(Received for publication, August 1, 1996)
From the Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, Carretera de Colmenar Km 15.5, Madrid 28049, Spain
Transcription factor MYB.Ph3 from
Petunia binds to two types of sequences, MBSI and MBSII,
whereas murine c-MYB only binds to MBSI, and Am305 from
Antirrhinum only binds to MBSII. DNA binding studies with
hybrids of these proteins pointed to the N-terminal repeat (R2) as the
most involved in determining binding to MBSI and/or MBSII, although
some influence of the C-terminal repeat (R3) was also evident.
Furthermore, a single residue substitution (Leu71 Glu)
in MYB.Ph3 changed its specificity to that of c-MYB, and c-MYB with the
reciprocal substitution (Glu132
Leu) essentially gained
the MYB.Ph3 specificity. Molecular modeling and DNA binding studies
with site-specific MYB.Ph3 mutants strongly supported the notion that
the drastic changes in DNA binding specificity caused by the Leu
Glu substitution reflect the fact that certain residues influence this
property both directly, through base contacts, and indirectly, through
interactions with other base-contacting residues, and that a single
residue may establish alternative base contacts in different targets.
Additionally, differential effects of mutations at non-base-contacting
residues in MYB.Ph3 and c-MYB were observed, reflecting the importance of protein context on DNA binding properties of MYB proteins.
One characteristic of most eukaryotic transcription factors is that they can be grouped into a small number of families, each including factors with sequence similarity over their DNA-binding domain (reviewed in Refs. 1-3). In a given species, different members of the same family usually regulate unique, often partially overlapping, groups of target genes, at least in part due to distinct, although related, DNA binding specificities (4, 5). The basis of distinctiveness/similarity in DNA binding specificity among members of each family of transcription factors is not yet fully understood, although some progress toward rationalizing this problem has already been made (Refs. 6 and 7 and references therein).
One of the families of transcription factors is that of MYB proteins, so named because the first member of the family to be discovered was the product of the avian myeloblastosis oncogene v-myb. Subsequently, members of this family, sharing the MYB DNA-binding domain, have been found in all eukaryotes investigated, from yeast to humans (reviewed in Refs. 8-10).
Structurally, the best characterized member of the family is c-MYB, the cellular homologue of v-MYB, for which the solution structure of its DNA-binding domain has been solved, both in the free form and in complex with DNA (11-15). The c-MYB DNA-binding domain consists of three imperfect repeats (R1, R2, and R3), each of which folds into a variant of the homeodomain helix-turn-helix motif, similar to that of the prokaryotic LexA protein (7, 11-13, 15, 16). The third helix of the R2 repeat, the recognition helix, however, shows certain conformational flexibility in the free form, which is stabilized upon binding to DNA, and the same is true for the equivalent helix of B-MYB (14, 15, 17, 18). MYB repeats are also characterized by the presence of three conserved tryptophan residues regularly spaced by 18 or 19 amino acids that play a relevant role in the folding of the hydrophobic core of the MYB domain (11, 12, 19, 20). In their interaction with DNA, the recognition helices of both R2 and R3 lie on the major groove of the DNA and interact with each other, resulting in a cooperative binding to DNA sequences with the consensus pentanucleotide core CNGTT (12). The R1 repeat, which is missing in all plant MYB proteins (for examples, see Ref. 21) has no observable effect on DNA binding specificity, although it contributes to the stability of the protein·DNA complex (12, 22-24). The three key base contacts are established by residues Lys128 (R2), Lys182 (R3), and Asn183 (R3), which are fully conserved in all known plant and animal MYB proteins (Refs. 12, 21, and 25 and references therein). However, whereas all known animal (R1, R2, and R3) MYB proteins recognize the same type of core sequence as c-MYB, in plants, there are at least some MYB proteins that show binding specificity differing from that of c-MYB (22, 25-33).
A striking case of this divergence in binding specificity is that of MYB.Ph3, which is a transcription factor predominantly found in epidermal cells of Petunia flowers. Like some other plant MYB proteins, such as the C1, Pl, and P proteins from maize, the Am305 protein from Antirrhinum, and others, MYB.Ph3 possibly regulates the flavonoid (phenylpropanoid) biosynthetic pathway (21, 31, 33-37). MYB.Ph3 has been shown to bind to two types of site: MBSI1 (A(a/D)(a/D)C(G/C)GTTA, where a/D is A, G, or T, A being the preferred base), which conforms to the core consensus sequence CNGTT and is bound by c-MYB; and MBSII (AGTTAGTTA), which resembles the binding site of P and Am305 proteins and which is not bound by c-MYB (30, 31, 33). Our previous studies support the idea that binding of MYB.Ph3 to MBSI and MBSII does not involve alternative orientations of its two MYB repeats, despite of the resemblance of these sites to inverted and direct repeats of the GTTA motif, respectively (33, 38).
Here we report on the analysis of the molecular determinants that
enable MYB.Ph3 to recognize two different types of sequence. Remarkably, a single residue substitution in the R2 repeat of MYB.Ph3
(Leu71 Glu) changes its specificity to that of c-MYB,
and that the reciprocal substitution in c-MYB, Glu132
Leu, essentially confers MYB.Ph3 specificity. We provide evidence that
the ability of a single residue substitution to have such great effects
on DNA binding specificity reflects the fact that certain residues
influence this property directly, through base contacts, and also
indirectly, through interactions with other base-contacting residues,
and that some residues can establish alternative base contacts in
different targets. In addition, we show that substitutions in
(presumably) non-base-contacting residues can also affect the DNA
binding properties of MYB proteins, and that their effect may be
different in c-MYB and MYB.Ph3, thereby underlining the importance of
protein context in determining DNA binding.
Constructs coding for Petunia MYB.Ph3C1,
murine c-MYB
R1C1, and Antirrhinum Am305
C1 have been
described previously (31, 33). Constructs coding for the mutant
derivatives of these proteins, used in this study, were obtained by
PCR-mediated, site-directed mutagenesis of the corresponding cDNAs
as described by Cormack (39). To prepare the constructs encoding MYB
chimeric proteins, the cDNA fragments corresponding to the parts of
the MYB proteins present in the chimeras were obtained by PCR
amplification with one phosphorylated oligonucleotide, that
corresponding to the internal part of the chimera. After ligation of
the two fragments present in each chimera, a second PCR was performed
with the oligonucleotides corresponding to the 5
and 3
ends of the
chimeric cDNA. All cDNA fragments coding for mutant or chimeric
proteins were cloned into the XbaI-BamHI sites
(or XbaI-PstI for P2A3 and M2A3 chimeras) of the
pBluescript vector. All PCR fragments used in the constructs were
confirmed by sequencing.
RNAs, obtained by in vitro transcription of the corresponding constructs using T7 or T3 polymerase, were used for in vitro translation in the flexi-rabbit reticulocyte system (Promega) supplemented with magnesium acetate and potassium chloride to final concentrations of 2.05 and 75 mM, respectively, in the presence of [35S]methionine, following the manufacturer's instructions. After in vitro translation, SDS-PAGE analysis of the reticulocyte extracts was performed to allow estimation of the amount of each translated protein by measurement of 35S cpm in the corresponding protein band and correction for methionine content.
DNA Binding Reactions and EMSAPCR labeling of MBSI, II, IG, and IIG oligonucleotides, DNA binding reactions, and electrophoretic mobility shift assays (EMSAs) were performed as described in Solano et al. (33). Each binding reaction (15 µl) contained 4 ng of labeled DNA, 400 ng of poly(dI·dC), 150 ng of denatured salmon sperm DNA, and rabbit reticulocyte lysate consisting of a measured amount of the in vitro translated protein, supplemented with lysate incubated in the absence of external RNA to give a final volume of 2 µl, so that all reactions had equimolar amounts of protein.
Molecular ModelingThe structures used for the analysis were the average NMR structure of c-MYB bound to DNA (GTCAGTTA), as deposited in Protein Data Bank (40) under code 1MSE by Ogata et al. (12), and the best 25 NMR solutions (Protein Data Bank code 1MSF). Modeling of MYB.Ph3 complexed with DNA (MSBI or MSBII) was carried out with the WHATIF package (41). The quality of the resulting structures was assessed by different standard structures based on normality of molecular contacts (42) and deviation from normal exposed hydrophobic surfaces (43). The analysis of alternative conformations for different residues corresponds to the WHATIF secondary structure-specific rotamer data base (version Feb. 1996).
To investigate
the molecular determinants that allow MYB.Ph3 protein to recognize two
different types of sequence, we first analyzed the role of R2 and R3
repeats on DNA binding. For this purpose, we took advantage of the
differential affinity of murine c-MYB and Antirrhinum Am305
proteins for both types of MYB.Ph3 consensus binding sites (28, 31,
33). As shown by EMSAs (electrophoretic mobility shift assays, Fig.
1), derivative MYB.Ph3C1 binds both types of
consensus sequence (MBSI and MBSII) with the same affinity, whereas
derivative c-MYB
C2R1 only binds to MBSI but not to MBSII, and
derivative Am305
C1 shows the opposite behavior. Protein Am305 also
differs from MYB.Ph3 in that it prefers a variant of MBSII with a G in
position +2 (MBSIIG; Ref. 31; Fig. 1), whereas an additional difference
between c-MYB and MYB.Ph3 is that a change of T for G at position +2 in
MBSI (MBSIG) still allows a certain binding by c-MYB and not by MYB.Ph3
(24).
We constructed hybrid proteins that combined R2 and R3 MYB repeats: P2
and P3 from MYB.Ph3; A2 and A3 from Am305; and M2 and M3 from c-MYB.
These chimeric proteins, like their progenitors (Fig. 1), also
contained amino acid sequences beyond the strict R2 and R3 repeats,
originating from the 5 or the 3
coding parts of the cDNAs, except
for M2, which only included an additional methionine from an engineered
initiation codon (33). However, previous work with c-MYB (12, 22, 28),
as well as the studies with site-directed mutants described in the next
sections, showed that the effect of these additional sequences on DNA
binding specificity was negligible. As shown in Fig. 2,
all chimeric proteins, except P2A3, recognized at least one of the four
sequences, albeit generally with lower affinity than their parental
proteins, particularly M2A3. The type of sequence recognized
(i.e. I or II) was mainly dependent on the type of R2 repeat
in the chimera. Thus, proteins with the R2 repeat of MYB.Ph3 (P2) were
able to bind type I and type II sequences, as can MYB.Ph3, whereas
proteins with the R2 repeat of c-MYB (M2) or Am305 (A2) showed a
preference for type I or type II, as found for A2A3 or M2M3,
respectively. Because type I and II sequences differ at their 5
halves, R2 should be mainly implicated in the interaction with the 5
half of the sites. On the other hand, M2P3 and M2A3, which share the
same R2 repeat, showed differential affinity for I and IG sequences,
respectively, thus implicating the R3 repeat in the interaction with
the 3
part of the targets. The same conclusion could be drawn from a comparison of A2P3 with A2A3.
However, there must be some functional interdependence between repeats
R2 and R3 in their interaction with the 5 and 3
halves of the
sequence, respectively. For instance, the A2P3 protein bound to MBSIIG
with higher affinity than P2P3 or M2P3, indicating some role of R2 (A2)
in the interaction with the 3
half of the sequence. The same
conclusion could be drawn by comparing A2M3 with P2M3 and M2M3, or P2P3
with M2P3. On the other hand, the higher binding affinity of A2P3
than A2A3 to MBSI can be taken as an example of the influence of the R3
repeat on the interaction with the 5
half of the sequence.
An analysis of R2-
and R3-specific residues responsible for the differential binding
specificity of MYB.Ph3 versus c-MYB and/or Am305 was then
undertaken. The amino acid residue of the R3 repeat determining the
preference for T rather than G at position +2 was investigated. A
comparison of the amino acid sequence of R3-recognition helices from
MYB.Ph3 and Am305, which respectively prefer T and G at position +2,
revealed several differences (Fig. 3). Among these,
residue Asn125 of MYB.Ph3, which is substituted by an Arg
residue in Am305, was selected for site-directed mutagenesis, based on
previous evidence that implicated the equivalent residue from c-MYB in the interaction with +2T (see Fig. 7; position 5T in Ref. 12). The
MYB.Ph3 (Asn125
Arg) mutant now preferred the MBSG to
the MBS sequences (Fig. 3), thereby revealing the influence of residue
Asn125 from MYB.Ph3 (or Arg from Am305) in the specificity
of +2 contacts. Mutations of residue Asn125 to Ser, Ile, or
His decreased overall affinity without affecting specificity,
indicating that this residue is not the only determinant of position
+2, in agreement with the studies using chimeric proteins described in
the previous section (see also Fig. 7).
Major Role of Residue Leu71 from the R2 Repeat in Dual Recognition by MYB.Ph3
Recognition determinants within the R2
repeat of MBSI and/or MBSII were investigated using chimeric proteins
obtained by full or partial replacement of the recognition helix of the
A2 repeat from protein A2M3 by its M2 counterpart, because repeats A2
and M2 determined the most extreme differences in binding to types I
and II sequences (Fig. 2). As shown in Fig. 4, full
substitution of the recognition helix (A2M3-3 protein) conferred the
c-MYB specificity, whereas the partial substitution in A2M3-2 did not greatly alter the A2M3 specificity. This suggested that the N-terminal half of the R2 recognition helix was the major determinant of binding
to MBSI or MBSII in the Am305/c-MYB context.
To confirm the importance of amino acid residues of the N-terminal part
of the R2 recognition helix in the MYB.Ph3 context, we performed EMSA
with mutant derivatives of the MYB.Ph3 and c-MYB proteins affecting the
three nonconserved positions in these two proteins (Fig.
5). Each replacement had a different effect on the DNA
binding properties of MYB.Ph3 and of c-MYB. Remarkably, a single
residue substitution in MYB.Ph3 (Leu71 Glu) conferred
c-MYB specificity (with respect to the targets used), and the
reciprocal change in c-MYB (Glu132
Leu) showed the
reverse behavior, because it rendered a c-MYB derivative able to bind
to MBSII sequences, although showing a slight preference for MBSI
sequences. An additional change in c-MYB (Gln-Glu
Ser-Leu) resulted
in a preference of this mutant protein for MBSII. It is noteworthy that
the MYB.Ph3 (Cys-Ser
Ile-Gln) also showed preference for MBSI
sequences. In fact, its relative affinity to MBSI compared to MBSII was
higher than that of the corresponding mutant of c-MYB, c-MYB (Glu
Leu), indicating that the effect of this residue is dependent on
protein context. A similar conclusion can be drawn by comparing the DNA binding properties of MYB.Ph3 (Ser-Leu
Gln-Glu) and MYB.Ph3 (Cys-Ser-Leu
Ile-Gln-Glu) with those of c-MYB, or of MYB.Ph3 (Cys
Ile) with those of c-MYB (Gln-Glu
Ser-Leu).
Mutational Analysis of the Conserved Lys Residue in the R2 Repeat
A major difference between the two types of MYB.Ph3
binding site is the greater sequence constraints on MBSII compared to MBSI at positions 4 and
3 imposed by the presence of T instead of C
at position
2 (33). Thus, for instance, exchanging T in MBSII with C
has only a moderate effect on its binding by MYB.Ph3, whereas the
reciprocal change in MBSI (i.e. C
T), results in a great
impairment of binding (Ref. 33; Fig. 6). Molecular
modeling predicted that the highly conserved Lys residue from MYB.Ph3
(Lys67) should interact with
2
G in MBSI, like the
equivalent Lys of c-MYB, but with
4G (and perhaps with
3T) in the
MBSII sequence (see "Discussion" and Fig. 7); thus,
the Lys67 residue may be responsible for these sequence
constraints. To test this prediction, the effect of mutating the
Lys67 residue (to Ala or Ser) on DNA binding specificity
was examined. As shown in Fig. 6, the two mutants had reduced DNA
binding affinity but bound better to MBSI and MBSII than to MBSIG,
MBSIA (AAAAGGTTA), and MBSIIG, indicating that the mutations did not
affect MYB.Ph3 specificity indiscriminately. However, in sharp contrast
to wild-type MYB.Ph3, these mutant proteins bound similarly to MBSI and
MBSIT (AAATGGTTA), in agreement with the prediction that the
Lys67 residue is responsible for sequence constraints at
positions
3 and
4 in MBSII. In fact, when Lys67 does
not impose constraints on positions
3 and
4 (e.g. when MYB.Ph3 binds to MBSI, or when MYB.Ph3 (Lys67
Ala/Ser)
binds to MBSI or MBSII), it appears that the preferred base at these
positions is an A, as in MBSI. It is also noteworthy that the Lys
Ala/Ser mutations showed higher overall affinity to MBSIT than MYB.Ph3.
This could indicate that when the (large and charged) Lys residue of
MYB.Ph3 does not establish a base-specific contact, it may perturb base
contacts by other residues.
DNA binding studies with hybrid MYB proteins and with
site-directed MYB mutants reported here indicate that the R3 repeat is
the most responsible for differential binding to MBSI and MBSII compared to MBSIG and MBSIIG, whereas the R2 repeat was primarily involved in determining the MBSI/MBSII specificity (Figs. 2, 3, 4, 5).
Additionally, these experiments indicated that both repeats influence
each other's primary effect; for instance, the higher relative
affinity for MBSIIG (versus MBSII) of the A2P3 chimera with
respect to that of P2P3 reveals a role of R2 (A2) in the interaction
with the 3 half of the sequence (Fig. 2).
The proposed primary roles of the R2 and R3 repeats and their
functional interdependence are in good agreement with the available structural information on c-MYB. Indeed, the NMR solution structure of
the complex between the c-MYB R2R3 domain and DNA shows that its
repeats physically interact and bind to DNA in a partially overlapping
way (12). In this context, it is not surprising that the R2 and R3
amino acid sequences of both Am305 and MYB.Ph3 fit well in the
structure of the R2R3 domain from c-MYB (data not shown). This
structural similarity is also manifest in the effects of particular
residue substitutions, like that of Asn125 Arg in
MYB.Ph3, which resulted in a specificity change at position +2 (Fig.
3), the position interacting with the equivalent residue from c-MYB,
Asn186 (Ref. 12; Fig. 7).
The physical interactions between repeats of c-MYB bound to DNA result in (intramolecular) cooperativity (12). In this scenario, it is conceivable that for a MYB domain to be functional, its R2 and R3 repeats must adapt to each other. Our results with R2/R3 MYB chimeras are in line with this suggestion because most of them displayed reduced DNA binding affinity with respect to their progenitors (most notably P2A3). Thus, it appears that co-evolution may have placed constraints on the compatibility between repeats from different MYB proteins.
Leu71, a Key Residue for MYB.Ph3 Dual DNA Binding SpecificityIn this study, we have found that a single residue
substitution within the recognition helix of the R2 repeat of MYB.Ph3, Leu71 Glu, switches the dual DNA binding specificity of
MYB.Ph3 to the c-MYB specificity, and that the reciprocal
(Glu132
Leu) change in c-MYB essentially confers the
MYB.Ph3 specificity. As discussed below, such drastic effects on
specificity caused by the Leu
Glu substitution most likely indicate
that there are key residues that influence binding specificity not only
directly, through base contacts, but remarkably also indirectly,
through interactions with other base-contacting residues, and that a
single residue can establish alternative base contacts in different
targets.
In the NMR average structure of the complex of the c-MYB minimal
DNA-binding domain (R2R3) with its target DNA (GTCAGTTA; Ref. 12),
Glu132 interacts weakly with DNA (positions 2C and +1
C
in our nomenclature, see Figs. 1 and 7; Ref. 12). Additionally,
Glu132 establishes an electrostatic interaction with
Lys128, a key base-contacting residue that interacts with
2
G. Using molecular modeling (see "Materials and Methods"), we
predicted that the change of Glu for Leu would have two consequences:
(i) Leu would be in a hydrophobic cavity adequate to allow interaction with C or T at position
2 (see also Ref. 6), the bases respectively present in MBSI and MBSII, whereas Glu would not specifically interact
with T, and (ii) the electrostatic interaction between Lys128 of c-MYB (Lys67 in MYB.Ph3) and Glu does
not occur, thereby facilitating the interaction of this Lys residue
with an alternative position,
4 and to some extent with
3. This is
particularly important in the binding to MBSII, which has A rather than
G at position
2
. The possibility that a single residue can establish
contacts at two alternative positions has been documented/invoked in
several instances (44-46).
Further evidence that Lys128 (c-MYB coordinates) can
establish alternative base contacts was obtained from the analysis of
the 25 available NMR solution structures of the c-MYB(R2R3)· DNA
complex. Indeed, in two solutions Lys128 was found to
interact directly with 4G, whereas Glu132 was located far
away from the average structure and did not interact directly with the
DNA (see alternate positions of Lys128 and
Glu132 in Fig. 7). Hence, the preferential interaction of
c-MYB residue Lys128 with
2G could be due to the
electrostatic attraction of Lys128 toward
Glu132 (as close as 2.64 Å in some of the NMR
solutions).
These interpretations are also supported by our results with the
Lys67 Ala/Ser substitutions in MYB.Ph3, which broadened
specificity at positions
4 and
3 (Fig. 6), and with missing
nucleoside assays, which have shown that nucleoside at position
2
(A) in MBSII is fully dispensable in binding by MYB.Ph3 (33).The
requirement for T at position
3 in MBSII (33) is not well understood,
although it could reflect that the methyl group of T pushes
Lys67 to
4G, or alternatively that Lys67
interacts with GT rather than only with G (see Ref. 6).
Base-contacting residues play a critical and direct role
in determining the specificity of DNA-binding proteins. This is evident from the fact that specificity can be explained to a significant extent
using simple rules: the base-contacting specificity of different
residues and the (usually) fixed position of base-contacting residues
within the DNA-binding domain in each protein family (6, 7, 47). For
instance, in MYB.Ph3, the effect of the Asn125 Arg
substitution does conform to these rules. However, there is strong
evidence that binding specificity in MYB proteins can also be
indirectly modulated by non-base and base-contacting residues. Thus,
Am305 shares all putative base-contacting residues with MYB.Ph3
(Asn125
Arg), but only the latter strongly binds to
MBSI (Figs. 3, 4, and 7). Likewise, the maize P protein shares all the
putative recognition residues with MYB.Ph3 and/or c-MYB, but it binds
to a different site (GGT(T/A)GGT(A/G); Refs. 30 and 35). Moreover, in
addition to the indirect effects of the Leu/Glu substitutions discussed
above, we have also shown that several substitutions in presumably
non-base-contacting residues alter specificity and/or affinity (see
Fig. 5), and in some instances (e.g. the Gln/Ser substitution) the degree of the effect on specificity was different in
the MYB.Ph3 and in the c-MYB contexts.
One possible explanation for these indirect effects on binding
specificity could be that residue substitutions affect conformational properties of the protein, thereby influencing the strength of possible
contacts by recognition residues or imposing constraints in the
structural properties of the DNA (3), because some MYB proteins induce
bending/distortions upon binding to DNA (38, 48). In this regard, note
the presumed structural flexibility of the R2-recognition helix, a
property expected to be very sensitive to mutations (14, 15, 17, 18).
Some of the specificity effects of substitutions of non-base-contacting
residues could simply be mediated by side-chain interactions with
base-contacting residues, such as that of Glu132 with
Lys128 in c-MYB. For instance, residue Gln129
(c-MYB coordinates) interacts in the average structure with the phosphate backbone of the DNA, but in some of the solutions, it interacts with Glu132 or with Lys128. In
solutions where Gln129 interacts with Glu132 or
with Lys128, Lys128 interacts very closely with
2
G (see Fig. 7). Hence, it seems that Gln129 contributes
to maintain Lys128 in the conformation that favors the
interaction with
2
G, and such effect could be accentuated when
Glu132 is missing (c-Myb (Glu132
Leu) and
MYB.Ph3 (Cys-Ser
Ile-Gln); Fig. 5).
The differential effects of some residue substitutions, such as Gln/Ser, in MYB.Ph3 and c-MYB further underline the importance of protein context in MYB DNA binding specificity, possibly involving interresidue interactions. Indeed, we noticed that, in c-MYB, Glu132 and Gln129 are part of a network with several residues (Asn179, Lys182, Asp178, Arg131, and His135), phosphates, and bases. In MYB.Ph3, one of these residues, His135, is substituted by Ala (Fig. 7), and consequently, the effect of substitutions involving residues at positions 129 and 132 (c-MYB coordinates) cannot be the same in the two protein contexts.
The notion that DNA binding specificity is best viewed as the result of a network of interactions of residue side chains with the DNA backbone and bases, as well as with other residues, rather than the simple and independent contribution of base-contacting residues has also been highlighted for other protein families, such as bZIP, ribbon-helix-helix, homeodomain, prokaryotic helix-turn-helix, and others (for examples, see Refs. 44-52). Obviously, the importance of interresidue interactions will be higher in proteins with physically interacting DNA-binding subdomains, such as the MYB and cut-homeodomain proteins (12, 53).
The numbers of myb genes in plant species are large, in contrast to those in other types of eukaryotes; for instance, there are at least 20-30 myb genes in Petunia (21), and Arabidopsis contains over 100 of these genes.2 However, 6 of 8 putative recognition residues are fully conserved among all plant MYB proteins with known sequence (30 in data bases; data not shown), and the remaining 2 residues are conserved in at least 80% of the proteins. Therefore, mutations in non-base-contacting residues must have greatly contributed to the generation of functional diversity among the members of the plant MYB family.
We are very grateful to Drs. Cathie Martin and Roger Watson for providing us with the Am305 and c-MYB progenitor constructs, respectively. We thank Drs. Francisco García-Olmedo, Cathie Martin, Joseph Ecker, Darío García de Viedma, and Antonio Leyva for critical reading of the manuscript. The excellent technical assistance of María Jesús Benito is gratefully acknowledged.
Additional information can be obtained from the authors at the following WWW site: http://gredos.cnb.uam.es/sanchez/Myb.html.