Article |
Address correspondence to Susan R. Wente, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 3120 Medical Research Building III, Nashville, TN 37232-8240. Tel.: (615) 936-3443. Fax: (615) 936-3439. E-mail: susan.wente{at}vanderbilt.edu
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Abstract |
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Key Words: Gle1; mRNA export; cell-permeable peptide; nuclear transport; shuttling
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Introduction |
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The molecular paradigms for this pathway were initially established from studies of viral RNA export (Cullen, 2000; Hammarskjold, 2001). For the HIV-1 pathway, a nuclear export sequence (NES) in the RNA-bound viral Rev protein is recognized by Crm1 (Fischer et al., 1999), a member of a conserved family of transport receptors called karyopherins (also designated exportins/importins/transportins) (Gorlich and Kutay, 1999). Through interaction with specific adaptor proteins, Crm1 also mediates the export of cellular RNAs, such as 5S RNA and U1 snRNA (Fischer et al., 1995), and the 60S ribosomal subunit (Ho et al., 2000). A different karyopherin (exportin-t/Los1) mediates the export of tRNA (Gorlich and Kutay, 1999).
Karyopherins may play multiple roles in the export of mRNPs (Yi et al., 2002), some being required for the export of cellular mRNAs from early response genes (Gallouzi and Steitz, 2001; Gallouzi et al., 2001). Distinct sets of importins are needed for the reimport of shuttling hnRNPs (Michael et al., 1995, 1997). However, studies in yeast, mammalian, and Xenopus oocyte systems have illustrated a central role for the novel Mex67/TAP/NXF1 protein (Segref et al., 1997; Gruter et al., 1998; Katahira et al., 1999). In higher eukaryotes, splicing and TAP/NXF1-mediated nuclear export are believed to be coupled (Maniatis and Reed, 2002). TAP/NXF1, together with its cofactor p15/NXT1 (Fribourg, et al., 2001), promotes the export of both spliced and intronless transcripts through interactions with both mRNPs and NPC proteins (Maniatis and Reed, 2002). Thus, TAP/NXF1 may act as a shuttling bridge between the mRNP complex and the NPC (Zenklusen and Stutz, 2001). Although TAP/NXF1 was shown to cooperate with karyopherins (Shamsher et al., 2002), TAP/NXF1 itself does not bind Ran, and RanGTP hydrolysis is not required for TAP/NXF1-mediated mRNA export (Bachi et al., 2000).
Several other factors are also required for mRNA export, including the DEAD-box RNA helicase Dbp5 and two NPC-associated factors Gle1 and Gle2/Rae1 (Zenklusen and Stutz, 2001; Reed and Hurt, 2002). Dbp5 may remodel hnRNP complexes at the NPC and mediate the release of nonshuttling hnRNPs in the nucleus and shuttling hnRNPs in the cytoplasm. This could serve a critical role in mRNP transport directionality (Snay-Hodge et al., 1998; Tseng et al., 1998; Schmitt et al., 1999; Zhao et al., 2002). Work from several groups strongly supports the hypothesis that Gle1 plays an essential role in mRNA export (Del Priore et al., 1996; Murphy and Wente, 1996; Noble and Guthrie, 1996; Watkins et al., 1998). Human Gle1 (hGle1) and Saccharomyces cerevisiae Gle1 (scGle1) have no consensus RNA binding sites, and no functional protein motifs have been definitely identified (Reed and Hurt, 2002). Moreover, no hGle1-interacting proteins have been identified. Genetic connections implicate scGle1 as a modulator of scDbp5 activity (Hodge et al., 1999), and scGle1 also interacts with Nup42 (Murphy and Wente, 1996; Stutz et al., 1997; Strahm et al., 1999), Dbp5, and a nonessential factor, Gfd1 (Hodge et al., 1999; Strahm et al., 1999). Recently, our studies in yeast have shown that soluble inositol hexakisphosphate production is required for efficient scGle1 function (York et al., 1999). Overall, how Gle1 mediates nuclear transport is far from being resolved.
A long-standing question regarding Gle1 function is whether it is a dynamic shuttling factor or a stable component of the NPC (a nucleoporin). Others have suggested that scGle1 is a static nucleoporin (Rout et al., 2000). However, the mammalian Gle1 protein does not cofractionate with NPCs (Cronshaw et al., 2002). Here, we have focused on the human protein. We demonstrate that two hGle1 protein variants (hGle1A and B) are encoded from a single gene. The hGle1B isoform associates with the NE/NPC in HeLa cells and is the predominant variant. We subsequently show that both proteins can shuttle between the nucleus and cytoplasm and that this shuttling activity requires the presence of a novel 39amino acid domain. Moreover, using a cell-permeable peptide strategy, we found that hGle1 shuttling is essential for poly(A)+ RNA export.
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Results |
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Identifying an hGle1 domain with nuclear export activity
We speculated that if hGle1 is dynamic and transiently associates with NPCs while entering and exiting the nucleus, it should contain domains/sequences that harbor nucleocytoplasmic transport activities. The region of hGle1 spanning amino acid residues 444606 was specifically targeted for internal in-frame deletion based on our previous studies of scGle1 (Murphy and Wente, 1996; Watkins et al., 1998). The COOH-terminal regions of scGle1 and hGle1A/B are highly similar, and the corresponding region in scGle1 contains a putative leucine-rich (LR) NES-like motif that is essential for scGle1 function in mRNA export (Fig. 1 D; see Discussion). As a tool to delineate the region(s) of hGle1 that may harbor nuclear export activity, we generated a panel of ectopically expressed FLAG-tagged hGle1A deletion proteins and analyzed their respective intracellular distributions independent of the endogenous hGle1 by indirect immunofluorescence (IIF) using anti-FLAG antibodies. To assay for export activity, we placed the SV40 large T antigen NLS in frame at the COOH terminus of FLAGhGle1A. Based on the hypothesis that hGle1 harbors nuclear export activity, it should balance the activity of the heterologous NLS and result in cytoplasmic FLAGhGle1ANLS localization. Results from these experiments are summarized in Fig. 2. Although the majority of the FLAGhGle1ANLS was detected in the nucleus, FLAGhGle1ANLS was also clearly present in the cytoplasm (Fig. 2, lower left). Strikingly, the FLAGhGle1A444606NLS protein was localized exclusively in the nucleus. Various NH2- and COOH-terminal deletions of the 444606 region were tested to map the export activity. Deletions from the NH2-terminal side eliminated cytoplasmic localization (
444511), whereas deletions from the COOH-terminal side up to residue 483 retained the export activity (
484606) (Fig. 2). Thus, a 39amino acid region from residues 444483 was necessary to confer cytoplasmic localization of FLAGhGle1ANLS.
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To independently test whether hGle1 shuttles, we analyzed the dynamics of EGFPhGle1B in live HeLa cells (Fig. 3 C). This was based on our observation that ectopically expressed EGFPhGle1B localizes like endogenous hGle1 (Fig. 1 A). Using the technique of fluorescence loss in photobleaching (FLIP), we analyzed whether nuclear EGFPhGle1B could exchange with the cytoplasmic pool of EGFPhGle1B. By repeatedly bleaching an area of the cytoplasm, the decrease in nuclear EGFP fluorescence over time was monitored. As controls, the same bleach protocol was conducted on EGFPhuman coilin (a nonshuttling protein) and EGFPhNup98 (a mobile nucleoporin) transfected cells (Griffis et al., 2002). Our results show that EGFPhGle1B nuclear fluorescence was lost by repetitive cytoplasmic bleaching, similar to EGFPhNup98. In contrast, <9% of nuclear EGFPcoilin fluorescence was lost in the same time frame. Thus, these experiments corroborate the microinjection results and demonstrate that hGle1B harbors nucleocytoplasmic shuttling activity.
To test whether the 39amino acid region from residues 444483 was required for hGle1 shuttling, we attempted to analyze the dynamics of EGFPhGle1BSD in live HeLa cells. Unfortunately, ectopic expression of EGFPhGle1B
SD was toxic to the cells (unpublished data). Thus, GSThGle1B
SD was purified and microinjected into one nucleus of binucleate HeLa cells (Fig. 3 D, left). After incubation at 37°C for 5 h, GSThGle1B
SD was detected only in the injected nucleus in 87.8% (±6.7%; n = 52) of injected cells. GSThGle1B
SD was also microinjected into the cytoplasm of HeLa cells and detected in the nucleus in 89.4% (±2.5%; n = 55) of cases (Fig. 3 D, right, asterisk). However, over the same time frame after injection, the nuclear signal was not nearly as intense as with GSThGle1B (Fig. 3, B and D, asterisk). We concluded that GSThGle1B
SD did not have nuclear export activity, and any remaining domain(s) had weak nuclear localization activity. The SD may provide the primary nuclear import pathway (see below). Similar experiments were conducted using GSThGle1A and GSThGle1A
SD and showed identical results (not depicted).
The SD of hGle1 is required for hGle1-dependent mRNA export
Our previous studies have shown that hGle1 is required for the export of poly(A)+ RNA in HeLa cells (Watkins et al., 1998). We speculated that hGle1 nucleocytoplasmic shuttling may be important for its transport function and that this role may be mediated by the interaction of the SD with another factor in the poly(A)+ RNA export pathway. To investigate this hypothesis, we used a cell-permeable peptide strategy that is based on inhibiting proteinprotein interactions (Pritchard et al., 1999). We reasoned that by competing for the factor(s) required for hGle1 transport, we would abrogate the shuttling of endogenous hGle1. We would then be able to study the effect of a "static" hGle1 on total poly(A)+ RNA distribution within the cell by in situ hybridization. A similar strategy was used by Gallouzi and Steitz (2001) in a study of mRNA export pathways.
To test this hypothesis, a peptide corresponding to the SD was synthesized as a COOH-terminal fusion to a 16amino acid portion of helix 3 from the homeodomain of the Drosophila transcription factor antennapedia (Prochiantz, 1999), designated APhGle1-SD (Fig. 4 A). The antennapedia peptide (AP) sequence mediates the uptake of peptides into cells and confers stability (Derossi et al., 1996, 1998). As a control, an AP fusion peptide was also synthesized with a randomly scrambled sequence of the same amino acid composition as the SD (designated APhGle1-scrSD; Fig. 4 A). As seen in Fig. 4 B, incubating cultured HeLa cells (at <10 passages, see Materials and methods) with media containing 5 µM APhGle1-SD peptide had distinct effects. After 4 h in the presence of APhGle1-SD peptide, 5% of cells appeared to undergo apoptosis (asterisk). Interestingly, all the remaining interphase cells showed marked accumulation of poly(A)+ RNA in the nucleus. In contrast, no cytotoxicity was detected with the APhGle1-scrSD incubation, and all the cells showed normal poly(A)+ RNA distribution by in situ hybridization (untreated control cells in Fig. 4 C). In culture media with <10% FBS or with APhGle1-SD peptide concentrations >5 µM, the viability of the cells was drastically reduced. However, if the inhibitory peptide was removed after the 4-h incubation, the cells recovered and showed normal poly(A)+ RNA distribution after 12 h (not depicted).
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To test whether the decrease in mRNA export resulted from a general perturbation of transport through NPCs, protein import and export activity was analyzed. For nuclear import, cells transiently expressing a glucocorticoid receptor (GR) GFP fusion protein were treated with APpeptides for 4 h (Fig. 5 A). In the absence of the agonist dexamethasone, a substantial GRGFP cytoplasmic pool was observed. When dexamethasone was added to induce import, nuclear accumulation of GRGFP was observed in the presence of either peptide. To determine if protein export from the nucleus was perturbed, the intracellular distribution of karyopherin/importin ß1 was monitored by IIF. We hypothesized that if the general nuclear protein export pathways were affected by APhGle1-SD peptide, karyopherin/importin ß1 would accumulate in the nucleus of treated cells. However, the cellular distribution of this shuttling karyopherin was not affected (Fig. 5 B). Thus, protein import and export are not markedly inhibited under conditions where APhGle1-SD decreases poly(A)+ RNA export. These data suggest that hGle1-SD function is specifically essential for the mRNP export process.
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To more directly address the effect of APhGle1-SD on hGle1 nucleocytoplasmic shuttling, we analyzed the distribution of microinjected GSThGle1A in cells treated with the APhGle1-SD peptide. Compared with the control APhGle1-scrSD cells, the presence of the APhGle1-SD peptide resulted in less efficient import of cytoplasmically injected GSThGle1A into the nucleus (90 ± 2.5%; n = 49 microinjected HeLa cells) (Fig. 7 A, left). Similarly, the APhGle1-SD peptide impaired the export of nuclear-injected GSThGle1A (88.5 ± 3%; n = 43 injected cells) (Fig. 7 A, right).
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Discussion |
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We also report that two hGle1 isoforms are expressed in HeLa cells, hGle1A and hGle1B. Both are identical except for their very COOH-terminal regions. Two lines of evidence suggest that the main hGle1 protein in HeLa cells is hGle1B. First, in contrast to EGFPhGle1A, EGFPhGle1B intracellular localization is highly similar to endogenous hGle1. Second, semiquantitative RT-PCR experiments show that hGle1B is the more abundant mRNA isoform in HeLa cells. We speculate that the unique 43amino acid region at hGle1B's COOH terminus is required for NPC/NE association. Interestingly, this region of hGle1B shares similarities with scGle1 (Fig. 1 D), which is also predominately localized at NPCs (Murphy and Wente, 1996; Rout et al., 2000). Our future work will address whether each hGle1 isoform plays distinct in vivo roles.
Based on recent proteomic analysis of the mammalian NPC (Cronshaw et al., 2002) and these studies, we conclude that hGle1 is not a stable component of the NPC and that hGle1 is not required for overall maintenance of NPC structure and function. In cells treated with APhGle1-SD peptide, no perturbations in the localization of nucleoporins recognized by mAb414, in GFPGR protein import, or in karyopherin/importin ß1 nuclear export were detected. In light of recent elegant reports on hNup153 (Nakielny et al., 1999; Daigle et al., 2001), hNup98 (Griffis et al., 2002), Npap60/Nup50 (Lindsay et al., 2002), and scNup2 (Dilworth et al., 2001), it is becoming evident that a subpopulation of factors with steady-state NPC localization have unexpected dynamic properties. We propose that hGle1B transiently associates with NPCs during its function as a shuttling mRNA export mediator.
Results from our photobleaching experiments and cell-permeable peptide studies strongly support the conclusion that endogenous hGle1 has a bona fide SD and dynamically enters and exits the nucleus. In the presence of competing APhGle1-SD peptide, the association of endogenous hGle1 with the NPC/NE was substantially reduced, and the shuttling of recombinant hGle1 was severely impaired. Coincidentally, APhGle1-SD peptide induced nuclear poly(A)+ RNA accumulation, suggesting that hGle1 is involved in a molecular cascade that leads to the export of mRNP complexes out of the nucleus. It is possible that APhGle1-SD peptide may compete for hGle1's nucleoporin binding site(s) or for an adaptor protein that bridges the interaction of hGle1 with nucleoporins at NPCs. The latter would be similar to the interaction networks of karyopherin/importin ß1 for (Gorlich et al., 1995a,b) or Crm1 for Rev (Fornerod et al., 1997). Alternatively, the SD peptide may affect more than Gle1. If the unidentified SD-binding factor has additional interacting partners, their interactions could be equally perturbed. Thus, isolating an hGle1-SDinteracting protein is of great interest. As a dynamic NPC-associated protein, we speculate that hGle1 either actively participates in the export process or facilitates the nuclear import of an unidentified factor that would mediate mRNA export.
Interestingly, the amino acid sequence of the hGle1 SD is not similar to any of the previously characterized nucleocytoplasmic SDs (Fig. 8). A direct alignment of the hGle1 SD with the M9 of hnRNP A1 suggests weak similarity, which could result in a potential interaction with the M9-binding karyopherin ß2A/transportin-1 (Siomi et al., 1997). However, the key consensus residues implicated in transportin-1 binding are not present in hGle1 (Pollard et al., 1996). We tested for an hGle1-SD and transportin-1 interaction and found that bacterially expressed GSTtransportin-1 does not directly bind immobilized hGle1-SD peptide (unpublished data). This suggests that transportin-1 is not directly involved in hGle1 shuttling. Further deletions within the hGle1 SD and selective point mutations based on interspecies homology analysis have not yet been successful in separating the nuclear import and export activities (unpublished data).
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Our work in this report strongly suggests that hGle1 dynamics are key to the poly(A)+ RNA export mechanism. It will be of great interest to identify which factor(s) interacts with hGle1A and B and, in particular, the hGle1 SD. We predict that this information will be valuable in ascertaining the molecular cascade leading to hGle1-mediated mRNP export.
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Materials and methods |
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Cell-permeable peptide assay and nuclear import assay
Peptides were synthesized by FAST-moc chemistry and purified by reverse-phase HPLC, and the sequence was confirmed by mass spectrometry. Each peptide harbors the antennapedia sequence at its NH2 terminus (RQIKIWFQNRRMKWKK) (Fig. 4 A). For assays, peptides were first diluted in complete DME and added cells (CCL-2 cells; lot no. 2462426; passage <10 after receipt from American Type Culture Collection) and grown to 7080% confluency. After 4 h incubation, cells were processed for in situ hybridization and/or IIF. We noted a decrease in the susceptibility of HeLa cells after passage 150 from reception; 10% of the population appeared to become resistant to the effects of APhGle1-SD peptide, likely due to diminished peptide uptake. This trend increases with passage number, ultimately resulting in APhGle1-SDresistant HeLa cell populations. Nuclear import activity in APpeptide-treated cells was determined after transfection with pK7.GR.GFP expression vector (Carey et al., 1996) and using dexamethasone treatment (10 µg/ml; Sigma-Aldrich).
IIF
HeLa cells were processed by three methods, Fix-Triton, Digitonin-Fix, and Digitonin-Fix-Triton, as described by Watkins et al. (1998). The primary antibodies used were affinity-purified rabbit anti-GST (1:200), affinity-purified rabbit anti-hGle1 (1:200; Watkins et al., 1998), rabbit antilamin B (1:50; antipeptide NC-6; gift of N. Chaudhary and G. Blobel, The Rockefeller University, New York, NY), mAb414 (1:10; Davis and Blobel, 1986), rabbit anti-FLAG (1:200), and mouse antiimportin ß (anti-p97; 1:1,000; Affinity BioReagents, Inc.). FITC-conjugated donkey antirabbit (1:200) or FITC-conjugated donkey antimouse (1:200; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Cells were examined using an Olympus BX50 microscope. Digital images were recorded with a Photometric CoolSNAP HQ camera (Roper Scientific) using MetaVue 6.0 software and digitally manipulated in Adobe Photoshop 7.0®.
In situ hybridization
Cells were processed as described by Watkins et al. (1998). 3'-digoxigeninlabeled oligo(dT)30 probe was synthesized (Wente and Blobel, 1993) and used at 7 µg/ml. The 3'-digoxigeninlabeled DHFR probe complementary to nucleotides 530569 of DHFR mRNA (Gallouzi and Steitz, 2001) was synthesized by Integrated DNA Technologies Inc. and used at 20 µg/ml. After hybridization, cells were blocked for 1 h with 6% nonimmune sheep serum (Jackson ImmunoResearch Laboratories), and hybridized probes were detected with 1:200 sheep antidigoxigenin Fabrhodamine (Roche Applied Science). For in situ/IIF double experiments, polyclonal anti-hGle1 antibodies (1:200) or mAb414 (1:10) were used in combination with 1:200 FITC-conjugated sheep antirabbit or sheep antimouse (Sigma-Aldrich), respectively.
Cytoplasm/nuclear RNA isolation and RT-PCR
HeLa cells (5 x 106) treated or untreated with APpeptides were scraped off culture plates and suspended in ice cold RNase-free PBS. Nuclear and cytoplasmic RNA fractions were isolated after the NP-40 lysis procedure (Greenberg and Ziff, 1984), except that the lysis buffer was supplemented with 1 mM DTT and 1 U/µL of RNAsinTM (Promega). Cell lysis was monitored by bright field microscopy, and the integrity of the isolated nuclei was confirmed by nucleoplasmic exclusion of the 70-kD TR-labeled dextran (not depicted). Total RNA from each fraction was isolated using TrizolTM reagent (Invitrogen). Genomic DNA contamination of the purified cytoplasmic and nuclear RNA was estimated by PCR before reverse transcription using the same conditions as below (not depicted). Each RNA fraction was subsequently reverse transcribed using Superscript IITM and random hexamers (Invitrogen) as per the manufacturer's recommendations. Serial dilutions of the resulting cDNAs were made, and 10% of each was used in PCR studies. For DHFR gene amplification (28 cycles), specific primers (forward, 5'-CAGAACATGGGCATCGGCAAGAACG-3'; reverse, 5'-AAACAGAACTGCCACCAACTATCCA-3') were used. 50% of each reaction was separated by electrophoresis through a 1.7% agarose gel stained with ethidium bromide. Amplification of hGle1A and hGle1B was conducted using the above conditions and a common forward primer, F (5'-ATACCAGGTCAGTTCTGGAAGATGC-3'), with the respective A-specific reverse primer (5'-TCAACGAGAGGGACTGGAC-3') or B-specific reverse primer (5'-AGGAGCGCCAGAAGGAGG-3').
Plasmid constructs
Cloning of hGle1 cDNA isoforms.
IMAGE clone no. 22734 was used to generate full-length hGle1A NH2-terminally tagged with EGFP (pSW1409; CLONTECH Laboratories, Inc.) or FLAG (pSW1102). Based on partial EST sequence information, hGle1B-specific primers were designed and used to generate a 2.3-kbp fragment by using the HerculaseTM polymerase mix (Stratagene) and PCR. hGle1B cDNA was subsequently cloned into pEGFP-C1 (pSW1482; CLONTECH Laboratories, Inc.).
FLAGhGle1NLS constructs.
Oligonucleotides encoding the SV40 large T antigen NLS were annealed and cloned at the 3' end of FLAGhGle1A, yielding FLAGhGle1ANLS (pSW1116). Digestion of pSW1116 with BglII generated FLAGhGle1444606NLS (pSW1127). Deletions of the hGle1 444606 region were made by PCR and cloned back into pSW1127, generating pSW1190 (FLAGhGle1
444541NLS), pSW1206 (FLAGhGle1
444511NLS), pSW1416 (FLAGhGle1
511606NLS), and pSW1415 (FLAGhGle1
484606NLS).
GST expression constructs.
Full-length hGle1A and B were cloned in frame with GST in pGex-3X series (AP Biotech) (pSW728 and pSW1487, respectively). These were used to generate pGex-hGle1A444483 (pSW1407) and pGex-hGle1B
444483 (pSW1496). An hGle1 DNA fragment coding for region 444483 was amplified by PCR from IMAGE clone no. 22734 and cloned into pGex 5X-2 (AP Biotech), yielding pGex-hGle1444483 (SD) (pSW1412).
Purification of recombinant proteins
GST fusion proteins were expressed in BL21 cells and purified according to the manufacturer's instructions (AP Biotech), except cells were lysed by French press (12,000 PSI). After affinity chromatography, fusion proteins were eluted with 15 mM reduced glutathione (Sigma-Aldrich) in 150 mM NaCl, 50 mM Tris, pH 8.5. Eluates were dialyzed against microinjection buffer (10 mM phosphate buffer, pH 7.6, 75 mM KCl) and stored at -70°C.
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Acknowledgments |
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This work was supported by funds from the National Institutes of Health (NIH) (GM-197190 to D.M. Barry, GM-59975 to M.A. Powers, and GM-51219 to S.R. Wente). E.R. Griffis is a predoctoral trainee of the NIH (T32 GM08367-13).
Submitted: 19 November 2002
Revised: 10 February 2003
Accepted: 10 February 2003
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