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Genetics, Vol. 178, 725-736, February 2008, Copyright © 2008
doi:10.1534/genetics.107.074799
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Department of Biochemistry and Molecular Biology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
5 Corresponding author: Ohio State University, Department of Biochemistry, Room 233, Biological Sciences Bldg., 484 W. 12th Ave., Columbus, OH 43210.
E-mail: hopper.65{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Gal3 is the galactose sensor of the GAL gene switch. Recessive gal3 mutations impair induction, whereas dominant GAL3C mutations encode proteins that bind to Gal80 independently of galactose and confer constitutive GAL gene expression (BLANK et al. 1997). Gal3 is a paralogue of the Gal1 galactokinase (BAJWA et al. 1988; WOLFE and SHIELDS 1997; PLATT et al. 2000). Unlike Gal3, Gal1 is not sufficiently expressed in the absence of galactose to serve as an inducer (TSUYUMU and ADAMS 1974; BROACH 1979; BHAT et al. 1990; HITTINGER and CARROLL 2007). However, when Gal1 is expressed from a surrogate promoter, it can substitute for Gal3 in activation of the GAL genes, and this does not require its galactokinase activity (BHAT and HOPPER 1992). Moreover, the sequence motifs of Gal1 that bind galactose and ATP are conserved in Gal3 (PLATT et al. 2000; THODEN et al. 2005). Although native Gal3 lacks galactokinase activity, the insertion of serine and alanine (Gal3-SA) at a single position within one of its galactokinase homology motifs results in the acquisition of galactokinase activity (PLATT et al. 2000). It has also been shown that a D62A substitution in Gal1 can impair its ability to catalyze phosphorylation of galactose, and the corresponding amino acid substitution in Gal3 impairs its capacity to induce expression of the GAL genes in response to galactose (SELLICK and REECE 2006). Thus, the binding of galactose and ATP to Gal1 or Gal3 affects their capacity to bind to Gal80 and also serves as the catalytic function of Gal1 or Gal3-SA.
Studies of the highly similar GAL gene switch of the yeast Kluyveromyces lactis (Kl) further support the notion that Gal3 is a galactose sensor (MEYER et al. 1991; ZENKE et al. 1996). K. lactis lacks a GAL3 gene (VOLLENBROICH et al. 1999) but expresses galactokinase (encoded by KlGAL1) at moderately high levels in the absence of galactose (DICKSON and MARKIN 1980; RILEY and DICKSON 1984; CARDINALI et al. 1997). Klgal1 recessive mutations confer the noninducible phenotype and impaired binding of KlGal1 to KlGal80, whereas dominant KlGAL1 mutations confer constitutivity and galactose-independent binding of KlGal1 to KlGal80 (VOLLENBROICH et al. 1999; MENEZES et al. 2003). Thus, KlGal1 is the galactose sensor in K. lactis.
The mechanism by which the binding of galactose and ATP triggers Gal3 interaction with Gal80 is unresolved. Gal3, Gal1, and KlGal1 belong to the GHMP superfamily of small-molecule kinases (BORK et al. 1993). Crystal structures are available for several family members including galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (ZHOU et al. 2000; KRISHNA et al. 2001; ROMANOWSKI et al. 2002; YANG et al. 2002; THODEN and HOLDEN 2003; THODEN et al. 2005). All GHMP superfamily members have conserved motifs for the binding of ATP and their specific substrates, and most have been crystallized with their substrates bound. Thus, the binding sites for galactose and ATP in galactokinase are well established.
All GHMP members share a similar overall protein fold (ANDREASSI and LEYH 2004). Taking advantage of this, MENEZES et al. (2003) used the structure of mevalonate kinase (YANG et al. 2002) as a template to model the structure of KlGal1. On the basis of the locations of KlGAL1 mutations they proposed that the binding of galactose and ATP to a cleft in front of a hinge region brings together the upper and lower lips of KlGal1 to create a docking surface for KlGal80 (MENEZES et al. 2003). Although attractive as a testable hypothesis, this proposal was based on a severely limited KlGal1 homology model due to the low level of sequence identity between KlGal1 and mevalonate kinase (13%). An additional limitation of the model of KlGal1 is that it lacks the insertion motif (97 residues in Gal3) that is common to Gal3, Gal1, and KlGal1. It is likely that the insertion motif plays an essential role in binding of Gal3 to Gal80 because Escherichia coli galactokinase, which lacks the insertion motif, provides galactokinase activity but not the Gal3-specified GAL gene induction activity in yeast cells (BHAT et al. 1990).
The crystal structure of the S. cerevisiae Gal1 bound with galactose and AMPPNP has recently been solved at 2.4-Å resolution (THODEN et al. 2005) and provides a reliable template for deriving models of Gal3 (THODEN et al. 2005; DIEP et al. 2006). Because Gal3 is 72% identical and 92% similar to Gal1, these Gal3 homology models provide excellent predictive values for the structure of Gal3 and facilitate structure/function analyses.
We previously carried out intragenic suppression analyses of GAL3C mutations to address the issue of how the binding of galactose and ATP affects the binding of Gal3 to Gal80 (DIEP et al. 2006). Gal3C proteins bind to Gal80 in the absence of galactose and cause constitutive expression of the GAL genes (BLANK et al. 1997). A suppressor (GAL3SOC) of GAL3C mutations and four suppressible alleles (GAL3C-F237Y, -V396A, and -S509P/L) colocalize to a region in the Gal3 model that contains a putative ligand-regulated hinge region previously identified in a KlGal1 homology model (MENEZES et al. 2003). In contrast, the single nonsuppressible GAL3C allele (GAL3C-D368V) is remote from the hinge region. The nonsuppressible nature of GAL3C-D368V and its location within the insertion motif suggested that D368 and other nearby residues might compose the surface that interacts with Gal80 (DIEP et al. 2006).
In this study we performed a genetic analysis to identify the Gal3 surface that docks with Gal80. We found that mutations that affect Gal3's binding to Gal80 alter residues in a well-defined composite surface composed of many noncontiguous residues. We propose that this surface of Gal3 is the docking site for Gal80.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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(GRIFFITH and GIETZ 2003) was grown at 37° in LB medium supplemented with 50 mg/liter of ampicillin.
Yeast strains:
Yeast strains and phenotypes used in this study are listed in Table 1. ScPD2 was derived from ScVP2-103 (PILAURI et al. 2005) by disruption of the GAL1 gene. Sc750 and Sc751 were derived from Sc724 (BLANK et al. 1997) by disruption of the GAL80 gene and integration of the GAL80S–0 and GAL80S–1 alleles, respectively. The GAL80S-2 mutation was not integrated into the genome, but instead was carried on a CEN plasmid (pMPW87). In this case the strain Sc787 (gal1
gal3gal80) was used to carry GAL80S-2 on pMPW87. Sc754 was derived from Sc723 (BLANK et al. 1997) by disruption of the GAL1 gene. Further details on the construction of these strains are available upon request.
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5000 gap-repaired colonies, 250 grew on plates containing 5-FOA. After screening by Western blots, 26 candidates had full-length proteins and showed repeated loss of interaction with DBD-GAL80 in both the reverse and the forward two-hybrid assays. The plasmids from these candidates were isolated and sequenced to identify the mutations. The candidate plasmids were transformed back into ScPD2 carrying DBD-GAL80 to reconfirm their phenotypes.
Plasmid construction:
The plasmid pCD107 was constructed by ligating the BglII/XhoI fragment from pAKS130 [GAL3-SA in the pTEB16 backbone (BLANK et al. 1997)] into the corresponding sites of pGAL3-VP16 (PILAURI et al. 2005). GAL3 fragments in pCD107-bearing mutations were transferred into pTEB16, using the gap-repair method (MUHLRAD et al. 1992). This was done by cotransformation of the EcoRI fragment of pCD107 with the backbone of pTEB16 after digestion with BstBI. Mutations from pTEB16 were transferred into pMPW60 by swapping the NsiI/KpnI fragments. All mutations were verified by sequencing.
Spot assay for cell growth:
Transformed yeasts were grown to late log phase in selective medium. Each culture was adjusted to the same number of cells and serial 10-fold dilutions were made with dH2O. Six microliters of 10–1, 10–2, 10–3, and 10–4 dilution were spotted onto the appropriate dropout plates and incubated for up to 6 days.
Pull-down assay for GST-Gal3 and Gal80 interaction:
Sc787 cells cotransformed with plasmids carrying GST-GAL3 and GAL80 were grown to midlog phase and whole-cell extracts containing GST-Gal3 and Gal80 were prepared as described (BLANK et al. 1997), using a modified lysis buffer (20 mM HEPES pH 7.4, 0.5% Triton X-100, 200 mM NaCl, 0.5 mM EDTA, 2 mM DTT, and 5 mM MgCl2). Protease inhibitor cocktails (PIC) (PIC-D, 88 mg/ml PMSF and 1 mg/ml pepstatin A in DMSO; PIC-W, 157 mg/ml benzamidine, 0.5 mg leupeptin, and 0.5 mg bestatin in water) were added to all lysis buffer and all subsequent solutions at 1/1000 dilutions. The whole-cell extract (1 mg) was brought up to a volume of 500 µl with lysis buffer containing 2 mM ATP and 25 mM galactose. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. The whole-cell extracts were then incubated with 50 µl of 50% glutathione Sepharose beads on a rotator at 4° for 2 hr. The beads were pelleted and washed three times with 500 µl of lysis buffer either with or without ATP and galactose. The beads were then boiled for 5 min in 40 µl of 1x SDS–PAGE sample loading buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 100 mM DTT, and 0.003% Pyronin Y) before analysis by standard SDS–PAGE and Western blot. Antibodies against GST and Gal80 were used together at dilutions indicated in the figure legends.
Homology model manipulation and bioinformatics:
Derivation of the Gal3 homology model was previously described in detail (THODEN et al. 2005; DIEP et al. 2006). Visualization of the model was carried out using PyMOL. The multiple sequence alignment was generated using the TCoffee web server (3DCoffee mode) and modified by GeneDoc and Microsoft Word.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In the presence of wild-type GAL80, wild-type GAL3 conferred GAL gene activation only in the presence of galactose as expected, whereas all five GAL3C mutations showed constitutive activation (Figure 1A). Of all the GAL80S and GAL3C combinations, only one showed intergenic suppression: GAL80S-1-G323R and GAL3C-D368V. Cells carrying both of these mutations exhibited GAL gene activation only in the presence of galactose. The GAL80S-1 mutation was epistatic to the remaining four GAL3C alleles, whereas the GAL80S-0 and GAL80S-2 mutations were epistatic to all five GAL3C alleles. This allele-specific interaction between the GAL80S-1-G323R and GAL3C-D368V mutations suggests that the Gal80-G323 and Gal3-D368 residues affect galactose-dependent interaction of these proteins.
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The noninducible phenotype due to gal3-D111C is suppressed by GAL80-M350C:
Intergenic suppression of a Klgal1 noninducible mutation by a KlGAL80 mutation was reported for the K. lactis GAL gene switch (MENEZES et al. 2003). To test if a similar suppression could be established in S. cerevisiae, we changed the corresponding residues in the S. cerevisiae Gal3 and Gal80 to cysteines and expressed the genes on plasmids (gal3-D111C on pCD123 and GAL80-M350C on pCD125). The gal3-D111C mutation combined with the wild-type GAL80 causes the GAL genes to be noninducible as determined by a colony-growth assay with the PGALHIS3 reporter (Figure 2, row 5), but this phenotype was suppressed by GAL80-M350C (Figure 2, row 6) and was indistinguishable from cells carrying wild-type GAL3 (pMPW66) and GAL80 (pMPW82) (Figure 2, row 2). The suppression of gal3-D111C by GAL80-M350C mirrors the Klgal1/KlGAL80 intergenic suppression and provides further support for the similarity of the pairwise interactions.
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) mutant for growth on galactose (PLATT et al. 2000). We used this in vivo assay to screen the isolates for those that are impaired in Gal80 interaction but retain the ability to complement a gal1, as those would likely be folded properly. GAL3-SA was fused to the VP16 activation domain to form the prey construct (pCD107). In the forward two-hybrid assay, Gal3-SA-VP16 interacted with the DBD-Gal80 bait (pAKS42) in a galactose-dependent manner, indicating that the SA insertion and the VP16 fusion did not perturb the galactose-induced interaction between Gal3 and Gal80 (data not shown). The interaction was also detected in the reverse two-hybrid assay, as the strain ScPD2 carrying both GAL3-SA-VP16 and DBD-GAL80 was unable to grow in the presence of galactose and 0.1% 5-FOA (data not shown). Mutagenesis of GAL3-SA-VP16 yielded 26 5-FOA-resistant single-amino-acid missense variants (Table 2). All 26 expressed full-length proteins at levels similar to wild-type Gal3-SA-VP16 (data not shown). The prey plasmids were isolated from these colonies and their DNA sequence was determined. All mutations resulted in single-amino-acid substitutions (Table 2). These plasmids were then reintroduced into strain ScPD2 carrying the DBD-GAL80 bait to determine their forward and reverse two-hybrid interactions in the presence of galactose. With the exception of three variant proteins (M71V, H199R, and N278Y), which were only mildly impaired, all of the remaining 23 mutations completely prevented Gal3 interaction with DBD-Gal80 in the forward two-hybrid assay (Figure 3). For most of the isolates the relative magnitudes of the interaction in the forward and reverse two-hybrid assays were similar. However, some mutations caused a complete loss of growth in the forward two-hybrid assay but allowed mild growth in the reverse two-hybrid assay (such as C429Y). When the forward two-hybrid assay plates were incubated longer (6 days, data not shown), these isolates eventually exhibited weak growth, consistent with retention of some interaction with DBD-Gal80. Importantly, all 26 Gal3 variant proteins were at least partially impaired in interaction with DBD-Gal80.
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The Gal380NB variants are defective in activation of a GAL gene promoter:
The capacity of the Gal380NB variants to activate the GAL gene switch was determined by assaying growth of cells containing the PGALHIS3 reporter gene on plates lacking histidine. Each gal380NB mutation in cis with the GAL3-SA mutation was expressed in a gal1gal3 strain (Sc781), and the cells were spotted on a plate containing galactose. Although cells with GAL3-SA showed appreciable activation of the GAL promoter, the level was reduced compared to cells with wild-type GAL3 (Figure 5), as might be expected since the SA insertion occurs at the highly conserved P-loop motif found within the active site where galactose and ATP bind (PLATT et al. 2000; THODEN et al. 2005). The SA insertion could cause a decrease in galactose affinity and/or impair the conformational change thought to be induced by the ligands and required for binding to Gal80. Most of the gal380NB-SA derivatives showed complete loss of GAL gene activation while some showed only weak to moderate levels of activation. For example, the M71V and R330C variants supported a weaker activation of the GAL gene promoter than did Gal3-SA. This was consistent with the relative levels of their physical and two-hybrid interactions with Gal80. In summary, our yeast reverse two-hybrid selection yielded Gal3 variants showing impaired interaction with Gal80 and impaired GAL gene induction.
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for galactokinase activity. Those gal380NB mutations that lose galactokinase activity might affect either the global integrity or the local conformation of the protein or both. Strain Sc754 (gal1) carrying a gal380NB mutation in cis with the GAL3-SA mutation was spotted onto plates with galactose as the sole carbon source. GAL3-SA, but not wild-type GAL3, was able to support cell growth on this medium, consistent with the observed galactokinase activity of the Gal3-SA protein (Figure 6) (PLATT et al. 2000). Two of the 26 gal380NB mutations complemented the gal1 (L103S and D111N, Figure 6). The remaining 24 gal380NB mutations did not support growth in this assay (data not shown). We discuss below how these 26 Gal380NB variants might be defective in binding to Gal80.
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for galactokinase activity and thus strongly implicate the L103 and D111 residues directly in either the allosteric communication or the docking surface. Remarkably, even though the remaining10 surface variants showed concordant loss of galactokinase-complementing activity and Gal80 binding, they colocalized in the same region of the homology model as L103 and D111 (Figure 7, green spheres). Moreover, this is the region that also includes D368, the site of the GAL3C-D368V suppressor of GAL80S-1-G323R (see above). This striking spatial colocalization of these 10 Gal380NB variant residues with L103, D111, and D368 highlights this region as a likely candidate for interaction with Gal80. The putative Gal80-interaction surface is localized on the Gal3 homology model on the opposite side of the hinge region (Figure 7, cyan spheres). We discuss possible implications of these observations and propose a mechanistic model for explaining how the binding of galactose and ATP induces Gal3 to interact with Gal80.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In our selection for Gal3 variants defective for binding to Gal80 we identified the L162P substitution that is likely to affect ligand binding since L162 resides within the P-loop, a conserved nucleotide-binding motif within the GHMP superfamily. Thirteen other substitutions affect residues buried in the hydrophobic core of the Gal3 homology model. Such buried residues could participate directly in allosteric communication between ligand binding and articulation of the Gal80-binding surface or could act indirectly by altering the overall fold or local conformation. The remaining 12 substitutions show striking colocalization to structural elements that form a single composite surface on a Gal3 homology model. This composite surface is localized opposite to a previously identified hinge region that has been implicated in the allosteric communication effected by ligand binding (MENEZES et al. 2003; DIEP et al. 2006).
Our intergenic suppression studies of GAL3 and GAL80 mutations strongly implicate the importance of the affected amino acids in the mutual docking of Gal3 and Gal80. Four observations strengthen this notion. First, the inability of Gal3 to bind to the Gal80S-1-G323R protein is restored with the Gal3-D368V substitution variant, thereby confirming the physical basis of genetic suppression observed with the GAL3C-D368V/GAL80S-1-G323R suppression pair. Second, the Gal3-D368 residue (of which D368V suppresses GAL80S-1-G323R) lies within one of the four contiguous helices that constitute a 97-residue insertion motif that is common to the Gal80-binding proteins, Gal3, Gal1, and KlGal1 (Figure 8A, blue helices). The E. coli galK protein, which is unable to bind to Gal80, lacks two of the four helices including the Gal3-D368 helix. Third, the fact that the Gal3-D111N and Gal3-L103S variants are defective in binding to Gal80 but retain the capacity to complement the gal1 strongly argues for the direct involvement of residues D111 and L103 in galactose-mediated interaction with Gal80. Furthermore, the overall colocalization of residues D368, D111, and L103 to a single composite face of the Gal3 homology model (Figures 7 and 8B) suggests that this is the Gal80-binding surface. Fourth, on the basis of the crystal structure of KlGal80, it was suggested that a large disordered region of KlGal80 consisting of residues 328–362 becomes ordered when bound with KlGal1 (THODEN et al. 2007). Importantly, this region is surrounded by KlGal80 residues equivalent to variant residues of the S. cerevisiae Gal80 that are defective in interaction with Gal3. Thus, Thoden et al. proposed that the KlGal80 residues 328–362 mark the binding surface for KlGal1. The corresponding disordered region of the S. cerevisiae Gal80 consists of residues 327–346, which are flanked by residues G323 and M350 that were identified in our intergenic suppression. This is consistent with our proposal that the Gal80-G323 and -M350 residues directly interact with Gal3 and in turn implicate the Gal3-D111 and -D368 residues as constituents of the Gal80-binding surface. On the basis of the convergence of evidence presented above, we refer to the identified Gal3 surface as a putative Gal80-docking surface.
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, indicating that the mutations at these residues specifically affected the Gal80-binding activity. How might the binding of galactose and ATP to Gal3 allosterically modulate the presentation of its Gal80-docking face? Gal3 is a member of the GHMP superfamily consisting of several members, including Gal1, whose crystal structures are known. The only member to have its apo- and holo-structures determined is homoserine kinase (HSK) (ZHOU et al. 2000; KRISHNA et al. 2001). Superimposition of the two structures identified conformational changes upon binding of ligands (homoserine and AMPPNP) (KRISHNA et al. 2001). Those studies revealed that a local conformational change occurred at the upper lip helix (residues 181–189) of HSK and predicted a direct contact between the side chain of the R187 residue and the substrate homoserine by hydrogen bonding (ZHOU et al. 2000; KRISHNA et al. 2001). The corresponding upper lip helix in Gal1 (residues 271–292) also contains a side chain (Y274) (Figure 8A, green) that could make direct contact with galactose by hydrogen bonding (Figure 8B, cyan) (THODEN et al. 2005). The corresponding upper lip helix of Gal3 (263–284) (Figure 8A, red helix) and Gal1 immediately precedes the insertion motif (Figure 8A, blue helices), which is topologically linked to the upper lip helix by folding around it and forming a cap-like structure (Figure 8B). The last two helices of the four-helical insertion motif contain residue D368 and are absent in bacterial galactokinases (Figure 8A). Thus, we propose that binding of galactose and ATP induces a local conformational change at the Gal3 upper lip helix that in turn directly modulates the conformation of the insertion motif and thereby articulates presentation of the candidate Gal80-binding face.
We speculate that in addition to the insertion motif other residues proximal to the insertion motif also contribute to the interaction. Specifically, the 12 surface-exposed Gal380NB variants that localize predominantly around the insertion motif could contribute to the interaction with Gal80. Thus, we propose that the Gal80-binding surface of Gal3 is composed of noncontiguous residues including L103, D111, D368, and residues represented by the surface-exposed Gal380NB variants.
In summary, we identified Gal3 residues that participate in intergenic suppression of impaired Gal3–Gal80 binding (D368 and D111) and Gal3 residues that are important for binding to Gal80 (Gal380NB variants). These distinct data sets converge to reveal a single composite surface of Gal3 as a likely Gal80-binding surface. We presented a testable hypothesis of how the various structural elements of Gal3 and specific residues within the candidate docking surface might constitute an allosteric mechanism. Deciphering this mechanism and obtaining physical/chemical evidence for which residues directly contribute to the binding energy of the Gal3–Gal80 interaction await detailed biochemical studies.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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2 Present address: Department of Biochemistry, Ohio State University, Room 233, Biological Sciences Bldg., 484 W. 12th Ave., Columbus, OH 43210.
3 Present address: Department Hematology and Oncology, Mount Sinai School of Medicine, Basic Sciences Bldg., 10 E. 101st St., Room 340, New York, NY 10029.
4 Present address: Bacteriology Division, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702-5011.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Communicating editor: M. JOHNSTON
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