Identification of a Soybean Protein That Interacts with
GAGA Element Dinucleotide Repeat DNA1
Indu Sangwan and Mark R. O’Brian*
Department of Biochemistry, State University of New York, Buffalo, New York 14214
Dinucleotide repeat DNA with the pattern (GA)n/(TC)n, so-called GAGA elements, control gene expression in animals, and
are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max)
cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)n/(CT)n DNA. Soybean GBP was dissimilar from the
GAGA factor of Drosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than
(GA)n/(CT)n. GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA
element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA
element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter
is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root
nodules elicited by the bacterium Bradyrhizobium japonicum. In addition, Gbp transcripts were much higher in leaves of
dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in
the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a
regulatory feature in plants.
Repetitive DNA sequences are found throughout
the genomes of higher eukaryotes. Satellite, minisatellite, and microsatellite DNAs are tandemly repeated sequences, with the latter comprising repeats
of two to five nucleotides (Charlesworth et al., 1994).
Although repeat DNA is primarily associated with
heterochromatin, microsatellite sequences can be
found within or near genes, and in some cases have
been shown to affect gene expression. In particular,
so-called GAGA elements comprising the dinucleotide repeat sequence (GA)n/(CT)n have been found
in the promoters of numerous genes in animals (Gilmour et al., 1989; Kerrigan et al., 1991; Li et al., 1998;
Simar-Blanchet et al., 1998; Bevilacqua et al., 2000;
Melfi et al., 2000; Wyse et al., 2000; Busturia et al.,
2001; Hodgson et al., 2001; Mishra et al., 2001).
GAGA elements have been most thoroughly examined in Drosophila melanogaster, where they are involved in the regulation of numerous developmental
genes. In those cases, GAGA elements repress gene
expression by stabilizing nucleosomes, and thereby
preventing transcription (Croston et al., 1991; Lu et
al., 1993). A protein called GAGA factor, encoded by
the trithorax-like gene in D. melanogaster, binds to
GAGA elements in promoters and, in most cases,
relieves repression. GAGA factor binding to the ele1
This work was supported by the National Science Foundation
(grant no. MCB– 0089928).
* Corresponding author; e-mail [email protected]; fax
716 – 829 –2725.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.002618.
1788
ment results in local nucleosome disruption to allow
gene expression (Tsukiyama et al., 1994; Tsukiyama
and Wu, 1995).
Dinucleotide repeat sequences and other microsatellite DNA are also found in higher plants (Lagercrantz et al., 1993; Bell and Ecker, 1994; Struss and
Plieske, 1998; Cardle et al., 2000; Casacuberta et al.,
2000). The soybean (Glycine max) Gsa1 gene encoding
the chlorophyll and heme synthesis enzyme Glu
1-semialdehyde aminotransferase has a (GA)9/(CT)9
GAGA element in its promoter that is implicated
to control that gene (Frustaci et al., 1995). Glu 1-semialdehyde aminotransferase catalyzes the formation of
the tetrapyrrole precursor ␦-aminolevulinic acid
(ALA); thus, Gsa1 is highly expressed in leaves and
symbiotic root nodules for chlorophyll and heme synthesis, respectively (Sangwan and O’Brian, 1993; Frustaci et al., 1995). Leghemoglobin is an abundant plant
heme protein in nodules that is not expressed in nonsymbiotic root tissue (for review, see O’Brian, 2000).
In accordance, Gsa1 is expressed at a very low level
in uninfected roots and GAGA-binding activity is absent in nuclear extracts of that tissue (Frustaci et al.,
1995).
In the present study, we identify a regulated gene
from soybean that encodes a protein that binds specifically to (GA)n/(CT)n DNA, including the Gsa1
promoter. The soybean GAGA-binding protein
(GBP) is dissimilar to the animal protein, but expressed homologs are found in other plants. It is
likely that interaction between GAGA elements and
its cognate protein is a regulatory feature in gene
expression in higher plants.
Plant Physiology, August
2002, Vol.
pp. 1788–1794,
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GAGA-Binding Protein in Soybean
RESULTS
Isolation of a Soybean Nodule cDNA That
Encodes a GBP
A yeast one-hybrid screen allows the isolation of
cDNAs that encode proteins that bind to a cis-acting
element. A perfect dinucleotide repeat element
[(GA)27/(CT)27] was cloned upstream of the HIS3
selectable marker gene in pHISi and subsequently
integrated into the genome of yeast (Saccharomyces
cerevisiae) strain YM4271. The HIS3 gene confers His
prototrophy on the strain, and is not expressed to
high levels unless the GAL4-activation domain (AD)
is brought to the promoter. The heme and chlorophyll gene Gsa1 from soybean contains a GAGA element in its promoter, and this gene is expressed in
leaves and root nodules (Frustaci et al., 1995). Thus, a
unidirectional soybean nodule cDNA library was
constructed in pGAD424, which results in protein
fusions of yeast GAL4-AD with the product of the
inserted cDNA. Thus, a fusion between a soybean
nodule GAGA-binding domain with GAL4-AD will
result in recruitment of the AD to the GAGA element
at the HIS3 promoter and activate transcription.
The one-hybrid screen yielded a nodule cDNA
clone named pNPGAD3 that conferred His prototrophy on yeast strain YM4271. Introduction of
pNPGAD3 into a yeast strain that had the GAGA
sequence upstream of the lacZ gene conferred a blue
cell phenotype in the presence of X-gal, indicative of
reporter gene activity (Fig. 1A). Northern-blot analysis using the nodule cDNA as probe yielded an
RNA about 1.2 kb in size, which was substantially
longer than the 776-bp cDNA insert of pNPGAD3.
An additional 5⬘ sequence was obtained by PCR using a nodule cDNA library constructed in pUC18 as
template and primers complementary to the vector
and to a portion of the pNPGAD3 insert cDNA.
The cloned cDNA encoded a protein 282 amino
acids in size (Fig. 1B) and contained a long 5⬘untranslated region of 237 nucleotides in length. The
encoded protein contained no substantial homology
to GAGA factor from D. melanogaster, nor was it
similar to any characterized protein in the databases.
However, it was similar to unknown proteins from
other plants (see below). The protein contained a
putative nuclear localization signal (Fig. 1B), consistent with its ability to bind DNA. Thus, this screen
identified a heretofore uncharacterized gene and protein that we designated Gbp and GBP, respectively
(GenBank accession no. AF502431).
GBP Binds (GA)n/(CT)n Dinucleotide Repeat DNA But
Not Other Dinucleotide Repeat DNA
Dinucleotide repeat DNA has been reported to take
on unique conformations (Hentschel, 1982); thus, it
was possible that GBP recognizes an overall DNA
conformation that may not be strictly sequence spePlant Physiol. Vol. 129, 2002
Figure 1. Identification of GBP. A, Activation of lacZ by pNPGAD3
in a yeast one-hybrid system. Yeast strain YM4271(placZGAGA)
harboring pNPGAD3 was spotted on a plate and grown, and then the
plate was flooded with X-gal. Blue color formation (dark color in
black and white image) indicates ␤-galactosidase activity (left spot).
pNPGAD3 encodes a GAL4-GBP fusion protein, indicating that GBP
binds to the GAGA element in the lacZ promoter. The vector
pGAG424 did not activate the lacZ gene under the control of the
GAGA element (middle spot). As a positive control, pGAD53 m,
which encodes a GAL4-p53 fusion protein, was introduced into
strain YM4271(p53Blue), which harbors a p53-binding site in the
lacZ promoter (right spot). B, The deduced protein sequence of GBP.
The underlined segment denotes a putative nuclear localization
signal.
cific. To address this question, we measured GBP
binding to (GA)27/(CT)27 double-stranded DNA and
other dinucleotide repeat DNA by electrophoretic
mobility shift assays (EMSA). Initial experiments indicated that pure recombinant GBP formed multiple
complexes with the (GA)27/(CT)27 DNA as observed
by multiple bands and an overall smear pattern on
the EMSA gels (data not shown). This was probably
the result of multiple binding sites on the DNA for
protein. However, a GBP-maltose-binding protein
(MBP) fusion gave a single band (Fig. 2), perhaps
because steric affects of the larger protein did not
allow multiple protein complexes to form on the
DNA. MBP alone did not form a complex (data not
shown); thus, the binding was specific to GBP. The
binding of GBP to (GA)27/(CT)27 DNA in vitro corroborated the yeast one-hybrid data. Electrophoretic
mobility shifts were not observed using other dinucleotide repeat DNA; specifically, (GT)27/(CA)27,
(AT)27/(TA)27, or (GC)27/(CG)27 double-stranded
DNA (Fig. 2). Thus, GBP is specific for (GA)n/(CT)n
DNA and is not a general dinucleotide repeat DNAbinding protein.
GBP Binds to the Soybean Gsa1 Promoter in Vitro
The soybean Gsa1 promoter contains a perfect
dinucleotide repeat sequence of (GA)9/(CT)9 (Frustaci et al., 1995; Fig. 3A). This GAGA element is
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1789
Sangwan and O’Brian
but expressed to a much lesser extent in those exposed to light, indicating that Gbp is a lightresponsive gene in etiolated plantlets. The expression
of Gbp in leaves of mature plants, which were harvested in the light, indicates that the effects of light
differ in the plants grown under different conditions.
Soybean root nodules from 24-d-old plants elicited
by infection with the bacterial endosymbiont Bradyrhizobium japonicum expressed Gbp mRNA at a level
comparable with leaves and substantially higher than
that found in uninfected roots (Fig. 4A). Thus, Gbp is
activated in symbiotic root nodules. The data show
that Gbp is a regulated gene in soybean.
Figure 2. Interaction of GBP with dinucleotide repeat DNA. EMSA
were carried out with a 54-bp double-stranded DNA comprising 27
repeating units of GA/CT (GA), TA/AT (TA), GT/CA (GT), and GC/CG
(GC). The GC/CG-unbound DNA ran faster than the other unbound
DNA fragments, and the image was moved for direct comparison
with the other free DNAs. The DNAs were run either free (⫺) or with
MBP-GBP fusion protein (⫹). The mobility of the DNAs were unaffected in the presence of MBP alone (data not shown).
immediately downstream of a (TA)5/(AT)5 sequence
that is presumably a TATA element. We addressed
the binding of GBP to the Gsa1 promoter by EMSA as
described above using a 60-bp DNA fragment corresponding to the Gsa1 promoter that includes the
GAGA element. Purified recombinant GBP bound to
the Gsa1 promoter DNA as seen by mobility shifts,
whereas a control 60-bp fragment corresponding to a
region of pBluescript SK (pSK) did not (Fig. 3C).
GBP-MBP fusion protein also bound to this element
as well (data not shown), which was consistent with
its binding to the longer dinucleotide (Fig. 2). The
18-bp GAGA element sequence within the Gsa1 fragment was removed and replaced with an 18-bp sequence corresponding to a portion of pSK (Fig. 3B).
This fragment did not bind to GBP (Fig. 3C), showing
that the dinucleotide repeat was necessary for GBP
binding to the Gsa1 promoter. It also demonstrated
the GBP did not bind to the TATA box in the Gsa1
promoter, which is consistent with the inability to
bind to a (TA)27/(AT)27 DNA sequence (Fig. 2). Finally, introduction of the GAGA element into the
pSK fragment resulted in binding of the DNA to
GBP. The data show that GBP binds to the Gsa1
promoter in vitro and that the GAGA element is both
necessary and sufficient for binding.
The Gbp Gene Is Expressed in Soybean Leaves and Is
Induced in Symbiotic Root Nodules
Expression of the Gbp gene was assessed at the
mRNA level by northern blots using a Gbp fragment
as a probe (Fig. 4). Gbp transcripts were observed in
leaves of 24-d-old plants (Fig. 4A). Gbp mRNA was
also examined in leaves of etiolated plantlets either
grown in the dark completely or exposed to light for
24 h immediately before harvesting the plants. Gbp
message levels were high in the dark-treated plants,
1790
Gbp Gene Homologs Are Found in Other Plants
We searched for Gbp gene homologs in other plants
in GenBank (http://www.ncbi.nlm.nih.gov) and from
expressed sequence tag (EST) cDNA databases compiled by The Institute for Genomics Research (TIGR;
Figure 3. Binding of GBP to the promoter of the Gsa1 gene. A, The
promoter region of Gsa1, including the GAGA element. The underlined region was used in the gel shift assays. The bolded nucleotide
shows the transcription start site determined previously (Frustaci et
al., 1995). The italicized codon shows the translation start site. B, The
four probes used in the analysis are as follows: I, 60-bp DNA
fragment corresponding to the multiple cloning site of pBluescript SK
used as a negative control; II, 60-bp DNA fragment within the Gsa1
promoter that includes the GAGA element, underlined in A; III, probe
II, except that the 18-bp GAGA element was removed and replaced
with an 18-bp sequence from pBluescript SK; and IV, probe I, except
18 bp was removed and replaced with the GAGA element. C, EMSA
were carried out with the four probes and purified GBP. ⫺, Free
probe; ⫹, presence of GBP in the binding reaction.
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Plant Physiol. Vol. 129, 2002
GAGA-Binding Protein in Soybean
similarly among legumes. It is likely that Gbp is an
expressed gene in many plants.
DISCUSSION
Figure 4. Northern-blot analysis of Gbp mRNA in soybean tissues. A,
Gbp mRNA was analyzed in poly(A⫹) RNA from leaves (L), roots (R),
and nodules (N). Ubiquitin (Ubi) was used as a control for a constitutively expressed gene. B, Leaves from illuminated (I) or dark-treated
(D) etiolated plantlets were analyzed for Gbp. Cab is a control for a
light-regulated gene.
http://www.tigr.org/tdb) using BLAST (blastp and
tblastn algorithms; Altschul et al., 1990). Genes encoding proteins with high similarity to GBP were found in
the dicots Arabidopsis, potato (Solanum tuberosum),
and tomato (Lycopersicon esculentum) and in the monocot rice (Oryza sativa; Fig. 5). Soybean GBP had the
greatest similarity to the predicted amino acid sequence of homologs in potato and tomato (54% identity, 67% similarity) and the least to that found in rice
(42% identity, 53% similarity). Furthermore, the proteins from the dicots were homologous over the entire
length, but the rice GBP homolog was less similar at
the N-terminal portion and is predicted to contain an
additional 53 amino acids at that end. Arabidopsis
contains two GBP homologs with 72% identity to each
other. Finally, the Gbp gene homologs have corresponding ESTs; therefore, they are expressed genes.
The Arabidopsis genome contains two additional
genes that encode hypothetical proteins (GenBank
accession nos. AAC36166 and AAF18661) with high
similarity (56% and 48% identity) to soybean GBP
at the C terminus (amino acids 166–282), but are
dissimilar at the N terminus. This made it difficult to
predict whether the numerous plant ESTs in the
databases that corresponded only to the 3⬘ end of
Gbp gene actually encode homologs of GBP. However, ESTs corresponding to the 5⬘ end were identified in soybean (BE660059), Medicago truncatula
(TIGR no. TC33063), and Lotus japonicus (GenBank
no. AW719515), raising the possibility of an additional Gbp homolog in soybean as well as in other
legumes. The Gbp-like M. truncatula cDNAs were
from nodules, nodulated roots, and leaves, and the
single similar cDNA from L. japonicus was from nodules, indicating that Gbp homologs are expressed
Plant Physiol. Vol. 129, 2002
In the present study, we identified a soybean
cDNA encoding GBP, a protein that specifically
binds the dinucleotide repeat DNA (GA)n/(CT)n and
binds to the GAGA element of the Gsa1 gene. The
findings implicate a functional interaction between
the dinucleotide repeat DNA and a specific protein in
plants; hence, this phenomenon is not confined to
animals, but rather it occurs in higher eukaryotes
more generally. Dinucleotide repeat DNA can form
cruciform structures (Hentschel, 1982). However,
GBP recognized only (GA)n/(CT)n repeat DNA and
not other dinucleotide repeats (Fig. 2); hence, the
basis of recognition is not a general feature of dinucleotide repeat sequences. Thus, GBP is not expected
to bind to the TATA box dinucleotide repeat element
found in most eukaryotic genes and does not bind to
the putative TATA box of Gsa1. Furthermore, the
GAGA element of Gsa1 was both necessary and sufficient for binding by GBP (Fig. 3), suggesting that
presence of the element in a gene promoter can be
taken as prima facie evidence for recognition by GBP.
The Gbp gene was expressed at a low level in roots,
but was elevated substantially in symbiotic root nodules (Fig. 4); thus, Gbp is a regulated gene with a
likely role in nodule function. The GAGA element of
the Gsa1 gene has been implicated in control of that
gene, and the expression pattern of Gbp is qualitatively similar to that of Gsa1 (Sangwan and O’Brian,
1993; Frustaci et al., 1995). Furthermore, GBP binds to
the Gsa1 promoter at the GAGA element. Collectively, the data suggest that GBP is involved in the
positive control of the Gsa1 gene.
High expression of Gsa1 is required for chlorophyll
synthesis in green tissues for synthesis of the tetrapyrrole precursor ALA. In accordance, ALA formation in
those tissues is controlled by, or coordinated with,
factors related to photosynthesis, particularly light
(Bougri and Grimm, 1996; Kumar et al., 1996; Tanaka
et al., 1996). Nodules are unusual in that a high level of
tetrapyrrole synthesis occurs in non-photosynthetic
tissue for heme formation, requiring induction of ALA
synthesis genes in the absence of light (Frustaci et al.,
1995; Sangwan and O’Brian, 1999). Similarly, Gsa1 is
expressed in leaves of etiolated plants for synthesis of
chlorophyll precursors (Frustaci et al., 1995). Here, we
found that Gbp expression does not require light, and
is actually higher in etiolated plants exposed to light
compared with the light-treated plants. Thus, it is
plausible that GBP compensates for the lack of light or
some other factor normally associated with photosynthesis to allow expression of Gsa1 in nodules and in
etiolated plant leaves.
Gbp gene homologs were identified in other dicots
and in a monocot as well; therefore, the gene may be
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1791
Sangwan and O’Brian
Figure 5. Alignment of soybean GBP with homologs from other plants. The GBP homologs were identified by BLAST searches
of databases. Arabidopsis sequences (protein identification nos. AAF63172 and AAF18588) and the rice sequence (protein
identification no. AAK52535) were identified in the GenBank database using a BLAST search. Corresponding ESTs have been
found for these sequences; thus, they are annotated as unknown proteins. The potato and tomato sequences were derived from
EST consensus sequences found in the TIGR database (http://www.tigr.org/tdb) using a BLAST search (identification nos.
TC85862 and TC23396 for tomato and potato, respectively). The sequences were aligned using Clustal W (version 1.81). The
stars represent identity at that position in all six sequences. The colon represents similarity at that position.
common in higher plants. Analysis of the Arabidopsis chromosome using the Patmatch program
(http://www.arabidopsis.org) revealed 813 perfect
GA/CT dinucleotide repeats of 18 nucleotides
[(GA)9/(CT)9)] or longer in the genome. Although
the analysis did not allow a practical determination
of the location of all the elements with respect to
genes, we could readily find (GA)n/(CT)n sequences
in the upstream regions of numerous genes. The gene
encoding geranylgeranyl reductase (Keller et al.,
1998) has a (GA)9/(CT)9 element immediately upstream of the transcription start site, and the same
element is found in the promoter of the GPA1 gene
encoding a G protein ␣-subunit (Ma et al., 1990).
Similarly, genes encoding unknown proteins were
found that have (GA)n/(CT)n sequences in their up1792
stream regions (e.g. protein identification nos.
AAF26463, AAG51765, and AAL08240). The present
work shows that soybean GBP binds to (GA)n/(CT)n
repeat sequences independent of the genetic context
(Fig. 3); therefore, GBP would very likely bind to
promoters containing that element, at least in vitro.
From this, we speculate that interactions between
GBP-like proteins and GAGA elements are a regulatory feature in higher plants.
MATERIALS AND METHODS
Plants and Bacteria
Soybeans (Glycine max cv Essex) were inoculated with Bradyrhizobium
japonicum strain I110 and grown in a growth chamber under a 16-h-light/
8-h-dark regime at 25°C and harvested after 24 d. Leaves, roots, and nodules
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Plant Physiol. Vol. 129, 2002
GAGA-Binding Protein in Soybean
were taken for RNA extraction and analysis. Etiolated plantlets were grown
in a growth chamber in complete darkness for 10 d and either left in the dark
or exposed to direct light to green for the final 24 h before leaves were
harvested for RNA isolation. Escherichia coli strains TB1 and DH5␣ were
used for propagation and handling of plasmids. Strains harboring plasmids
used in this study were grown in Luria-Bertani (LB) medium supplemented
with 50 to 200 ␮g mL⫺1 ampicillin.
RNA Isolation and Analysis
RNA was isolated from tissues of 24-d-old plants or from leaves of
etiolated seedlings. Tissues were excised, frozen in liquid N2, and homogenized in a blender with buffer and phenol (2:2:3 [w/v/v]). The homogenized buffer contained 500 mm Tris (pH 8), 10 mm EDTA, 100 mm NaCl,
0.5% (w/v) deoxycholate, and 1 mm ␤-mercaptoethanol. Total RNA was
isolated from the homogenate and poly(A⫹) RNA prepared as described
(Frustaci et al., 1995). Northern-blot analysis of poly(A⫹) RNA was carried
out as described previously (Sangwan and O’Brian, 1999) using cDNAs as
probes. Five micrograms of RNA was used in each lane.
Construction of a Soybean Nodule cDNA Library for
Yeast One-Hybrid Screening
Five micrograms of poly(A⫹) RNA isolated from 24-d-old nodules was
used for cDNA synthesis using a cDNA synthesis kit according to the
manufacturer’s instructions (Stratagene, La Jolla, CA). The resultant cDNA
had an EcoRI site at the 5⬘ end and a XhoI site at the 3⬘ end for unidirectional
cloning. cDNA greater than 400 bp in size was ligated into the EcoRI/SalI
site of the GAL4-AD vector pGAD424 (CLONTECH Laboratories, Palo Alto,
CA), and ligated DNA was used to transform E. coli strain XL1-Blue MRF⬘,
and transformants were selected on LB plates containing 200 ␮g mL⫺1
ampicillin. Approximately 106 colonies were scraped off the plates and
plasmids isolated from the cells. Over 90% of the plasmids contained inserts
as estimated from miniplasmid preps of 20 clones. The library should
encode fusion protein of the GAL4-AD with products of the nodule cDNA.
Construction of Yeast Strains and Selection for cDNA
Encoding GBP Using a One-Hybrid Screen
Double-stranded DNA containing (GA)27/(CT)27 flanked by EcoRI and
SalI on the 5⬘ and 3⬘ ends [with respect to the (GA)27 strand] were constructed by annealing commercially synthesized oligonucleotides together.
The DNA was ligated into the EcoRI/SalI of pLacZi or the EcoRI/MluI site
of pHISi, which contain a lacZ and HIS3 gene, respectively. In the latter case,
the MluI and SalI sites of the insert and vector, respectively, were filled in
with the Klenow fragment of DNA polymerase before ligation. The plasmids were linearized by digestion with XhoI for pHISiGAGA or NcoI for
pLacZGAGA, introduced and integrated into the genome of yeast (Saccharomyces cerevisiae) strain YM4271.
The nodule cDNA library was screened for clones encoding proteins that
interacted with GAGA element DNA by introducing the library into
YM4271(pHISiGAGA) and selecting for colonies that grew in the absence of
Leu and His and in the presence of 50 mm 3-amino-1,2,4-triazole. 3-Amino1,2,4-triazole is a competitive inhibitor of the HIS3 gene and eliminates
leaky expression. Strain YM4271 is a His auxotroph; thus, growth requires
recruitment of the GAL4-AD to the HIS3 promoter as a result of interaction
of fusion protein with the GAGA element in the HIS3 promoter. Colonies
arising after 2 d were restreaked on the selective media, and plasmids were
isolated and transformed into YM4271(pLacZGAGA) to test for the ability to
activate another gene by development of blue color in the presence of X-gal.
As a positive control, YM4271(p53BLUE), which contains three tandem
copies of a p53-binding site upstream of lacZ, was activated by pGAD53 m,
which encodes the mouse p53. The selection identified three identical clones
that strongly activated the reporter genes; one of them, pNPGAD3, was
used in this study.
pNPGAD3 did not contain the entire cDNA as judged by comparing the
insert size with the mRNA size on northern blots. Thus, an additional 5⬘
sequence was obtained by PCR using the pGAD424 nodule library as
template, and primers corresponding to the vector and to the insert. The
vector primer used was 5⬘-GCGATAACGCGTTTGGAAT-3⬘ and the insert
Plant Physiol. Vol. 129, 2002
primer used was 5⬘-GGCCAGATGACCATAGAGGA-3⬘. A BstXI restriction
site was present in the DNA that overlapped the original pNPGAD3 and the
PCR product, which was used to construct a single, complete cDNA that
contained the complete open reading frame and flanking DNA.
Overexpression of Gbp cDNA in E. coli and
Purification of the Recombinant Protein
The coding region of the GBP was amplified by PCR using primers that
added restriction sites of BamHI and SalI to the 5⬘ and 3⬘ ends, respectively.
The forward and reverse primers were 5⬘-ATCAGTTGGTGGATCCATGGATGGTGATAA-3⬘ and 5⬘-CCATAGAGTCGACCTACCTGATAGTGACAA3⬘. The PCR product was ligated into the BamHI/SalI site of pMalC2 (New
England Biolabs, Beverly, MA) and transformed into E. coli strain TB1. The
plasmid encodes a fusion of MBP with GBP. The cells were grown at 37°C
in LB medium to an optical density of 600 of 0.5, then put on ice for 30 min.
Afterward, 2% (v/v) ethanol was added to cultures, and the cells were
induced with 0.5 mm isopropylthio-␤-galactoside and continued to incubate
with shaking at 20°C overnight. The cells were broken with a French
pressure cell and cleared by centrifugation at 8,000g for 20 min. The fusion
protein was purified from the extract using an amylose resin according to
the manufacturer’s instructions. The protease Xa was used to cleave the
MBP from GBP, and GBP was purified from the other proteins by the
amylose resin and size fractionation.
EMSA
Binding of GAGA protein to various DNA elements was carried out by
EMSA as described previously (Frustaci et al., 1995). The binding buffer
reaction mixture contained 10 mm bis tris borate (pH 7.5), 1 mm MgCl2, 40
mm KCl, 5% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, and 1 mm dithiothreitol. To a 25-␮L reaction mix, the following was added: 2.5 ␮g of bovine
serum albumin, 1 ␮g of unlabeled poly dI-dC as nonspecific competitor
DNA, 50 fmol of 32P-labeled DNA (approximately 6 ⫻ 106 becquerels), and
10 or 100 pmol of GBP. Samples were run on 5% (w/v) non-denaturing
PAGE and exposed to autoradiography as described previously (Frustaci et
al., 1995).
Received January 14, 2001; returned for revision March 17, 2002; accepted
May 8, 2002.
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