(Received for publication, August 10, 1995; and in revised form, September 15, 1995)
From the
TATA-binding protein (TBP) gene promoter binding factor (TPBF) is a transactivator which binds to the TBP promoter element (TPE) sequence of the Acanthamoeba TBP gene promoter and stimulates transcription in vitro. We have isolated a cDNA clone encoding TPBF. TPBF is a polypeptide of 327 amino acids with a calculated molecular mass of 37 kDa. The predicted amino acid sequence of TPBF shows no significant homology to other proteins. TPBF has two potential coiled-coil regions, a basic region, a proline-rich region, a histidine-rich N terminus, and a nuclear targeting sequence. The recombinant protein has an apparent molecular mass of 50 kDa, identical with that of TPBF purified from Acanthamoeba. Recombinant TPBF is able to bind DNA and activate transcription with the same specificity as natural Acanthamoeba TPBF, demonstrating the authenticity of the clone. Mobility shift assays of co-translated TPBF polypeptides and chemical cross-linking demonstrate that TPBF is tetrameric in solution and when bound to DNA. Analyses of TPBF mutants show that Coiled-coil II is essential for DNA binding, but Coiled-coil I and the basic region are also involved. TPBF is thus a novel DNA-binding protein with functional similarity to the tumor suppressor protein p53.
Accurate transcription initiation of all three classes of genes
in eukaryotic cells requires stepwise assembly of several general
transcription factors and the appropriate RNA polymerase on promoter
DNA. The TATA-binding protein, TBP, ()is involved in
transcription by all three RNA polymerases both in vitro and in
vivo(1, 2, 3, 4, 5) .
TBP is complexed into SL1(1) , TFIID(6) , and TFIIIB (7, 8, 9, 10) for its function in
RNA polymerase I, II, and III systems, respectively. These
TBP-containing initiation factors are recruited to the different
classes of promoters by specific protein-DNA (11, 12) and/or protein-protein
interactions(1, 7, 8, 9, 10, 13) .
In the case of TATA-containing class II promoters, TFIID, consisting of TBP and a large number of associated factors (TAFs)(5, 14, 15, 16) , binds directly to DNA through specific interactions between TBP and the TATA box (11, 12) as the first step in formation of the initiation complex. This TFIID-DNA complex then recruits other general transcription factors, such as TFIIB, TFIIE, TFIIF, TFIIH, and RNA polymerase II to form a complete initiation complex(17) .
An additional class of transcription factors, known as sequence-specific transcription activators, is involved in efficient transcription by RNA polymerase II. These activators bind specifically to promoter sequences and modulate levels of expression of the selected genes, providing a regulatory strategy for eukaryotic cells to control development, differentiation, and their responses to extracellular stimuli. Evidence obtained in recent years suggests that sequence-specific activators stimulate transcription through direct or indirect (via coactivators) communication with the general transcription factors. Interactions between activators and general transcription factors TFIIA(18) , TFIIB(19, 20, 21) , TBP(22) , TAFs(23, 24) , TFIIF(25) , and TFIIH (26) have been reported. However, the mechanism of transcription stimulation is not well understood.
Typical eukaryotic transcription activators are composed of discrete structural domains that have specific functions(27) , for example, in multimerization, DNA binding, and transcription activation or repression. Different structural motifs involved in DNA binding and activation have been identified and used to classify transcription activators. A fully functional activator can be constructed by combination of functional domains from different activators(28) .
The Acanthamoeba TBP gene promoter contains two major elements that are necessary for efficient transcription. The TATA box at -30 functions by binding TFIID, which is necessary for basal transcription(29, 30) . The TPE is a 23-base pair element centered around -90, which stimulates basal transcription up to 10-fold in vitro. The TPE binds a regulatory factor called TPBF (31) , which was identified and purified previously in this laboratory (31, 32) . TPBF is of interest because it regulates TBP gene transcription, but also because it is an apparently novel type of DNA-binding protein. Chemical interference assays demonstrated protein-DNA contacts on opposite faces of the DNA helix(32) . This pattern, while reminiscent of the proposed model for p53 tetramer bound to DNA(33) , is distinct from that produced by other factors(32) . Although TPBF was found by gel filtration to be oligomeric(32) , the resolution was not sufficient to distinguish trimeric and tetrameric forms of the protein. Finally, TPBF is phosphorylated, and removal of phosphate increased DNA binding, suggesting that phosphorylation could play a regulatory function in vivo.
In order to determine the basis for these properties of TPBF and to permit further characterization of its role and mechanism in TBP gene expression, we isolated cDNA and genomic DNA clones encoding TPBF. Expression and analysis of cloned TPBF and mutant derivatives demonstrate that TPBF is a novel tetrameric DNA-binding protein. It contains a C-terminal coiled-coil domain necessary for tetramerization, as well as an apparently large central region involved in DNA binding. Other structural features of TPBF are discussed.
Figure 1:
Nucleotide sequence, predicted amino
acid sequence, and schematic diagram of the TPBF gene. A,
nucleotide and deduced amino acid sequences of the TPBF cDNA. The underlined sequences correspond to the sequenced peptides. Open triangles show the two arrays of heptad repeats of
hydrophobic amino acids. B, schematic presentation of TPBF
indicating presumptive functional domains (see text). Relative
positioning of the domains is shown by the scale above the gene. These
data have been submitted to GenBank under accession number
L46867.
Amplification of Acanthamoeba castellanii genomic DNA (37) by PCR was performed under the following cycle conditions: the first cycle at 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 48 °C for 1 min, and 72 °C for 2 min, and the last cycle at 72 °C for 10 min. Several PCR products were generated using either primer combination (data not shown). The products were subcloned into the pSK(-) vector (Stratagene) and sequenced. One subclone, encoding the TPBF peptides, was obtained.
In vitro protein syntheses from plasmids encoding full-length and mutant
TPBF were carried out with the Single Tube Protein System 2 (Novagen)
in which transcription driven by T7 RNA polymerase is coupled to
translation by a rabbit reticulocyte lysate (42) .
[S]Methionine (DuPont NEN) was incorporated into
synthesized proteins. All procedures were performed according to the
manufacturer's recommendations.
Figure 4: DNA binding and transactivation activities of purified recombinant TPBF. A, silver staining of an SDS-PAGE containing purified TPBF proteins used in EMS and in vitro transcription assays. Lane 1, TPBF purified from Acanthamoeba nuclear extracts. Lane 2, full-length recombinant TPBF. Lane 3, mutant TPBF deleting amino acids 254-296. B, DNA binding activity and specificity of recombinant TPBF analyzed by EMS assay with 4% native polyacrylamide gel. 5 ng of proteins was used in each reaction. Lane 1, natural Acanthamoeba TPBF. Lane 2, full-length recombinant TPBF. Lanes 3 and 4, full-length recombinant TPBF plus 50 ng of unlabeled specific and nonspecific competitor DNA, respectively. Lane 5, mutant TPBF. Lane 6, control DNA without protein. C, transcription stimulation by purified recombinant TPBF. In vitro transcription assay was performed using HeLa cell nuclear extracts with the template containing an intact TPE. Lane 1, HeLa nuclear extracts alone. Lanes 2 and 3 had 100 ng of full-length and mutant TPBF included, respectively.
Figure 8: DNA binding activity of TPBF deletion mutants. 5 ng of natural Acanthamoeba TPBF or 2 µl of in vitro synthesized TPBF polypeptides indicated in Fig. 7were analyzed. A, EMS assay of TPBF mutants on a 4% native polyacrylamide gel. Lane 1, Acanthamoeba TPBF. Lanes 2-9 show DNA binding activities of various TPBF polypeptides as indicated on the top of the panel. Lane 10 was a control without protein. Nonspecific binding from reticulocyte lysate is indicated by an open triangle. B, EMS assay of TPBF mutants on a 6% native polyacrylamide gel. Mutants whose DNA binding activities were too faint to be detected in A were assayed. The mutant assayed in each lane is indicated on the top of the panel. The specific protein-DNA complex is indicated by a solid triangle, the nonspecific ones by an open diamond.
Figure 5:
Recombinant TPBF binds to DNA as a
tetramer. TPBF polypeptides were synthesized by in vitro coupled transcription-translation at the ratios indicated on the top of the panel. 1 µl of each reaction mixture was
analyzed by EMS assay with 6% native polyacrylamide gel.
Homo-oligomeric complexes are indicated by open triangles while hetero-oligomeric complexes by solid triangles. A
minor complex likely caused by the breakdown product of 1-76
is indicated by a solid diamond. The band shown by an open
diamond is related to nonspecific DNA binding from the
reticulocyte lysate.
Figure 7:
Structures and synthesis of TPBF mutants. A, map of TPBF deletion mutants. Deletion mutants were
generated as described under ``Experimental Procedures.''
Mutants with sequences between amino acids X and Y removed are denoted as X-Y on the left side of the panel. The column on the right side summarizes DNA binding activities as determined by EMS assays
shown in Fig. 8. B, SDS-PAGE analysis of in vitro synthesized TPBF polypeptides. An autoradiogram of
S-labeled full-length TPBF (lane 1) and its
deletion mutants (lanes 2-8) is
shown.
Figure 2:
Analysis of the TPBF gene and its
transcript. A, Southern blot analysis of genomic DNA. 2 µg
of Acanthamoeba genomic DNA was digested with the indicated
restriction enzymes and probed with radiolabeled TPBF cDNA as described
under ``Experimental Procedures.'' Size markers are indicated
on the left side of the panel. B, Northern blot analysis of Acanthamoeba mRNA. 10 µg of poly(A) mRNA
was probed with radiolabeled TPBF cDNA. The size of TPBF mRNA was
estimated by comparison to TBP mRNA and shown on the left margin.
C, primer extension of Acanthamoeba mRNA. 2 µg of
poly(A)
mRNA was used in primer extension with a
primer whose sequence was derived from TPBF cDNA (see
``Experimental Procedures''). The size of the primer
extension product was determined by sequencing a TPBF genomic clone
with the same primer.
We used ``rapid amplification of cDNA ends'' (43) to obtain the missing 5` end of the cDNA. In order to find a rapid amplification of cDNA ends primer, we isolated a genomic clone encoding TPBF and partially sequenced it. Two in-frame methionines were found in the 50-bp sequence preceding the 5` end of the cDNA within the genomic copy of the TPBF gene. Using the primer starting from the first in-frame methionine in combination with primer RT2, we successfully obtained the missing part of the cDNA from mRNA by reverse transcription-PCR and reconstructed the full-length cDNA. The complete (both strands) sequence of the reconstructed TPBF cDNA is presented in Fig. 1A.
The complete cDNA comprises 1088 bp and contains an open reading frame of 327 amino acids with a predicted molecular mass of 37 kDa (Fig. 1A). There are several sequence motifs of potential importance to the function of TPBF as a transcription activator. First, it bears a putative nuclear localization signal of 5 consecutive basic residues KKRRK (residues 132-136, Fig. 1), which appears in many transcription factors(44) . Second, there are two segments containing heptad repeats of hydrophobic residues in the sequence (indicated by open triangles, Coiled-coil I and II). Coiled-coil II contains a perfect hydrophobic 4-3 repeat that could form a coiled-coil structure and drive oligomerization of TPBF(45, 46) . Third, the region between residues 40 and 85 is proline-rich, which, by analogy to other factors, might be important in mediating transcription activation(47) . Fourth, the N-terminal 24 amino acids of TPBF is unusually histidine-rich, containing 10 histidine residues. However, data base searches showed that TPBF lacks significant sequence homology to any other known genes(48) . These features are considered further under ``Discussion.''
Northern
analysis (Fig. 2B) indicates that TPBF is transcribed
into a single mRNA with a size of about 1,100 nucleotides. Primer
extension of Acanthamoeba mRNA generated one single band of
the expected size (Fig. 2C), indicating that the
transcript begins about 30 bp downstream from an imperfect TATA box
within the genomic copy of the TPBF gene. The TPBF transcript appears
to be extremely rare. 10 µg of mRNA was required to obtain a clear
signal in Northern blot analysis. Similarly, only 3 plaques from 1
10
were obtained, suggesting that the level of TPBF
expression in Acanthamoeba is very low. The low abundance of
the TPBF message is in contrast to that of TPBF protein in nuclear
extracts, which contain
200 ng of TPBF/mg as judged by Western
blotting (data not shown).
Figure 3: Purification of recombinant TPBF expressed in E. coli. Recombinant TPBF at various stages of purification was analyzed by SDS-PAGE and stained with Coomassie Blue. Lane 1, E. coli cells transformed with pET3a vector. Lane 2, E. coli cells transformed with pET3a containing TPBF cDNA. Lane 3, lysate from E. coli containing recombinant TPBF. Lane 4, flow-through of E. coli lysate over nickel affinity column. Lane 5, TPBF following elution from nickel affinity column. Lane 6, TPBF following elution from DEAE-cellulose.
The existence of histidine-rich
sequences at the N terminus of full-length or deleted TPBF enabled us
to purify them by Ni affinity chromatography (Fig. 3, lane 5). The recombinant proteins were further
purified by DEAE-cellulose chromatography, yielding a single major band (Fig. 3, lane 6).
The molecular mass of the full-length TPBF as measured by SDS-PAGE is 50 kDa, which differs significantly from the predicted mass of 37 kDa. However, the SDS gel mobility of recombinant TPBF perfectly matches that of natural Acanthamoeba TPBF purified from nuclei (Fig. 4A). The apparent discrepancy between SDS gel mobility and the predicted molecular mass of TPBF may be due to the abundance of positively charged amino acids in the protein. Natural TPBF migrates as a doublet on SDS gel due to phosphorylation(32) . Surprisingly, recombinant TPBF showed the same mobility as the phosphorylated form of TPBF (Fig. 4A, lanes 1 and 2).
Since natural Acanthamoeba TPBF is able to
transactivate the TBP gene promoter in HeLa cell nuclear
extracts(32) , we tested whether TPBF expressed in E. coli could substitute for natural TPBF in transcription activation. 100
ng of recombinant TPBF was added to in vitro transcription
reactions carried out with HeLa cell nuclear extract. The results
showed that recombinant TPBF is fully active for transcription
activation (Fig. 4C, lane 2). However, a
10-fold greater amount of recombinant TPBF was required to achieve the
same level of activation as stimulated by natural TPBF, as determined
by titration with rTPBF. This may suggest that a significant portion of
recombinant TPBF that is active in DNA binding is deficient in
transactivation. As expected, the TPBF mutant 254-296 is
unable to stimulate transcription (Fig. 4C, lane
3). To ensure that the observed transactivation was TPE-dependent,
parallel experiments were done using a TBP promoter that lacks the TPE
element. As expected, TPBF was not able to stimulate transcription in
the absence of the TPE sequence (data not shown). We have also obtained
similar results using Acanthamoeba extracts immunodepleted of
TPBF (data not shown).
The formation of five major different complexes
by the cotranslated proteins strongly suggests that TPBF forms a
tetramer when bound to DNA. The two outer bands correspond to
homo-oligomeric complexes (L and S
), while the
three inner bands correspond to hetero-oligomeric complexes
(L
S
, L
S
, and
L
S
).
Figure 6: Both recombinant and natural TPBF form tetramers in the absence of DNA. Purified recombinant TPBF and partially purified natural Acanthamoeba TPBF were chemically cross-linked with DTSSP or glutaraldehyde, resolved by SDS-PAGE, and followed by silver staining or immunoblotting. A, silver staining of an SDS-PAGE containing cross-linked recombinant TPBF. Lane 1, TPBF alone. Lanes 2 and 3 contained 25- and 50-fold molar excess of DTSSP over TPBF, respectively. Lane 4 contained 0.001% of glutaraldehyde. B, immunoblotting of chemically cross-linked natural TPBF. Lane 1, control without cross-linker. Lanes 2 and 3 show cross-linking with 25- and 50-fold molar excess of DTSSP, respectively. Lane 4 was treatment of the reaction shown in lane 3 with 50 mM DTT. The positions of monomeric and cross-linked TPBF are indicated.
DTSSP cross-linking of partially purified Acanthamoeba TPBF detected by immunoblotting produced two cross-linked products apparently identical with those produced by recombinant protein (Fig. 6B), indicating that recombinant TPBF has the same structure as natural TPBF. This result also demonstrates that both cross-linked bands contain TPBF. Cross-linked bands were removable by treatment with 50 mM DTT, which cleaves the disulfide bond within DTSSP linking the monomers (Fig. 6B, lane 4).
Cross-linking also showed that mutant 254-296 is unable
to form a tetramer in solution (data not shown). Loss of
multimerization of TPBF mutant
254-296 is likely due to loss
of the Coiled-coil II structure. Multimerization of TPBF is thus
evidently necessary for binding to DNA (see also below).
To examine the role of Coiled-coil II in DNA
binding, we checked the internal deletion 254-296 in this
system and again found it was inactive in DNA binding (Fig. 8A, lane 7, and Fig. 8B, lane 3). This deletion removes two heptads from Coiled-coil
II, presumably preventing tetramerization. We also tested two
C-terminal deletion mutants. Interestingly, removal of 7 amino acid
residues from the C terminus increased the DNA binding activity
severalfold (Fig. 8A, lanes 2 and 9).
Removal of Coiled-coil II (mutant
278-327) resulted in the
production of a polypeptide unable to bind DNA (Fig. 8A, lane 8, and Fig. 8B, lane 4). These results indicated that regions essential for
DNA binding are distributed between amino acid residues 123 and 320.
Presumably, the C-terminal Coiled-coil II drives tetramerization and is
therefore necessary for DNA binding; while the regions that make DNA
contact are located between amino acid residues 123 and 280.
We have isolated full-length cDNA encoding TPBF, an Acanthamoeba transcription activator, which regulates expression of the TBP gene(31, 32) . To our knowledge, TPBF is the first regulatory protein isolated and cloned that controls expression of a basal transcription factor. Several criteria establish that the cDNA encodes authentic TPBF. First, the cDNA encodes the peptides sequenced from TPBF purified from nuclei. Second, recombinant TPBF expressed in E. coli comigrates with natural Acanthamoeba TPBF (Fig. 4A). Third, recombinant TPBF shows the same DNA binding specificity and activity as natural TPBF. Fourth, recombinant TPBF is able to stimulate transcription in a TPE promoter-dependent fashion. Fifth, recombinant TPBF and natural TPBF bind avidly to a nickel affinity column due to the histidines present in the N terminus. Finally, antibody raised against recombinant TPBF recognizes natural TPBF in Acanthamoeba nuclear extracts. There are, however, some physical differences between recombinant and natural TPBF. For example, TPBF produced in E. coli, which is presumably not phosphorylated, comigrates during SDS-PAGE with the phosphorylated form of natural TPBF. It is thus possible that either recombinant TPBF has been modified or natural TPBF has additional unidentified modifications. Similarly, recombinant TPBF is somewhat less active in stimulating transcription than natural TPBF.
The predicted amino acid sequence of TPBF has no significant
homologues in the public data bases(48) , in accord with its
unusual DNA binding properties. Many sequence-specific transcription
factors have been grouped into several distinct families, such as basic
helix-loop-helix, zinc finger, homeodomain, helix turn helix, or
leucine zipper proteins (50) . TPBF does not fall into any of
these families, as judged by alignment between TPBF and the consensus
sequences that characterize each family. However, the TPBF sequence
contains several regions that suggest a function (Fig. 1B). The N-terminal domain is remarkably
histidine-rich, and these histidines can coordinate with chelated
nickel. While we do not yet know whether TPBF requires metal for any of
its activities, it conceivably contains Zn or
Ni
, perhaps arranged in a configuration similar to
the metal ions in urease(51) . Metal coordination by histidines
might stabilize a particular protein conformation analogous to zinc
fingers or zinc clusters(50) . While the histidines are not
required for DNA binding based on mutagenesis, they could be involved
in another function that our assays did not assess, for example,
transcription activation. A similar possibility exists for the
proline-rich region between amino acid residues 40 and 85. Proline-rich
domains can function as activation regions in some transcription
factors such as CTF(47) . However, we have been unable to
localize the transactivation domain directly since we have been unable
to establish a homologous system to assay mutant TPBFs. We failed to
recover activated transcription by simply adding back-purified
recombinant or native TPBF into a nuclear extract in which TPBF has
been sequestered by specific TPE DNA, or nickel affinity chromatography
(data not shown). All these results suggest TPBF might employ a
coactivator or adaptor to mediate its transactivation
activity(18, 52) . It will be of considerable interest
to identify the transactivation domain of TPBF as well as its target.
Adjacent to and overlapping the proline-rich domain, there is a potential coiled-coil domain (Coiled-coil I) comprising a heptad repeat of hydrophobic residues. While this region could potentially contribute to tetramerization (see below), deletion mutagenesis suggests it is not necessary, but instead may have a stabilizing effect on the overall structure. However, because this region contains two proline residues which are likely to destabilize or prevent helix formation, the importance of Coiled-coil I is somewhat unclear.
In the central portion of TPBF, there is a putative nuclear localization signal and a region that is rich in basic residues. By analogy with leucine zipper proteins or basic helix-loop-helix proteins, it is possible that this latter region may be involved in DNA binding.
At the C terminus of TPBF there is an additional coiled-coil domain (Coiled-coil II), containing hydrophobic 4-3 repeats. Positions a and d (the positions within a heptad repeat are conventionally referred to as a, b, c, d, e, f, and g, see (53) ) of the heptads in the array named Coiled-coil II are almost perfectly hydrophobic. Although this region resembles a leucine zipper, its perfectly amphipathic hydrophobic character suggests that it is likely to form higher order oligomers, since perfect coiled-coil domains can form trimeric or tetrameric bundles(54) . In accord with this prediction, direct chemical cross-linking and cotranslation experiments establish that TPBF exists as a tetramer both in solution and when bound to DNA.
Analyses of several TPBF deletion mutants supports the
predictions made from inspection of its sequence. Although our analyses
were constrained by an inability to express all mutants in E.
coli, several important conclusions were reached. Chemical
cross-linking and binding studies of mutant proteins demonstrate that
Coiled-coil II is essential for tetramerization and therefore DNA
binding. Coiled-coil II is the major, if not the only, region driving
tetramerization of TPBF. Although Coiled-coil I is not essential for
DNA binding (Fig. 6B), removal of the region greatly
reduces DNA binding activity, suggesting its involvement in either
stabilizing tetramer or tetramer-DNA complex. Other proteins with two
or more separate coiled-coil domains have been
reported(55, 56) . The reovirus cell attachment
protein 1 has two coiled-coils, one is involved in the formation
of a loose multimer while the other stabilizes the multimer (56) . Our prediction of how the two coiled-coils function in
TPBF is similar to this model, i.e. Coiled-coil II mediates
tetramerization of TPBF, and the tetramer is further stabilized by
Coiled-coil I. This idea is supported by the preferential formation of
homotetramer of wild type TPBF over a truncated version lacking
Coiled-coil I (Fig. 5, lane 2). It is likely that the
association of cotranslated polypeptides is not random. Instead, it
favors the formation of tetramers containing Coiled-coil I, which
provides additional stabilization. However, Coiled-coil I may also be
involved in stabilizing tetramer-DNA interactions.
TPBF requires a
relatively large domain for efficient, sequence-specific DNA binding.
Unlike GCN4, for example, which only requires the C-terminal 56 amino
acid residues for DNA binding(57) , the region in TPBF required
for efficient DNA binding spreads from 20 to 320. Since the mutant
1-122 had very weak DNA binding activity, the region from 20
to 122 is most likely involved in determining the binding efficiency
but not specificity. The region involved in determining DNA sequence
specificity is therefore contained within amino acids 123 to 281. We
have preliminary evidence suggesting the basic region from 123 to 194
is in fact necessary for DNA binding. (
)However, finer
mapping needs to be done to further define the region necessary for
sequence-specific DNA binding.
Previous studies showed that TPBF makes numerous symmetrical contacts with TPE(32) . The overall pattern of the base and phosphate contacts, suggesting that TPBF contacts DNA symmetrically on opposite faces of the helix, is unique. This binding pattern is in keeping with the novel structural features of TPBF, especially its divergent coiled-coil region and the widely spread basic region. One of the unique features of TPBF is that it is tetrameric, which is probably determined by the arrangement of hydrophobic residues in positions a and d in Coiled-coil II(54) . Tetramerization of TPBF can explain the protein-DNA contacts inferred from chemical interference assays. First, TPBF is able to occupy a large region of DNA since it contains a four-stranded helical bundle. Second, the region for tetramerization is located at the C terminus of TPBF allowing four DNA binding domains of TPBF, located more than 100 amino acid residues from the tetramerization domain, to reach relatively distal binding sites. It is possible that the tetramerized TPBF contacts the DNA helix perpendicularly from one side. Thus, its four DNA binding domains can make symmetrical contacts on opposite faces of the DNA helix. Interestingly, this DNA binding pattern resembles the way tumor suppressor p53 binds to its specific DNA recognition sequence(33) . Although no obvious similarities exist between these two proteins in amino acid sequence or specific DNA binding sites, it is possible that they belong to a novel family of DNA-binding proteins that function in regulating expression of genes involved in growth and differentiation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L46867[GenBank].