional response results from rapid functional activation/inactivation of a transcriptional repressor/activator (perhaps in response to occupancy of a regulatory metal binding site).
A well characterized example of a gene that is copper regulated at the transcriptional level is the Saccharomyces cerevisiae CUPl gene, which encodes copper-metallothionein (Karin et al., 1984). Transcription of this gene is activated in response to elevated concentrations of copper ions (Karin et al., 1984). This metal-dependent transcriptional activation is mediated by a factor (encoded by the ACEl gene) that, when tJ. Quinn and S. Merchant, unpublished results. bound to copper, interacts with tandemly repeated DNA sequence elements found upstream of the CUPl gene (Thiele and Hamer, 1986;Thiele, 1988;Furst et al., 1988;Evans et dl., 1990). The ACEl gene product was also demonstrated to display appropriate sequence-specific DNA-binding activity in vitro in response to occupancy, by Ag(I), of its metal binding site (Furst et al., 1988;Buchman et al., 1989). Additionally, the metal-activated CUPl gene was found to be equally responsive to both copper and silver ions in vivo (Furst et al., 1988;Buchman et al., 1989). It seemed germane therefore to examine the metal specificity of the regulatory system described here, particularly in light of the observation that the depletion of Cyt cfi in another alga, S. acutus, is reported to be affected by either copper or silver ions Sandmann et al., 1981). We find that in our system silver ions are not capable of substituting for copper ions in the repression of Cyt cs expression at the level of mRNA accumulation. We therefore suggest that the metal specificity of this response is unique in its preference for copper over other transition metals.
We have suggested in earlier work that a putative copperresponsive factor controlling expression of the C. reinhardtii gene for Cyt ce competes with plastocyanin for copper ions, since cells are able to repress the gene for Cyt cfi only when the level of available copper ions exceeds that required for stoichiometric plastocyanin synthesis (Merchant et al., 1991).
T o develop this model it was appropriate to examine and compare the physiological metal specificity of the transcriptional response with that of type I copper-proteins (Engeseth and McMillin, 1986). We find that whereas Ni(I1) and Zn(I1) salts are completely ineffective at regulating accumulation of Cyt cs (not shown), mercury ions are indeed capable of reducing the levels of Cyt cs-specific transcripts. It is unlikely that the repression of transcription of the gene encoding c y t c6 is merely a consequence of the general toxic effect of mercury ions since transcripts for plastocyanin, the small subunit of ribulose-bisphosphate carboxylase, and P-tubulin (in the same RNA preparations) are either unaffected or only minimally affected by these concentrations of mercuric salts. We cannot, however, distinguish between functional replacement of copper by mercury versus nonspecific inactivation of an essential Cyt cs-specific, thiol-containing transcriptional activator. At any rate, our results do support the general concept of an intracellular metal ion sensor that is specific for the Cyt csencoding gene and that reacts with a transcriptional response. Although the Cyt c6 regulatory response in vivo requires higher levels of mercury ions than copper ions, the affinity/stability of the metal site in the regulatory protein awaits in vitro characterization since the effective intracellular concentration of mercury ions may well be much lower than that of copper ions supplied at equivalent extracellular concentrations (because of stimulation of glutathione synthesis by mercury ions and the resulting accumulation of stable mercaptides)." It is interesting to note in this regard that mercury ions, although capable of replacing bound copper ions in holoplastocyanin in vitro (Kimimura and Katoh, 1972;Colman et al., 1978;Church et al., 1986), do not allow accumulation of (mercury)plastocyanin in vivo. One possible explanation for this discrepancy is that mercury ions supplied in the medium might not be accessible for holoplastocyanin synthesis in vivo. We know that mercury ions indeed enter the cell since they induce changes in mRNA steady-state levels (Figs. 6 and 8) and elicit a heavy metal-dependent stress response.5 However, the metal ions may not be transported to the lumen of the thylakoid membrane where holoplastocyanin formation is believed to occur (Merchant and Bogorad, 1986b;Li et al., 1990). Alternatively, it is possible that holoplastocyanin formation is catalyzed in vivo and that the enzyme responsible for metal attachment to apoplastocyanin cannot use mercury ions as a substrate. We also cannot exclude the possibility that mercury-plastocyanin is indeed formed in vivo but does not accumulate, because of rapid degradation, as is the case for apoplastocyanin (Merchant and Bogorad, 1986a). Ongoing work in this laboratory is attempting to distinguish between the above possibilities. In any event, the metal specificity of plastocyanin is greater i n vivo than it is i n vitro.
A more direct analysis of the putative C. reinhardtii copperresponsive transcription factor and its metal binding site(s) awaits its isolation. With the advent of a reliable and efficient method for the nuclear transformation of C. reinhardtii (Debuchy et al., 1989;Kindle et al., 1989;Kindle, 1990) we plan to proceed toward the identification of copper-responsive DNA sequence elements associated with the gene encoding Cyt cs as well as the isolation of the regulatory metalloprotein(s) that control(s) its transcription.