dnas diphosphate-diacylglycerol synthases of two plant species · diphosphate-diacylglycerol...

Plant Physiol. (1 997) 11 3: 997-1 002

Rapid Communication

Complementary DNAs Encoding Eukaryotic-Type Cytidine-5’- Diphosphate-Diacylglycerol Synthases of Two Plant Species

Joachim Kopka, Marion Ludewig, and Bernd Miiller-Rober*

Institut für Genbiologische Forschung Berlin, lhnestrasse 63, D-I 41 95 Berlin, Germany (J.K., M.L.); and Max- Planck-lnstitut für Molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, Haus 20, D-14476

Golm/Potsdam, Cermany (B.M.-R.)

Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triph0sphate:phosphatidate cytidyltransferase, EC 2.7.7.41) catalyzes the formation of CDP-diacylglycerol, which is the precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We report the first cloning, to our knowledge, of two plant cDNAs, StCDSl and AtCDS1, coding for CDP-diacylglycerol synthases from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively. The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthases and contain eight predicted transmembrane-spanning domains. We analyzed gene expression in shoot and root tissues of potato plants and demonstrated enzyme activity by expression of N-terminally truncated, recombinant StCDSl in Escherichia coli.

CDS (cytidine triph0sphate:phosphatidate cytidyltransferase, EC 2.7.7.41) catalyzes the synthesis of CDP-DG and PPi from PA and CTP. CDP-DG is the precursor of the minor lipids PI, PG (via PG phosphate), cardiolipin, and possibly phosphatidylserine (Moore, 1982). CDS activity is present in most plant membrane systems. Located at the inner envelope membrane of chloroplasts, CDS may be mainly engaged in PG synthesis (Andrews and Mudd, 1985). In mitochondria, CDP-DG is the substrate for PG phosphate synthase (EC 2.7.8.5) and cardiolipin synthase. CDS and other enzymes involved in cardiolipin biosynthesis are enriched at the inner mitochondrial membrane (Frentzen and Griebau, 1994). Microsomes isolated from castor bean endosperm contain approximately 75% of total extractable CDS activity (Kleppinger-Sparace and Moore, 1985). Microsomal membranes might account for most of the PI synthesis in plant cells (Moore, 1982). Studies on microsomes isolated from spinach leaves also suggest the presence of phosphatidylserine synthase (EC 2.7.8.8), which utilizes CDP-DG (Marshall and Kates, 1974). More- over, CDS and other enzymes necessary for PI resynthesis from phospholipase C and D reaction products are present in plant plasma membranes (Wissing et al., 1992). As demonstrated by the recent discovery of an eye-specific CDS from Drosophila melanogaster, CDS activity is essential for IP,-mediated light perception in insect eyes (Wu et al.,

* Corresponding author; e-mail berndampimp-golm.mpg.de; fax 49-331-977-2301.

997

1995). Ir,-mediated signal transduction is operative in plant cells (reviewed by Dr~bak, 1992). For this reason, plant CDS might not only be involved in the biosynthesis of minor phospholipids, but might also modulate Ir,- mediated signal transduction in plants.

To investigate the role of CDS in plants we isolated and characterized the first plant cDNA clones of this enzyme. We present the predicted primary structures and properties of CDS from potato (Solanum tuberosum) (StCDSl) and Arabidopsis tkaliana (AtCDSl), a preliminary analysis of gene expression in potato plants, and enzyme activity of recombinant StCDSl.

MATERIALS A N D METHODS

Cloning and Sequencing of cDNAs

A TBLASTN search (Altschul et al., 1990) for CDS homologous plant sequences was performed in the EST database at the National Center of Biotechnology Information (Bethesda, MD). Two overlapping ESTs from Arabidopsis tkaliana, T45653 and N97146, were found with a “bait“ amino acid sequence of CDS from Esckerickia coli. The ESTs contained a continuous open reading frame that encoded a partia1 protein with highest sequence homology to the CDS of Drosophila melanogaster (Wu et al., 1995) and Sacchnromy- ces cerevisiae (Shen et al., 1996). A 317-bp molecular probe was generated by PCR with forward primer 5’- GGCCTTTAATAAAGCTGTCTCC-3’ and reverse primer 5’-TACCGAAAAACCCCCCAAAAGG-3’ from 50 ng of first-strand cDNA, which was prepared from poly(A)+ RNA of rosette leaves of A. tkaliana ecotype C-24. The PCR fragment was subcloned into the pCR plasmid (Invitrogen, San Diego, CA) and sequenced. A [ c~-~*P]dCTP-Iabeled probe (Random Primed DNA labeling kit, Boehringer Mannheim) was used to isolate full-Iength cDNAs from two AZAPII cDNA libraries. These libraries were prepared from poly(A)+ RNA of flowers and siliques of A. tkaliana ecotype C-24 or from poly(A)* RNA of epidermal fragments isolated from potato (Solanum tuberosum L. cv Dé-

Abbreviations: CDS, CDP-diacylglycerol synthasets); DG, diacylglycerol; EST, expressed sequence tag; GST, glutathione S-transferase; IP,, inositol-1,4,5-triphosphate; IPTG, isopropyl-p-D-galac- topyranoside; PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol.

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998 Kopka et al. Plant Physiol. Vol. 11 3 , 1997

sirée) leaves (Miiller-Rober et al., 1995). Hybridization of plaque lifts was performed at 42°C overnight in PEG buffer (Amasino, 1986). Filters were washed either at high stringency (65°C in 3X SSC, 0.5% SDS for 20 min, and in 0.2X SSC, 0.5% SDS for 30 min) or at low stringency (45°C in 5X SSC for 15 min, and in 5X SSC, 0.5% SDS for 30 min) when screening the cDNA libraries of Arabidopsis or potato, respectively. pBluescriptI1 SK+ plasmids containing target inserts were obtained from isolated phages by in vivo excision according to the manufacturer's protocol (Strat- agene). The longest cDNAs encoding CDS from Arabidop- sis and potato, AtCDSl and StCDSZ, respectively, were sequenced (T7 sequencing kit, Pharmacia). Standard molecular biology methods were performed as described previously (Sambrook et al., 1989).

Computational Analysis of Predicted Amino Acid Sequences

The computational services and options of the Wisconsin Package Version 8.1 (Genetics Computer Group, Madison, WI) were used with default parameters. Analysis of amino acid sequence homology was performed with the BLAST program. Multisequence alignments were created by the PILEUP option, and amino acid conservation was analyzed by the PRETTYBOX program. The dendrogram illustrating the degree of homology between CDS proteins of different organisms (Fig. 1) was calculated by the GROWTREE algorithm according to the unweighted pair group method. The calculation was based on the distance matrix of a multisequence alignment and was corrected by the Kimura method for protein sequences. Percentage of sequence identity and similarity was determined by pairwise alignment using the BESTFIT program. Hydropathy plots were generated according to the algorithm of Kyte and Doolittle (1982), and transmembrane spanning domains were predicted by submitting multisequence alignments to TMAP- analyses at the World Wide Web server of the European Molecular Biology Laboratory (Heidelberg, Germany). Subcellular sorting was predicted at the PSORT World Wide Web server for analyzing and predicting protein- sorting signals at the Institute for Molecular and Cellular Biology (Osaka, Japan).

249aa Escherichia coli P06466 288aa Haemophilus influenzae P44937 271aa Pseudomonas aeruginosa D50811 270aa Brucella aboltus U51683 447aa Drosophila melanogaster S52437 455aa Caenorhabditis e/egans U41007 421 aa Arabidopsis thaliana X94306

424aa Solanum tuberosum X91909 457aa Saccharomyces cerevisiae P38221 rl' 305aa Mycoplasma genifalium U39730

Figure 1. Dendrogram illustrating the degree of homology between presently known CDS proteins. The number of amino acids (aa), source organisms, and database accession numbers are listed.

RNA Blot Analysis

RNA blot analysis was performed as described previously (Fieuw et al., 1995). The following DNA probes were used for hybridizations: (a) the 2.0-kb KpizI/BamHI fragment of the StCDSl plasmid, containing the complete StCDSZ insert; (b) the 0.52-kb Eco47III/XhoI fragment of the StCDSl plasmid, containing the 3' untranslated region of the cDNA; and (c) the 3.5-kb KpnIIBamHI fragment of plasmid efEST G56 (accession no. R28706), coding for 25s rRNA of potato. Labeling of cDNA fragments, hybridization, and wash at high stringency was performed as described above. Autoradiography was performed with X-Omat AR film (Kodak) at -70°C.

Analysis of Recombinant StCDSl Expressed in E. coli

Recombinant plant CDS was expressed in E. coli BL21 cells as a GST-CDS fusion protein using the pGEX-4-T2 fusion protein vector (Pharmacia), which provides an N-terminal GST tag. DNA fragments of the complete coding regions of StCDSl and AtCDSZ and of codons 108 to 424 of StCDSZ were amplified by PCR using Taq DNA polymerase. The forward and reverse PCR primers intro- duced BamHI and XkoI restriction sites, respectively, which were used for oriented, in-frame cloning of the PCR fragments into pGEX-4-T2. The cloning strategy preserved the native stop codons of CDS, but replaced the N-terminal Met residue by a Gly-Ser dipeptide, which linked GST to recombinant CDS. The pGEX-4-T2 vector containing the truncated StCDSl cDNA (corresponding to amino acids 108424) was designated pGEXstCDS1. Four to eight inde- pendently transformed E. coli BL21 lines for each of the three vectors were screened for expression of fusion protein. To demonstrate CDS activity, an E. cozi BL21 line, which carried the pGEXstCDSl plasmid, was selected. A 1000-mL culture was grown at 28°C to A,,, = 0.3 and divided into two equal cultures, one of which was induced with 5 mM IPTG. Both cultures were subsequently incu- bated at 28°C and harvested at = 0.7. A 500-mL culture of an E. coli BL21 line transformed with control plasmid pGEX-4-T2 was also induced with IPTG and grown under identical conditions. The three E. coli preparations were designated pGEXstCDSl - IPTG, pGEX- stCDSl + IPTG, and pGEX-4-T2 + IPTG (see Table I). The recombinant proteins were affinity-purified according to the manufacturer's instructions (Pharmacia). The volume of the three final glutathione eluates containing the affinity-purified proteins was 0.3 mL.

CDS was assayed according to the method of Sparrow and Raetz (1985). The reaction was performed in a volume of 0.05 mL, and contained 0.1 M Tris-HCI, pH 7.5, 0.2 M

KCl, 10 mM MgCI,, 1 mg/mL BSA, 5 mM Triton X-100,0.25 mM DTT, 1 mM PA (P9511, Sigma), and 5 mM [a-32P]CTP (2,000-10,000 cpm/nmol). The reaction was stopped by 1 mL of ch1oroform:methanol (1:1, v/v), and phase separa- tion was achieved by the addition of 0.5 mL of 10 mM HCI and 1 M NaCl. The aqueous supernatant was aspirated, and 0.4 mL of the organic phase was subjected to scintillation counting. The labeled lipid product was analyzed by TLC



Plant CDP-Diacylglycerol Synthase 999

Table 1. Enzymatic analysis of representative glutathione eluates prepared from E. coli harboring plasmids pGEX-4-TZ or pGEXstCDS1 E. colicultures were either kept without IPTG (-IPTG) or treated with 5 mM IPTC (+IPTG) to induce production of recombinant CST-StCDS1

fusion protein. Unmodified CST was produced from pCEX-4-T2-containing E. coli cultures treated with IPTG. Clutathione eluates, which contained affinity-purified protein, were prepared as described in ”Materials and Methods.” Note that affinity-purified fusion proteins always contained monomeric GST (see Fig. 4).

Clutathione Eluates

pCEX-4-T2 + IPTC pCEXstCDSl - IPTC pCEXstCDSl + IPTC Parameter

Total protein (mg) 0.580 0.024 0.1 70 GST specific activity (pmola min-’ mg-’) 5.05 3.10 2.93 CDS specific activity (pmolh min-’ mg-’ X 106) 0.57 76.0 1200.0

a Reaction product, 2,4-dinitrobenzene-I -glutathione conjugate. ’ Reaction product, CDP-DC.

on silica gel plates (type Si 250 PA, J.T. Baker) and co- migration with authentic lipid standards. Reference lipids were PA (P9511, Sigma), 1,2-dioleoyl-sn-glycerol (D0138, Sigma), and CDP-1,2-dipalmitoyl-sn-glycerol (C3263, Sig- ma). The solvent system of Ganong et al. (1980), i.e. chloroform:methanol:acetic acid:water (25:15:4:2, v/v), was applied.

Protein concentration was determined by the bicincho- ninic acid method after masking sulfhydryl groups with iodoacetamide (Hill and Straka, 1988). Expression of fusion protein was monitored by GST activity with 1-chloro-2,4- dinitrobenzene (Habig et al., 1974). SDS-PAGE was performed on 12.5% homogenous gels according to the method of Laemmli (1970). Polyacrylamide gels were either silver-stained (Gottlieb and Chavko, 1987) or blotted onto nitrocellulose membranes. Western blots were performed according to the method of Landschütze et al. (1995) with a polyclonal anti-GST antibody (Pharmacia).

RESULTS AND DlSCUSSlON

The first evidence for the presence of a CDS homolog in plants was found in an EST database search using the amino acid sequence of CDS from E. coli as bait. Two overlapping ESTs from A. thaliana that encoded a partia1 protein highly homologous to known eukaryotic CDS were identified. The EST sequence information was used to am- plify a corresponding PCR fragment from first-strand cDNA of A . thaliana. This molecular probe was then suc- cessfully employed to clone a complete cDNA from A. thaliana, A t C D S l , and a homologous cDNA from potato, StCDSZ. The StCDSl and AtCDSZ cDNA clones were com- posed of 2014 and 1843 bp, respectively. Both cDNAs contained one long, continuous open reading frame, which was preceded by a stop codon in the same reading frame as the first ATG codon. This observation indicated that the cDNAs were full-length. The StCDSl cDNA coded for a 424-amino acid protein with a predicted molecular mass of 49.1 kD. The deduced AtCDSl protein consisted of 421 amino acids and had a putative molecular mass of 48.6 kD. The size of the predicted plant CDS was similar to that of other eukaryotic CDS proteins, whereas prokaryotic CDS proteins were smaller (Fig. 1). Like most CDS proteins, StCDSl and AtCDSl had high calculated pI values: 10.4 and 9.9, respectively. As illustrated in Figure 1, StCDSl and AtCDSl were highly homologous to each other, exhibiting 75.2% amino acid identity. Both proteins shared approxi-

mately 40% amino acid identity with eukaryotic CDS proteins from yeast, Caenorhabditis, and Drosophila. In contrast, plant CDS had only 22.1 to 31.3% amino acid identity with prokaryotic CDS. However, 21 amino acid residues were invariable in all presently known CDS proteins (Fig. 2). Analysis of the hydropathy profile of StCDSl (Fig. 2) and AtCDSl (not shown) indicated the presence of eight transmembrane-spanning regions (TM1-TM8). This observation is in agreement with previous protein biochemical data, which indicated that CDS is an integral membrane protein (Sparrow and Raetz, 1985; Wissing et al., 1992).

Comparison with prokaryotic CDS demonstrated that eukaryotic CDS proteins contain a highly variable, hydro- philic N-terminal extension. These comparisons also showed that conservation of amino acid residues coincide mainly with the linking regions between TM5 and TM6 and those between TM7 and TM8, and with the predicted transmembrane domain, TM7 (Fig. 2). Residues 317 to 382 of plant CDS (representing TM7-TM8) shared 21% amino acid identity (48% similarity) with residues 31 to 90 of PG phosphate synthase of E. coli. This region of PG phosphate synthase contains a CDP-alcohol phosphatidyltransferase signature that is present in a number of phosphatidyltrans- ferases (Hjelmstad and Bell, 1991). However, within plant CDS, this consensus pattern itself is not conserved. The sequence homology detected between plant CDS and E. coli PG phosphate synthase might nevertheless indicate that the TM7 to TM8 region of the plant enzymes forms part of the reaction center.

Analysis of protein-sorting signals with the PSORT program (see “Materials and Methods”) did not reveal poten- tia1 mitochondrial or chloroplast targeting sequences in either of the plant CDS proteins. In contrast, the PSORT program predicted possible localization within the eukaryotic membrane system, with the highest probability for accumulation at the plasma membrane (not shown). How- ever, we were not able to reliably identify endoplasmic signal sequences in plant CDS.

RNA blot analysis of StCDSZ gene expression showed a transcript size of 2.3 kb (Fig. 3). StCDSl transcripts were detectable in all preparations of total RNA from various potato tissues. The highest steady-state mRNA levels were in roots and sink leaves. Epidermal fragments, which are strongly enriched in guard cells (Miiller-Rober et al., 1995), contained low amounts of StCDSl mRNA. We initially used the complete StCDSl cDNA as a molecular probe.



1000 Kopka et al. Plant Physiol. Vol. 113, 1997

AtCDS . .StCDS. .ScCDS. .

DmCDSM t

AtCDS L V NSICDS L V NScCDS A T K

DmCDSECCDS

I I S A F V MA N 1 H|G R F PI I S A F L HA N M WQ R F O W

W F F MA L A S I I BT R I MJS P Y T YG G F AjHiv L F G 1 L jgS Y V He N Y O Y FG L A MA A VI S W G Y

AtCDS LStCDSHScCDS

DmCDSI S _EcCDS F R T

Figure 2. Hydropathy plot of the deduced amino acid sequence of CDS from potato (StCDSl) and multisequence alignmentof StCDSl with CDS protein sequences from A. thaliana (AtCDSI), 5. cerevisiae (ScCDS), D. melanogaster (DmCDS), andE. coll (EcCDS). Predicted transmembrane-spanning domains (TM1-TM8) are indicated by horizontal bars. Amino acidresidues conserved in more than half of the shown sequences are shaded in black; conservative substitutions are marked ingray. Amino acid residues, which are invariably present in all CDS proteins (see Fig. 1), are indicated by arrows.

However, human ESTs indicated that eukaryotic organ-isms might express at least two different CDS isoforms (forexample, compare ESTs R27966 and W26557). For this rea-son, we repeated RNA blot hybridizations with a morespecific probe generated from the 3' untranslated region ofthe StCDSl cDNA. This probe exhibited the same hybrid-ization pattern (not shown) as the full-length probe andthus confirmed that the expression pattern shown in Figure3 was specific for StCDSl. The 25S rRNA signal intensities

StCDSl

25S rRNA

- 2.3kb

- 3.5kb

Figure 3. RNA blot analysis of StCDSl gene expression in varioustissues of flowering potato plants. Total RNA (50 /xg/lane) was probedwith the complete StCDSl cDNA and with a cDNA encoding 25SrRNA of potato (see "Materials and Methods"). Sizes of transcripts areindicated.

of each sample demonstrated that approximately equalamounts of total RNA were analyzed (Fig. 3).

From preliminary experiments it appeared that the Nterminus of StCDSl suppressed efficient protein expressionin yeast. For this reason, we decided to express recombi-nant StCDSl and AtCDSI in E. coli. Because £. coli containsendogenous CDS activity (Sparrow and Raetz, 1985), weselected an N-terminal GST tag for affinity purification ofrecombinant plant CDS (see "Materials and Methods").When the complete StCDSl and AtCDSI proteins werefused to GST, SDS-PAGE analyses showed only short, trun-cated fusion proteins. Translation appeared to terminatewith high probability after the first 3 and 2 kD of StCDSland AtCDSI, respectively (not shown). Because both plantCDS coding regions contained multiple AGA and AGGcodons, which are low-usage codons for Arg in E. coli, wecloned the GST fusion constructs into the pSBET vectordesigned for concomitant overexpression of the argU geneof the rare tRNAarg4/ which mediates translation of theAGA and AGG codons in £. coli (Schenk et al., 1995). Thisattempt increased the amount but not the size of GSTfusion proteins (not shown). We were finally successful bydeleting the N terminus of StCDSl. Comparison with pro-karyotic CDS indicated that the N termini of eukaryoticCDS proteins might not be required for enzyme activity. Afusion protein of GST and residues 108 to 424 of StCDSl,



Plant CDP-Diacylglycerol Synthase 1001

£

B

Figure 4. SDS-PACE analysis of the glutathione eluates analyzed inTable I. The eluates contain recombinant GST (pGEX-4-T2 + IPTG)and GST-StCDS1 fusion protein prepared from E. coll cultures with-out (pGEXstCDSI - IPTG) and with (pGEXstCDSI + IPTG) inductionby IPTG. A, Silver stain; B, western blot using a polyclonal anti-GSTantibody (Pharmacia). Arrows indicate positions of two GST-StCDS!fusion proteins (61 and 70 kD).

which was produced by E. coli cells transformed with theIPTG-inducible plasmid pGEXstCDSI (see "Materials andMethods"), was active (Table I). When glutathione eluatescontaining affinity-purified fusion protein were preparedunder identical conditions, induction of protein expressionwith IPTG resulted in a 112-fold increase in total CDS activ-ity (not shown) and a 16-fold increase in specific CDSactivity of the glutathione eluate, whereas specific GSTactivity remained unchanged (Table I). This observationwas made in three independent purification experiments.The phenomenon can be explained by assuming that underboth conditions a certain amount of the endogenous GST ofE. coli (Nishida et al., 1994) is co-purified. Under non-IPTG-induced conditions, GST from E. coli should make no dif-ference to specific GST activity, but, rather, should exert astrong effect on specific CDS activity of the preparation.

1200

1000

f 'a, 800*- E

600

E

200

0,5phosphatidic acid [mM]

Figure 5. Substrate dependence of CDS activity in glutathione eluatecontaining recombinant GST-StCDS1 . Reactions were performed asdescribed in "Materials and Methods" in the presence of varyingconcentrations of PA. Data represent means ± SE (n = 3).

A B1 2 2

(t=0) (t=180) (t=180) Rf

0.98 DG

0.90 PA

0.63 CDP-DG

Figure 6. Thin-layer chromatograms of the lipophilic reaction prod-uct of GST-StCDS1 activity. CDS reactions were started with a-32P-labeled CTP and either stopped immediately (lane 1, t = 0 min) orafter 3 h (lanes 2, t = 180 min). The reaction product was extractedwith chloroform:methanol (1:1, v/v). To each extract was added 60j^g of a lipid mixture containing approximately equal amounts ofnonlabeled DG, PA, and CDP-DG. Preparations were dried under N2

and applied quantitatively onto TLC plates. Lipids were separated bychloroform:methanol:acetic acid:water (25:15:4:2, v/v) and visual-ized by autoradiography (A, lanes 1 and 2) or subsequent treatmentwith 50% H2SO4 (B, lane 2). CTP was immobile under the conditionsapplied (not shown). Rf, Retention factor.

For comparison with the GST-StCDSl fusion protein, un-altered, recombinant GST was prepared under identicalconditions from IPTG-induced cells transformed with thepGEX-4-T2 plasmid. The resulting glutathione eluate con-tained only GST (Fig. 4) and had high specific GST activity,but CDS activity was hardly detectable (Table I), demon-strating that unmodified GST had no CDS activity. CDSactivity of the glutathione eluate containing GST-StCDSlfusion protein was absolutely dependent on the presence ofPA (Fig. 5). In addition, a lipophilic product accumulatedwith reaction time (Fig. 6A). This lipid co-migrated withauthentic CDP-DG on thin-layer chromatograms (Fig. 6B).SDS-PAGE and western blot analysis of the affinity-purified fusion proteins (Fig. 4) exhibited the presence oftwo inducible fusion proteins with apparent molecularmasses of 61 and 70 kD, respectively.

In conclusion, to our knowledge, we have cloned the firstplant cDNAs encoding CDS. In exemplary studies we dem-onstrated SfCDSI gene expression and enzyme activity of aplant CDS. Thus, it is now possible to investigate the role ofCDS in plants by overexpression and antisense inhibitionof CDS gene expression. With the help of transgenic plantswe might be able to obtain new insights into the function ofthe minor lipids PI and PG and into the role of CDS insignal transduction events in higher plants.

ACKNOWLEDGMENTS

The authors wish to thank Ursula Uwer and Nicholas J. Provart,(Institut fur Genbiologische Forschung, Berlin, Germany) for pro-



1002 Kopka et al. Plant Physiol. Vol. 11 3 , 1997

viding the AZAPII cDNA libraries. We wish to thank Lothar Willmitzer for his support in recent years.

Received August 16, 1996; accepted November 20, 1996. Copyright Clearance Center: 0032-0889/97/ 113/0997/06. The accession numbers for the sequences reported in this article

are X94306 (A tCDS1) and X91909 (StCDS1).

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