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First published online August 10, 2007; 10.1105/tpc.107.052076 The Plant Cell 19:2557-2568 (2007) © 2007 American Society of Plant Biologists MYB98 Positively Regulates a Battery of Synergid-Expressed Genes Encoding Filiform Apparatus–Localized Proteins[W]Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840 1 Address correspondence to drews{at}bioscience.utah.edu.
The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98–green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.
The angiosperm female gametophyte contains two synergid cells that lie adjacent to the egg cell and are essential for reproduction. During the angiosperm fertilization process, a pollen tube grows into one of the synergid cells, ceases growth, ruptures, and releases its two sperm cells into this cell. The synergid cell that interacts with the pollen tube typically undergoes cell death before or upon pollen tube penetration. The two sperm cells then migrate to and fuse with the egg cell and central cell, and ultimately, the fertilized female gametophyte gives rise to the embryo and endosperm of the seed (reviewed in Lord and Russell, 2002  ; Weterings and Russell, 2004).
During the final stages of pollen tube growth, a pollen tube grows along the placental surface, along the surface of the ovule's funiculus, into the ovule's micropyle, and finally into the female gametophyte. To determine whether the female gametophyte plays a role in pollen tube guidance, several groups have analyzed pollen tube growth in Arabidopsis thaliana mutants defective in embryo sac development. These studies showed that the female gametophyte is required for pollen tube guidance at two stages: guidance from the placenta to the funiculus (funicular guidance phase) and guidance from the funiculus to the micropyle (micropylar guidance phase) (reviewed in Higashiyama, 2002
To determine which cells within the female gametophyte produce the pollen tube attractant(s), Higashiyama and colleagues (2001)
At the micropylar pole, the synergid cell wall is extensively thickened and elaborated, forming a structure referred to as the filiform apparatus (see Supplemental Figure 1 online). In Arabidopsis and many other species, the filiform apparatus has numerous finger-like projections into the synergid cytoplasm. Several functions for the filiform apparatus have been proposed, including pollen tube reception, import of metabolites, and export of the pollen tube attractant(s). However, a specific function of the filiform apparatus in pollen tube reception or transport has not been demonstrated (reviewed in Willemse and van Went, 1984
Genetic screens have identified several mutants that appear to be defective in synergid cell function. In sirene (Rotman et al., 2003
Genes functioning in the synergid cells have also been identified through expression-based screens. The Arabidopsis synergid-expressed genes include MYB98 (Kasahara et al., 2005
MYB98 encodes a MYB family protein and is predicted to function as a transcriptional regulator. Within the female gametophyte, MYB98 is expressed predominantly in the synergid cells. myb98 female gametophytes are defective in pollen tube guidance: wild-type pollen tubes grow from the placenta to the funiculus but then fail to grow into the micropyle, indicating that the myb98 mutation affects the micropylar guidance phase of pollen tube guidance. In addition, the filiform apparatus of myb98 mutants lacks the finger-like projections observed in wild-type synergid cells. However, with the exception of the filiform apparatus defect, myb98 synergid cells appear normal, suggesting that MYB98 regulates the expression of a specific set of genes that function in pollen tube guidance and filiform apparatus formation (Kasahara et al., 2005 ). Here, we provide evidence for this model. We show that MYB98 is localized to the nuclei of the synergid cells, binds DNA at a specific sequence, and is required for the expression of a battery of synergid-expressed genes. We also show that at least one of the downstream genes is likely a direct target of MYB98. Finally, we show that many of the downstream genes encode proteins that are secreted into the filiform apparatus. Together, these data suggest very strongly that MYB98 activates a branch of the synergid gene regulatory network that is required for pollen tube guidance and filiform apparatus formation.
MYB98 Binds DNA and Is Localized to the Nucleus MYB98 is predicted to encode a MYB family transcription factor (Stracke et al., 2001 ). If this prediction is correct, MYB98 should bind DNA and be localized to the nucleus. To determine the subcellular localization of MYB98, we analyzed transgenic plants containing a construct (ProMYB98:GFP-MYB98) comprising the MYB98 promoter, a green fluorescent protein (GFP) coding sequence, and the entire MYB98 coding sequence. This construct was sufficient to rescue the myb98-1 mutant phenotype, suggesting that localization of this fusion protein mimics that of endogenous MYB98. Rescue was established by generating plants homozygous for both the myb98-1 mutation and the ProMYB98:GFP-MYB98 construct (see Methods); these plants had full seed set.
As shown in Figures 1A and 1B
, in a mature, unfertilized, wild-type female gametophyte (stage FG7) (Christensen et al., 1997
We used electrophoretic mobility shift assays (EMSAs) to determine whether MYB98 binds DNA. We previously showed that MYB98's Myb domain (R2 and R3 Myb repeats) is highly similar to that of mammalian c-Myb (Kasahara et al., 2005 ), suggesting that MYB98 binds DNA to a sequence similar to that of c-Myb. Several studies have shown that c-Myb binds to the consensus sequence YAACNG (Y = thymine or cytosine, N = any nucleotide) and that the AAC residues are critical for binding specificity (Biedenkapp et al., 1988; Weston, 1992; Tanikawa et al., 1993; Oda et al., 1998). To determine whether MYB98 binds to the c-Myb consensus sequence, we generated the MYB98 Myb domain in Escherichia coli and analyzed interaction with a labeled oligonucleotide containing the sequence TAACGG using EMSAs. As shown in Figure 2A , addition of the MYB98 Myb domain caused a shift in the mobility of the labeled oligonucleotide, indicating that MYB98 binds to DNA. To determine whether this binding is specific for the sequence TAACGG, we added unlabeled oligonucleotides to the binding reaction. As shown in Figure 2A, the wild-type oligonucleotide competed fully, whereas an oligonucleotide containing mutations in the core AAC residues (M1) competed much less effectively. These data indicate that MYB98 binds to DNA in a sequence-specific manner.
To further define the MYB98 DNA binding sequence, we performed a series of competition assays in which binding of MYB98 to a labeled oligonucleotide containing the sequence TAACGG was competed with a 25-fold excess of unlabeled oligonucleotides containing mutations at various positions. As shown in Figure 2B, oligonucleotides containing mutations at positions 5 and 6 (M2, M3, and M4) competed as effectively as the wild-type oligonucleotide, an oligonucleotide containing a cytosine at position 1 (M7) competed but did so less effectively than the wild-type oligonucleotide, and oligonucleotides containing an adenine or a guanine at position 1 (M5 and M6) did not compete with the wild-type oligonucleotide. Together, these data suggest that MYB98 binds to the sequence YAAC with a preference for TAAC. In summary, MYB98 is localized to the nucleus of the synergid cells and binds DNA at a specific sequence. These data suggest very strongly that MYB98 functions as a transcriptional regulator in the synergid cells.
MYB98 Is Not Autoregulated
Identification of 16 Genes Downstream of MYB98
To validate these expression patterns, we analyzed the expression of the corresponding promoter:GFP constructs for these 17 genes in MYB98 and myb98-1 female gametophytes. Using crosses, we generated plants hemizygous for the promoter:GFP construct and homozygous for the myb98-1 mutation. We then scored the number of female gametophytes expressing GFP in the synergid cells in these plants as well as in the parental line. As shown in Figure 3 , the number of female gametophytes expressing ProDD27:GFP in the synergid cells was approximately equal in MYB98/MYB98 and myb98-1/myb98-1 pistils. By contrast, the other 16 genes showed reduced expression of the corresponding promoter:GFP construct in myb98-1/myb98-1 pistils (Figure 3). Together, these data indicate that MYB98 is required for the expression of these 16 genes during synergid cell development.
Genes Downstream of MYB98 Encode Proteins That Localize to the Filiform Apparatus The predicted functions of the 16 genes downstream of MYB98 are listed in Table 1. More than half (9 of 16 genes) encode proteins of unknown function. All except one (DD34) encode proteins with predicted N-terminal signal peptides. Most (11 of 16 genes) of the genes downstream of MYB98 encode small, Cys-rich proteins (Table 1), 6 of which belong to two families: DD11, DD18, and DD31 are within the CRP3700 subgroup; and DD4, DD17, and DD34 are within the CRP3740 subgroup (Silverstein et al., 2007 ).
To gain insight into the functions of the genes downstream of MYB98, we obtained and analyzed T-DNA mutants from the Salk Institute Genomic Analysis Laboratory collection (Alonso et al., 2003 As a second approach, we selected six genes (DD2, DD4, DD11, DD12, DD32, and DD34) and determined the subcellular localization of the encoded proteins. DD34 was chosen because it lacks a putative signal peptide (discussed above). The other five have putative signal peptides. We analyzed transgenic lines expressing C-terminal GFP fusion proteins in the synergid cells. Each transgene construct contained the upstream regulatory sequences and the complete coding region fused with a GFP coding sequence. We analyzed the localization patterns in mature, unfertilized female gametophytes (stage FG7). As shown in Figures 1C and 1D, DD34-GFP, which lacks an N-terminal signal peptide, is distributed throughout the cytoplasm. By contrast, as shown in Figures 1E to 1H and Supplemental Figures 2A to 2F online, with the other five fusion proteins, which all contain N-terminal signal peptides, strong GFP signals were associated with the filiform apparatus and GFP was not detected in the cytoplasm. These data suggest that DD34 is a cytoplasmic protein, whereas the DD2, DD4, DD11, DD12, and DD32 proteins are secreted into the filiform apparatus. The results presented above suggest that within the synergid cells, secretion into the filiform apparatus is the default secretory pathway. To test this, we analyzed transgenic plants containing a protein fusion construct (ProMYB98:SP-GFP) comprising the MYB98 promoter, the coding sequence of a signal peptide (from DD11), and a GFP coding sequence. As shown in Supplemental Figures 2G and 2H online, this fusion protein was localized to the filiform apparatus. Thus, an N-terminal signal peptide appears to be sufficient to localize a protein to the filiform apparatus in the synergid cells.
The DD11 Promoter Contains a MYB98 Binding Site Necessary for Synergid Expression
To determine whether any of these YAAC sites, or any other sequences, are necessary for DD11 expression, we generated a series of 5' (Figure 4B) and 3' (Figure 4C) deletions of the DD11 promoter, fused these with a GFP coding region, introduced these promoter:GFP constructs into wild-type Arabidopsis plants, and analyzed  10 transgenic lines per construct. With the 5' deletion series, a promoter fragment containing 174 bp upstream of the translational start site was sufficient to drive the expression of GFP in the synergid cells, but removal of an additional 37 bp (to position –137) abolished expression (Figure 4B). With the 3' deletion series, a promoter fragment containing –352 to –139 bp relative to the translational start site was sufficient to drive the expression of GFP in the synergid cells, but removal of an additional 40 bp from the 3' end (to position –179) abolished expression (Figure 4C). The regions identified in the 5' and 3' deletions are compared in Figure 4D. These regions overlap by 36 bp, at positions –174 to –139, suggesting that this 36-bp region contains sequences necessary for synergid expression. As shown in Figure 4D, this region contains a single putative MYB98 binding site (TAAC) at position –144. We refer to this site as TAAC–144. To determine whether MYB98 can bind to TAAC–144, we performed EMSAs with the MYB98 Myb domain and an oligonucleotide containing a region of the DD11 promoter encompassing this site. As shown in Figure 2C, MYB98 bound to this oligonucleotide, and this binding was competed with the wild-type competitor and competitors with mutations outside the core AAC residues (oligonucleotides M9 and M10) but not with competitors bearing mutations within the core AAC residues (oligonucleotide M8). These data indicate that MYB98 binds to TAAC–144 in vitro. To determine whether TAAC–144 is necessary for DD11 expression, we generated a construct (mTAAC–144) in which this site is mutated in the context of a promoter fragment containing 174 bp upstream of the translational start site and introduced this construct into wild-type plants. As shown in Figure 4E, the wild-type promoter fragment was sufficient to drive the expression of GFP in the synergid cells. By contrast, the same construct containing a mutation in TAAC–144 was not expressed in the synergid cells. Together, these data indicate that the DD11 promoter contains a MYB98 binding site necessary for synergid expression.
The synergid cells are required for pollen tube guidance and fertilization and have several structural specializations, including a filiform apparatus. We are dissecting the gene regulatory network of the Arabidopsis synergid cell to understand how this important cell type acquires its unique features and functions. Here, we show that MYB98 functions as a transcription factor, that 16 genes lie downstream of MYB98 in the synergid gene regulatory network, and that at least 1 of these genes, DD11, is a direct target of MYB98. We further show that many of the genes downstream of MYB98 encode proteins that are secreted into the filiform apparatus, suggesting that they play a role in the formation or function of this structure. Together, these data establish that MYB98 activates a branch of the synergid gene regulatory network required for pollen tube guidance and filiform apparatus formation.
MYB98 Encodes a Synergid Cell Transcription Factor
MYB98 Positively Regulates a Battery of Genes within the Synergid Cells Five of the genes downstream of MYB98 (DD3, DD4, DD31, DD39/QRT3, and DD67) are expressed in the absence of MYB98, albeit at much reduced levels (Figure 3; see Supplemental Figure 3 online). The dramatically reduced expression of these genes in myb98 female gametophytes indicates that MYB98 is a central regulator of these genes. The low but detectable expression of these genes in myb98 female gametophytes indicates that other synergid factors can compensate, in part, for the absence of MYB98. Thus, these genes appear to be activated in the synergid cells by multiple factors.
MYB98 Binds to the Sequence TAAC and Directly Regulates DD11 To identify in vivo MYB98 binding sites, we dissected the promoter of the DD11 gene. This analysis identified a 36-bp region that contains a MYB98 binding site at position –144 (TAAC–144). Mutations in the core AAC residues of TAAC–144 abolish both MYB98 binding (Figure 2C) and synergid expression (Figure 4E). These data suggest very strongly that MYB98 directly regulates the DD11 gene, although we cannot eliminate the possibility that MYB98 activates the expression of another MYB protein that binds to TAAC–144 in the DD11 promoter.
The MYB98 binding site (TAAC) is unlikely to confer synergid expression on its own. Analysis using Patmatch (http://www.Arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl) indicates that this sequence is present one or more times in the promoter of essentially every gene within the Arabidopsis genome. Therefore, it is likely that additional cis elements are required to confer synergid-specific expression. Consistent with this, the DD11 promoter contains six TAAC sites (Figure 4A), and only TAAC–144 was shown to be necessary for synergid expression. None of the other TAAC sites appear to be functional: TAAC–77, TAAC–70, and TAAC–36 do not compensate for the loss of TAAC–144 in construct mTAAC–144 (Figure 4E); TAAC–237 does not compensate for the loss of TAAC–144 in the –179 3' promoter deletion construct (Figure 4C); and deletion of TAAC–390 does not affect expression (Figure 4B). These observations suggest that only TAAC–144 is functional. TAAC–144 may contain adjacent sequences required for optimal MYB98 binding or may be located at an optimal distance from other cis elements. Several other R2R3-MYB proteins, including WEREWOLF and GLABRA1 (Schiefelbein, 2003 Currently, we do not know whether the other 15 genes are regulated directly or indirectly by MYB98. All of these genes contain multiple TAAC sites in their promoters. However, as discussed above, the same is true for essentially every gene in the Arabidopsis genome, and our analysis of the DD11 promoter has shown that not all TAAC sites are functional; thus, the presence of the TAAC sites in the downstream genes is not indicative of direct regulation. Promoter analysis of the other downstream genes will be required to determine which, if any, are direct targets.
Functions of the Genes Downstream of MYB98
Many of the MYB98-regulated genes could feasibly encode a pollen tube attractant. Several of these genes are predicted to encode proteins involved in cell signaling. DD32 encodes a protein similar to the S proteins from Papaver. These proteins are expressed in the stigma, interact with incompatible pollen, and induce a Ca2+-dependent signaling pathway that results in cell death of the incompatible pollen tubes (Thomas et al., 2003
Several of the MYB98-regulated genes may play a role in filiform apparatus formation. Two of these genes are predicted to encode cell wall–modifying proteins. DD39/QRT3 encodes a polygalacturonase involved in the degradation of pectin in plant cell walls (Rhee et al., 2003
The filiform apparatus dramatically increases the surface area of the plasma membrane at the micropylar pole of the synergid cells (see Supplemental Figure 1 online) and is associated with an elaborated endoplasmic reticulum, Golgi stacks, and numerous vesicles. Based on these structural features, it has been proposed that the filiform apparatus facilitates the transport of substances into and out of the synergid cells (Willemse and van Went, 1984
Plant Materials and Growth Conditions Arabidopsis thaliana seeds were surface-sterilized with chlorine gas and germinated on plates containing 0.5x Murashige and Skoog salts (Sigma-Aldrich M-9274), 0.05% MES, 1.0% sucrose, and 0.8% Phytagar (Life Technologies). Ten-day-old seedlings were transferred to Scott's Redi-Earth or Sunshine Mix No. 2 supplemented with MiracleGro and grown under 24 h of illumination. The following T-DNA lines were used: DD4 (SALK_073152), DD18 (SALK_117305), DD31 (SALK_114435), DD39/QRT3 (SAIL_912_G05), DD56 (SALK_023946), and DD67 (SALK_067858).
Plant Transformation
Expression and Purification of MYB98 The transformed bacteria were grown to a density of 1.5 at OD600 at 37°C in 50 mL of Luria-Bertani medium, isopropyl-1-thio-ß-galactopyranoside was added to a final concentration of 100 nM, and the bacterial culture was incubated at room temperature for 1 h. The culture was then centrifuged to pellet the bacteria, the supernatant was removed, and the bacterial pellet was resuspended in 2 mL of HIS-Bind buffer (20 mM Tris-Cl, 0.5 M NaCl, and 5 mM imidazole, pH 8.0) plus 25 µL of E. coli protease inhibitors (Sigma-Aldrich). To isolate the recombinant protein, the cells were disrupted using a probe sonicator and centrifuged at 20,000g for 15 min. One hundred microliters of nickel-nitrilotriacetic acid agarose slurry (GE/Amersham) was then added to the supernatant. This mixture was incubated with gentle rocking for 20 min at 4°C. The mixture was then centrifuged, the supernatant was removed, and the pellet was washed three times, once with 1 mL of HIS-Bind buffer followed by two washes with 1 mL of HIS-Wash buffer (20 mM Tris-Cl, 0.5 M NaCl, and 60 mM imidazole, pH 8.0). The pellet was then resuspended in 200 µL of HIS-Elute buffer (20 mM Tris-Cl, 0.5 M NaCl, and 1 M imidazole, pH 8.0) for 10 min at room temperature. This solution was then centrifuged, and the supernatant was dialyzed in storage buffer (20% glycerol, 0.5 mM DTT, 6 mM MgCl2, 50 mM KCl, 0.1 mg/mL BSA, 10 mM Tris-Cl, and 1 mM EDTA, pH 8.0) using a Centriprep centrifugal filter unit with an Ultracel YM-3 membrane (Millipore).
EMSAs
Analysis of GFP Expression Patterns
Protein Fusion Constructs To determine whether the GFP-MYB98 protein fusion complements the myb98-1 phenotype, we introduced the ProMYB98:GFP-MYB98 construct into myb98-1/myb98-1 plants by Agrobacterium-mediated transformation. In the T1 generation, we observed rescue of the pollen tube guidance defect in at least 50% of ovules in 17 independent transformants. In addition, in the T2 generation, we identified plants homozygous for both the myb98-1 mutation and the ProMYB98:GFP-MYB98 construct; these plants had full seed set. To generate the protein fusion constructs for DD2, DD4, DD11, DD12, and DD32, we amplified the promoter regions and coding regions using the following primers: DD2F and DD2R for DD2, DD4F and DD4R for DD4, DD11F and DD11R for DD11, DD12F and DD12R for DD12, and DD32F and DD32R for DD32. Sequences of primers are listed in Supplemental Table 1 online. In all cases, the PCR primers added unique restriction enzyme sites that were used to ligate the PCR products into the pBI101.gfp vector. These constructs were introduced into wild-type Arabidopsis plants by Agrobacterium-mediated transformation, and at least 10 transgenic lines were analyzed per construct.
To generate the protein fusion construct for the DD11 signal peptide (ProMYB98:SP-GFP), we amplified 700 bp of the MYB98 regulatory region using the following primers: 5941-98F and 5941-98R3. This PCR product was cloned into pFGC5941 (Kerschen et al., 2004
Analysis of Promoter:GFP Expression in MYB98/MYB98, MYB98/myb98, and myb98/myb98 Pistils
Plants homozygous for the promoter:GFP transgenes were used in a cross with myb98-1/myb98-1 pollen. The resulting F1 plants were hemizygous at the promoter:GFP locus and heterozygous at the MYB98 locus. F1 plants were used as pollen donors in a second cross with myb98-1/myb98-1 females. The resulting F1 plants were genotyped by PCR to identify plants hemizygous for the promoter:GFP locus and homozygous for the myb98-1 mutation using primers described by Kasahara et al. (2005) When scoring the percentage of female gametophytes with GFP fluorescence in the synergid cells, at least three pistils were analyzed from at least three individuals per generation.
Promoter Deletion Constructs
Real-Time RT-PCR RNA was extracted from the pistil tissue using the Qiagen RNeasy kit following the manufacturer's protocol (www.qiagen.com). DNA contamination was removed from samples using the Ambion TURBO DNA-free DNase kit following the manufacturer's protocol. After DNase treatment, RNA samples were repurified using the Qiagen RNeasy kit following the manufacturer's protocol. Aliquots of RNA (1 µg) were reverse-transcribed using the RETROscript kit (Ambion) following the manufacturer's protocol.
For real-time RT-PCR, PCR was performed using the Roche FastStart DNA Master SYBR Green I master mix (www.roche.com) in a volume of 10 µL on a Roche LightCycler system. The PCR mixture consisted of 0.5 µL of cDNA, 0.5 µM primers, and 1x master mix. For each real-time RT-PCR run, ACTIN2 (At3g18780) was used as an internal control to normalize for any pipetting error of the cDNA template. The PCR program consisted of 95°C for 5 min followed by 45 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 10 s. To determine the specificity of the PCR, the amplified products were subjected to melt curve analysis using the machine's standard method as well as run on a gel. The reported threshold cycle values are averages of two independent trials (biological replicates). For DD18 and DD32, expression was not detected in myb98-1 ovules. In these cases, a threshold cycle value of 40 was used to calculate fold change. All real-time RT-PCR primers were the same as reported by Steffen et al. (2007)
Identification of TAAC Sites within the Arabidopsis Genome
Supplemental Data
We thank Ramin Yadegari, Jaimie Van Norman, and members of the Drews laboratory for critical review of the manuscript. We thank Ramin Yadegari for providing the pBI101.GFP and 35SmpGFP vectors. We thank the ABRC for providing the T-DNA insertion alleles and pFGC5941. This work was supported in part by a National Science Foundation grant (Grant IOB-0542953) to G.N.D. and by a National Institutes of Health Developmental Biology Training Grant (Grant 5T32 HD-07491) appointment to J.A.P.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Gary N. Drews (drews{at}bioscience.utah.edu).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.052076 Received April 4, 2007; Revision received July 20, 2007. accepted July 25, 2007.
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ABSTRACT
INTRODUCTION
30 min). Double-stranded oligonucleotides were labeled with digoxigenin using the Roche digoxigenin gel shift kit, second generation (Roche), following the manufacturer's protocol. The binding reactions were performed in binding buffer (10% glycerol, 0.5 mM DTT, 6 mM MgCl2, 50 mM KCl, 0.1 mg/mL BSA, 10 mM Tris-Cl, and 1 mM EDTA, pH 8.0) for 15 min at room temperature. For all reactions, 77 fM of digoxigenin-labeled oligonucleotides, the appropriate concentration of unlabeled oligonucleotides, and 20 ng of protein were mixed in a 10-µL binding reaction. The binding reactions were then incubated on ice for 5 min and were resolved on 1-mm-thick 8% native polyacrylamide gels in 0.5x TBE buffer (44.5 mM Tris base, 44.5 mM boric acid, and 1 mM EDTA, pH 8.0) at 250 V for 20 min using the mini Protean 3 system (Bio-Rad). The oligonucleotides were transferred to Hybond-N+ nylon transfer membranes (GE/Amersham) by electroblotting at 400 mA for 30 min in 0.5x TBE. The oligonucleotides were cross-linked to the nylon membranes using a UV Stratalinker 1800 (Stratagene) at the standard setting (120,000 µJ/cm2). Digoxigenin-labeled oligonucleotides were detected according to the manufacturer's protocol (Roche). 