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(Received for publication,
December 8, 1994; and in revised form,
March 3, 1995 ) From the
We have identified in the promoter of the FBP1 gene
from Saccharomyces cerevisiae, which codes for
fructose-1,6-bisphosphatase, two elements which can form specific
DNA
The yeast Saccharomyces cerevisiae is able to utilize a
variety of carbon sources, but the preferred one, at least in
laboratory conditions, is glucose. Glucose produces a variety of
physiological effects in S. cerevisiae(1) , among them
a strong repression of many enzymes not required for growth on glucose.
The glucose repression system in yeast (for reviews, see Refs.
2-4) is quite complex, and its mechanism is far from understood.
It operates mainly at the level of gene transcription, although effects
on mRNA stability have been also
reported
(5, 6, 7, 8) . Studies with
different mutants have clearly established that not all the genes
affected by catabolite repression are controlled by the same set of
regulatory proteins but that there are different circuits of
repression. Therefore, to understand the mechanisms of catabolite
repression in yeast, a variety of systems ought to be studied.
Up to
now, most studies have been directed to the elucidation of the control
of the genes necessary for the utilization of sugars like galactose or
sucrose. It has been found that expression of the GAL genes is
dependent on the transcriptional activator Gal4 (9). Glucose represses
the GAL4 gene through the action of Mig1
(10, 11) and also facilitates the binding of Mig1 to upstream
repressing sequences in the promoters of the GAL genes
(11-13). In the case of the SUC2 gene, Mig1 is also able
to bind to the promoter and to inhibit transcription
(14) , but a
transcriptional activator binding to the upstream activating sequences
(UAS)
While different mutations, including
mig1, have been identified which relieve the SUC2 and
GAL genes from catabolite repression, none of them allows the
expression, in the presence of glucose, of gluconeogenic genes such as
FBP1, PCK1, and ICL1, coding for
fructose-1,6-bisphosphatase (Fru-1,6-P
In the present
study, we have undertaken a detailed analysis of different regions of
the FBP1 promoter. We have investigated the behavior of these
regions both with respect to their ability to act as UAS in the
CYC1-lacZ reporter system
(18) and to their capacity to
form specific complexes with nuclear proteins.
We conclude that the
FBP1 promoter contains at least two activating regions
regulated by glucose; one of them appears to require the binding of
proteins at two different sites to fully activate transcription.
Figure 1:
Structure
of the FBP1 promoter and of the oligonucleotides OL1 and OL2.
The approximated position of the DNA
Figure 4:
DNase I protection analysis of the
DNA
Since an activating sequence appeared to be located between
positions -527 and -480 of the FBP1 promoter
(17) , we examined the effect of its deletion on a
fusion gene FBP1-lacZ. To avoid possible artifacts due to
variations in the number of copies of the gene, we placed the
constructions in a centromeric plasmid. We found that the level of
The -527 to -480 sequence
could have activating capacity by itself or reinforce the action of a
downstream activator. To evaluate the capacity of this and other
regions of the FBP1 promoter to activate transcription,
fragments of the promoter (Fig. 1) were inserted into a UAS-less
reporter plasmid
(18) , and the capacity of the plasmids to
direct the synthesis of
As Fru-1,6-P
The fragment -317 to -176
of the FBP1 promoter was also inserted in the reporter plasmid
and did not show UAS activity. Such an activity could have been masked
by the negative regulatory protein Mig1 binding to a site around
position -200
(16) . However, we found that even in a
mig1 background, the fusion gene was not expressed (data not
shown).
We had observed that a fusion gene FBP1-lacZ required part of the coding sequence of FBP1 to be
expressed, the first 57 base pairs being sufficient
(15) . This
suggested the existence of a downstream activating sequence and
therefore we tested the fragment -53 to +57 for UAS
activity. No expression of
To
look for a possible correlation between the effect of different
fragments of the FBP1 promoter on transcription and their
capacity to bind specific proteins, we performed band-shift assays. The
DNA segment from -527 to -476 yielded a single major
retarded band (E) as shown in Fig. 2a. This is
in contrast with the results of Schöler and Schüller
(30) who observed 4 specific DNA
Figure 2:
Gel mobility shift analysis with fragments
-527/-476 (a) and -479/-313
(b) from the FBP1 promotor. The DNA fragment was
labeled and incubated as described under ``Experimental
Procedures.'' Samples in lanes 1 were incubated without
added protein, and samples in lanes 2 and 3 were
incubated with the amount of nuclear protein indicated. The protein
extract was from derepressed cells (D), from repressed cells
(R), or from a derepressed cat1 mutant strain
(Dcat1).
In the case of the fragment -316 to
-176, two complexes were observed which were in fact two
doublets. They did not depend on the state of repression or
derepression of the cells (Fig. 3). All of them are designated
collectively as complex A and were not detectable when a nuclear
extract from repressed cells of a mig1 mutant was used. In the
absence of Mig1, a faint band remained which was much stronger when
derepressed cells from a mig1 mutant were used. Competition
assays indicated that this new complex, C, is specific (results not
shown). In a cat1 background, the two doublets corresponding
to complex A showed changes in mobility, while in a cat1/mig1 double mutant, complex C was barely visible.
Figure 3:
Gel mobility shift analysis with the
fragment -316/-176 from the FBP1 promotor. General
experimental conditions were as described in Fig. 2. Samples in
lanes 1 were incubated without added protein, and samples in
lanes 2-4 were incubated with the amount of nuclear
protein indicated. The protein extract was from derepressed cells
(D), from repressed cells (R), from a repressed
mig1 mutant strain (Rmig1), from a derepressed
cat1 mutant strain (Dcat1), from a derepressed
cat1/mig1 double mutant strain (Dcat1 mig1), or from
a derepressed mig1 mutant strain
(Dmig1).
When the fragments
of the FBP1 promoter -179 to -73 and -76 to
+57 were used in electrophoretic mobility shift assays, no clear
specific band shifts were observed.
To identify the DNA sequences
where nuclear proteins bind specifically, footprinting experiments were
carried out. For the -527 to -476 fragment, indirect
footprinting was performed in both the coding and noncoding strands
with the results shown in Fig. 4a. The different
footprints on the two strands of the promoter indicated that the
protein(s) in the complex E bound asymmetrically to the DNA.
For the
-479 to -313 fragment, indirect footprinting was attempted
with both B and D complexes. For B, the results for both strands are
shown in Fig. 4b. They show that protection from DNase I
cleavage is completely asymmetric, from -431 to -424 in the
coding strand and from -422 to -417 in the noncoding
strand. In the case of the D complex, no protection could be observed
in our experimental conditions. In an attempt to define more precisely
the region of the promoter where this complex could be formed, a band
shift was performed with a DNA fragment from -480 to -413.
As shown in Fig. 5b, this fragment was sufficient for
the formation of both the B and D complexes. An oligonucleotide
covering the region -432 to -415 acted as a competitor for
the formation of the B complex, but did not affect the formation of the
D complex, which therefore is likely to be in the region between
-480 and -432.
Figure 5:
Gel
mobility shift analysis with the oligonucleotide OL1 (a), the
fragment -480/-413 from the FBP1 promotor
(b), and the oligonucleotide OL2 (c). General
experimental conditions were as described in Fig. 2. Samples in
lanes 1 were incubated without added protein, and samples in
lanes 2-6 were incubated with nuclear protein from
derepressed cells (a, 32 µg; b, 8 µg;
c, 24 µg). Competition experiments were performed with a
50-fold or 100-fold excess of the unlabeled fragment used as probe (*)
or of a different unlabeled fragment (OL1 or
OL2).
Using extracts from a mig1 mutant, we could localize the DNA region where complex C is formed
by indirect footprinting. A clear footprint was observed at positions
-239 to -232 on the noncoding strand
(Fig. 4c). Although this sequence is very similar to the
consensus sequence for the binding of the protein complex
Hap2-Hap3-Hap4, ACCAAYNA
(31) , we could still observe complex C
when extracts from a double mutant mig1/hap2 were used
(results not shown).
The results from the footprinting experiments,
together with those of sequential deletions
(15, 17) ,
suggested that the regions -507 to -489 and -432 to
-415 of the FBP1 promoter contained upstream activating
sequences. We therefore tested the synthetic oligonucleotides OL1 and
OL2 (Fig. 1) which include these sequences, both for their
capacity to activate transcription and to bind proteins in a specific
manner.
As shown in Table III, OL1 inserted in a UAS-less
fusion gene activated transcription to the same degree as the fragment
-527 to -476 (see Table II), and this activation
occurred only in the absence of glucose. It appears, therefore, that
the sequences within the larger fragment responsible for the regulated
activation of transcription are all included in the oligonucleotide. In
contrast, the oligonucleotide OL2 activated transcription only weakly,
and the effect of glucose on transcription was also weak, only about
2.5-fold repression. This suggests that other regions outside the
-432 to -415 sequence are required for proper control of
transcription. We therefore tested the fragments -450 to
-413 and -480 to -408. The first one was only
slightly more active than the oligonucleotide while the larger fragment
acted as a strong UAS. In all cases, the activation was observed only
in the absence of glucose. We checked also the fragment -480 to
-448 and found that, by itself, it did not stimulate
transcription at all.
In band-shift assays, OL1 was able to bind
nuclear proteins to form complex E. The binding was specific as shown
in Fig. 5a, and the complex was also completely
dependent on a functional CAT1 gene (Fig. 6a).
In the case of OL2, two strong complexes were formed, but only one of
them was specific and likely corresponded to complex B
(Fig. 5c). The nonspecific complex was independent of
CAT1, while the specific one was only formed in a CAT1 background (Fig. 6b).
Figure 6:
Gel mobility shift analysis with the
oligonucleotides OL1 (a) and OL2 (b). General
experimental conditions were as described in Fig. 2. Samples in
lanes 1 were incubated without added protein, and samples in
lanes 2-4 were incubated with the amount of nuclear
protein indicated. The protein extract was from wild-type yeast
derepressed cells (WT) or from a derepressed cat1 mutant strain (cat1).
The FBP1 promoter appears to be composed of a
variety of elements. An element located between positions -507
and -489 can act as UAS, and our footprint experiments situate
the sequence involved in the protein-DNA interaction between positions
-506 and -492 (Fig. 1). Very similar sequences are
found in the promoters for FBP1, ICL1, and PCK1 (Fig. 7); they include the carbon source-responsive element,
CSRE, previously described by Schöler and Schüller
(30) but also three additional bases, TTC, common to the three
promoters. Since it has been reported
(30) that the sequence
from -506 to -493 works as a strong UAS while the sequence
from -503 to -488 is inactive, it can be concluded that the
G at position -504 cannot be replaced by a T, while the T at
position -492 is dispensable. Therefore, we suggest that the
consensus sequence for an activating element in gluconeogenic genes
should include two additional bases as shown in Fig. 7. This
sequence has no homology with consensus sequences for known activating
factors. We have tried to identify a protein able to bind to this
sequence by screening a
Figure 7:
Comparison of sequences from the promoters
of genes encoding gluconeogenic enzymes. Sequences from the promoters
of FBP1 (36), ICL1 (30), and PCK1 (15) are
shown. Bases which differ from the FBP1 sequence are written
in lowercase. The carbon source-responsive element, CSRE (30),
is shown in brackets. R stands for A or G, W for A or T, and Y
for C or T.
A
second activating element is located in the region -480 to
-408 of the FBP1 promoter. This element can form two
complexes, B and D, and acts as a strong UAS completely repressed by
glucose. It contains the sequence -432 to -415, sufficient
to form complex B, but which acts only as a weak UAS. This sequence
presents some similarities with those recognized by Mig1
(33) ,
Reb1 (=Grf2)
(34) , and Rap1 (=Grf1) (35). However,
it has been observed previously that the protein or proteins binding
this sequence and called Dap1 could not be Rap1 or Mig1
(17) . As
for Reb1, it is a large protein (128 kDa) which would form a
DNA
We have found
that neither complex E nor complex B is formed when nuclear extracts
from repressed cells are used in a band-shift assay, in agreement with
observations from other groups
(17, 30) . When the
extract is from cells with an interrupted CAT1 gene, E is not
formed and B is either not formed or has an altered mobility, depending
on the DNA probe used ( Fig. 2and Fig. 6). All these
results are consistent with the in vivo situation where the
sequences containing the -507 to -489 or -432 to
-415 elements act as an UAS only in derepressed cells with an
intact CAT1 gene. Hap2 which is required for maximal
derepression of Fru-1,6-P
The identification in the region -243 to -228
of a complex C, which is formed only when extracts from derepressed
cells are used, suggests the presence of a potential UAS. While the
large region -317 to -176 has no UAS activity in a reporter
plasmid, when tested as a potential upstream repressing sequence, it
represses much less in the absence of glucose than in its
presence
(21) . A possible interpretation of these results is
that complex C facilitates transcription in the absence of glucose
through a modification of the chromatin structure.
Up to now, the
only protein able to bind to the FBP1 promoter which has been
characterized is the repressing protein Mig1. We have found that the
mobility of the Mig1
Although in the gluconeogenic
gene ICL1 all the stimulatory influence of the promoter
appears to act via a single upstream element (30), a variety of
regulatory elements are present in the FBP1 promoter. These
elements, however, seem to play similar roles, since they are affected
in the same way by the presence of glucose and by the Cat1 protein. It
cannot be yet ascertained whether they are truly redundant or serve for
a fine tuning of Fru-1,6-P
The plasmids were
constructed as described under ``Experimental Procedures''
and used to transform strains W303-1A (WT), H366
(
The plasmids
were constructed as described under ``Experimental
Procedures'' and used to transform strain W303-1A. The sequence of
the oligonucleotides OL1 and OL2 is shown in Fig. 1.
Volume 270,
Number 21,
Issue of May 26, 1995 pp. 12832-12838
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
protein complexes and which confer glucose-repressed
expression to an heterologous reporter gene. Complex formation and
activation of transcription by either element require a functional
CAT1 gene and are not blocked by a hap2-1 mutation,
although this mutation interferes with maximal expression of the
FBP1 gene. A sequence from one of the elements acts as a weak
upstream activating sequence, but its activity can be stimulated up to
10-fold by neighboring sequences. A further element of the promoter has
been characterized, which forms a specific DNAprotein complex
only when a nuclear extract from derepressed cells is used. This
element does not activate transcription in a heterologous promoter. The
DNA sequences of the three elements involved in protein binding,
defined by DNase I footprinting, have no homology with consensus
sequences for known activating factors.
()
of the promoter has not been yet
identified
(4) . 
ase),
phosphoenolpyruvate carboxykinase, and isocitrate lyase, respectively.
Since this indicates that the mechanisms of regulation of these genes
have characteristics different from those of the SUC2 and
GAL genes, we have undertaken a study to define the
cis- and trans-acting control elements of FBP1 and PCK1. Deletion analysis of the FBP1-lacZ and
PCK1-lacZ fusion genes showed a complex picture of both
promoters, with regions of apparent functional redundancy
(15) .
We have also identified in the FBP1 promoter two sites able to
bind nuclear proteins
(16) . The capacity of the region
-431/-420 to bind proteins was subsequently confirmed,
using a synthetic oligonucleotide
(17) . In this later work, it
was also shown that a region further upstream, at -506/-477
was required for maximal activation of transcription.
Yeast Strains and Media
Strains of S.
cerevisiae used in this work are listed in Table I.
Escherichia coli TG1 was used for plasmid manipulations. For
the preparation of nuclear extracts, the yeasts were grown on 1% yeast
extract, 1% peptone, and 2% glucose. Repressed cells were collected
when the culture reached 2-4 mg of wet yeast/ml. Derepressed
cells were prepared by overnight incubation of repressed cells in 1%
yeast extract, 1% peptone, and 2% ethanol at a cell density of 20
mg/ml. Yeasts carrying plasmids were grown on Difco yeast nitrogen base
supplemented with 2% glucose. Repressed cells were obtained by
harvesting them in the exponential phase of growth (below 2.5 mg of wet
yeast/ml). Derepressed cells were prepared as described above. Oligonucleotides
Oligonucleotides were purchased
from MedProbe A.S. (Oslo, Norway) or synthesized in house. To obtain
synthetic double-stranded DNA fragments (shown in Fig. 1),
single-stranded oligonucleotides of reverse complementary sequence were
mixed in equimolar amounts.
protein complexes observed in
the FBP1 promoter in vitro (A, B,
C, D, and E) is shown. Oligonucleotides: the
sequence of the FBP1 promoter is written in
uppercase; the additional SalI protruding ends are
written in lowercase; protected bases in the corresponding
footprint of the FBP1 promoter are shown in
brackets.
Recombinant DNA Methods
Cloning procedures and
additional recombinant DNA techniques were performed according to
established protocols
(19) . Plasmid Constructions
Plasmid pJJ11b was obtained
from plasmid pJJ11
(16) by substituting the fragment containing
the FBP1 promoter until the position -480 by a fragment
from pRG6
(20) containing the FBP1 promoter until the
position -530. To obtain the plasmid pLG669-ZX, a
XhoI-XhoI 0.4-kilobase fragment containing the two
UAS of the CYC1 gene promoter was removed from plasmid
pLG669-Z
(18) . To insert different fragments of the FBP1 promoter into the unique XhoI site of pLG669-ZX, the
auxiliary plasmid pOV10, which has a polylinker with a variety of
unique sites between a XhoI and a SalI site, was
used
(21) . Sequences of the FBP1 promoter were taken
from plasmid pRG6 and subcloned into pOV10. Restriction ends were
blunt-ended in the case where compatible sites were absent from the
polylinker. The 5`-phosphorylated double-stranded oligonucleotides had
protruding ends which allowed direct insertion in the XhoI
site of pLG669-ZX. Orientation and copy number of the oligonucleotide
inserts were determined by DNA sequencing. Transformation
Bacterial competent cells were
prepared, stored, and transformed by standard techniques
(22) .
Yeast transformations were performed according to Ito et
al.
(23) .
Yeast extracts were
prepared by shaking with glass beads as described
(24) omitting
the centrifugation step, and the enzyme was assayed as in Ref. 25,
samples being centrifuged before reading. Protein was determined by the
method of Lowry et al.
(26) using the BCA Protein Assay
Reagent (Pierce). 
-Galactosidase AssayPreparation of Yeast Nuclear Extracts
Nuclear
extracts were obtained as described in Schneider et al. (27)
using repressed or derepressed cells. Band-shift Assays
Protein-nucleic acid complexes
were allowed to form in 10 mM Tris-HCl, pH 7.5, 5 mM
MgCl, 2 mM EDTA, 10 mM 2-mercaptoethanol,
1 mM phenylmethylsulfonyl fluoride, 50 mM NaCl, and
12.5% glycerol (v/v). In a volume of 20 µl, nuclear extract
(4-30 µg of protein) was preincubated with 1 µg of
poly(dI-dC) for 15 min in ice. The DNA probe, labeled with the Klenow
fragment of DNA polymerase I
(19) , was then added (1 ng,
approximately 40,000 cpm), and incubation was continued for 30 min at
room temperature. Electrophoresis was performed in 4% polyacrylamide
gels in Tris borate, pH 7.6
(19) , at 12.5 V/cm for 90 min to 4 h
at 4 °C. Indirect DNase I Footprint Assay
Six parallel
samples were prepared, as described above, using 2 ng (80,000 cpm) of
DNA probe, 1 µg of poly(dI-dC), and protein at the concentration
indicated in the legend for Fig. 4, in a final volume of 40
µl. After the 30-min incubation, 1 µl of 200 mM
MgCl, 100 mM CaCl, and 1 µl of
DNase I (10 to 100 ng) were added to each sample, which was incubated
for 60 s at room temperature. Digestion was stopped in ice by the
addition of 1/10 volume of 0.5 M EDTA, and samples were loaded
onto a 4% polyacrylamide gel and electrophoresed as above. The
positions of free and protein-bound DNA were identified by
autoradiography, the corresponding fragments were excised, the
equivalent fragments of the six samples were pooled together, and DNA
was isolated by electrophoresis in a gel of agarose and binding to a
DEAE-membrane
(28) . After elution, the DNA was precipitated with
ethanol, denatured, and applied to an 8% polyacrylamide sequencing gel.
protein complexes E (a, fragment
-527/-476), B and D (b, fragment
-479/-313), and C (c, fragment
-316/-176). The T+C and A+G lanes correspond to the Maxam-Gilbert sequencing ladder. Free DNA
(a, lanes 1 and 3; b, lanes 2 and 5; c, lane 1) and DNAprotein
complexes (a, lanes 2 and 4; b,
lanes 1 and 4 (complex D) and lanes 3 and
6 (complex B); c, lane 2) were separated in
a nondenaturing 4% polyacrylamide gel after incubation with a nuclear
protein extract from wild-type yeast derepressed cells (a, 1
mg/ml; b, 0.4 mg/ml) or from mig1 derepressed cells
(c, 0.8 mg/ml) and DNase treatment. Protected bases are
marked; hypersensitive bases are shown with a +
sign.
-galactosidase was 2.5-fold higher in a yeast carrying the fusion
gene pJJ11b, containing the promoter up to position -527, than in
a yeast carrying plasmid pJJ11
(16) , which contains a promoter
starting at position -480. In both cases, expression occurred
only in derepressed cells. -galactosidase was tested (Table II).
Both fragments -527 to -475 and -480 to -312
were able to activate transcription and this only in the absence of
glucose. The orientation of the inserted DNA had a moderate effect on
expression for the smaller fragment, but a very strong one for the
larger fragment. This could indicate an influence of the distance
between the UAS and the TATA box on the rate of transcription; an
alternative possibility would be the presence of a repressing sequence
which would be more effective when placed between the UAS and the TATA
box. ase is not expressed in a cat1/snf1 mutant
(29) , and levels of Fru-1,6-Pase are
decreased in hap2 and hap4 mutants,()
the effects of cat1 and hap2 mutations in
the expression of the fusion genes were tested. No expression at all
was seen in cat1 mutants while in hap2 mutants there
was no decrease in -galactosidase levels, but rather an increase
of about 50% (Table II). -galactosidase was observed, showing
that this fragment could not activate transcription by itself. protein complexes in their
band-shift experiments. This could be due to the fact that they used
whole yeast extracts while we use nuclear proteins. The gel retardation
pattern observed by Niederacher et al.(17) using an
oligonucleotide which covers the same region comprises also a single
band of low mobility in addition to some blurred bands which do not
seem specific. The DNAprotein complex E was observed only when
nuclear extracts from derepressed cells were used for the band shift,
it was also absent when the nuclear proteins used were from a cat1 mutant. In competition experiments, a 50-fold molar excess of the
unlabeled DNA fragment prevented the formation of the complex, while
the same amount of the fragment -479 to -313 had no effect
(results not shown). The DNA fragment from -479 to -313
gave two strong specific retardation bands, B and D,
as shown in Fig. 2b. Complex B was observed only when a
nuclear extract from derepressed cells was used, while complex D was
also formed with the protein extracted from repressed cells. Both
complexes were specific, competition was observed with an excess of the
corresponding unlabeled DNA, while the DNA from -527 to
-476 did not affect the formation of the complexes (not shown).
When extracts from a cat1 mutant were used, complex D
decreased or almost disappeared while complex B appeared to change its
mobility (Fig. 2b). A weaker specific complex migrating
between B and D was observed only when extracts from a derepressed,
wild-type yeast were used.
gt11 expression library with a labeled DNA
probe (results not shown). Such an experiment has been unsuccessful, as
well as our attempt to perform a Southwestern blot
(32) .
protein complex with a lower mobility than the B complex. As
in the case of complex E, neither a Southwestern probe nor a screen of
a gt11 expression library with a labeled DNA probe allowed the
identification of a protein taking part in complex B (results not
shown). When band-shift experiments are performed with the
oligonucleotide OL2, two protein-DNA complexes are formed, but only one
of them is specific (Fig. 5c). As the nonspecific
complex is not detected with the larger fragment -480 to
-413, it would seem that sequences adjacent to the region
-432 to -415 are not required for the formation of complex
B but play a role in avoiding the formation of nonspecific complexes.
In addition, the neighboring sequences could allow the formation of
complex D which may act as a coactivator. We have examined whether the
sequence -450 to -413, which includes a potential site for
the activator of transcription Yap1 (15), was a stronger UAS than the
shorter -432 to -415 sequence (Table III). The
difference is small, thus indicating that nucleotides upstream of
-450 are required to activate transcription. ase is not necessary
for the activity of the two upstream elements of the FBP1 promoter. In fact, their activity was slightly increased in a
hap2 background, but the significance of this observation is
not clear. DNA complexes changes when extracts from a
cat1 mutant are used in the band-shift assay but is the same
for extracts from repressed or derepressed cells. This observation
could indicate that, in the absence of Cat1, Mig1 exists in a modified
form but that in wild-type cells the same form of Mig1 is found,
whether glucose is present or not. ase expression.
Table:
Regulation of the expression of fusion genes
containing fragments of the FBP1 promoter
cat1) and W303 hap2 (hap2-1).
Table:
Effect of the insertion of different fragments
of the FBP1 promoter into a UAS CYC1-lacZ plasmid
)ase,
fructose-1,6-bisphosphatase. )
We thank L. Guarente for plasmid pLG669-Z, H. Ronne
for yeast strains H190, H366, and H368, and P. Santisteban and M. J.
Mazón for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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