Plant Physiol. (1991) 95, 264-2680032-0889/91 /95/0264/05/$01 .00/0

Received for publication April 10, 1990Accepted September 23, 1990

Isolation and Characterization of a cDNA Coding for PeaChloroplastic Carbonic Anhydrase'

Nathalie Majeau and John R. Coleman*

Centre for Plant Biotechnology, Department of Botany, University of Toronto, Toronto, Ontario, Canada M5S 3B2

ABSTRACT

Using a polyclonal antibody generated against the purified pea(Pisum sativum) carbonic anhydrase (CA) monomeric species,we have isolated and characterized a cDNA coding for thisenzyme. Protein sequence analysis was used to confirm theidentity of the clone. The presence of a large transit peptidesuggests that CA is transported into the chloroplast and thenprocessed to the mature size of approximately 26 kilodaltons.Northern hybridization, using the CA cDNA as a probe of totalleaf RNA, revealed a single transcript of 1.45 kilobase pairs. Thistranscript was not detected in RNA extracted from root or etio-lated leaf tissue. Comparison of the deduced amino acid se-quence with that of spinach CA showed approximately 68%identity over the length of the nascent protein but with greatersimilarity observed within the mature protein sequences. In ad-dition, regions of the pea and spinach CA proteins were found tobe significantly similar to the Escherichia coli cyanate permease.

One of the more abundant soluble proteins in the leaves ofhigher plants is the enzyme CA2 (carbonate dehydratase4.2.1.1), which catalyzes the reversible hydration of CO2 toHCOy. Although it may represent as much as 1 to 2% of thesoluble leaf protein, there are many aspects of higher plantCA structure, localization, regulation, and role in metabolismthat are not well understood (16, 18). For example, significantdifferences appear to exist between monocot- and dicotyle-donous plants with respect to molecular mass of the holoen-zyme. Molecular sizes can range from 42 to 45 kD in monocotspecies to 140 to 250 kD when isolated from a number ofdicot species (1, 2, 18). The relatively small range in theobserved subunit size (26-34 kD) for all higher plant CAproteins suggests that some variation in subunit number ofthe native protein occurs. Most localization studies indicatethat CA is found in the chloroplasts of C3 plants and withinthe cytosol of C4 species; however, other reports indicate thatadditional isozymes and tissue locations are possible (1, 13,18, 24). Within the C3 chloroplast, CA is thought to speed thedehydration of HCOY and thus maintain the supply of CO2for Rubisco (18). It has also been postulated, however, thatCA activity could directly facilitate diffusion of CO2 across

' Supported in part by a grant to J. R. C. from the Natural Sciencesand Engineering Council ofCanada. N. M. is a recipient ofan NSERCpost-graduate scholarship.

2 Abbreviations: CA, carbonic anhydrase; kb, kilobase pair; Rub-isco, ribulose bisphosphate carboxylase/oxygenase.

the chloroplast envelope by maintenance of the equilibriumbetween the inorganic carbon species (18, 24). In addition,CA may play a role in the buffering capacity ofthe chloroplaststroma by enhancing the rates of the dehydration/hydrationreactions.As an initial step in the study ofCA activity and regulation

of expression in higher plants, we have isolated and charac-terized a cDNA clone coding for pea CA. We have alsodetermined CA transcript abundance and compared the de-duced amino acid sequence with another higher plant CA andwith an Escherichia coli polypeptide which exhibits somesequence similarity to the pea CA.

MATERIALS AND METHODS

Plant Material

Peas (Pisum sativum var Little Marvel) and corn (Zeamays var Early King) were grown in growth rooms for either14 or 21 d in vermiculite, with a day length of 16 h, illumi-nation of 700 ,uE m-2-s-' and a day/night temperature re-gime of 20°C/16°C. Plants were watered every 3 d and pro-vided with a nutrient solution weekly. Etiolated tissue wasproduced by germination and growth in total darkness usinga similar temperature, watering, and nutrient regime.

Isolation of Pea Leaf Carbonic Anhydrase

Leaves and stems (250 g) of 21 -d-old, light-grown peas werehomogenized in ice-cold extraction buffer (0.3 M Tris-SO4[pH 8.3] containing 5 mM DTT, 1 mm PMSF, 1 mm benz-amidine) using a Waring blender for 1 min and filteredthrough Miracloth, and the soluble fraction was clarified bycentrifugation at 4°C (40 min, 30,000g). The supernatant wascollected and subjected to (NH4)2SO4 fractionation. The 30to 60% saturation precipitate was resuspended, dialyzedagainst 5 mM Tris-SO4 (pH 9.5) buffer, and then charged ontoan affinity column (p-methylaminobenzene sulfonamide sub-stituted Sepharose 4B) synthesized as previously described(26). After extensive washing with 25 mM Tris-SO4 (pH 8.3),the CA containing fraction was eluted with 25 mm Tris-SO4(pH 8.3) containing 25 mm NaClO4 and then was dialyzedextensively against 5 mM Tris-SO4 (pH 8.3). Fractions con-taining CA activity, as determined by an electrometric assay(25), were analyzed by SDS-PAGE as described previously(6). In situ CA activity, after separation of total soluble peaprotein on nondenaturing polyacrylamide gels, was detectedby ultraviolet fluorescence of bromocresol purple after expo-sure to CO2 at low temperature (1).

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SEQUENCE AND EXPRESSION OF PEA CARBONIC ANHYDRASE

Production of Antibodies Against Carbonic Anhydrase

Affinity column-purified CA was subjected to SDS-PAGE(7-15% gradient gel), and the CA monomeric species (pri-marily a 25.5 kD polypeptide and a minor 27.5 kD band)were electoeluted from the gel slices. After collection ofpreim-mune serum, the isolated CA was combined with Freund'sadjuvant and was injected into rabbits using a previouslydescribed immunization schedule (5). Western blotting tech-niques were used to test the specificity ofthe rabbit polyclonalantisera and to identify the CA monomer in total solubleprotein extracts (3).

Isolation and Characterization of cDNA Clones

A X gtl 1 cDNA library, synthesized from light-grown, pealeaf mRNA (23), was screened using the anticarbonic anhy-drase serum as previously described (3). Inserts from theimmunopositive recombinant phage were generated by EcoRIdigestion and then subcloned directly into the plasmid vectorpBS (Stratagene Inc.). Double-stranded DNA sequencing ofthe inserts was performed using synthesized oligonucleotideprimers and the dideoxy method of Sanger et al. (19). DNAsequence analysis was performed by using the Pustell Se-quence Analysis Software (International Biotech. Inc.).

RNA Isolation and Northem Hybridization

Total RNA was isolated from leaves, roots and etiolatedleaves of 14-d-old plants that were first frozen and powderedin liquid nitrogen, prior to extraction using the method de-scribed in (6). Equal amounts of RNA from each tissue weredenatured and electrophoresed on a 1.5% agarose gel contain-ing 0.66 M formaldehyde. Transfer to nitrocellulose, prehy-bridization and hybridization conditions were as previouslydescribed (17). The cloned 0.95 kb EcoRI cDNA insert usedas a probe was labeled with [a-32P]dCTP using a randomprimer procedure (9). The hybridization pattern was deter-mined by autoradiography and the sizes of the stained ribo-somal bands and transcripts estimated by comparison withknown standards.

Protein Sequence Determination

An internal peptide fragment, generated by cyanogen bro-mide digestion of affinity column and SDS-PAGE purifiedCA was sequenced by Edman degradation using an AppliedBiosystems Sequenater, model 477A.

RESULTS

Carbonic anhydrase, isolated from the green tissue ofPisumsativum, has been purified by a combination of affinity chro-matography and gel electrophoresis. After separation on anSDS-polyacrylamide gel, the protein fraction eluted from thesulfonamide affinity column contained an abundant 25.5 kDpolypeptide and a minor band of 27.5 kD (Fig. 1, lane 3).This same protein fraction exhibited maximal levels of CAactivity prior to SDS-PAGE. CA activity was also detectedafter nondenaturing gel electrophoresis of soluble pea poly-peptides and staining of the gel with bromocresol purple. The

1 2 3

KD

97.4- 1.7I

66.2-

45.0-

' 1 n_

I 4 5

J *n .v 'K

21.5-

14.4-

27.5

-\ 25.5

Figure 1. Purification of pea CA and Western blot analysis. Totalsoluble proteins isolated from corn (lane 1), pea (lane 2), and specifi-cally bound pea proteins eluted from a CA affinity column (lane 3)were separated by SDS-PAGE (7-15% linear gradient) and stainedfor protein with Coomassie brilliant blue (lanes 1-3). A replicate gelof lanes 1 and 2 was electrotransferred to nitrocellulose (lanes 4 and5) and incubated with pea CA antiserum. Specifically bound CAantibody was detected with goat anti-rabbit IgG conjugated horse-radish peroxidase acting upon 4-chloro-1-napthol. The positions ofthe molecular mass markers (BRL Inc.) and the pea CA monomericspecies are indicated.

fluorescent band which appears after exposure of the gel toCO2 was excised, electroeluted, and the extracted proteinssubjected to SDS-PAGE. The same abundant 25.5 kD poly-peptide and minor 27.5 kD protein were observed (data notshown).The 25.5/27.5 kD doublet was excised from SDS-polyacryl-

amide gels and used to generate a polyclonal antibody inrabbits. A Western blot of total soluble protein isolated fromPisum sativum and Zea mays is shown in Figure 1. Theantibody cross-reacted strongly with a 25.5 kD polypeptide inthe total soluble protein isolated from pea (Fig. 1, lane 5). Avery faint immunosignal from the 27.5 kD protein couldoccasionally be detected in the pea soluble protein extracts.The antibody failed to cross-react with any of the soluble

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MAJEAU AND COLEMAN

5' GTTTTGAATCTGCATTGCACCA ATG TCT ACC TCT TCA ATA AACM S T S S I N 7

GGC TTT AGT CTT TCT TCT TTG TCC CCT GCC AAA ACT TCT ACC AAAG F S L S S L S P A K T S T K 22

AGA ACT ACA TTG AGA CCC TTT GTT TTT GCA TCT CTT AAC ACT TCTR T T L R P F V F A S L N T S 37

TCT TCT TCA TCT TCT TCC TCG ACT TTC CCT TCT CTT ATT CAA GACS S S S S S S T F P S L I Q D 52

AAG CCG GTT TTC GCT TCT TCT TCT CCT ATC ATC ACC CCA GTT TTGK P V F A S S S P I I T P V L 67

AGA GAA GAA ATG GGA AAG GGC TAT GAT GAA GCT ATT GAA GAA CTCR E E M G K G Y D E A I E E L 82

CAA AAA TTG TTG AGG GAG AAG ACT GAA CTG AAA GCC ACA GCT GCTQ K L L R E K T E L K A T A A 97

GAG AAG GTT GAG CAA ATC ACA GCT CAG CTA GGA ACA ACA TCA TCAE K V E Q I T A Q L G T T S S 112

TCT GAT GGC ATT CCA AAA TCT GAA GCC TCT GAA AGG ATC AAA ACTS D G I P K S E A S E R I K T 127

GGT TTC CTT CAC TTC AAG AAA GAG AAA TAT GAC AAG AAT CCA GCTG F L H F K K E K Y D K N P A 142

TTG TAT GGT GAA CTT GCC AAA GGC CAA AGC CCT CCG TTT ATG GTGL Y G E L A K G Q S P P F M V 157

TTT GCA TGT TCA GAC TCA AGA GTC TGC CCA TCT CAT GTG CTA GATF A C S D S R V C P S H V L D 172

TTC CAG CCA GGT AAA GCC TTT GTG GTC AGA AAT GTT GCT AAC TTGF Q P G K A F V V R N V A N L 187

GTT CCA CCA TAT GAC CAG GCA AAA TAT GCC GGA ACT GGT GCT GCAV P P Y D Q A K Y A G T G A A 202

ATT GAG TAC GCA GTT CTG CAT CTC AAG GTT TCC AAC ATT GTT GTCI E Y A V L H L K V S N I V V 217

ATT GGA CAC AGT GCT TGT GGT GGT ATT AAG GGA CTT TTG TCC TTTI G H S A C G G I K G L L S F 232

CCA TTT GAT GGA ACC TAC TCC ACT GAT TTC ATT GAG GAG TGG GTCP F D G T Y S T D F I E E W V 247

AAA ATT GGT TTA CCT GCA AAG GCG AAG GTG AAA GCA CAA CAT GGAK I G L P A K A K V K A Q H G 262

GAT GCA CCT TTT GCA GAG CTA TGC ACA CAC TGT GAG AAG GAA GCTD A P F A E L C T H C E K E A 277

GTG AAT GCT TCC CTT GGA AAC CTT CTC ACC TAC CCA TTT GTG AGAV N A S L G N L L T Y P F V R 292

GAG GGA TTG GTG AAC AAG ACA TTG GCA CTC AAA GGA GGA TAC TATE G L V N K T L A L K G G Y Y 307

GAC TTT GTG AAA GGA TCC TTT GAG CTT TGG GGA CTT GAA TTT GGCD F V K G S F E L W G L E F G 322

CTT TCG TCC ACT TTC TCC GTA TGA ACATCAACCATATATCAATGACCACATL S S T F S V --- 329

CTTGATTACTAAGTCAAAGATGTGGCCACGATTCTACATTGGAAGCTATAATCTCGTGGTTAAAGGTCGTGTGTCTGCAATGAAGAAGCCTACCAACTTTCATCATATGATATTTTATATATATGAATCTATGAACTTGTATTATGTACTATATATCATGTATCCCATCTTAAATAAGAGTTTCTAATTCCTATTGAGGCAAAAAAAAAAAAAAAAAAAA 3

Figure 2. Nucleotide and deduced amino acid sequence of the pealeaf carbonic anhydrase cDNA. The amino acids identified by proteinsequencing of an internal peptide fragment are underlined. The under-lined region at the 3' end of the cDNA represents a putative polyad-enylation signal sequence.

proteins isolated from corn (Fig. 1, lane 4) and from etiolatedpea leaf or root tissue (data not shown).

Approximately 200,000 plaques obtained from the pea leafcDNA library were screened with the pea CA antibody. Atotal of 10 individual plaques were found to be true immu-nopositive clones after rescreening, and these clones were alsofound to exhibit strong sequence homology as determined bySouthern hybridization techniques (data not shown). Afterdigestion by EcoRI, the two largest inserts, 0.95 and 1.3 kb,were subcloned into the vector pBS. Partial nucleotide se-quence analysis revealed that these two clones were identical.The 1.3 kb fragment was sequenced completely in both direc-tions and is presented along with the deduced amino acidsequence in Figure 2. The sequence encodes an open readingframe for a polypeptide of 35.7 kD and a segment of thededuced amino acid sequence was found to match exactly theamino acid sequence of CNBr-generated peptide fragmentisolated from the purified protein (Fig. 2). N-Terminal proteinsequencing of the purified polypeptide did not provide con-clusive data.The 1.3 kb cDNA insert was hybridized to total pea RNA

isolated from light grown leafand stem tissue, root tissue, andetiolated leaf tissue (Fig. 3). A single 1.45 kb transcript wasidentified in the RNA isolated from light grown leaf and stemtissue (Fig. 3, lane 4) but was not present in RNA isolatedfrom etiolated leaf or root tissue (Fig. 3, lanes 5 and 6).The deduced pea CA amino acid sequence is compared

with spinach CA (4, 8) (Fig. 4) and with the deduced aminoacid sequence of the cyanate permease gene isolated fromEscherichia coli (20) (Fig. 5). The permease sequence wasidentified as having significant homology with carbonic an-hydrase using Genbank and the Pustell sequence analysisprogrammes. An examination of a number of animal CAamino acid sequences (7, 10, 22) using the same software didnot reveal any significant regions of similarity with the peasequence.

4 5 6KB

5.60..4.27 -3.48 --

1 "98,1-1 .901.59 -

1.37 --

0.94 -

0.82 --

Figure 3. Determination of pea CA transcriptabundance. Total RNA isolated from light-grownleaves (lane 1), dark-grown (etiolated) leaves(lane 2), and root tissue (lane 3) was separatedby electrophoresis on a 1.5% agarose gel con-taining 0.66 M formaldehyde, transferred to nitro-

- 1 .45 cellulose, and probed with the 32P-labeled CAcDNA. The ethidium-stained gel (lanes 1-3) andthe resulting hybridization profiles (lanes 4-6)are shown. The size of the transcript was deter-mined by its position relative to the mobility ofdenatured X-DNA fragments after digestion withHindlll and EcoRI, as well as Escherichia coliribosomal RNA.

i.1 2 3

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SEQUENCE AND EXPRESSION OF PEA CARBONIC ANHYDRASE

Pea MSTSSINGFSLSSLSPAKTSTKRT . TLRPFVFASLNTSSSSSSSSTFPSLIQDKPVFASS 59

Bpi MST. ING. CLTSISPSRTQLKNTSTLRP. TFIA. NSRVNPSSSVP. PSLIRNQPVFAAP 54

Pea SPIITPVLREEMGKGYDEAIEELQKLLREKTELKATAAEKVEQITAQLGTTSSSDGIPKS

Bpi APIITPTLKEDMAY ..EEAIAALKKLLSEKGELENEAASKVAQITSELAD .... GGTPSAA

Pea E . ASERIKTGFLHFKKEKYDKNPALYGELAKGQSPPFMVFACSDSRVCPSHVLDFQPGKA

Bpi SYPVQRIKEGFIKFKKEKYEKNPALYGELSKGQAPKFMVFACSDSRVCPSHVLDFQPGEA

119

108

178

168

Pea FVVRNVANLVPPYDQAKYAGTGAAIEYAVLHLKVSNIVVIGHSACGGIKGLLSFPFDGTY 238

Bpi FMVRNIANNVPVFDKDKYAGVGAAIEYAVLHLKVENIVVIGHSACGGIKGLMSFPDAGPT 228

Pea STDFIEEWVKIGLPAKAK . VKAQHGDAPFAELCTHCEKEAVNASLGNLLTYPFVREGLVN 297

Bpi TTDFIEDWVKICLPAKHKVL. AEHGNATFAEQCTHCEKEAVNVSLGNLLTYPFVRDGLVK 287

Pea KTLALKGGYYDFVKGSFELWGLEFGLSSTFSV 329

Bpi KTLALQGGYYDFVNGSFELWGLEYGLSPSQSV 319

Figure 4. Comparson of the deduced amino acid sequences for peaand spinach CA. Numbering for the pea (Pea) and spinach (Spi) CAcDNAs start at the initiating methionine. Identity at the amino acidlevel is indicated by the symbol *. The N-terminus of the purifiedspinach protein, as determined by protein sequencing (4), is indicated.

DISCUSSION

In this study, we identified and characterized a cDNA clonecomplementary to the mRNA for pea leaf carbonic anhy-drase. The isolation of this clone was accomplished by im-munological techniques using an antibody directed againstthe purified CA monomeric species. The purified CA was

resolved as a 25.5 kD polypeptide with a minor band of 27.5kD. Previous studies have indicated that both chloroplasticand cytosolic forms of CA exist in pea; however, only singlemonomeric species of approximately 25 to 30 kD have beenreported when the isolated protein was electrophoresed underdenaturing conditions (2, 15). It is possible that the formationof the smaller, more abundant polypeptide in the doublet isthe result of proteolysis despite the presence ofprotease inhib-itors during the isolation procedure. An alternative explana-tion is that the 27.5 kD polypeptide represents a less abundant,additional CA isozyme (possibly cytosolic) with limited anti-genicity to the polyclonal antibody. The inability of the peaCA antibody to cross-react with a corn polypeptide supportsthe view that significant taxonomic diversity exists amonghigher plant carbonic anhydrases (16, 18). The identity of thetwo largest cDNA clones detected by cross-reactivity with thepea CA polyclonal antibody was verified after nucleotidesequence analysis and comparison with the amino acid se-

quence of an internal peptide fragment generated by CNBrdigestion of the purified enzyme. In addition, the recentlypublished sequence of a cDNA clone coding for spinach CA(4, 8) was found to be similar to the pea sequence (approxi-mately 76.5% identity at the amino acid level over the lengthof the mature spinach protein).Northern analysis of total mRNA indicates that the ap-

pearance of the pea CA transcript is light-regulated and leaf-tissue specific. Western blot analysis of green and etiolatedleaf tissue using the pea CA antibody supports these obser-vations. Previous studies have reported the presence of CAactivity in etiolated or root tissue (1 1, 13, 18). It is possiblethat species-specific variation or low light contamination ofgrowth conditions may account for these differences. It is alsopossible that our cDNA probe or polyclonal antibody are

unable to identify additional (nonchloroplastic) pea CAisozymes.

Several regions of the two higher plant CA proteins containstretches of amino acids which are identical and these maycontain functional domains such as zinc, inorganic carbon,or anion binding sites. The plant carbonic anhydrase aminoacid sequences were not found to exhibit significant homologyto a number of animal CA sequences. In particular, the activesite arrays of histidine residues, common to animal CA pro-teins, were not present (22). It is possible that until the activesite configuration of the plant protein is determined, similar-ities between plant and animal carbonic anhydrases will re-main elusive. Although we were unable to determine withcertainty the N-terminal sequence of the purified pea CA, thesize of the mature pea CA protein as well as N-terminalsequence analysis of the spinach CA indicate that both poly-peptides have very long putative transit peptides, in excess of100 amino acids (12, 14). In addition to the length, an arrayof eight consecutive Ser residues flanked by Thr residues, isanother unusual feature of the pea pre-sequence. It has beensuggested that the large number of acidic residues betweenamino acids 60 and 100 of the spinach polypeptide areunlikely components of a transit peptide and that this portionofthe pre-sequence is removed after uptake by the chloroplastand cleavage of the transit peptide (8). Similarly, the peasequence contains a large number of charged residues withinthis region (AA 60-104) and the typical Arg residue enrich-ment of the putative transit peptide C-terminus (immediatelyupstream of AA 104, Fig. 4) is also not apparent (12). Thesedata support the notion that the site oftransit peptide cleavagemay reside within the first 60 amino acids of the nascentpolypeptide (8).The observation of similarities between the plant CA and

the Escherichia coli cyanase permease sequences was verysurprising and is not yet fully understood. The permease isresponsible for transport of the cyanate ion (OCN-) acrossthe bacterial membrane, after which the cytosolic enzymecyanase catalyses its decomposition to bicarbonate and am-monia (20, 21). The structure of the cyanate ion may besufficiently similar to one of the inorganic carbon species oranions capable of binding to carbonic anhydrase. Cyanate is

CA QLGTTSSSDGIPKSEASERIKTGFLHFKKEKYDKNPALYGELAKGQSPPFMVF . ACSDSR 164

Per M.K .....E.I . ID.GFLKFQREAFPKREALFKQLATQQSPRTL. FISCSDSR 43

CA VCPSHVL.DFQPGKAFVVRNVANL.VPPYDQAKYAGTGAAIEYAVLHLKVSN.IVVIGHS 221* ** ** ** * ** * * * **** * ** ** ***

Per LVP. ELVTQREPGDLFVIRNAGN. IVPSYGPEP. GGVSASVEYAVAALRVS . DIVICGHS 99

CA ACGGIKGL..... LS.FPFDGTY.S.TD... FIEE.WVKIGLPAKAKVKA.QHGDAPFAE 268

Per NCGAMTAIASCQCMDHMPAVSHWLRYADSARVVNEARPHSDLPSKAAAMVRENVIAQLAN 159

CA LCTH. CEKEAVNASLGNLLTYPF. VREGLVNKTLALKGGYYDF . VKGSFELWGL. EFGLS 324

Per LQTHPSVRLALEEG. GSLHGWVYDIESGSI. . A. AFDGATRQFVPLAA. NPR. VCAIRLR 213

CA STFSV 329

Per QPTAA 218

Figure 5. Comparison of the deduced amino acid sequences for peaCA (CA) and the E. coli cyanate permease gene (Per). Numbering forthe pea CA mature protein is as described in Figure 3. Numberingstarts at the beginning of the open reading frame for the E. colipermease gene product. Identity at the amino acid level is indicatedby the symbol *.

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MAJEAU AND COLEMAN

known to be a potent inhibitor of mammalian CA (7 andreferences within) and does inhibit pea CA activity (JR Cole-man, unpublished data). We are currently investigating therelationship between CA activity and the ability to assimilateor metabolize cyanate.

Note Added in Proof

C. A. Roeske and W. L. Ogren have also recently identifiedand sequenced a pea cDNA clone coding for chlorplasticcarbonic anhydrase. The nucleotide and deduced amino acidsequences are presented in Nucleic Acids Research 18: 3413(1990).

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. Gloria Coruzzi, The Rock-efeller University, for the generous gift of the pea leaf cDNA library.

LITERATURE CITED

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2. Atkins CA, Patterson BD, Graham D (1972) Plant carbonicanhydrases. II. Preparation and some properties of monoco-tyledon and dicotyledon enzyme types. Plant Physiol 50: 218-223

3. Bailly J, Coleman JR (1988) Effect of CO2 concentration onprotein biosynthesis and carbonic anhydrase expression inChlamydomonas reinhardtii. Plant Physiol 87: 833-840

4. Burnell JN, Gibbs MJ, Mason JG (1990) Spinach chloroplasticcarbonic anhydrase. Plant Physiol 92: 37-40

5. Chua N-H, Bartlett S, Weiss M (1982) Preparation and charac-terization of antibodies to chloroplast proteins. In M Edelman,RB Hallick, N-H Chua, eds, Methods in Chloroplast MolecularBiology. Elsevier Biomedical Press, Amsterdam, pp 1063-1080

6. Coleman JR, Grossman AR (1984) Biosynthesis of carbonicanhydrase in Chiamydomonas reinhardtii during adaptationto low CO2. Proc Natl Acad Sci USA 81: 6049-6053

7. Deutsch HF (1987) Carbonic anhydrases. Int J Biochem 19: 101 -113

8. Fawcett TW, Browse JA, Volokita M, Bartlett SG (1990) Spin-ach carbonic anhydrase primary structure deduced from thesequence of a cDNA clone. J Biol Chem 256: 5414-5417

9. Feinberg AP, Vogelstein B (1984) A technique for radiolabelingDNA restriction endonuclease fragments to high specific activ-ity. Anal Biochem 137: 266-267

10. Fernley RT (1988) Non-cytoplasmic carbonic anhydrases. TrendsBiochem Sci 13: 356-359

11. Goushtina LM, Fournadjieva ST, Pesheva IS, Kudrev TG (1988)Two forms ofcarbonic anhydrase in pea roots. Comptes rendusde l'Acadamie bulgare des Sciences 41: 103-105

12. Heijne von G, Steppuhn J, Herrmann RG (1989) Domain struc-ture of mitochondrial and chloroplast targeting peptides. EurJ Biochem 180: 535-545

13. Kachru RB, Anderson L (1974) Chloroplast and cytoplasmicenzymes. V. Pea-leaf carbonic anhydrase. Planta 118: 235-240

14. Keegstra K, Olsen IJ, Theg SM (1989) Chloroplast precursorsand their transport across the envelope membranes. Annu RevPlant Physiol 40: 471-501

15. Kisiel W, Graf G (1972) Purification and characterization ofcarbonic anhydrase from Pisum sativum. Phytochemistry 11:113-117

16. Okabe K, Yang S-Y, Tsuzuki M, Miyachi S (1984) Carbonicanhydrase: Its content in spinach leaves and its taxonomicdiversity studied with anti-spinach leaf carbonic anhydraseantibody. Plant Sci Lett 33: 145-153

17. Owttrim GW, Coleman JR (1989) Regulation of expression andnucleotide sequence of the Anabaena variabilis recA gene. JBacteriol 171: 5713-5719

18. Reed ML, Graham D (1981) Carbonic anhydrase in plants:Distribution, properties and possible physiological roles. In LReinhold, JB Harborne, T Swain, eds, Progress in Phytochem-istry, Vol 7. Pergamon Press, Oxford, pp 47-94

19. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing withchain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467

20. Sung Y-C, Fuchs JA (1988) Characterization of the cyn operonin Escherichia coli K- 12. J Biol Chem 263: 14769-14775

21. Sung Y-C, Fuchs JA (1989) Identification and characterizationof a cyanate permease in Escherichia coli K-12. J Bacteriol171: 4674-4678

22. Tashian RE (1989) The carbonic anhydrases: Widening perspec-tives on their evolution, expression and function. BioEssays10: 186-192

23. Tingey SV, Walker EL, Coruzzi GM (1987) Glutamine synthe-tase genes of pea encode distinct polypeptides which are differ-entially expressed in leaves, roots and nodules. EMBO J 6: 1-9

24. Tsuzuki M, Miyachi S, Edwards GE (1985) Localization ofcarbonic anhydrase in the mesophyll cells of terrestrial C3plants in relation to CO2 assimilation. Plant Cell Physiol 26:881-891

25. Wilbur KM, Anderson NG (1948) Electrometric and colorimetricdetermination of carbonic anhydrase. J Biol Chem 176: 147-154

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