Identification and expression of a cDNA encoding cystathionine γ-synthase in soybean

Plant Science 146 (1999) 69 – 79 www.elsevier.com/locate/plantsci

Identification and expression of a cDNA encoding cystathionine g-synthase in soybean Cleo A. Hughes a, Joan S. Gebhardt b, Andree´ Reuss b, Benjamin F. Matthews b,* b

a Department of Biology, Morgan State Uni6ersity, Baltimore, MD 21251, USA U.S. Department of Agriculture, Agricultural Research Ser6ice, Soybean and Alfalfa Research Laboratory, Building 006, Belts6ille, MD 20705, USA

Received 16 December 1998; received in revised form 19 March 1999; accepted 25 March 1999

Abstract Methionine is one of the essential amino acids that are synthesized in low amounts in seeds of many agronomically important crop plants such as soybean. Cystathionine-g-synthase (CS; EC 4.2.99.9) is the branch point enzyme leading to methionine synthesis. We isolated a 1023 nucleotide cDNA-encoding soybean CS from a leaf cDNA library using a 672-nucleotide Arabidopsis cDNA probe. The complete cDNA contains a single open reading frame of 1608 bp that encodes a 536 amino acid protein with a predicted molecular mass of 58 090 Da. The amino terminal portion of the deduced amino acid sequence is rich in threonine and serine, suggesting the presence of a chloroplast transit peptide (155 amino acids). The soybean CS amino acid sequence shares sequence identity with several CS proteins, Arabidopsis thaliana, Z. mays, M. crystallium, and Escherichia coli. The coding region minus the transit peptide was cloned in-frame into pUC18. This construct was used to transform and complement an E. coli methionine auxotroph, AB301. Northern analysis revealed that a 1.9 kb soybean CS mRNA was expressed at the highest level in 8-day light-grown cotyledons and the lowest level in 8-day dark-grown leaves. Southern analysis suggests that the multiple banding patterns may be indicative of several restriction sites within the genomic sequence or CS may be part of a small gene family. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cystathionine g-synthase; Methionine; cDNA; Complementation; Expression; Glycine max

1. Introduction In plants, the essential amino acids, methionine, lysine, threonine and isoleucine are all synthesized from aspartate via a common pathway. These amino acids are required in the diets of nonruminant animals to ensure proper growth and development of these animals [1,2]. Legumes, such as soybean, are used as feed for nonruminant animals, but are nutritionally low in the amino acid methionine. In order to compensate for the low methionine content, methionine or methioninecontaining supplements are mixed with soybean meal to improve the nutritional quality of the * Corresponding author. Tel.: +1-301-5045730; fax: + 1-3015045320. E-mail address: [email protected] (B.F. Matthews)

meal. Supplementation of the meal with amino acids is costly and therefore, increasing the amount of methionine through genetic engineering may be a more cost-effective means of providing meal with the proper balance of amino acids. The essential amino acids lysine, threonine, isoleucine, and methionine share the first two steps in the aspartate pathway. Aspartate is converted into aspartate semialdehyde which serves as a common substrate for two enzymes: dihydrodipicolinate synthase, DHPS [3–5] and homoserine dehydrogenase, HSDH [3,6] (Fig. 1). Homoserine is phosphorylated by homoserine kinase to produce o-phospho-homoserine. Cystathionine-g-synthase (CS; EC 4.2.99.9) is the first enzyme in the pathway dedicated to the synthesis of methionine (Fig. 1). CS catalyzes the condensation of o-phospho-homoserine with cysteine to form cystathion-

0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 0 5 2 - 7

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ine. Following the condensation reaction, cystathionine b-lyase divides cystathionine into homocysteine and serine. Finally, methionine is synthesized formed by methylation of homocysteine from N 5-methyltetrahydrofolate [3,6,7]. Lysine is produced when aspartate semialdehyde is converted to dihydrodipicolinic acid via the branch point enzyme, DHPS. Aspartate semialdehyde can also be converted into homoserine, which is a precursor for both methionine and threonine synthesis. Threonine and isoleucine production diverge from that of methionine at ophosphohomoserine. Threonine synthase is the first enzyme committed to threonine and isoleucine production [3,6]. In higher plants, CS has been partially characterized at the biochemical and genetic levels. CS is localized in the chloroplast [8] and was purified to apparent homogeneity from spinach chloroplasts [9]. Under nondenaturing conditions the enzyme has an apparent molecular weight of 215 000 Da and consists of subunits ranging in size of 50–53 kDa [9]. CS also was partially purified from an acetone powder prepared from wheat. The wheat enzyme is a tetramer of 155 kDa and apparently

consists of four identical subunits of 34.5 kDa [10]. Studies by Kreft and others [10] demonstrated that the wheat CS enzyme requires pyridoxal phosphate as a cofactor for activity. At the genetic level, an Arabidopsis genomic clone containing a portion of the gene encoding CS was isolated, but the clone lacked the 5% end and contained 669 nucleotides of the coding region which included the 3% end of the gene [11]. Recently, two cDNAs were reported to encode Arabidopsis CS [12]; GenBank accession numbers U43709 and U62147. The cDNAs are almost completely identical and U62147 is referred to as the precursor CS gene whereas U43709 represents the cDNA that was further characterized. The complete Arabidopsis thaliana CS cDNA was isolated by complementation of a CS deficient mutant of Escherichia coli. The CS coding region was 1692 bp and encodes a 60 kDa protein [12]. More recently, the cDNA encoding CS from Arabidopsis was cloned and used to overexpress the CS enzyme in E. coli [13]. The recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5% phosphate. The biochemical [8–10,13] and molecular evidence ([12]; this paper) indicate that pyridoxal phosphate is an important cofactor for CS enzyme activity. Since the levels of methionine are limiting in soybean, we decided to study the first enzyme, CS, dedicated to methionine synthesis at both the genetic and biochemical levels in soybean. The information gathered may be used to devise ways of increasing methionine levels in soybean. Herein, we report the isolation and expression of a soybean (Glycine max) cDNA encoding CS and demonstrate its complementation of an E. coli auxotroph lacking CS activity.

2. Materials and methods

2.1. Material

Fig. 1. Pathway leading to the synthesis of methionine from aspartate. Abbreviations: AK, aspartokinase; ASDH, aspartate semialdehyde dehydrogenase; DHPS, dihydrodipicolinic acid synthase; HSDH, homoserine dehydrogenase; CS, cystathionine g-synthase; TS, threonine synthase.

Soybean (Glycine max L. cv. Century) seeds were germinated in vermiculite in the greenhouse. Dark-grown seedlings were grown in vermiculite in boxes wrapped in dark plastic bags. The leaf tissue used as the source of mRNA for cDNA synthesis was grown on water-saturated paper towels in the green house [14]. The Arabidopsis

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‘Columbia’ cDNA was synthesized from mRNA extracted from whole plants [15]. E. coli cells, XL1Blue, pBluescript, and Pfu polymerase were obtained from Stratagene (La Jolla, CA) and the cystathionine g-synthase auxotroph (AB301, metB relA spoT1) [16] was obtained from E. coli Genetic Stock Center [17]. DH5a cells, restriction enzymes, T4 DNA ligase, alkaline phosphatase from calf intestine, and Taq DNA polymerase were purchased from Life Technologies (Gaithersburg, MD). pCRBlunt was purchased from Invitrogen (Carlsbad, CA). pT7Blue was obtained from Novagen (Madison, WI). [a32 P]dCTP was purchased from DuPont–New England Nuclear (Boston, MA). Marathon kit was obtained from Clontech, Palo Alto, CA. Oligonucleotides were synthesized on an DNA synthesizer (Applied Biosystems). Common chemicals were purchased from Amresco (Solon, Ohio).

2.2. Isolation of soybean CS cDNA clone Oligonucleotide primers [708 5%-GTT TCA AAA ATA TGC CAC AAG AGG GG-3%] and [707 5%-GAT GGC TTC GAG AGC TTG AAG AAT GTC-3%] were synthesized to regions that corresponded to the ends of the published, partial Arabidopsis CS DNA sequence [11]. Amplification was conducted by dissociating Arabidopsis cDNA at 94°C for 2 min, then the reaction was cycled 35 times at 92°C for 45 s, 60°C for 15 s and 72°C for 1 min with a final extension time of 6 min. A 672 nucleotide amplification product was generated using primer 708 and primer 707 and was partially sequenced to confirm its identity. A soybean cDNA library was synthesized from mRNA isolated from 14-day-old leaves was constructed in lZap as described previously [18]. A portion of the coding region of Arabidopsis CS was amplified from cDNA by PCR and used as a probe to screen 500,000 clones from the soybean cDNA library. The product was radiolabeled with [a-32P]dCTP (3000 Ci/mmol) by the random primed labeling method [19]. The DNA sequences of the inserts from cDNA clones that hybridized to the Arabidopsis CS probe were determined by the using the Dye terminator method [20]. One clone CS2.2A which hybridized to the Arabidopsis CS cDNA probe was chosen for further study. Phagemid DNA (pBluescript) containing the pCS2.2A cDNA insert was isolated from

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lambda CS2.2A through the excision process by co-infection with helper phage (M13KO7). DNA sequencing analysis was performed in both directions on double stranded plasmid DNA. Sequence data comparisons with the Arabidopsis CS cDNA sequence [12] revealed that the 5% end of the gene was missing. The 5% end of the soybean CS cDNA was isolated by 5% RACE (Rapid Amplification of cDNA Ends) [21] using the Marathon kit (Clontech, Palo Alto, CA). cDNA was synthesized from mRNA isolated from 7-day dark-grown cotyledon. Following cDNA synthesis, the Marathon cDNA adapter was ligated to ds cDNA. After the ligation of the adapter, 1 ml of the reaction was diluted in 50 ml of Tricine–EDTA buffer. Five microliters of adapter ligated cDNA was amplified using adapter primer AP1 [5%-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3%) and gene specific primer 824 [5%-GCA GCA TTC GGG TTA AGT GTA CC-3%]. A nested PCR was performed using 10 ml of primary reaction along with the nested adapter and gene-specific primers AP2 [5%-ACT CAC TAT AGG GCT CGA GCG GC3%) and 823 (5%-GAG ACA CAT TGT GCT GCT CCA ATG C-3%]. The PCR product generated was approximately 411 bp. The product was cloned into pT7 vector (Novagen, Madison, WI) and sequenced. This product only added 338 bp to the 5% end of the soybean CS gene and the ATG start site was still missing. In comparison to the Arabidopsis CS sequence, about 500–550 bp were lacking. Another 5% RACE procedure was performed using cDNA that was synthesized from mRNA extracted from 4-day leaf and cotyledon. The adapter-ligated cDNA was amplified using primers AP1 [5%-CCATCCTAATACGACTCACTATAGGGC-3%] and 922 [5%CCCAGTCTTTCAGCGGC-3%]. The 5% RACE PCR product was about 567 bp in length. This product was cloned and sequenced. This provided an additional 522 bp from the 5% end. This sequence included the ATG start site and the remainder of the transit peptide sequence. DNA and amino acid sequences were analyzed by computer using the Wisconsin Genetics Computer Group programs: GAP [22] and PILEUP [23].

2.3. Construction of cystathionine-g-synthase expression plasmid The region encoding the predicted mature CS protein was amplified by polymerase chain reac-

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tion (PCR). One oligonucleotide primer, 998 [5%CAG CAT CTA GAG GCG GCT GAA AGA CTG GGT AGG-3%], was synthesized to a region that corresponded to the predicted amino terminus of the mature CS protein (553–570 nt) with a 5% extension of 15 nucleotides containing a XbaI restriction endonuclease site for cloning. The inverse oligonucleotide primer 960 [5%-GAC AAG CTT GAG CAG GAA AAC TGT CTA TAT AGC TTC C-3%], (1686–1710 nt) was designed to have a 5% extension of 12 nucleotides including a HindIII restriction site. The PCR reaction, 50 ml, contained 100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris – HCl pH 8.75, 20 mM MgSO4, 1% Triton X-100, 1000 mg/ml BSA, 200 mM dATP, dTTP, dCTP and dGTP, 200 nM of each primer 998 and 960, 100 ng of CS plasmid DNA and 2.5 U of Pfu polymerase. PCR cycling conditions were 95°C for 45 s for initial denaturation followed by 95°C for 45 s, 55°C for 45 s, and 72°C for 2 min (30 cycles) with a 10 min final extension at 72°C. The 1145 bp product was gel-isolated, digested with the restriction enzymes XbaI and HindIII and cloned into XbaI and HindIII sites of plasmid pUC18 vector. The resulting plasmid, SCS1E, was used to transform E. coli DH5a cells.

2.4. Complementation experiments An E. coli CS auxotroph AB301 [16], (metB relA, spoT1) was used in complementation studies. AB301 was transformed with pUC18 (control) or pSCS1E and plated onto LB plates containing 100 mg/ml ampicillin (AMP) and 1 mM isopropylthio-b-D-galactoside (IPTG). Approximately 200 colonies from each transformation were replica plated onto several plates: (1) LB containing 100 mg/ml AMP and 1 mM IPTG; (2) M9 plates supplemented with 40 mg/ml of 16 amino acids (excluding cys, met, tyr, trp), 100 mg/ml AMP and 1 mM IPTG; (3) M9 plates supplemented with 40 mg/ml of 16 amino acids (excluding cys, met, tyr, trp), 100 mg/ml AMP; (4) M9 plates containing 40 mg/ml of 16 amino acids (excluding cys, tyr, trp) 100 mg/ml AMP, 1 mM IPTG, and 40 mg/ml met. One clone SCS1E145 was used for growth studies in liquid M9 media. AB301 cells containing pUC18 (40 mg/ml of 17 amino acids and excluding cys, tyr, trp) and pSCS1E (40 mg/ml of 16 amino acids, excluding met, cys, tyr, trp) were grown in liquid M9 media with their respective supplements

overnight. Cultures were diluted 1–25 in fresh M9 media with 40 mg/ml of 16 amino acids (excluding met, cys, tyr, trp) and 100 mg/ml AMP and further supplemented with either: (+) 40 mg/ml methionine; (+) 40 mg/ml methionine and 1 mM IPTG; ( −) 40 mg/ml methionine; (− ) 40 mg/ml methionine and (+ ) 1 mM IPTG to determine the growth of these cells containing pUC18 and pSCS1E. Growth was measured as an increase in optical density of 550 nm at 0 and after 2 and 8 h of growth.

2.5. Southern blot analysis Genomic DNA was isolated as previously described [14]. Genomic DNA (10 mg) was digested with restriction enzymes, BamHI, BglII, EcoRI, HincII, according to manufacturer’s instructions. The digested DNA was resolved by electrophoresis on a 1% agarose gel and transferred to nylon membrane by capillary action [15]. The 1023 bp CS cDNA was used as the hybridization probe and was labeled with [a-32P]dCTP (3000 Ci/mmol) by the random primed labeling method [19]. The genomic blot was hybridized in standard hybridization buffer (Boehinger Mannheim, Indianapolis, IN) for 16 h and 45 min at 60°C. The blot was washed at a final stringency of 0.1X SSC, 0.1% SDS at 60°C and exposed to XOMAT AR film for 15 days.

2.6. Northern blot analysis Northern analysis was performed using polyA + mRNA isolated from 8-day-old soybean leaves and cotyledons as described above. PolyA + RNA was prepared as described by Maniatis et al. [15]. Five micrograms per lane of polyA + RNA was separated on a denaturing agarose–formaldehydegel. Equal loading of polyA + RNA was confirmed by ethidium bromide staining. PolyA + RNA gel was transferred to a positively charged nylon membrane. The CS probe (1023 bp) was labeled with [a-32P]-dCTP (3000 Ci/mmol) by the random primed labeling method [19]. The polyA + RNA blot was hybridized in 50% formamide, 5X SSC, 5X Denhardt’s solution [24], 0.1% SDS, 5% dextran sulfate and 100 mg/ml denatured salmon sperm DNA for 18 h at 42°C and washed in 0.1X SSC, 0.1% SDS for 1 h at 42°C and placed on Kodak MS film for 18 h. Hybridization signals

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were quanitified using a densitometer, Lab scan program and 1 D Elite Imagemaster software from Amersham Pharmacia Biotech, Piscataway, NJ.

3. Results

3.1. Isolation and characterization of a cDNA encoding CS A cDNA clone was isolated from a lZap cDNA library synthesized from mRNA extracted from leaves using an Arabidopsis CS cDNA as a probe. Oligonucleotide primers 708 and 707 derived from the genomic sequence of Arabidopsis were used to amplify a 672 bp fragment. This fragment was sequenced to confirm its identity. The 672 bp fragment was used as a probe to identify the soybean CS cDNA. The 1023 bp cDNA (864 – 1887 bp) contained the 3% end of the gene, but not a 5% start codon (Fig. 2). The 5% end of the CS gene was isolated by the 5% RACE procedure. The initial 5% RACE experiment added 338 bp to the 5% end (525–863 bp), but the ATG start site was still missing. The second 5% RACE procedure provided an additional 525 bp (5% untranslated (1 – 87 bp) and 88–525 bp) which contained the encoded ATG start and the remainder of the deduced chloroplast transit peptide (88– 552 bp). The nucleotide and deduced amino acid sequences are shown in Fig. 2. The 1887 nucleotide cDNA contains 87 nucleotides of 5% untranslated leader sequence which is followed by a single open reading frame of 1608 nucleotides. The inframe stop codon (TAA) is located 45 nucleotides upstream of the in-frame ATG start codon which indicates that the translation of the CS gene could not have occurred 5% to the ATG start codon. Nucleotides 1695–1887 represent the 3% untranslated sequence which includes a polyA + tail. The first translational initiation codon occurs at nucleotide 88 and a termination codon that lies at the end of the reading frame at nucleotide 1695 – 1698. The open reading frame encodes a protein of 536 amino acids with a calculated predicted molecular weight of 58 kDa which represents the size of the precursor protein. The deduced amino acid sequence of the encoded CS soybean protein corresponds well to the reported

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plant CS sequences of A. thaliana (85% similarity and 80% identity), Z. mays (78% similarity and 72% identity), M. crystallinum (87% similarity and 82% identity), but is less similar to CS from E. coli (63% similarity and 50% identity; Fig. 3) and other microorganisms (data not shown). The first 155 amino acids are rich in serine and threonine which compose 20% of the amino acids. This probably represents a transit peptide targeting the protein to the chloroplast, since CS activity is known to be associated with the chloroplast [8]. Furthermore, comparison of the amino acid sequence of the soybean CS putative transit peptide with the transit peptides of Arabidopsis, Z. mays, and M. crystallinum revealed 77, 78 and 77% similarities, respectively. Comparisons of the mature soybean CS protein with those from Arabidopsis and M. crystallinum showed slight increases in similarity (88 and 90%) and identity (83 and 85%) than with the precursor proteins, except with Z. mays where the percent similarity remained the same at 77% and the percent identity was reduced to 71%. One region that is more highly conserved among the six amino acid sequences compared is the proposed pyridoxal phosphate binding domain LGADLXXHSXTKY (amino acids 341– 353; Fig. 3). This region is shared by several pyridoxal phosphate dependent enzymes such as cystathionine b-lyase [25], methionine gamma lyase [26], and o-acetyl-homoserine –o-acetyl-serine sulfhydrylase [26]. These amino acids residues Leu-341, Gly-342, Ala-343, Asp-344, His-348, Ser-349, Thr-351, and Lys-352 are conserved in soybean, Arabidopsis [12], corn [27], ice plant [28], and E. coli [29]. The amino acids Leu-345 and Try-353 are conserved in all of the CS sequences except in ice plant where they are replaced by the amino acid Phe. Whereas, Val-346 is conserved in all the other CS sequences except soybean CS where Val-346 is replaced by Ile. At amino acid 347, Val is conserved in both Arabidopsis CS sequences and ice plant, but in soybean and E. coli Val is replaced by Leu and by Ile in the corn sequence. There is some divergence at amino acid 350, where in both Arabidopsis sequences and corn Ala replaces the Leu residue in soybean. In both ice plant and E. coli, the Leu residue is replaced by Met and Cys, respectively.

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Fig. 2. Nucleotide sequence and deduced amino acid sequence of a cDNA encoding CS from soybean G. max cv. Century. The in-frame stop is underlined. The stop codon is indicated by an asterisk (*). The possible chloroplast cleavage site is double underlined.

3.2. Complementation studies The coding region of pSCS1 cDNA corresponding to the putative mature protein was placed

in-frame in pUC18 and expressed in an E. coli auxotroph AB301 (metB, relA, spoT1) which lacked CS activity (Fig. 4) [16]. AB301 cells, containing or lacking pUC18 grew extremely well in

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the presence of methionine, but grew very poorly in the absence of methionine. Also, on solid M9 media without methionine no growth was observed in these cells. Initially, growth of AB301 cells was dependent on the residual methionine in the inoculum which contained methionine and was required for growth overnight of AB301, the mutant and AB301 containing pUC18. On the other hand, AB301 transformed with pSCS1E grew very well in the absence of methionine and similar growth was observed on solid M9 media.

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In the presence and absence of 1 mM IPTG, very similar growth patterns were seen, suggesting that the lacZ promoter does not tightly regulate CS expression. Thus, soybean CS gene was able to complement a methionine auxotroph of E. coli.

3.3. Southern and Northern analyses The genomic blot, digested with several restriction enzymes, was hybridized to a portion of the

Fig. 3. Amino acid alignment of deduced CS peptides. The alignment was performed using the Genetics Computer Group PILEUP program of Feng and Doolittle (1987) [23]. Comparison of amino acid sequences from soybean G. max; Arabidopsis (A. thaliana U43709 and U62147, Kim and Leustek, 1996 [12]); Z. mays (AF007786, Locke et al., 1997 [27]); M. crystallium (AF069317, Michalowski et al., 1998 [28]); E. coli (P24601). Dashes represent regions of identical amino acids whereas dots indicate gaps to optimize the alignment of the CS peptides. The conserved pyridoxal phosphate-binding region 341 – 353 is underlined.

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Fig. 4. Complementation studies using an E. coli CS mutant (AB301). The CS mutant, AB301 was transformed with either plasmids pUC18 (control) or pSCS1E. Cultures were grown in the presence and absence of 40 mg/ml methionine and 1 mM IPTG. Growth was monitored at 550 nm at 0, 2, 4, 6 and 8 h. , AB301 ( +) met; , AB301/pUC18 ( + ) met; , AB301/pSCS1E (+) met; , AB301 ( −) met; , AB301/ pUC18 ( − ) met; , AB301/pSCS1E (−) met; ", AB301 ( +) met (+) IPTG; 2, AB301/pUC18 ( +) met (+ ) IPTG; × AB301/pSCS1E ( −) met (+) IPTG.

and not the 5% end of the gene. Two separate 5% RACE experiments were performed to complete the coding region of the soybean CS gene. The complete cDNA contains 1887 nucleotides including the 5% noncoding region. The coding region of the soybean CS cDNA clone contains 1608 nucleotides. The presence of an in frame termination codon upstream of the ATG start site indicates that the 1608 nucleotides open reading frame represents the full coding region. The soybean CS gene encodes a protein of 536 amino acids with a predicted molecular mass of 58 kDa which is similar in size to the molecular mass of CS from Arabidopsis (60 kDa) [12], corn (54.8 kDa) [27], ice plant (58.5 kDa) [28]. Likewise, the deduced amino acid sequence of soybean CS was compared to both Arabidopsis CS1 and CS2, corn, ice plant, and to E. coli CS (Fig. 3). Soybean CS had 85% similarity and 80% identity to both Arabidopsis CS genes (U43709 and U62147; [12]), 78% similarity and 72% identity for corn [27], and 87% similarity and 82% identity for ice plant [28] when comparing the precursor proteins; however, when comparing the mature

CS gene (1023 bp) (Fig. 5). Several fragments of genomic DNA hybridized to the CS DNA probe: two fragments with BamHI (10 and 7 kb), four fragments with BglII (11, 10, 3, 1.6 kb) EcoRI (5.5, 2.3, 1.8, 0.6 kb), and HincII (6, 4, 2.5, 0.8 kb). Analysis of the DNA sequence of the cDNA clone indicates that BglII, EcoRI, and HincII cleave the CS cDNA clone, whereas BamHI does not cleave the cDNA. Therefore, the complex banding pattern of the genomic blot analyzed under high stringency suggests that there are several restriction sites within the genomic sequence or CS may be part of a gene family. The effect of light on CS expression was examined using polyA + RNA isolated from soybean leaves and cotyledons grown under light and dark conditions for 8 days (Fig. 6). A 1.9 kb CS transcript was detected in extracts of 8-day-old leaves and cotyledons grown under both conditions; however, light-grown material contained higher levels of CS mRNA, especially light-grown cotyledons.

4. Discussion The cDNA clone l SCS1 was first isolated as a partial cDNA (1023 bp) and contained the 3% end

Fig. 5. Southern blot analysis of soybean genomic DNA digested with BamHI (B1), BglII (B2), EcoRI (E) and HincII (H), separated on a 1% agarose gel. DNA was transferred to nylon membrane and hybridized to radiolabeled CS cDNA (1023 bp); 1 kb marker located in lane 1.

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Fig. 6. Northern blot analysis of CS mRNA levels in 8-dayold light and dark-grown leaves and cotyledons. Each lane contains 5 mg of polyA + RNA and lane (1) dark-grown leaves; (2) dark-grown cotyledons; lane (3) light-grown leaves; (4) light-grown cotyledons. PolyA + RNA was separated on a formaldehyde-containing gel and equal loading was determined by ethidium bromide staining. Following transfer to nylon membrane, hybridization was performed with the CS probe (1023 bp). Hybridization signals were quantified using a densitometer, Lab scan program and the 1D Elite Imagemaster software from Amersham Pharmacia Biotech.

proteins, the percent similarity (88%) and identity (83%) increased slightly for Arabidopsis, corn and ice plant. E. coli had 63% similarity and 50% identity (K01546; [30]). Further, the CS gene encodes a protein that contains a 155 amino terminal extension. The amino terminal extension shares several characteristics with other known chloroplast transit peptides [31,32]. The CS transit peptide is rich in hydroxylated amino acids, serine and threonine (20%) and small hydrophobic amino acids such as alanine and valine (21%) [31,32]. Chloroplast transit peptides contain three fairly well conserved homology blocks; however, the CS transit peptide shares homology with the amino terminal block MAXSXMXSS (X denotes any amino acid residue) which is shown in Fig. 2, (amino acids 1–9), but the other two blocks, the central and carboxyterminal, are not conserved very well

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[31,32]. Also, based on the consensus sequence for the chloroplast transit peptide cleavage site (V/IX-A/C¡A; X denotes a basic amino acid) [33], the soybean CS transit peptide contains the cleavage site at amino acids 153–156 (IHA¡A) (this paper) which is similar to the arabidopsis CS, amino acids 180–183 (VHA¡G) [12]. Cleavage of the soybean and Arabidopsis transit peptides at the IHA¡A and VHA¡G sites, respectively, would produce predicted CS proteins of that are identical in size, 42 kDa. The soybean and A. thaliana predicted mature CS proteins are very similar in size to the purified protein from E. coli (40 kDa) [29] and Bacillus sphaericus (41 kDa) [34]. However, purified CS proteins from plants such as spinach is somewhat larger (50 and 53 kDa) [9] whereas the wheat protein (34.5 kDa) [10] is smaller than the other CS proteins. All of the CS sequences identified thus far and the enzymes associated with the methionine pathway contain a highly conserved domain referred to as the pyridoxal phosphate binding domain, LGADLXXHSXTKY (amino acids 341–353; Fig. 3). This region has been identified in several pyridoxal phosphate dependent enzymes such as cystathionine gamma synthase ([12,27,28,35] and this paper]; cystathionine b-lyase [25], methionine glyase [26], and o-acetyl-homoserine –o-acetyl-serine sulfhydrylase [26]. These enzymes all require pyridoxal phosphate as a cofactor whereby pyridoxal phosphate is transiently bound at the active sites of these enzymes. Genomic analysis of the CS gene suggests that the multiple banding pattern maybe indicative of the multiple restriction sites for the BglII, EcoRI and HindII enzymes within the genomic sequence or that CS maybe part of small gene family. Northern analysis does not rule out the possibility of another gene nor does the genomic Southern analysis.

Acknowledgements The authors thank Drs Ann Smigocki, Kenneth P. Samuel, Gregory Wadsworth, and Donald Keister for critical review of the manuscript and Margaret McDonald, Hunter Beard and Andrea Samuels for their technical assistance. This work was supported by National Science Foundation, NSF grant no. IBN-9602190 and in part by grants

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NIH/RIMI 5P20RR-11606-02 MBRS: 5S06GM51971-03.

and NIH/GM/

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