©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The DpsA Protein of Synechococcus sp. Strain PCC7942 Is a DNA-binding Hemoprotein
LINKAGE OF THE Dps AND BACTERIOFERRITIN PROTEIN FAMILIES (*)

(Received for publication, May 15, 1995; and in revised form, July 11, 1995)

Maria Marjorette O. Peña George S. Bullerjahn (§)

From the Department of Biological Sciences, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403-0212

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps protein family have been shown to form large (150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the dpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA protein revealed that a carboxyl-terminal domain of the protein was >60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the protein complex comigrated with both the heme and the catalase activity, and O(2) evolution measurements yielded a maximal specific activity of 1.7 µmol of H(2)O(2) consumed/µg of protein min. We speculate that the protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps protein family and the bacterioferritins is discussed.


INTRODUCTION

The Escherichia coli Dps protein(1) , also known as the PexB protein(2) , was first identified as a DNA-binding polypeptide that accumulated to high levels during stationary phase, and analysis of E. coli mutants lacking Dps function suggested strongly that the function of the protein is to protect DNA from oxidative damage(1) . Additional work has established that the E. coli Dps/PexB protein accumulates under a number of different regimes of oxidative stress and nutrient limitation(2) . The expression of dps/pexB during oxidative stress in the growth phase is dependent on positive regulation by OxyR(3) , although under stationary phase, dps/pexB transcription is dependent on the alternative factor RpoS and the DNA-binding protein IHF(3, 4) . Ultrastructural studies have also shown that the protein forms a complex composed of hexagonal, hexameric rings both in the presence and absence of DNA(1) . In a recent publication(5) , we described the purification and characterization of a 22-kDa polypeptide from nitrogen-starved Synechococcus sp. PCC7942 that is structurally and functionally similar to the E. coli Dps protein. From our studies, the Synechococcus sp. strain PCC7942 Dps homolog (henceforth named DpsA) was first purified from nitrogen-limited cells as a large polypeptide having an approximate molecular mass of 150 kDa; subsequent peptide sequencing and electrophoretic studies under denaturing and nondenaturing conditions indicate that this polypeptide is a complex of the DpsA protein containing single stranded DNA(5) . Denaturing PAGE (^1)indicated that the DpsA monomer had an apparent molecular mass of 22 kDa(5) . Due to the chemical properties of this polypeptide and its accumulation in nutrient-limited and stationary phase cells, we suggested that this polypeptide serves a similar function to that proposed for the Dps protein in E. coli(1, 5) . Many diverse bacteria also express Dps homologs, although only the E. coli, Bacillus subtilis, and Synechococcus sp. proteins have been well characterized with respect to DNA-binding activity and accumulation under stress conditions(1, 5, 6) . (^2)In this paper, we have cloned and sequenced the Synechococcus sp. dpsA gene, and the deduced amino acid sequence suggested that the DpsA protein is a hemoprotein due to the presence of a COOH-terminal domain homologous to the COOH termini of bacterioferritins(8, 9) . Other members of the Dps protein family do not exhibit this extensive degree of similarity to bacterioferritin. Characterization of the purified DpsA protein revealed that the complex exhibits a weak catalase activity, and spectroscopic studies revealed the presence of heme in the complex. This raises the possibility that a heme-dependent enzymatic activity plays a role in protecting DNA from peroxide damage in oxygenic phototrophs. Furthermore, the sequence similarity between the DpsA protein and the bacterioferritins suggests that they share a common evolutionary origin.


EXPERIMENTAL PROCEDURES

Strain and Growth Conditions

Synechococcus sp. strain PCC7942 was grown in BG-11 medium (10) at 25 °C under constant illumination by fluorescent lights (General Electric Cool White). The fluence rate was maintained at 150 µmol quanta m s. Under these growth conditions, nutrient-replete cells had a doubling time of approximately 6 h. Nitrogen-free BG-11 was prepared by omitting the nitrate stock solution from the culture medium. Nitrogen-stressed cultures were initiated by collecting 15-liter volumes of exponential phase nutrient-replete cells by centrifugation and resuspending the cells to the same initial density in nitrogen-free BG-11. Nitrogen-stressed cells were harvested after 72 h in nitrogen-free medium.

Standard Methods

Oligolabeling of DNA probes, restriction enzyme digestions, agarose gel electrophoresis, and library screening were performed according to the methods compiled by Sambrook et al.(11) . Nucleotide sequencing by the chain termination method (12) was performed with the Sequenase kit (Amersham Corp./U.S. Biochemical Corp.) according to the manufacturer's instructions.

Purification of the Synechococcus sp. DpsA Protein Complex

The 150-kDa DpsA protein complex was purified from 15-liter cultures of nitrogen-starved Synechococcus sp. exactly as described previously (5) except that beta-mercaptoethanol was omitted from the buffer during the final purification steps. Specifically, protein in the soluble extract of nitrogen-starved cells was sequentially precipitated by adding ammonium sulfate to 30 and 45% saturation; the 150-kDa DpsA protein complex was collected in the 45% ammonium sulfate pellet. The pellet was resuspended in a minimal volume of TMN (50 mM Tris HCl, pH 7.5, 5 mM MgCl(2), 10 mM NaCl) buffer containing 4 M urea and loaded on a 2.5 times 100-cm Sephadex G-200 column (Pharmacia Biotech Inc.) equilibrated with the same buffer. The 150-kDa DpsA complex eluted immediately following the void volume. Fractions containing the DpsA complex were dialyzed against distilled water, lyophilized, resuspended in TMN buffer, and loaded on a 1 times 15-cm DE23 anion exchange column (Whatman, Hillsboro, OR). The DpsA complex eluted at 1.5 M NaCl in TMN buffer following stepwise elution of the column with TMN/NaCl. Following dialysis against water and lyophilization, the protein was loaded onto a 0.5 times 15-cm phenyl-agarose column (Sigma) and eluted with a gradient of 0-70% (v/v) acetonitrile/H(2)0; the protein eluted from the column at approximately 50% acetonitrile. Following lyophilization, the material was dissolved in a minimal volume of TMN buffer and passed through a Centricon-100 ultrafiltration device (Amicon, Beverly, MA). Under these conditions, the complex retained a yellow/orange color indicative of the heme prosthetic group.

Cloning of the dpsA Gene

From the NH(2)-terminal peptide sequence data obtained from the purified DpsA protein complex, we cloned the gene by first making a small DNA probe by degenerate PCR of Synechococcus DNA. Custom oligonucleotide primers (Genosys, The Woodlands, TX) were designed to amplify the sequence encoding amino acids 43-65 of the Dps-like protein (summarized in Fig. 1). While we had a peptide sequence from which to derive the degenerate oligonucleotide encoding residues 43-48 (peptide sequence, QYQKHH; derived oligonucleotide sequence, 5`-CARTAYCARAARCAYCA-3`), the other primer was derived in part by knowing the consensus sequence of other Dps family members along with our incomplete peptide sequence of the DpsA protein covering residues 60-65 (Synechococcus DpsA peptide sequence, XEFFEX; reverse complemented oligonucleotide sequence, 5`-TCYTCRAARAAYTCRTG-3`). The synthesis of the second oligonucleotide was thus based on the assumption that residue 60 of the Synechococcus DpsA protein was histidine and that residue 65 was glutamic acid. In reality, the complete nucleotide sequence of dpsA later revealed that amino acid residue 65 was aspartic acid (see Fig. 2). Nevertheless, PCR amplification of Synechococcus sp. genomic DNA employing these primers yielded a 69-base pair probe (Fig. 1) that was suitable for P end-labeling and subsequent screening of a ZAP library of Synechococcus sp. PCC7942 DNA(13) . The PCR was performed by using the GeneAmp kit (Perkin-Elmer, Norwalk, CT) with 30-nmol primers, 50 ng of genomic DNA for 40 cycles of the following temperatures: 94 °C, 1 min; 36 °C, 5 min; 36 72 °C, 5 min; 72 °C, 3 min. The PCR product was cloned into the pCR1 vector (TA Cloning Kit, Invitrogen, San Diego, CA) vector and sequenced to confirm its identity. Of 30,000 plaques screened with the PCR probe, 6 positive clones were obtained. Following ZAP autoexcision subcloning(13) , nucleotide sequencing confirmed that each clone contained the region covering the dpsA gene. One of the plasmids, pMP1.2, was selected for complete sequencing with custom synthetic oligonucleotide primers. Translation of the DNA sequence revealed a 528-nucleotide open reading frame encoding all of the amino acid sequences determined by automated Edman degradation of the purified protein, including a COOH-terminal endoproteinase Lys-C fragment (Fig. 2, underlined).


Figure 1: Synthesis of the 69-nucleotide double-stranded dpsA probe by degenerate PCR. From both the N-terminal peptide sequence of the Synechococcus sp. DpsA protein (5) and the consensus peptide sequence for other Dps family members(2) , two degenerate oligonucleotides were synthesized that amplified a 69-base pair fragment encoding residues 43-65 of the DpsA protein (bottom). The asterisk designates the glutamic acid residue that later proved to be an aspartic acid residue following complete sequencing of the dpsA gene.




Figure 2: Nucleotide sequence of the Synechococcus sp. The PCC7942 dpsA gene and the deduced primary structure of its product are shown. Nucleotide sequences underlined include the putative ribosome binding site and rho-independent transcriptional terminator. Peptide sequences underlined correspond to sequences obtained by automated Edman degradation of NH(2)-terminal and endoproteinase Lys-C fragments of the purified protein.



Catalase Assays

Catalase activity was measured in solution as oxygen evolution in the presence of hydrogen peroxide. Ten micrograms of the DpsA protein complex was routinely added to TMN buffer containing a range of H(2)O(2) concentrations in a 1.5-ml sample chamber fitted with a Clark-type oxygen electrode (YSI, Yellow Springs, OH). The catalase reaction was stopped by the addition of sodium azide to 6 mM. The electrode system was calibrated by zeroing the instrument in the presence of sodium dithionite. Additionally, catalase activity staining of nondenaturing polyacrylamide gels employed the method described by Woodbury et al.(14) .

Gel Electrophoresis and Blotting

Nondenaturing PAGE was performed as described by Laemmli for denaturing electrophoresis(15) , except that SDS was omitted from both the sample and slab gels. Additionally, the sample was prepared for electrophoresis at 20 °C, and beta-mercaptoethanol was omitted from the sample buffer. Immunoblots of nondenaturing gels were prepared as described by Towbin et al.(16) ; protein assays were performed as described by Bradford (17) , employing bovine serum albumin as a standard.

Spectroscopic Estimation of Heme Content

Pyridine hemochrome derivatives of porphyrins were generated according to Falk (18) , and the heme content estimated by absorption at 409 nm. Visible absorption spectra were obtained at 20 °C with a Shimadzu UV160 spectrophotometer.


RESULTS

Screening a ZAP Synechococcus genomic library with the 69-base pair PCR product encoding amino acid residues 43-65 of the DpsA protein resulted in the retrieval of several ZAP clones covering the dpsA gene. DNA sequencing revealed a 528-nucleotide open reading frame encoding all of the peptide sequences determined by Edman degradation of the purified protein (Fig. 2). The molecular mass of the deduced DpsA protein was 19,692 Da, slightly smaller than the estimates from denaturing PAGE; the isoelectric point was calculated to be 5.15. Lipman and Pearson (19) protein sequence analysis confirmed that the DpsA protein is homologous to other members of the Dps protein family (Fig. 3, residues in blue). Other members of this small family include the B. subtilis MrgA protein (6, 7) an antigen from Treponema sp.(20) , a cold-shock-inducible protein from the filamentous cyanobacterium, Anabaena variabilis(^3)and a putative product of an open reading frame from Streptomyces aureofaciens(21) . Comparison of the DpsA protein to other members of the Dps family indicated that the protein has 19% identity, 38% similarity to the E. coli Dps/PexB protein and 20% identity, 40% similarity to the homolog from A. variabilis.^3 All the Dps polypeptides are most likely stress-inducible DNA-binding proteins as suggested by the DNA-binding activity demonstrated for the E. coli, B. subtilis, and Synechococcus sp. homologs(1, 5, 6) .^2 What was unexpected was the presence in the Synechococcus sp. DpsA polypeptide of a COOH-terminal domain highly similar in sequence to a COOH-terminal sequence of bacterioferritin (Fig. 4, (8) ). Comparison to the Azotobacter vinelandii bacterioferritin (8) exhibits 55% similarity (27/49 residues starting with Met-116 of the DpsA protein). Additionally, there is a detectable, albeit lower, degree of sequence similarity between the DpsA protein and bacterioferritin NH(2) termini (Fig. 3, residues in red). Comparing the COOH termini, virtually all the residues that represent the bacterioferritin consensus in this sequence are conserved (Fig. 4), and over the entire DpsA polypeptide, 55% of the bacterioferritin consensus sequence is conserved (Table 1). Overall, the DpsA protein is 39% similar to the A. vinelandii bacterioferritin (Fig. 4). It should be pointed out that the other members of the Dps protein family do not exhibit this high degree of sequence similarity to the bacterioferritins (Fig. 3). However, alignment of the bacterioferritin consensus sequence (7, 8, 9) to the Dps protein family indicates shared residues across both groups, suggestive of a common evolutionary origin (Fig. 3; residues of the bacterioferritin consensus shown in blue). Furthermore, individual members of the Dps family also yield similarities to the bacterioferritin consensus (Fig. 3, residues in red). Also included in this analysis are sequences of two proteins from Helicobacter pylori and Hemophilus ducreyi that were retrieved from the GenBank data base but had not been previously assigned to the Dps family (Fig. 3). The overall percentage of similarity of all these proteins to the bacterioferritin consensus is summarized in Table 1.


Figure 3: Comparison of the DpsA protein to the bacterioferritin (bottom lines, italics) and Dps family (top lines, italics) consensus sequences. Residues of the shared Dps family signature sequence (1) are marked in blue; those shared with the bacterioferritin consensus (7) are shown in red. Those residues represented in both the Dps and bacterioferritin consensus are indicated in blue in the bacterioferritin consensus sequence (italics, bottomlines). Dps/PexB, E. coli Dps protein; S. aure, S. aureofaciens Dps homolog; A. var, A. variabilis Dps homolog; MrgA, B. subtilis MrgA protein; TYF1 An, Dps homolog (TYF1 antigen) from Treponema sp.; H. ducreyi, putative neutrophil activating protein from H. ducreyi^4; H. pylori, putative pilin precursor protein^5; Bfr, A. vinelandii bacterioferritin. The H. ducreyi and H. pylori proteins have not been previously assigned to the Dps protein family. The asterisk at Met-116 of the DpsA sequence indicates the start of the region of strong homology to Azotobacter bacterioferritin. Overall, 55% of the bacterioferritin consensus sequence is represented in DpsA (see Table 1for summary).




Figure 4: Comparison of the DpsA primary structure to A. vinelandii bacterioferritin. The residues similar to the domain covering residues 86-135 of A. vinelandii bacterioferritin are indicated in boldface. Those positions corresponding to the bacterioferritin consensus sequence (7) are indicated with plus (+) symbols.





The sequence similarity of DpsA to A. vinelandii bacterioferritin ( Fig. 3and Fig. 4) led us to determine whether the Synechococcus sp. DpsA protein could bind heme, as has been shown for the bacterioferritin proteins(7, 8, 22) .^2 Indeed, purification of the DpsA protein complex yielded an orange preparation having spectroscopic properties of bound heme; UV/visible absorption spectra yielded a complex spectrum having putative Soret absorbance maxima at 352-410 nm and an additional peak at 552 nm (Fig. 5A). Furthermore, treatment of the material with pyridine at alkaline pH yielded a pyridine hemochrome derivative having absorbance maxima around 410 and 550 nm (Fig. 5B). Such absorbance bands are diagnostic for heme and are likely indicative of protoporphyrin IX(17) . Based on the molar absorptivity of proto IX, we estimate there is approximately 1 mol of heme bound per mol of DpsA hexamer. This low heme content may indicate that the complex lost heme during purification; this is not very surprising, given that the purification scheme depends on gel filtration in 4 M urea and elution from DEAE-cellulose at 1.5 M NaCl. Preparations of the DpsA complex reported initially (5) did not have an obvious color, most likely because the complex was dissolved in buffers containing beta-mercaptoethanol. Addition of reducing agents allowed for the complex to be dissociated more easily into the 20-22-kDa DpsA monomer (5) , and such treatment probably aided in extracting any prosthetic groups from the protein complex.


Figure 5: A, visible absorption spectrum of the DpsA protein complex; B, visible absorption spectrum of the pyridine hemochrome derivative of the DpsA preparation.



Since the Dps proteins are associated with resistance to peroxide stress, these data raised the possibility that the prosthetic group could serve to consume peroxide by a heme-dependent peroxidase or catalase mechanism. Indeed, testing the complex for catalase activity yielded modest rates of oxygen evolution when assayed in the presence of 9 mM H(2)O(2) (Fig. 6); however, the complex did not yield detectable peroxidase activity in the presence of substrates such as guaiacol (2-methoxyphenol). More detailed analysis revealed that the activity had an apparent K(m) of 11 mM, and a V(max) of 1.7 µmol of H(2)O(2) consumed µg protein min. Additionally, activity staining of gels in which the purified DpsA protein complex had been electrophoresed under nondenaturing conditions revealed that the complex retained catalase activity (Fig. 7); the zone of clearing associated with catalase activity (laneC) identified the position in the gel to which both the orange color (laneA) and the immunoreactive Dps protein complex comigrated (lanesB and D). Thus, the gel data demonstrate that under conditions of low denaturation, heme remains bound to the protein and the protein complex retains a low level of catalase activity. These data argue against the possibility that a minor contaminating catalase activity copurified with the DpsA protein complex.


Figure 6: Catalase activity of the DpsA protein complex. Ten micrograms of protein were assayed in 9 mM H(2)O(2); oxygen production was monitored by a Clark-type electrode. The protein complex was added at the time point indicated by the arrow, and the reaction was stopped by the addition of sodium azide to 6 mM. Analysis of the pen deflection yielded a O(2) evolution rate of 410 nmol/µg protein/min (corresponding to 820 nmol of H(2)O(2) consumed/µg protein/min).




Figure 7: Electrophoresis of the DpsA protein complex. Left to right, photograph of the unstained gel after nondenaturing PAGE showing the orange-colored DpsA complex (laneA); identical gel following silver staining (laneB); parallel sample stained for catalase activity (laneC); parallel sample immunoblotted and probed with the DpsA antibody (laneD). The darkerregion in the middle of the zone of catalase activity is contributed by the orange color in the sample. The arrow next to the rightpanel indicates the migration of the prestained 97-kDa molecular mass marker in this PAGE system.




DISCUSSION

In this paper we report that the Dps homolog from Synechococcus sp. PCC7942 has a weak catalase activity in vitro. This activity is consistent with the proposal that the Dps proteins function to protect the chromosomal DNA from peroxide damage(1) . Of course, it is not possible at this time to state for certain whether this catalase activity is present in vivo, but it is attractive to speculate that the bound heme is involved in a peroxide-consuming mechanism located at the chromosome. A problem with this proposal is that the activity of the purified complex is very low, yielding a K(m) that is likely not physiological. However, it is possible that this low activity reflects the loss of heme during purification, as suggested by the low heme content determined by spectroscopy. Additionally, the DpsA complex has been purified as a 150-kDa soluble protein-DNA-heme complex that is quite different in organization from the high molecular mass supramolecular arrays of the E. coli Dps protein complexed to linear DNA seen by electron microscopy(1) . Perhaps similar large arrays of the Synechococcus sp. DpsA protein complexed to large chromosomal DNA fragments are required for maximum activity.

We should stress that other members of the Dps protein family do not exhibit the similarity in sequence to the bacterioferritins exhibited by the DpsA protein (Fig. 3). Thus, other members of the Dps family may lack this heme-binding function. If so, perhaps the endogenous oxidative stresses associated with photosynthetic metabolism in Synechococcus sp. require additional function(s) provided by the heme-binding structure. Additionally, since both the DpsA protein and bacterioferritins form extremely stable oligomeric complexes(1, 5) ,^2 perhaps this similar domain is also involved in forming or stabilizing these quaternary structures. Knowing that the DpsA protein binds heme may help explain why the protein accumulates under all nutrient stresses tested except iron limitation (5) . It is possible that in Synechococcus sp., the functional assembly and stability of the protein-DNA-heme complex is dependent on the presence of iron in the porphyrin ring. Alternatively, iron limitation may alter dpsA expression at the transcriptional level.

The data presented here also suggest strongly that the DpsA protein is a divergent member of the bacterioferritin/ferritin superfamily (8) .^2 Previous studies have yielded an evolutionary link between bacterioferritins and eukaryotic ferritins(8) , and thus we propose that the Dps protein family shares a common evolutionary origin with this group. We argue that the Dps proteins may have evolved initially as heme-binding or metal-binding complexes that later acquired DNA-binding activity. However, the other members of the Dps family lack the strong similarity to bacterioferritin, and to date no one has reported a heme-binding activity associated with other Dps proteins. Examining the bacterioferritin and Dps consensus sequences does reveal some sequence conservation across the Dps family (Fig. 3, Table 1), and this includes bacteria of divergent lineage (e.g.Streptomyces and E. coli). Making the assumption that the bound heme has some enzymatic function in vivo, we suggest that among the heterotrophic bacteria, the heme-binding domain became dispensable, yet was retained in a cyanobacterium whose obligate oxygenic metabolism would likely yield greater demands for oxidative protection. Extensive sequence analysis and biochemical studies of Dps family members from representative phylogenetic groups will be necessary to test this hypothesis.

We also note that the Dps protein family may include other gene products that are listed in the available data bases and may be assigned other putative functions. A polypeptide described as a neutrophil-activating protein from H. pylori (25) having properties of a novel bacterioferritin was retrieved from our search of the GenBank data base with the dpsA sequence, and such comparisons indicate that the H. pylori protein has the peptide signature diagnostic for the Dps family (see Fig. 3). Additionally, a protein described as a pilin precursor from H. ducreyi (26) similarly has the Dps signature sequence (Fig. 3). Determining the precise roles for these polypeptides awaits further investigation.


FOOTNOTES

*
This work was supported by a grant from the Ohio Board of Regents Research Challenge Program and National Science Foundation Instrumentation Grant BIR-9205431. 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) U19762[GenBank].

§
To whom correspondence should be addressed. Tel.: 419-372-8527; Fax: 419-372-2024; bullerjahn{at}opie.bgsu.edu.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

(^2)
Chen, L., and Helmann, J. D.(1995) Mol. Microbiol., in press.

(^3)
N. Sato(1991) PIR accession number JU0384.

(^4)
D. J. Evans, D. G. Evans, H. C. Lampert, and H. Nakano(1994) GenBank accession number U16121[GenBank].

(^5)
R. J. Brentjens and S. M. Spinola(1995) GenBank accession number U18769[GenBank].


ACKNOWLEDGEMENTS

We thank Dr. John Helmann for helpful discussions on the B. subtilis MrgA protein, for the bacterioferritin/Dps connection, and for sharing data on MrgA prior to publication. We acknowledge the assistance of Dr. Kshitij Dwivedi during the latter parts of this study.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.