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Genetic Interactions of Yeast Eukaryotic Translation Initiation Factor 5A (eIF5A) Reveal Connections to Poly(A)-Binding Protein and Protein Kinase C Signaling
Sandro R. Valentinia,b, Jason M. Casolaria, Carla C. Oliveirac, Pamela A. Silvera, and Anne E. McBride2,aa Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115,
b Department of Biological Sciences, School of Pharmacy, São Paulo State University, Araraquara, SP, 14801-902, Brazil
c Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-900, Brazil
Corresponding author: Pamela A. Silver, 44 Binney St., SM922, Boston, MA 02115., pamela_silver{at}dfci.harvard.edu (E-mail)
Communicating editor: L. PILLUS
| ABSTRACT |
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The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.
REGULATION of the fate of cytoplasmic mRNA involves a delicate balance between its recruitment to ribosomes for translation and its degradation by nucleolytic enzymes. As researchers have studied separately the factors that are important for translation and those that are required for mRNA turnover, it has become clear that these two processes are intimately linked. eIF5A is an enigmatic protein that has been implicated in several steps of RNA metabolism including both translation and mRNA degradation.
eIF5A is a highly conserved protein encoded in the genomes of eukaryotes and archaebacteria (![]()
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Alternatively, it was suggested that eIF5A could be involved in the translation of a specific subset of mRNAs, for example, those involved in the cell cycle progression (G1/S transition; ![]()
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The yeast Saccharomyces cerevisiae contains two 90% identical genes encoding eIF5A, TIF51A (HYP2), and TIF51B (HYP1) (![]()
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haploid strain (![]()
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Although eIF5A has been suggested to participate in the nucleocytoplasmic trafficking of the HIV-1 Rev protein/RRE complex (![]()
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Recently it was shown that a yeast mutant harboring a temperature-sensitive allele of TIF51A exhibits a defect in mRNA decay, accumulating uncapped mRNAs at the restrictive temperature. In addition, this strain shows an
30% decrease in protein synthesis at high temperature (![]()
Here we have characterized three novel alleles of TIF51A and have used them to address the proposed functions of eIF5A. We also present data linking eIF5A with both poly(A)-binding protein and protein kinase C. The isolation of PAB1 and PKC1 as multicopy suppressors of a temperature-sensitive allele of TIF51A, tif51A-1, suggests important connections between these three proteins and their roles in RNA metabolism, including translation, mRNA decay, and ribosome biogenesis.
| MATERIALS AND METHODS |
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Yeast strains, growth conditions, and plasmids:
S. cerevisiae strains and their genotypes used in this work are listed in Table 1. Procedures for cell growth and genetic manipulations were according to standard protocols (![]()
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Construction of a marked TIF51A allele and linkage assays:
To mark TIF51A with the URA3 gene, a 1.4-kb PstI fragment containing TIF51A was removed from the plasmid pPS1483 and ligated into the NsiI site of the vector YIp5, generating the plasmid pPS1591. This plasmid was linearized using HpaI, which cleaves within the TIF51A gene, and used to transform a W303a/
diploid strain. After selection on uracil dropout plates, transformed diploids were sporulated. Genomic DNA from the selected diploids and their haploid progeny was digested with PstI and subjected to Southern blot analysis using the 1.4-kb PstI fragment as a probe. One of the haploid cells that contained the URA3 gene integrated next to TIF51A was chosen for linkage assay (PSY1241). To examine linkage of the tif51A temperature-sensitive (ts) alleles and TIF51A, the tif51A mutants (PSY1242PSY1244) were initially crossed to this strain containing a TIF51A-marked allele (PSY1241). The diploids resulting from these crosses were sporulated and subjected to tetrad analysis.
Amplification and sequencing of the tif51A temperature-sensitive alleles:
Genomic DNA was prepared from the tif51A temperature-sensitive strains (PSY1245, PSY1248, and PSY1249). The TIF51A gene was amplified by PCR using Taq DNA polymerase (Perkin-Elmer, Norwalk, CT) and the primers NPL2-A and NPL2-B. PCR products were purified and sequenced using the same primers at the Dana-Farber Cancer Institute Molecular Biology Core Facility.
Cloning TIF51A and the tif51A temperature-sensitive alleles:
TIF51A was cloned from genomic DNA purified from a wild-type strain (FY23) and the ts alleles from the tif51A ts strains (PSY1245, PSY1248, and PSY1249). PCR reactions were performed using primers NPL2-C and NPL2-D, which contain a BamHI site. PCR products were digested with BamHI and gel purified. The fragment containing wild-type TIF51A was cloned into the BamHI site of pRS316, generating the plasmid pPS1592. For the mutated alleles, PCR fragments were transferred into pRS315, resulting in plasmids pPS1593 (tif51A-1), pPS1594 (tif51A-2), and pPS1595 (tif51A-3). All constructs were confirmed by sequencing.
Disruption of TIF51A:
A PCR strategy (![]()
Anti-eIF5A Western blotting:
To produce anti-eIF5A antisera, a plasmid to express GST-eIF5A in Escherichia coli (pPS1596) was constructed as follows. The TIF51A gene was amplified by PCR from genomic DNA of a wild-type strain (FY23) using primers TIF51A-1 and TIF51A-2. The PCR product was digested with BamHI and AvaI, gel purified, and ligated into pGEX-4T-1 (Pharmacia, Piscataway, NJ) that had been digested with BamHI and SalI. GST-eIF5A was bacterially expressed from this plasmid, the fusion protein purified, and eIF5A released from GST-eIF5A by thrombin (Sigma, St. Louis) cleavage essentially as described (![]()
To determine eIF5A levels in tif51A mutant strains, cells were grown to midlog phase at 25°, shifted to 37° or left at 25° for 3 hr, and then lysed in radio-immune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) with 1 mM phenylmethylsulfonyl fluoride as previously described (![]()
Localization of eIF5A:
Indirect immunofluorescence was used to localize endogenous eIF5A using the polyclonal anti-eIF5A at a dilution of 1:1000. This experiment was performed essentially as described (![]()
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To localize eIF5A in living cells, a plasmid that expressed a fusion between green fluorescent protein (GFP) and eIF5A was transformed into a wild-type strain (FY23). This plasmid, pPS1597, was generated by PCR amplification of TIF51A from FY23 genomic DNA using primers NPL2-L and NPL2-M. The PCR product was digested with BamHI and HindIII and ligated into the pCGF-1a vector (![]()
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mRNA stability assay:
Exponentially growing yeast cultures were shifted from 25° to 37°. At various times, samples were collected and quickly frozen in a dry ice-ethanol bath. Total RNA was isolated from yeast cells by a modified hot phenol method (![]()
High-copy suppressor screen:
A URA3/2µ genomic yeast library (![]()
High-copy suppressor genes were characterized by subcloning different segments of the original clone into pRS426 (URA3/2µ) and testing in PSY1252. The minimal suppressing subclone containing the PKC1 open reading frame (ORF) is pPS1600. Also, TIF51A and the five other high-copy suppressor genes were cloned into pRS426 using the following primers containing a BamHI site: NPL2-C and NPL2-D to clone TIF51A (pPS1598); 137-E and 137-F to clone YOR137C (pPS1599); PAB1-C and PAB1-D to clone PAB1 (pPS1601); WSC1-A and WSC1-B to clone WSC1 (pPS1602); WSC2-A and WSC2-B to clone WSC2 (pPS1603); and WSC3-A and WSC3-B to clone WSC3 (pPS1604). In addition, hemagglutinin (HA)-tagged forms of wild-type and K853R mutant PKC1 were subcloned into pPS1600 by digestion of pGAL[PKC1::HA] and pGAL[pkc1-K853R::HA] (![]()
| RESULTS |
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Characterization of three new temperature-sensitive alleles of TIF51A:
In a screen for conditional mutants defective in nuclear protein localization in the yeast S. cerevisiae, several complementation groups were obtained (![]()
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Using one of the original mutants (PSY8) and a yeast genomic library, TIF51A was cloned by complementation of the temperature-sensitive phenotype (![]()
These three TIF51A temperature-sensitive alleles were cloned by PCR into pRS315 (CEN/LEU2) using genomic DNA obtained from the tif51A strains. To test whether the cloned mutant alleles of TIF51A were functional, we generated a strain in which TIF51A was disrupted. Sporulation of a heterozygous TIF51A/tif51A
::HIS3 diploid revealed that TIF51A is an essential gene since only two viable his- spores were obtained in 24 tetrads analyzed. This result agreed with ![]()
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The three TIF51A alleles demonstrate varying degrees of temperature sensitivity upon growth at 37° on rich media, YEPD (Fig 1A). While the allele tif51A-2 displays a moderate reduction in growth at the nonpermissive temperature, alleles tif51A-1 and tif51A-3 display more pronounced growth defects. To determine the time of onset of the growth defect in the tif51A mutant strains, midlog phase cultures were monitored at different times after shift to the nonpermissive temperature (Fig 1B). Although tif51A mutant strains showed slightly slower growth than the wild-type strain at 25° (left), they showed a significant decrease in growth rate by 23 hr after shift to 37° (right). The discrepancy in the relative growth defect between tif51A-1 and tif51A-2 on plates vs. liquid may reflect a growth defect that is overcome after longer incubation times.
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To define the mutations in the TIF51A gene present in the tif51A temperature-sensitive mutants, genomic DNA was isolated from the tif51A strains and the TIF51A gene was amplified by PCR. The 0.7-kb fragment containing TIF51A was purified and then sequenced. These mutations were confirmed by sequencing the cloned alleles in pPS1593, -1594, and -1595. A single mutation of the same residue was detected for the tif51A-1 and tif51A-2 alleles. The proline at position 83 was changed to serine (P83S) or leucine (P83L), respectively. Two mutations were mapped in the tif51A-3 allele, a cysteine-to-tyrosine change at position 39 (C39Y) and a glycine-to-aspartic acid change at position 118 (G118D).
Two crystal structures of archaebacterial eIF5A proteins have been solved (![]()
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To determine eIF5A protein levels in the tif51A mutant strains, a rabbit polyclonal antiserum against eIF5A was produced using recombinant yeast eIF5A expressed in E. coli. This antiserum was used to detect eIF5A by Western blot in cell lysates prepared from wild-type and temperature-sensitive TIF51A strains grown at the permissive and nonpermissive temperature (Fig 3). At 25°, eIF5A of the expected molecular mass (
20 kD) was detected in the wild-type (WT) and in the tif51A-1 and tif51A-2 strains. In the case of tif51A-3, eIF5A migrates slightly more slowly, due to the G118D mutation in eIF5A in the tif51A-3 strain (data not shown). After a 3-hr shift to 37°, levels of mutant eIF5A proteins are significantly lower than that of the wild-type protein, suggesting that these are loss-of-function alleles. Indeed, after 12 hr at 37°, eIF5A mutant protein is no longer detectable by Western blotting (data not shown).
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Localization of eIF5A in the yeast S. cerevisiae:
eIF5A has been proposed to be an export factor for the human immunodeficiency virus (HIV) Rev protein, by virtue of acting as an adapter to the transporter exportin/CRM1 (![]()
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To ascertain whether this localization reflected eIF5A distribution in live cells, TIF5A was inserted downstream of a galactose-inducible green fluorescent protein gene. This plasmid produces functional eIF5A in that it restores the ability of tif51A-1 to grow at the restrictive temperature after galactose induction (data not shown). This plasmid was transformed into a wild-type strain and after 12 hr induction with galactose, GFP-eIF5A localization was determined by fluorescence microscopy (Fig 4B). As expected, GFP-eIF5A was detected predominantly in the cytoplasm with a stronger signal in the perinuclear region (Fig 4B, left). These results conclusively show that eIF5A is a cytoplasmic protein and accumulates in the perinuclear region.
If eIF5A were to facilitate HIV Rev export by binding both Rev and the Xpo1/Crm1 exporter, export of eIF5A itself would be expected to be blocked by mutations in the export machinery. To test if eIF5A is exported by Xpo1/Crm1, its localization was monitored in xpo1-1 cells, which are temperature sensitive for export by this pathway. Xpo1-1 cells carrying GFP-eIF5A or GFP-Rev on 2µ URA3 plasmids under the galactose-inducible promoter were grown to midlog phase and then induced with galactose for 3 hr, shifted to 37° for 1 hr, fixed, and then visualized by fluorescence microscopy (Fig 4C). GFP-eIF5A localization was predominantly cytoplasmic in xpo1-1 cells at 37°. Comparison with DAPI staining shows no marked increase in nuclear localization of eIF5A, which would be expected if eIF5A shuttled in an Xpo1-dependent manner, suggesting that eIF5A is not exported by Xpo1 in yeast. In contrast, GFP-Rev showed a marked accumulation of fluorescent signal in nuclei of xpo1-1 cells after 1 hr at the nonpermissive temperature (Fig 4C).
To further test the dependence of Rev export upon eIF5A, GFP-Rev localization was examined in strains containing the TIF51A temperature-sensitive alleles. After a 3-hr shift to the nonpermissive temperature, GFP-Rev displayed cytoplasmic localization similar to that seen in the wild-type control (Fig 5). The slight increase in signal in the mutant alleles after temperature shift does not coincide with the nucleus as determined by DAPI staining (Fig 5). These data are consistent with a recent study in mammalian cells that revealed that exportin 4, rather than CRM1, exports eIF5A (![]()
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mRNA decay defect of tif51A alleles:
eIF5A has also been proposed to play a role in mRNA decay (![]()
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Suppression of the temperature-sensitive phenotype of tif51A-1:
To expand our understanding of the function of eIF5A, we wished to identify other genes that encode proteins that work similarly or together with eIF5A, and therefore we performed a high-copy suppressor screen. A yeast genomic library was transformed into the tif51A-1 strain and transformants that could grow at 36° were selected. Twenty-three plasmids that still suppressed temperature sensitivity after retransformation into tif51A-1 were characterized by DNA sequencing. Six clones, representing 15 of these plasmids, were selected and used to define the ORF that suppresses the temperature-sensitive phenotype of tif51A-1. These clones are presented in Table 4. One clone contained the entire PKC1 gene, which was cloned twice in this screen, without flanking ORFs. Subcloning revealed the other suppressing ORFs to be the following: YOR137C, WSC2, WSC1, WSC3, and PAB1. To eliminate partial flanking ORFs, these five genes were also cloned by PCR and the constructs tested for suppression of the temperature-sensitive phenotype of tif51A-1 strain. Fig 7 shows that the ability of tif51A-1 to grow at 36° was restored by expression of these genes in high copy. In addition, all tif51A-1 suppressors were allele specific, as they were unable to rescue growth of tif51A-3 at high temperatures (data not shown). The plate phenotype of tif51A-2 at 36° was too weak to test suppression in this manner.
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To test whether the two most-well-studied suppressor proteins, poly(A)-binding protein (Pab1) and protein kinase C (Pkc1), acted by increasing the levels of mutant eIF5A protein, mutant strains bearing the multicopy PAB1 and PKC1 plasmids were grown at 25° or 37° for 3 hr and eIF5A levels were monitored by Western blotting (Fig 8). Overexpression of PAB1 and PKC1 did not have a significant effect on steady-state levels of eIF5A at either permissive or nonpermissive temperatures. The slight increase in eIF5A-3 levels in the presence of excess Pkc1 was not reproducible.
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Connections between PKC1 signaling and eIF5A:
To examine the importance of PKC1 signaling in tif51A mutant strains, we tested whether a kinase-inactive form of PKC1 could suppress the tif51A-1 temperature sensitivity. Strain PSY1245 was transformed with high-copy plasmids encoding wild-type Pkc1, HA-tagged wild-type Pkc1, or HA-tagged Pkc1 with a lysine-to-arginine change in the active site (K853R). Serial dilutions of each transformed strain were then plated at 25° or 37° (Fig 9). Whereas the ability of PKC1 to suppress tif51A-1 temperature sensitivity was not impaired by the addition of an HA tag (WT vs. WT::HA), the active site mutation (K853R::HA) eliminated suppression. Growth of the pkc1
strain, which requires PKC activity, is shown as a control (![]()
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Although PKC1 is involved in multiple signaling pathways in yeast, the additional isolation of three WSC genes as high-copy suppressors of tif51A-1 suggested a connection between eIF5A and a specific pathway involved in stress response and maintenance of cell wall integrity (for review see ![]()
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| DISCUSSION |
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In spite of two decades of research on eIF5A, the role of this ubiquitous protein remains mysterious. In this report, three temperature-sensitive alleles of TIF51A were characterized (tif51A-1, tif51A-2, and tif51A-3). While mRNA decay experiments indicated that the stronger mutations led to stabilization of mRNAs, localization studies of wild-type eIF5A suggested that this protein is unlikely to be involved in HIV Rev export. The tif51A-1 allele was used to contribute further to the search for a function for this highly conserved protein. Screening of a 2µ plasmid library containing genomic DNA uncovered six genes that allowed growth of tif51A-1 strains at high temperature. Further experiments supported the importance of the PKC/WSC signaling pathway for cell wall integrity in tif51A-1 mutant cells.
In fluorescence microscopic experiments, eIF5A was detected primarily in the cytoplasm with a concentration in the perinuclear region (Fig 4). This result agrees with the subcellular localization of mammalian eIF5A (![]()
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The identification of conditional tif51A alleles in a screen for mislocalization of a nuclear localization sequence (NLS)-reporter protein initially would seem to suggest that eIF5A is crucial for nuclear import (![]()
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A high-copy suppressor screen with the tif51A-1 allele revealed six genes that could suppress the temperature sensitivity of this mutant. These suppressors are PAB1, YOR137C, PKC1, WSC1, WSC2, and WSC3. The most frequent suppressor in the screen, YOR137C [named here suppressor of eIF5A (SIA1)], encodes a protein with a peptide signal for membrane localization and was recently associated with activation of the yeast plasma membrane H+-ATPase by glucose (![]()
Another high-copy suppressor of tif51A-1, PAB1 has been shown to be involved in multiple aspects of RNA metabolism, including 3'-end processing, translation, and mRNA decay. The yeast gene, PAB1, is essential but the lethality caused by deletion of PAB1 can be suppressed by mutations in a number of other genes, including genes that encode components of the translational and mRNA degradation machinery (![]()
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Like Pab1, eIF5A has been implicated in both translation and mRNA decay. Although eIF5A was first thought to be a translation initiation factor (![]()
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The high-copy suppressor focused on in this work, PKC1, is the yeast homolog of the metazoan PKC gene and encodes a regulator of one of the four mitogen-activated protein-kinase cascades characterized so far in S. cerevisiae (![]()
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Although PKC signaling has not been shown to have a direct role in mRNA translation or decay, recent studies have revealed the importance of PKC/WSC signaling in the regulation of ribosome synthesis (![]()
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The ability of tif51A-1 temperature sensitivity to be partially suppressed either by overexpression of PKC or WSC genes (Fig 6) or by the addition of sorbitol (Fig 9) suggests that eIF5A may act either in the PKC pathway for cell integrity or in a parallel pathway. eIF5A is phosphorylated in vivo, but the kinase responsible for this nonessential modification has not been identified. PKC1 overexpression may suppress tif51A-1 by increasing phosphorylation of the mutant protein. Alternatively, PKC1 could phosphorylate a protein that interacts with eIF5A and that is important for its function. One candidate eIF5A-interacting protein is deoxyhypusine synthase, the essential enzyme that catalyzes the first step in the lysine-to-hypusine modification (![]()
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The isolation of PAB1, PKC1, and the WSC genes as high-copy suppressors of tif51A-1 points to different roles for eIF5A in RNA metabolism from mRNA decay to ribosome synthesis. The allele specificity of suppression suggests that eIF5A may have more than one important role in the cell, leading to functional differences between the alleles. It is interesting to note that PKC1 was identified in a Pab1p two-hybrid screen (![]()
| FOOTNOTES |
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2 Present address: Department of Biology, Bowdoin College, Brunswick, ME 04011. ![]()
| ACKNOWLEDGMENTS |
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We are grateful to Dr. Sung-Hou Kim for use of the M. jannaschii eIF5A crystal structure and Dr. David Levin for PKC1 reagents. We thank Valerie Weiss for her help in analyzing the crystal structure of eIF5A and Allyson Hatton and Tracy Stage-Zimmermann for critical reading of the manuscript. Sandro Valentini thanks Mario H. Bengtson and Patricia Munerato for technical assistance. This work was supported by grants to S.R.V. and C.C.O. from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), a training grant from the National Eye Institute to J.M.C., a National Institutes of Health grant to P.A.S., and a National Institutes of Health postdoctoral fellowship to A.E.M.
Manuscript received April 25, 2001; Accepted for publication October 30, 2001.
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