©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning, Heterologous Expression, and Characterization of Human Glyoxalase II (*)

(Received for publication, June 27, 1995; and in revised form, September 6, 1995)

Marianne Ridderström (1) Franca Saccucci (2) Ulf Hellman (3) Tomas Bergman (4) Giovanni Principato (2) Bengt Mannervik (1)(§)

From the  (1)Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, S-751 23 Uppsala, Sweden, the (2)Istituto di Biologia e Genetica, Facoltà di Medicina e Chirurgia, Università di Ancona, Via Ranieri, Montedago, I-601 00 Ancona, Italy, the (3)Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden, and the (4)Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5` and 3` cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5` coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.


INTRODUCTION

The glyoxalase system (1, 2, 3) consists of two distinct enzymes, glyoxalase I (EC 4.4.1.5., lactoylglutathione lyase) and glyoxalase II (EC 3.1.2.6., hydroxyacylglutathione hydrolase). Glyoxalase I (4) catalyzes the isomerization of the hemimercaptal adduct, formed spontaneously from methylglyoxal and glutathione, to S-D-lactoylglutathione. This product is hydrolyzed by glyoxalase II into D-lactic acid and glutathione. The biological substrate methylglyoxal is produced mainly from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in glycolysis, but can derive also from aminoacetone and hydroxyacetone formed in the catabolism of threonine and acetone, respectively(3, 5) .

Glyoxalases I and II have been found in most tissues of mammals, as well as in other species such as bacteria and plants. The enzymes have broad substrate specificities for 2-oxoaldehydes and their corresponding S-2-hydroxyacylglutathione derivatives, respectively. Although the glyoxalase system has been studied for a long time, the biological role of these ubiquitous enzymes is still unclear. They are probably involved in detoxication of 2-oxoaldehydes, which can be formed from both xenobiotics and endogenous compounds(4) . Research areas of current interest include diabetes (6) and cancer therapy(7) . cDNA encoding glyoxalase I has been isolated from human colon and U937 cells (8, 9) and a corresponding DNA sequence has been identified in Pseudomonas putida(10) .

Glyoxalase II activity has been found in the cytosol fraction and in the mitochondria (11) of higher eukaryotes. The enzyme is a monomer with a molecular mass of 29 kDa. It is a basic protein with an isoelectric point of 8.4(12, 13) . Human glyoxalase II has been reported to be essentially monomorphic, but a rare variant has also been observed in certain populations(14, 15) .

In this paper we describe the cloning of a cDNA coding for glyoxalase II from human liver. This is the first DNA sequence reported for the enzyme from any species. A high level expression clone was constructed for heterologous expression in Escherichia coli. The recombinant enzyme was characterized and showed a kinetic behavior indistinguishable from that of the native enzyme.


EXPERIMENTAL PROCEDURES

Materials

A human adult liver cDNA library was obtained from Clontech Laboratories, Inc. Oligonucleotides were custom synthesized by Operon Technologies Inc. (Alameda, CA). Enzymes used for PCR (^1)and cloning were purchased from Boehringer Mannheim (Mannheim, Germany). ProBlott membranes were obtained from Applied Biosystems Inc. LambdaSorb and the vector pGEM-3Zf(+) were bought from Promega Corp. Sequenase version 2.1 and [alpha-P]dCTP were purchased from Amersham International (Amersham, Buckinghamshire, United Kingdom). The expression vector pKK223-3 was obtained from Pharmacia Biotech (Uppsala, Sweden). The vector was modified by digestion with AccI to eliminate the second restriction site for SalI and called pKK-D(16) . Affi-Gel 10 and protein assay reagent were bought from Bio-Rad. 5,5`-Dithio-bis(2-nitrobenzoate), glutathione, and other chemicals were purchased from Sigma. Thiol esters of glutathione were enzymatically synthesized (17) and S-p-nitrocarbobenzoxyglutathione chemically synthesized and purified as described(18) . Antiserum against rat glyoxalase II was prepared as described(19) .

Purification of Glyoxalase II from Human Erythrocytes

About 2 liters of red blood cells (from a blood bank) were treated with 6 liters of cold acetone (4 °C). The mixture was kept in the cold room overnight and then centrifuged at 5,000 times g for 15 min at 4 °C. The sediment (about 1.2 liter) was recovered and 3 volumes of 10 mM MOPS buffer, pH 7.1, containing 100 µM phenylmethanesulfonyl fluoride were added. The mixture was kept in the cold room with vigorous stirring for 2 h and then centrifuged at 15,000 times g for 15 min at 4 °C. The sediment was discarded and the supernatant (about 3 liters) was added to 20 ml of Affi-Gel 10 with immobilized glutathione(20) . The mixture was kept overnight in the cold room under shaking and then filtered under vacuum. The affinity matrix was resuspended in 10 mM MOPS buffer, pH 7.1, and poured into a column. After extensive washing with 10 mM MOPS buffer, pH 7.1, glyoxalase II activity was eluted with the same buffer containing 3 M NaCl. The active fractions were pooled (about 50 ml), diluted 1:6 (v/v) with 10 mM MOPS buffer, pH 7.1, and filtered through a column of hydroxyapatite (8 ml). Under these conditions, glyoxalase II activity is not bound to the column (less than 5%). The filtrate (hemeoglobin-free) was diluted 1:4 (v/v) and rechromatographed on Affi-Gel 10 with immobilized glutathione. Glyoxalase II activity was eluted with 2 mMN,S-di-Fmoc-glutathione (N-(9-fluorenyl)methoxycarbonyl) in 10 mM MOPS buffer; this thiol ester analog is a more effective eluant than is free glutathione.

Determination of Amino Acid Sequences

The protein sample was reduced with dithiothreitol and alkylated with 4-vinylpyridine. After SDS-PAGE and Coomassie Brilliant Blue staining on a 12% (w/v) gel, the band corresponding to glyoxalase II was excised, and the protein in the gel was subjected to in situ tryptic digestion(21) . Briefly, the gel was washed twice with 0.2 M ammonium bicarbonate, 50% (v/v) acetonitrile, then completely dried and finally rehydrated with a buffered solution (21) containing modified trypsin, sequence grade (Promega Corp.). After incubation overnight at 30 °C, the fragments generated were extracted with 0.1% trifluoroacetic acid, 60% acetonitrile and subsequently separated by reverse-phase liquid chromatography on a µRPC C2/C18 SC 2.1/10 column, operated in the SMART System (Pharmacia Biotech). A 160-min gradient (0-40%) of acetonitrile in 0.06% trifluoroacetic acid was developed at a flow rate of 100 µl/min. Several fractions were selected for automated sequence analysis in an Applied Biosystems Model 470A sequencer. Phenylthiohydantoin derivatives from the degradations were analyzed by reverse-phase high performance liquid chromatography as described(22) .

Purification of cDNA Library and Nested PCR

For the purification of cDNA, 10 ml (5.5 times 10^9 plaque-forming units) of phage lysate of the human liver library was used. The purification was carried out with LambdaSorb phage adsorbent in accord with the manufacturer's instructions. The DNA was dissolved in 500 µl of water.

For PCR amplification, 5 µl of the liver cDNA library was used in a 100-µl reaction mixture with 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl(2), 50 mM KCl, 0.2 mM of each dNTP, and 0.8 µM 5` and 3` primers. The mixture was overlaid with 100 µl of mineral oil. Incubation at 95 °C for 10 min was followed by a decrease to 70 °C and 2.5 units of Taq DNA polymerase was added. Amplification with specific primers involved 30 cycles at 95 °C for 1 min, 55 °C for 2 min, and 72 °C for 2 min. For degenerate primers, 3 cycles of denaturation at 95 °C for 1 min, annealing at 35-45 °C for 3 min, and extension at 72 °C for 2 min were performed, followed by 30 cycles with the annealing temperature increased to 45-55 °C. The -specific primers V1 and V2 had the following sequences 5`-CG GAATTC GAG CTC ACA CCA GAC CAA CTG GTA ATG-3` and 5`-CTC GAATTC ACC AAC TGG TAA TGG TAG CG-3`, respectively. The underlined sequence is the endonuclease EcoRI restriction site used for cloning of the PCR product.

The amplified DNA was digested with restriction enzymes at sites introduced via the PCR primers, and ligated to the vector pGEM-3Zf(+). After transformation to E. coli XL-1, clones harboring the DNA fragments were sequenced (23) on both strands. The clone was called pGHGII.

Vector Construction and Screening for High Level Expression in E. coli

The primer GII15Ex (5`-T CTA GAATTC ATG AAR GTD GAR GTD CTB CCD GCN CTB ACY GAY AAC TAY ATG TAY CTG GTC ATT GAT GAT-3`, including restriction sites for EcoRI, underlined) was designed for optimal expression in E. coli(24) by randomizing the silent positions in some of the triplets coding for the first 14 amino acid residues following the initiator methionine (Fig. 1). In the 3` end of the coding region, two stop codons (TAA) in tandem were introduced via the primer GIIstop (5`-T AAGCTTGTCGAC TTA TTA GTC CCG GGG CAT CTT GA-3`) which included restriction sites for HindIII and SalI (underlined).


Figure 1: Nucleotide sequence and deduced amino acid sequence of cDNA encoding human glyoxalase II. The termination codon is marked ``END.'' The dotted lines correspond to amino acid sequences determined for the peptides from glyoxalase II from erythrocytes. The altered bases in the 5`-region of the cDNA used for protein expression are indicated. The regions covered by the primers used in the PCR for the expression construct are marked with dashes.



PCR was performed as described (above) using the liver cDNA library as DNA template. After PCR, the fragments were digested with the restriction endonucleases EcoRI and SalI and ligated into the expression vector pKK-D. The resulting library of variant cDNA sequences for expression was transformed to E. coli XL-1.

For identification of clones expressing glyoxalase II, antiserum against the rat erythrocyte protein was used for immunoscreening on nitrocellulose filters(25) . The clone selected, pKHGII, was transformed into E. coli JM 109 for large scale expression.

Purification

A 3-liter bacterial culture (grown in 2% (w/v) tryptone, 1.5% (w/v) yeast extract, 0.5% (w/v) NaCl, and 1% (w/v) glycerol) was induced with 0.2 mM isopropyl-beta-D-thiogalactoside at OD 0.25 and cultured overnight at 37 °C. The bacteria were harvested by centrifugation, resuspended in 10 mM MOPS, pH 7.2, 0.1 mM phenylmethanesulfonyl fluoride containing 10 mg of lysozyme and incubated on ice for 30 min. Sonication was performed three times for 30 s followed by centrifugation. The bacterial lysate was applied to a column of Affi-Gel 10 with immobilized glutathione. After washing with 10 mM MOPS, pH 7.2, the enzyme was eluted with 3 M NaCl in the same buffer. Fractions with glyoxalase II activity were pooled. Protein concentration was estimated using the method of Bradford(26) .

Characterization of Purified Recombinant Glyoxalase II

Recombinant glyoxalase II, the erythrocyte enzyme, and a mixture of the two were analyzed by SDS-PAGE in a 12% (w/v) acrylamide gel(27) . The protein bands were stained with silver(28) .

Isoelectric focusing was performed using a model 8101 column (110 ml) with 1% (w/v) Ampholine, pH 7.0-9.0, at 5 °C and 450 V for 48 h (Pharmacia Biotech); 300 ng of recombinant glyoxalase II was analyzed and measurements were made to monitor active fractions in which pH was determined. The six first residues of the N terminus of the recombinant protein were determined after electroblotting to polyvinylidene difluoride membranes.

Assay for Glyoxalase II Activity and Kinetic Measurements

Specific activity was measured at 37 °C in a 1-ml reaction volume containing 900 µMS-D-lactoylglutathione, and 200 µM 5,5`-dithiobis(2-nitrobenzoate) in 100 mM MOPS, pH 7.2. The formation of glutathione in the presence of 5,5`-dithiobis(2-nitrobenzoate) (29) was monitored spectrophotometrically at 412 nm ( = 13.6 mM cm). As an alternative, the glutathione thiol ester hydrolysis was followed spectrophotometrically at 240 nm ( = 3.1 mM cm)(1) .

The kinetic determinations were carried out at 37 °C in 1 ml of 100 mM MOPS, pH 7.2. The amount of enzyme in the assay ranged from 7.4 to 150 ng/ml. The concentrations of S-D-lactoylglutathione and S-D-mandeloylglutathione were in the intervals 17.4-1840 µM and 0.52-520 µM, respectively.


RESULTS

Peptide Sequence Analysis

The fragments generated by tryptic digestion of glyoxalase II were separated by reverse-phase liquid chromatography. Edman degradation revealed the 12 peptide sequences shown in Table 1. The corresponding positions in the cDNA encoding glyoxalase II subsequently isolated are indicated in Fig. 1. The fragment sequences were used for primer design in the PCR reactions and, subsequently, for identification of the PCR products.



Cloning of cDNA Encoding Human Glyoxalase II

The N-terminal sequence of peptide a (Table 1) was used for design of primer number 1 (5`-ATAC GAATTC GTCGAC ATG AAR GTN GAR GTN YTN CCN GCN YTN ACN AC-3`), in which all the possible mRNA triplets coding for the amino acids were realized by randomizing some of the ``wobble base'' positions in the primer.

In a similar manner, primers number 2 (5`-ATAC GAATTC TTY TAY GAR GGN ACN GCN GAY GAR ATG-3`) and number 3 (5`-TCAA CTGCAG RTT NCC NGG YTC NAC RTG-3`), corresponding to peptides f and h, respectively, were designed as primers directed downstream and upstream, respectively. A primary PCR was performed with number 1 and oligo-dT. A second PCR followed to increase the specificity, using the primers number 2 and oligo-dT with the first PCR product as a template. Finally, a third PCR was carried out with primers numbers 2 and 3. This final combination of nested primers yielded a DNA fragment of 163 bp, which was digested with EcoRI and PstI, cloned, and sequenced. The sequence corresponded to positions between 430 and 570 in the finally determined cDNA sequence. The 96 bp between the primers contained codons corresponding to the amino acid sequence of peptide g.

The partial sequence cloned was used for design of primer number 4 (5`-ACTC GTCGAC TTG AGG TTG TTG ATG GTG TA-3`), directed upstream (Fig. 2), which in combination with number 1, allowed the isolation of the first 542 bp of the 5` part of the coding sequence. For the 5`-noncoding region, primer number 5 (5`-TCAA GAATTC GTCGAC CGG ATC CAC AAT GGC AGC-3`), directed upstream and located close to the 5` part of the coding region, was used together with two nested primers against the gt11 vector.


Figure 2: Isolation of cDNA via nested PCR. Localization of the primers designed from peptide sequences in Table 1. Three consecutive nested PCR yielded a 167-bp fragment (A). The sequence information of fragment A was used for the isolation of the 5`-coding region (B). The 5`-coding and noncoding region (C) was isolated with a primer close to the start codon and nested vector specific primers. A fragment (D) of 450 bp which corresponded to the 3`-coding and noncoding region was isolated with a specific primer, number 6, and nested vector specific primers. For both the 5` end and 3` regions two consecutive nested PCR were performed.



The remaining 3` part of the cDNA was isolated using primer number 6 (5`-ATAC GAATTC GTCGAC TAC ACC ATC AAC AAC CTC AA-3`) (Fig. 2) and primers directed to the vector. A fragment of 500 bp was cloned and sequenced.

The isolated cDNA contained 1011 bp (Fig. 1) including a coding region of 780 bp. The 5`-noncoding region consisted of 36 bp and the 3`-noncoding region of 195 bp.

The cDNA encodes a protein of 260 amino acid residues. The calculated molecular mass of the protein is 28,861 Da.

Comparison with Other DNA Sequences

A search for homology with other DNA sequences in the GenBank/EMBL Data Bank revealed 91-99% identity to 5` and 3`(^2)(^3)(^4)(^5)(^6)partial human cDNA sequences. These sequences cover about one-third of the glyoxalase II cDNA from each end and contain a few undetermined nucleotides.

Two partial 5` and 3` cDNA sequences from Arabidopsis thaliana,(^7)(^8)were shown to overlap each other and revealed 57% identity to human glyoxalase II. The deduced amino acid sequences were about 51% identical and 68% similar. Some regions of the primary structure showed significantly higher degree of identity (100% for residues 50-65, and 82% for residues 128-149).

The human glyoxalase II shares some sequence similarity with corn glutathione transferase I (30) in an overlap of 178 bp (including gaps, data not shown). Nucleotides 200-373 in glyoxalase II and 394-565 in glutathione transferase I are 56% identical.

Expression and Purification

Ten clones were isolated after screening the library of expression clones with antisera against rat glyoxalase II. In all cases the expressed protein had activity with S-D-lactoylglutathione. The clone giving the highest activity with S-D-lactoylglutathione, pKHGII, was chosen for large scale purification involving affinity chromatography with immobilized glutathione. From a 3-liter culture, 70 mg of enzyme was recovered in pure form. The specific activity of the purified enzyme was 1,400 µmol/min/mg of protein as determined with S-D-lactoylglutathione.

Characterization of the Recombinant Protein

The apparent molecular mass of recombinant glyoxalase II and the enzyme prepared from human erythrocytes were the same as judged from SDS-PAGE (Fig. 3). The silver-stained gel showed a single protein band of 29 kDa for both preparations (Fig. 3), indicating no major post-translational modifications in the enzyme from the natural source. The isoelectric point of the recombinant enzyme was determined as 8.5 by use of isoelectric focusing.


Figure 3: Silver-stained SDS-PAGE. From left to right: recombinant glyoxalase II, mixture of recombinant enzyme, and enzyme purified from erythrocytes, glyoxalase II from erythrocytes.



N-terminal sequence analysis of the purified recombinant glyoxalase II revealed a sequence MKVEVL identical to that determined for the protein prepared from erythrocytes and to the amino acid sequence deduced from the cDNA. The Nterminal methionine was 100% retained in the recombinant protein.

Kinetic Studies

Kinetic parameters for the recombinant enzyme were determined with S-D-lactoylglutathione and S-D-mandeloylglutathione (Table 2). For S-D-lactoylglutathione, the K(m) value was 187 µM and the k value 780 s. For the more hydrophobic S-D-mandeloylglutathione, the K(m) value was 29 µM and the k value 201 s. These values are in good agreement with those estimated for glyoxalase II purified from erythrocytes (Table 2).




DISCUSSION

The sequence of the cDNA encoding human glyoxalase II reported here provides the primary structure of a new member of the large group of glutathione-linked enzymes(31) . The nature of the protein as revealed by the nucleotide sequence is unequivocally glyoxalase II. This was further confirmed by peptide analysis of the purified protein and by the heterologous expression of a protein with full glyoxalase II activity. The cDNA sequence encodes a 260-amino acid residue protein with a calculated molecular mass of 28,861 Da, which is in accordance with the mass estimated in earlier studies(32) . The protein is identified as the major variant of glyoxalase II (14, 15) based on its isoelectric point (8.5) and the finding that several cDNA isolates had the same sequence. No evidence for a second variant was found in the cDNA library studied.

The optimized expression clone for glyoxalase II was found to have six alterations in the 5`-coding region in comparison with the wild-type sequence (Fig. 1). Sequence analysis of the entire coding region demonstrated that no additional mutations were present in the expression clone. Thus, the overall change in the new cDNA template made it compatible with the requirements for expression of the protein in E. coli without altering the amino acid sequence of the translation product. The original ``wild-type'' cDNA did not produce any detectable amount of enzyme in E. coli (data not shown). The yield of recombinant glyoxalase II (70 mg/3-liter culture) is approximately 100-fold higher than that from human erythrocytes (0.3 mg/liter hemolysate, cf. (32) ).

The relative migration in SDS-PAGE of the recombinant glyoxalase II and the protein purified from erythrocytes further confirmed the expected molecular mass of the enzyme. Isoelectric focusing of the purified recombinant protein was carried out to confirm that no mutations or post-translational modifications influencing the isoelectric point were present. In addition, direct N-terminal sequence analysis demonstrated the presence of the first six amino acid residues deduced from the cDNA sequence. Many recombinant proteins have their N-terminal methionine removed when expressed in a prokaryotic host. In the case of glyoxalase II, the initiator methionine is present to 100%. This might be due to the penultimate residue lysine, which does not promote removal of methionine in bacteria(33) .

The catalytic properties of the recombinant glyoxalase II are of obvious importance for further studies. Table 2shows that the kinetic constants for glyoxalase II with the standard substrate, S-D-lactoylglutathione, and for the more hydrophobic S-D-mandeloylglutathione were in good agreement with those obtained for the enzyme purified from human erythrocytes(32) . Thus, the protein appears properly folded and fully functional as required for more incisive mechanistic studies in the future.

Data base homology searches showed that two groups independently have determined the 5` and 3` regions of human cDNA sequences. Although there are some ambiguities in the deposited sequences, they resemble the first one-third of the human glyoxalase II cDNA sequence in the 5` end and the last one-third in the 3` end. These cDNA sequences have not been assigned to any protein.

Interestingly, also two partial cDNA sequences from A. thaliana are structurally similar to the 5` and 3` ends of the glyoxalase II cDNA and overlap each other. They were isolated by two groups independently, but not related to any known protein. The overlapping cDNA sequences of 762 bp show 57% identity with that of human glyoxalase II. The deduced amino acid sequences share 51% identity and are 68% similar. Some regions of the sequences are partly ambiguous, but the cDNA sequences from A. thaliana are clearly related and most probably represent glyoxalase II.

From an evolutionary perspective, it is interesting to note that not only mammalian and plant glyoxalase II sequences show extensive sequence similarities, but also that the maize glutathione transferase I appears to have a substantial, but spatially restricted, sequence similarity with glyoxalase II. At the DNA level, glutathione transferase I from corn (30) shares sequence similarity with human glyoxalase II in an overlap of 178 bp. Among glutathione transferases, the enzymes from plants have primary structures that differ strongly from those of the mammalian enzymes(34) . However, certain residues of importance for glutathione binding are fairly well conserved between mammalian and corn sequences and include residues 65-70, represented by QSNAIL in several mammalian enzymes(34) .

Although the glyoxalase system has been studied for a long time, its biological function remains unclear. Cloning of the cDNA encoding human glyoxalase II and expression of the protein in large amounts will facilitate studies of structural and functional aspects of the enzyme as well as the transcriptional regulation of its gene.


FOOTNOTES

*
This project was supported by grants from the Swedish Natural Science Research Council, Swedish Medical Research Council Projects 13X-3532 and 13X-10832, and the Swedish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X90999[GenBank].

§
To whom correspondence should be addressed. Tel.: 46-18-174539; Fax: 46-18-558431.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; bp, base pair; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-[N-morpholino]propanesulfonic acid.

(^2)
M. D. Adams, et al., GenBank accession number HS68512[GenBank].

(^3)
M. D. Adams, M. B. Soares, A. R. Kerlavage, C. Fields, and J. C. Venter, GenBank accession number TO8256.

(^4)
M. D. Adams, et al., GenBank accession number HS20015[GenBank].

(^5)
M. B. Soares, M. F. Bonaldo, P. Jelenc, L. Su, L. Lawton, and A. Efstratiadis, GenBank accession number HS8897.

(^6)
M. B. Soares, GenBank accession numbers HS9865 and HS9366.

(^7)
M. Krivitzky, I. Jean-Jacques, M. Kreis, and A. Lecharny, GenBank accession number Atts2378.

(^8)
T. Newman, GenBank accession number At9873.


ACKNOWLEDGEMENTS

We thank Drs. Ulf Landegren and Maria Lagerström-Fermer, Uppsala University, Uppsala, for valuable advice and Dr. Mikael Widersten of our laboratory for kind help with computer searches. We also thank Dr. Helena Danielson for helpful advice.


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