(Received for publication, January 29, 1997, and in revised form, March 4, 1997)
From the Neurobiotechnology Center, Ohio State University, Columbus, Ohio 43210
Hydroxy fatty acids from plant cutin were shown
previously to induce the expression of the cutinase gene via a
palindromic sequence located at 159 base pairs of the cutinase gene
in Fusarium solani f. sp. pisi (Nectria
hematococca mating type VI). Of the two overlapping palindromes
in this sequence, palindrome 2 was found to be essential for the
inducibility of cutinase by hydroxy fatty acids. Screening of a phage
expression library with the concatenated palindrome 2 as probe detected
a distinct cDNA clone encoding a polypeptide designated cutinase
transcription factor 1
(CTF1
) with a calculated molecular weight
of 101,109. This protein contains a Cys6Zn2
binuclear cluster motif sharing homology to the
Cys6Zn2 binuclear cluster DNA-binding domains
of transcription factors from Saccharomyces cerevisiae,
S. carlsbergensis, Kluyveromyces lactis,
Neurospora crassa, Aspergillus nidulans, and A. flavus. CTF1
, expressed in Escherichia coli,
showed specific binding to the palindrome 2 DNA fragment but not to
palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific
binding of CTF1
to palindrome 2. When CTF1
was expressed as a
fusion protein with the nuclear localization sequence of SV40 in yeast,
it transactivated the native cutinase promoter fused to the
chloramphenicol acetyl transferase (cat) gene. Mutation of
palindrome 2 but not palindrome 1 abolished this transactivation. Thus,
CTF1
positively acts in vivo by binding selectively to
palindrome 2 of the cutinase gene promoter.
Extracellular cutinases can help fungi to penetrate through the
outermost cuticular barrier of the host plant and, thus, play a role in
pathogenesis (1). When conidia of highly virulent fungal strains that
carry low levels of cutinase (2) contact the host cuticular surface,
small amounts of cutin hydrolysate would be produced, and the monomers
thus released are known to trigger expression of the cutinase gene in
the germinating conidia, leading to targeted secretion of cutinase at
the contact site (2). It is known that 10,16-dihydroxy C16
fatty acid and 9,10,18-trihydroxy C18 fatty acid, the
unique monomers of cutin, are the best inducers of cutinase (3). The
cis-elements responsible for this inducible expression of
the cutinase gene were identified previously using Fusarium
solani f. sp. pisi transformants containing segments of
the 5-flanking regions of the cutinase gene and their mutants fused to
the chloramphenicol acetyl transferase (cat) gene (4). The
element essential for the inducible expression of cutinase gene was
found to be located at
159 bp1 (4). Of
the two overlapping palindromes in this region, palindrome 2 was the
element necessary for the inducible expression of the cutinase gene.
Gel retardation studies showed that glucose-depleted cultures contained
a protein factor that specifically bound palindrome 2, and this factor
was designated cutinase transcription factor 1 (CTF1) (4).
Our efforts to identify transacting factors involved in the regulation
of cutinase gene transcription led to the isolation of a zinc finger
protein that specifically bound to the overlapping palindromic sequence
designated palindrome-binding protein (PBP) (5). Here we report that
further examination of the specificity of this binding by PBP revealed
that this protein bound only to palindrome 1 but not to palindrome 2. We, therefore, further examined expression libraries for a transacting
factor that would specifically bind palindrome 2 and report here the
isolation of a clone that encodes a distinct protein that specifically
binds to palindrome 2 but not to palindrome 1. This protein, designated
CTF1, shows a low degree of sequence identity to GenBank sequences,
but it contains a Cys6Zn2 binuclear cluster
motif similar to those found in positively acting transcription factors
in other fungi. That CTF1
is a transcriptional activator in
vivo for palindrome 2 of the cutinase gene promoter was further
demonstrated by its ability to transactivate the cutinase promoter
fused to the cat gene in yeast and by the observation that
this transactivation was abolished by mutation of palindrome 2 but not
palindrome 1.
Restriction and
modification enzymes were from Life Technologies, Inc. Chemicals were
from Sigma or Amresco. Poly(deoxyinosinic-deoxycytidylic) acid was from
Boehringer Mannheim and labeled nucleotides were from Amersham Corp.
Nitrocellulose filter discs were from Schleicher & Schuell. Duralon-UV
membranes were from Stratagene. Escherichia coli DH5
(Life Technologies, Inc.) was used for propagating all plasmids
(6).
The 27 discrete
clones identified in the original tertiary screening for PBP (5) were
tested by polymerase chain reaction, and those homologous to PBP clone
were not tested further. Phage DNAs were purified from the remaining
clones and double digested with either EcoRI and
BamHI, or EcoRI and SalI, or
EcoRI and SstI, or EcoRI and
XhoI. Subsequently, those phage clones that differed in
restriction patterns were probed (7) with 32P-labeled
palindrome 2 fragment that was concatenated to an average size of 316 bp or 17 copies of the palindrome 2 fragment as described (5). A
polypeptide encoded by one of the phage clones (designated ctf-15)
that bound the concatenated palindrome 2 was designated CTF1
. The
cDNA insert from this phage clone was subcloned into pBluescript
KS
vector (Stratagene) to produce pCTF1-15. Sequence
analyses indicated that this clone was not full-length. Additional
clones for CTF1
were isolated by screening a
gt11 library
constructed previously with random primers (5) and another
gt11
library constructed similarly with oligo(dT)
(5)2 with the cDNA inserts from
pCTF1-15 or its terminus as probe using standard procedures (6).
DNA inserts in gt11
phage clones were subcloned into KS
vector. Plasmid DNAs
were prepared (6) and used for automated sequencing with a model 373A
sequencer (Applied Biosystems). The putative subcellular location for
the polypeptide was predicted with PSORT (8) (version 6.3) in Pedro's
Biomolecular Research Tools from the World Wide Web using Netscape.
Protein homology search was conducted with the Blast program from NCBI
(9).
The cDNA insert from
ctf-15 was directly cloned into the EcoRI sites of a
glutathione S-transferase fusion vector, pGEX-4T-1 (Pharmacia Biotech
Inc.) to generate pGEX-4T-1/CTF1
(1-526). The resulting plasmid was
introduced into BL21 cells for expressing glutathione
S-transferase-CTF1
(1-526) fusion protein. Additionally, a BamHI fragment from pGEX-4T-1/CTF1
(1-526) encoding the
DNA-binding domain of CTF1
was subcloned into a thioredoxin fusion
vector, pET-32a(+) (Novagen) to generate pET-32a/CTF1
(DBD), which
was introduced into BL21(DE3) to express TRX-CTF1
(DBD) fusion
protein.
TRX-CTF1(DBD) fusion protein was induced (5),
partially purified with a Ni2+-NTA agarose column (Qiagen),
under denaturing conditions (10) and renatured in a dialysis bag as
described (7). The dialyzed protein samples were concentrated with
Centricon-30 filtration units pretreated overnight with 5% Tween 20 (Amicon).
The 37-mer palindromic element (159 to
178 bp) containing both overlapping palindromes 1 and 2 was prepared
as described (5). To prepare palindrome 1 fragment (pal 1),
oligonucleotides aat tcG GAT CGC GAG CCg and aat tcG GCT CGC GAT CCg
were annealed. To prepare palindrome 2 fragment (pal 2),
oligonucleotides aat tCG AGC CGA GGC TCG and aat tCG AGC CTC GGC TCG
were annealed. The annealed fragments contained palindrome 1 of 12 nucleotides and palindrome 2 of 14 nucleotides, each flanked on both
ends by EcoRI sites (shown in lowercase). These
EcoRI sites served for concatenation and fill-in labeling
with [
-32P]dATP. DNA-binding assays by gel retardation
with about 0.5 µg of partially purified recombinant PBP (5) and
CTF1
were done essentially as described (4, 5).
For
expression of the full-length polypeptide of CTF1 (1-909),
fragments for full-length cDNA clone were first amplified by
polymerase chain reaction as separate 5
-end and 3
-end fragments with
Pfu polymerase (Stratagene). These two fragments were then joined by SOEing polymerase chain reaction (11) and cloned in-frame into the PstI site of the yeast expression vector pGAD424, a
GAL4 DNA-activation domain hybrid cloning vector
(CLONTECH), to produce pGAD424/CTF1
. To delete
the GAL4 DNA-activation domain from this chimeric plasmid,
pGAD424/CTF1
was first digested with KpnI and SmaI. The large fragment containing the DNA segment for
CTF1
was gel-purified by Geneclean (Bio 101) and ligated to a
KpnI-SmaI linker that was prepared by annealing
two oligomers, CGC CGC CGC and GCG GCG GCG GTA C. The resultant plasmid
was designated pLEU/CTF1
. This plasmid allows for leucine selection
in yeast and would produce a fusion polypeptide of SV40 NLS and
CTF1
. As a negative control vector, the fragment for GAL4
DNA-activation domain in pGAD424 was also replaced as above with the
KpnI-SmaI linker to produce pLEU.
For expression of the N-terminal 526 amino acids of CTF1, an
expression vector that allows for tryptophan selection in yeast was
first modified from pGAD424 as follows. pGBT9, a GAL4 DNA-binding domain hybrid cloning vector from CLONTECH, was
digested with PstI and PvuII. The small fragment
coding for tryptophan was isolated and ligated to PstI and
EcoRV-cut pGAD424 to produce expression vector pTRP-AD that
allows for tryptophan selection in yeast. To remove the GAL4
DNA-activation domain from pTRP-AD, the plasmid was digested with
KpnI and EcoRI. The large fragment was isolated and ligated to a KpnI-EcoRI linker that was
prepared by annealing two oligomers, CGC GGC GGC GG and AAT TCC GCC GCC
GCG GTA C. The resultant plasmid was designated pTRP, and it allows for
tryptophan selection in yeast. To express the N-terminal 526 amino
acids of CTF1
, the EcoRI fragment of
ctf1-15 was
directly cloned into the EcoRI site of pTRP to generate
pTRP/CTF1
(1-526).
To introduce into yeast the wild-type and mutant cutinase gene
promoter/cat gene fusions, three yeast centromeric plasmids were constructed from pCAT360, pCAT433pal1, and pCAT433
pal2 (4).
The plasmid pCAT360 is a pBluescript KS vector that contains the
E. coli gene for hygromycin phosphotransferase
(hph) under the regulation of a Cochliobolus
heterostrophus promoter and the gene for chloramphenicol acetyl
transferase (cat) under the control of the wild-type
cutinase gene promoter. pCAT433
pal1 and pCAT433
pal2 are similar
to pCAT360 with the exception that in pCAT433
pal1, the sequence
GGATCG in the first half of palindrome 1 was mutated to ATGAGC, and in
pCAT433
pal2, the sequence GGCTCG in the second half of palindrome 2 was mutated to TATGGC (4). pCAT360 was digested with SalI
and NotI to release the DNA fragment for the wild-type
cutinase gene promoter/cat gene, and this fragment was cloned into the SalI and NotI sites of pRS413, a
yeast centromere plasmid (YCp) that allows for histidine selection in
yeast (Stratagene). The resultant plasmid is designated pYCAT.
Similarly, DNA fragments for mutant cutinase gene
promoter/cat gene fusions were isolated from pCAT433pal1
and pCAT433
pal2 by digesting the plasmids with XbaI and
SalI. The isolated DNA fragments were then cloned into
XbaI- and SalI-digested pRS413 to generate
pY
pal1 and pY
pal2, respectively.
Plasmids were introduced with manufacturer's protocols
(CLONTECH) into yeast strain YPH499 (MATa
ura3-52 lys2-801amber
ade2-101orche trp1-63
his3-
200 leu1-
1) (Stratagene) in the
following combinations, pLEU and pYCAT, pTRP and pYCAT,
pTRP/CTF1
(1-526) and pYCAT, pLEU/CTF1
and pYCAT, pLEU/CTF1
and pY
pal1, and pLEU/CTF1
and pY
pal2. Yeast transformants were
selected on tryptophan- and histidine-lacking or leucine- and
histidine-lacking minimal medium (CLONTECH)
according to the above combinations of plasmids. For long-term storage
at
80 °C, the resultant transformants were scraped from plates and resuspended in 15% glycerol in screw-capped tubes.
Yeast transformants were grown in 6.5-ml minimal medium in 50-ml conical tubes for 36-48 h (CLONTECH). Cells were harvested by centrifugation, washed with 10 ml of H2O once, and resuspended in 0.4 ml of 0.1 mM Tris·HCl, pH 7.8, and cells were disrupted for 3 min in a Mini-Beadbeater (Biospec Products, Bartlesville, OK) in the presence of 100 mg of glass beads (710-1180 µm) (Sigma). The mixture was transferred to a new tube, incubated for 10 min at 65 °C, and then centrifuged at 16,000 × g for 10 min at ambient temperature. Protein concentration of the supernatant was determined with a Bio-Rad protein assay kit (Bio-Rad) with bovine serum albumin as standard. CAT assays were done as described with D-threo-[dichloroacetyl-1,2-14C]chloramphenicol (DuPont NEN) as substrate (4). To obtain the linear rate of acetylation of chloramphenicol, the yeast protein supernatant was diluted appropriately, and different aliquots were assayed for CAT activity. The products were separated and quantitated with a PhosphorImager (Molecular Dynamics) (4). Relative CAT activity was expressed as a percentage of chloramphenicol acetylated.
Previously, we isolated a zinc finger protein, PBP, that
specifically bound to the palindromic fragment (5) containing both of
the overlapping palindromes present in the cutinase gene promoter.
Because palindrome 2 is the one essential for induction of cutinase by
hydroxy fatty acids, we tested to determine its binding specificity of
PBP to the individual palindromes by gel retardation assay. PBP bound
only to the palindrome 1 DNA fragment but not to palindrome 2 DNA
fragment (data not shown). Therefore, additional protein factors that
specifically bind to this palindrome 2 were sought. We screened gt11
expression library again to search for clones encoding CTF1 that
specifically binds palindrome 2 (4). Because the phage clones encoding
PBP were isolated using the fragment containing both palindromes, the
phage clones obtained during that screening may also contain clones
that would encode CTF1. During the tertiary screening for PBP clones,
27 discrete clones were obtained. Polymerase chain reaction tests
indicated that 10 of the phage clones belonged to those encoding PBP
(data not shown). The remaining 17 clones showed five distinctly
different restriction patterns, and the representative clones from each group were designated as
ctf1-8,
ctf1-11,
ctf1-15,
ctf1-22, and
ctf1-26. When tested by Southwestern hybridization (7), all five
clones showed binding to the concatenated palindrome 2 fragment (data
not shown). DNA inserts from these phage clones were subcloned into pBS
KS
to generate pCTF1-8, pCTF1-11, pCTF1-15, pCTF1-22, and
pCTF1-26. Initial sequencing of these clones indicated that pCTF1-8 was a partial clone of pCTF1-15, pCTF1-22 was a partial clone of pCTF1-26, and pCTF1-11 was a distinct clone.
The inserts in pCTF1-15, pCTF1-11, and PCTF1-26 were completely
sequenced, and the deduced polypeptides revealed the presence of
Cys6Zn2 binuclear cluster DNA-binding motifs.
We chose to characterize ctf1-15 here because it produced the
strongest signal in the Southwestern hybridization (7) (data not
shown). The polypeptide encoded by the DNA insert in pCTF1-15 was
designated CTF1
(Fig. 1). The lack of in-frame stop
codon for the DNA insert indicated that this clone represented a
partial open reading frame. Further screening of
gt11 libraries
identified four additional overlapping clones for CTF1
(Fig. 1). The
complete sequencing of the five overlapping clones for CTF1
revealed
a contiguous cDNA sequence of 3271 bp containing a complete open
reading frame (Fig. 1).
The 3271-bp contiguous sequence for CTF1 contained a single open
reading frame of 2727 bp that would encode a protein of 909 amino acids
with a calculated molecular weight of 101,109 (Fig. 1). CTF1
contains 11 potential consensus sequences for phosphorylation by casein
kinase II (12), 10 potential sites for phosphorylation by the cyclic
AMP-dependent protein kinase A (13), and 6 potential
phosphorylation sites for protein kinase C (14). Two consensus sites,
PQTP and PATP, for potential phosphorylation by mitogen-activated
protein kinase were also present (15). Six potential
asparagine-glycosylation sites (16) were observed. Three plausible
nuclear localization signals with the residue patterns of KRKK, KRHRK,
and PKRK(17) were identified in CTF1
, with a certainty of 0.7 (on a
scale of 1) of it being localized in the nucleus as predicted by PSORT.
Homology searches identified only a Cys6Zn2
binuclear cluster domain located at the N terminus from amino acid
residues 59 to 92 of CTF1
(Fig. 2).
As an initial step to test the function of the identified CTF1,
segments of the DNA fragment coding for CTF1
were cloned into
expression vectors. When the glutathione S-transferase
fusion vector was used to express the first 526 amino acids of CTF1
, no protein was detected (data not shown). When thioredoxin (TRX) fusion
vector was used to express the Cys6Zn2
binuclear cluster DNA-binding domain of CTF1
, fusion protein of
TRX-CTF1
(DBD) was detected (data not shown). Even though the
expressed TRX-CTF1
(DBD) fusion protein was insoluble, solubilization
with 6 M guanidine hydrochloride, partial purification, and
renaturation yielded a soluble protein preparation. DNA-binding assays
by gel retardation were performed with this renatured protein. As shown
in Fig. 3A, a single retarded band of
palindrome fragment-CTF1
complex was observed. That the binding of
CTF1
to the palindrome was specific was indicated by the observation
that unlabeled palindromic fragment but not an unrelated DNA fragment
competed for the binding by the labeled palindromic fragment. In
addition, CTF1
showed binding only to palindrome 2 but not to
palindrome 1 (Fig. 3B). Mutations in the first half of
palindrome 2 that overlaps with palindrome 1 drastically reduced
CTF1
binding (Fig. 3C). Mutations in the second half of
palindrome 2 abolished CTF1
binding. Substitution of the
G-nucleotides previously found to be at the binding site for the
naturally occurring palindrome 2 (4) also abolished binding of
palindrome 2 to the recombinant CTF1
(Fig. 3C). These results demonstrate the sequence specificity of palindrome 2 involved in binding CTF1
.
The above tests indicated that CTF1 is a protein factor that binds
the cutinase promoter; therefore, we tested whether it could function
as a cutinase transcriptional activator in vivo. Plasmid
constructs were made to express a hybrid fusion protein in which
CTF1
was fused to the nuclear localization sequence of SV40. The
CTF1
hybrid protein was tested for activation of transcription of
the cat gene fused to the cutinase promoter. Its
transactivating capability was indicated by the production of high
levels of CAT activity when the full-length cDNA clone for CTF1
was introduced into yeast cells carrying the wild-type cutinase gene
promoter/cat gene fusion cassette (Fig. 4B,
lanes WT and pLEU/CTF1
). Mutations in the
first half of palindrome 1 did not change the CAT activity appreciably
(Fig. 4B, lane
pal 1), whereas mutations in the second
half of palindrome 2 resulted in a drastic decrease of CAT activity
(Fig. 4B, lane
pal 2). That the C-terminal segment of
CTF1
was essential for the transactivation was shown by the
observation that expression of the hybrid protein containing the
N-terminal 526 amino acids of CTF1
fused to the nuclear localization
sequence of SV40 failed to activate the cutinase gene
promoter/cat gene (Fig. 4B, lane WT and
pTRP/CTF1
(1-526)).
Of the two overlapping palindromes in the palindromic DNA segment,
palindrome 2 is the one that confers inducibility of the cutinase gene
expression by plant cutin monomers. Therefore, we sought a
transcription factor specific for palindrome 2 by screening phage
clones originally obtained during the screening of the gt11 expression library for PBP clones (5). This screening identified three
protein factors, and one of them was designated CTF1
.
Mutations in the first half of palindrome 2 that overlaps with
palindrome 1 reduced the inducibility of the promoter by over 60-fold,
and mutations in the second half of palindrome 2 abolished any
inducibility of the promoter in vivo (4). The specific binding of the expressed CTF1 to palindrome 2 indicated by the gel
shift assays suggest that the cloned protein is one of the factors
involved in the regulation of cutinase gene expression in
vivo. In further support of this conclusion is the finding that
substitution of the G residues in palindrome 2, previously found to be
involved in the binding of the naturally occurring cutinase
transcription factor, abolished the binding of the expressed CTF1
.
CTF1 was demonstrated to be capable of functioning in
vivo as a positively acting transcription factor of the cutinase
gene promoter in yeast. CTF1
fused to the nuclear localization
sequence was able to activate transcription of the cat gene
(Fig. 4B). That this transactivation involved specific
binding to palindrome 2 was strongly suggested by the observation that
mutation of palindrome 2, but not palindrome 1, abolished the
transactivation (Fig. 4B). The notion that the
Cys6Zn2 DNA-binding domain in the cutinase transcription factor is in the N terminus and the activation domain would be expected to be in the C terminus is consistent with the finding that expression of the hybrid protein containing the N-terminal 526 amino acids of CTF1
fused to the nuclear localization sequence of SV40 failed to activate the cutinase gene promoter/cat
gene (Fig. 4B). These results demonstrate that CTF1
functions as a transcriptional activator for palindrome 2 of the
cutinase gene promoter in vivo. Mutations in palindrome 1 elevated inducibility of the cutinase gene promoter by 2-fold in
F. solani f. sp. pisi in vivo (4), suggesting
that PBP may function as a repressor by competing for binding the
overlapping palindromes.
The presence of putative nuclear localization signals suggested that
CTF1 may be a nuclear protein. The N-terminal
Cys6Zn2 binuclear cluster motifs found in
CTF1
is probably involved in the binding to the palindrome. Such
DNA-binding motif is characteristic of other regulatory proteins such
as GAL4 (18), ARGRII (19), PPR1 (20), PDR1 (21), PUT3 (22), HAP1 (23),
and UGA3 (24) of Saccharomyces cerevisiae; LAC9 of
Kluyveromyces lactis (25); MAL63 of S. carlsbergensis (26); NIT4 of Neurospora crassa (27); NIRA (28), UAY (29), QUTA (30), and AMDR (31) of Aspergillus nidulans; and AFLR of A. flavus (32). However, CTF1
does not share homology to any other regions of those factors.
Functionally, GAL4 is a positive activator that regulates the
transcription of the galactose-inducible genes GAL1, GAL2, GAL7,
GAL10, and MEL1 (18). PPR1 positively regulates
transcription of the genes URA1, URA3, and URA4
involved in controlling pyrimidine levels (20). Some of these protein
factors recognize a DNA sequence with two inverted repeats of CGG
elements separated by a spacing characteristic of the specific protein
factor (33). For example, PPR1 recognizes 5
-CGG(n6)CCG
with a spacer of 6 nucleotides, whereas the spacing for GAL4, PUT3,
PPR1, and LEU3 is 11, 10, 6, and 4 nucleotides, respectively (34).
HAP1, on the other hand, binds a direct repeat of CGG triplet with a
spacer of six nucleotides (33). Interestingly, CTF1
binds to a
palindrome with an oppositely oriented 5
-GCC(n2)GGC
(4).
The presence of nuclear localization signals, sequence-specific binding
to the region of the cutinase gene that is involved in the induction of
cutinase gene by hydroxy fatty acid, and the demonstrated capability of
the cloned factor to function as a transcription factor suggests that
CTF1 is a DNA-binding transcriptional activator involved in
mediating the inducibility of the cutinase gene promoter by plant
cutin.
Phosphorylation of DNA-binding protein factors play an important role
in nuclear localization (35), DNA binding, and transactivation (36,
37). Previously, it was shown that cutin monomers caused phosphorylation of nuclear proteins (38) and when nuclear extracts from
cutin monomer-induced culture were treated with immobilized phosphatase
binding to the cutinase gene promoter was abolished (38). In addition,
protein phosphorylation was detected only in the presence of cutin
monomers. Furthermore, transcription of the cutinase gene induced by
hydroxy fatty acids in a nuclear preparation from F. solani
f. sp. pisi showed a 30-min lag period during which
phosphorylation of a 50-kDa protein occurred and inhibition of
phosphorylation by protein kinase inhibitors strongly inhibited
cutinase gene transcription induced by the hydroxy fatty acids (38). On
the basis of such results, it was suggested that protein
phosphorylation may be involved in the activation of cutinase gene
transcription by hydroxy fatty acids. In fact, CTF1 contains numerous putative consensus sites for phosphorylation by CKII, protein
kinase C, and two possible consensus sites for mitogen-activated protein kinase. Whether any of these consensus sites are phosphorylated in vivo remains to be elucidated.
Activation of the cutinase gene transcription by hydroxy fatty acids
occurs only when glucose in the culture medium is depleted (39). This
catabolite repression could involve cyclic AMP; experimental evidence
had suggested that the involvement of cyclic AMP in cyclic AMP levels
in the fungus was elevated almost 3-fold upon glucose depletion, and
exogenous cyclic AMP could derepress the glucose effects.2
CTF1 contains several potential sites for phosphorylation by protein
kinase A, although it is not known whether they are actually involved in cutinase gene activation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U51671[GenBank].
We thank Kristi Rupert for help with DNA preparations.