From the
Institut für Physiologische Chemie,
Ruhr-Universität Bochum, D-44780 Bochum, Germany, and the
Max F. Perutz Laboratories, University
Departments at the Vienna Biocenter, Department of Biochemistry and Molecular
Cell Biology, University of Vienna, and Ludwig Boltzmann-Forschungsstelle
für Biochemie, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria
Received for publication, April 18, 2003
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ABSTRACT |
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INTRODUCTION |
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In addition to containing an ORE, the promoters of several fatty acid-responsive genes also have a type 1 upstream activation sequence (UAS1) that acts as target for the Adr1p transcription factor (11). The UAS1 consensus sequence consists of CYCCR(A/T/G)N436(T/A/C)YGGRG (12). Adr1p has been identified previously as a regulator of the glucose-repressible alcohol dehydrogenase gene ADH2 (13, 14) and was additionally implicated in the growth of yeast cells on oleic acid medium (11, 1517). Adr1p was subsequently shown to regulate directly the transcription of the ORE-dependent genes CTA1, SPS19, and POX1 (15, 18, 19).
Investigations into the expression of SPS19 and POX1 have
exposed a strict adherence to regulation by both Adr1p and Pip2p-Oaf1p. The
fact that Pox1p represents peroxisomal acyl-CoA oxidase, which is the first
and the rate-limiting enzyme of the -oxidation spiral, elucidated the
reason for the requirement for Adr1p during the growth of yeast cells on fatty
acids (Ref. 19, and references
therein). The promoters of these two genes exhibit an ORE/UAS1 overlap, which
is postulated to be significant for transcriptional up-regulation. Studies on
this type of overlap in the CTA1 promoter revealed a synergy between
the oleic acid-specific Pip2p-Oaf1p transcription factor and the more general
sensor of less favored carbon sources, Adr1p
(19).
Several mechanisms ensure that Adr1p initiates transcription of its target genes only in the presence of non-fermentable carbon sources. With respect to ORE-regulated genes, chromatin immunoprecipitations revealed that Adr1p binds to the UAS1 of CTA1 and POT1/FOX3 in vivo but does so only in the absence of glucose (20). Pip2p-Oaf1p is also tightly regulated by multiple layers of control. Although glucose inhibition is a feature common to both partners of this transcription factor, activation of the constitutively expressed Oaf1p depends on fatty acids (21), whereas PIP2 transcription is thought to be primarily regulated in response to its own abundance through an autoregulatory loop based on an ORE in the promoter of the PIP2 gene (21).
A potential ORE/UAS1 overlap has since been identified in the promoter of PIP2 (19), which raises the issue of the relationship between Adr1p and Pip2p-Oaf1p with respect to bulk gene transcription in cells grown on oleic acid. Here, the role of Adr1p in regulating PIP2 was investigated, and the results are discussed in terms of a transcriptional program that culminates in the induction of genes involved in fatty acid breakdown.
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EXPERIMENTAL PROCEDURES |
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Plasmid ConstructionsDouble-stranded oligonucleotides representing the Adr1p elements in the promoter of PIP2 were ligated to the matching SalI site in the plasmid vector pBluescript® SK+ (Stratagene, La Jolla, CA), resulting in plasmid pLW82. The nucleotides of all cloned inserts were confirmed by automatic sequencing. Plasmid pHPR184 was created by exchanging the ClaI-SalI fragment of pHPR183 with that of pYIMPIP2-HA3, followed by an exchange of the NotI-demarcated hemagglutinin A epitope cassette with a similarly delineated cassette containing the sequence for 9x Myc.
Media and Growth ConditionsProduction of Adr1p-LacZ in
E. coli cells was performed as described
(22). For RNA isolations,
cultures of the wild-type strain MF14 or the otherwise isogenic mutants devoid
of Adr1p or Pip2p were shifted to YP medium (1% w/v yeast extract, 2% w/v
peptone) containing the indicated carbon sources and grown for a further 16 h
(1). For -galactosidase
measurements, cells were induced in oleic acid medium as follows. Late
exponential phase cells from overnight pre-cultures consisting of YP medium
and 5% (w/v) D-glucose were transferred to 100-ml conical flasks
with 50 ml of YP medium containing both 0.2% (w/v) oleic acid (Merck) and
0.02% (w/v) Tween 80 (Sigma-Aldrich) that were adjusted to pH 7.0 with NaOH,
0.05% D-glucose, and 75 µg/ml ampicillin to an absorbance of
A600 = 0.2. Cultures were grown at 30 °C with vigorous
aeration for the periods indicated.
For immunoblotting, BJ1991 wild-type cells and otherwise isogenic
adr1 and pip2
oaf1
mutants were
grown in liquid YP medium containing 2% D-glucose and shifted to YP
media supplemented with either 4% D-glucose, 2% (v/v) ethanol, or
pH-adjusted 0.2% oleic acid and 0.02% Tween 80. Cultures were aerated
vigorously for 8 h in the case of cells grown on glucose (to
A600 < 1.0) and for 20 h in the cases of growth on
ethanol or oleic acid. For assays using culture drops, cells were grown in
liquid rich glucose YPD medium (YP and 2% D-glucose) overnight to
late exponential phase, diluted serially, and applied to solid YPD medium
solidified with 2% (w/v) agar. For qualitative estimates of
-galactosidase expression levels on solid YPD medium, 50 µl of 4%
(w/v) 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal)
in dimethyl formamide was added to the plates. Oleic acid plates contained a
0.67% (w/v) yeast nitrogen base with amino acids added, 0.1% yeast extract,
0.5% (w/v) potassium phosphate buffer at pH 6.0, 2% agar, autoclaved with 0.5%
Tween 80 and 0.125% oleic acid. Plates were prepared by pouring a thin layer
at 55 °C. These were used to assess fatty acid breakdown by clear zone
formation.
Enzyme Assays, Protein Extract Preparation, Immunoprecipitation, and
Immunoblotting-Galactosidase activities were assayed in
soluble protein extracts prepared by breaking cells with glass beads
(26) and expressed as nanomole
of O-nitrophenyl-
-D-galactopyranoside hydrolyzed per
minute and milligram of protein. Catalase measurements were performed as
described (27). Unless stated
otherwise, values reported here are the average of three experiments ±
S.D. Whole-cell extracts were prepared according to a published protocol
(28). Preparation of soluble
protein extracts and immunoprecipitation of a Myc-tagged protein were
performed essentially as described
(29). Briefly, soluble protein
extracts obtained from yeast cells (2 g wet weight) were incubated with
Dynabeads goat anti-mouse IgG (Dynal Biotech, Oslo, Norway) coated with
anti-Myc (9E10) antibodies. Following triple washing, the beads were boiled in
50 µl of 1x SDS sample buffer. The anti-Kar2p antibody was described
previously (30). The
polyclonal rabbit antibody against Cta1p was a gift from Dr. H. F. Tabak, and
the monoclonal antibody 9E10 (c-myc) was obtained from Berkeley
Antibody, Richmond, CA. Immunoreactive complexes were visualized using
anti-rabbit or anti-mouse IgG-coupled horseradish peroxidase in combination
with the ECLTM system from Amersham Biosciences.
MiscellaneousThe following procedures were performed
according to published methods: nucleic acid manipulations, formaldehyde gel
electrophoresis, blotting and hybridization
(31), yeast transformation
(32), yeast RNA preparation
for Northern analysis (33),
determination of protein concentration
(34), and electrophoresis
(35). Transformants containing
single SPS19-lacZ integrations were verified previously by Southern
analysis (36). The use of
PIP2, POT1/FOX3, CTA1, and ACT1 as probes has been
described previously (1).
Fragments containing UAS1CTA1, UAS1PIP2, or
FOX3 ORE were isolated from plasmids pLW81, pLW82, and pSKFOX3ORE
(Table II), respectively,
following digestion with EcoRI and XhoI. DNA fragment
isolations were performed using QIAEX II (Qiagen Inc., Valencia, CA) according
to the manufacturer's instructions. The [-32P]dATP-labeled
probes were generated with a Prime-a-GeneTM random primer labeling kit
(Promega, Madison, WI). Electrophoretic mobility shift analysis (EMSA) was
carried out according to a published protocol
(1).
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RESULTS |
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Incubation of labeled UAS1CTA1 with recombinant Adr1p-LacZ formed a high molecular weight complex (Fig. 1B, lane 2) that was specific to Adr1p elements, because the addition of excessive amounts of UAS1CTA1 (Fig. 1B, lane 3) or the corresponding element in the ADH2 promoter (18), UAS1ADH2 (Fig. 1B, lane 4), caused the intensity of the signal due to this complex to diminish. The results also showed that the intensity of the Adr1p signal could be reduced following the addition of excess DNA representing UAS1PIP2 (Fig. 1B, lane 5), although this reduction was less pronounced compared with those associated with UAS1CTA1 and UAS1ADH2. On the other hand, excess PIP2 ORE, which flanks the half-site of UAS1PIP2 (Fig. 1A), did not weaken the signal's intensity (Fig. 1B, lane 6). UAS1PIP2 could also be shown to be targeted directly by Adr1p (Fig. 1B, lanes 712). The top two bands seen in Fig. 1B, lane 2 (marked with arrows and asterisks) were reasoned to not represent genuine complexes because they could be competed by a nonspecific competitor DNA that failed to reduce the signal of the predominant complex (Fig. 1B, lane 6). Hence, EMSAs performed using a recombinant Adr1p-LacZ fusion protein revealed that UAS1PIP2 was able to recruit the transcription factor in vitro.
Adr1p Influences PIP2 Transcription on Oleic AcidNorthern
analysis was performed to determine whether Adr1p actually affected
PIP2 transcription (Fig.
2A). The results demonstrated that levels of
PIP2 transcripts were higher in wild-type cells grown on oleic acid
medium compared with the situation on ethanol
(Fig. 2A; top pair
of panels), albeit the PIP2 signal was weaker compared with
those corresponding to the two peroxisomal enzyme genes,
POT1/FOX3 and CTA1. Nevertheless, the Northern blot
could expose a reduction in the transcription of PIP2 by the
adr1 mutant under oleic acid medium conditions
(Fig. 2A, top
right hand panel) even when considering the moderately unequal loading
seen using ACT1 (encoding actin), which served as a control gene that
is not affected by Adr1p (Fig.
2A, bottom pair of panels). Hence, the reduced
PIP2 signal resembled the situation with the two Adr1p-dependent
positive control genes (11,
17) seen in the middle
panels.
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Regulation of PIP2 was also investigated in response to excess
levels of Adr1p (Fig.
2B). Multiple ADR1 copies have been reported
previously to result in increased transcription of certain genes whose
promoters contain a UAS1, including ADH2, CTA1, and SPS19
(15,
18,
24). Northern analysis was
performed on immobilized RNA extracted from haploid 16xADR1 cells
containing multiple copies of the ADR1 gene
(24) as well as from isogenic
ADR1 cells containing a single wild-type ADR1 gene copy, adr1-1 cells
with an adr1-1 mutation, or ADR15C cells expressing
a partially constitutive ADR1
(23). The results showed that
PIP2 transcription was elevated in oleic acid-grown cells harboring
16 ADR1 copies compared with the other cell types
(Fig. 2B). In
addition, a reduction in transcript accumulation could also be seen in the
adr1-1 mutants (Fig.
2B), which was in agreement with the corresponding
adr1 lane in Fig.
2A. Signal intensity notwithstanding, the RNA profile of
PIP2 under these oleic acid-medium conditions was essentially
identical to that of POT1/FOX3 and CTA1
(16,
17). Hence, PIP2
transcription appeared to rely on Adr1p.
To confirm the dependence of PIP2 on Adr1p, transcription was
studied using a PIP2-lacZ reporter construct
(1). Levels of
-galactosidase activity were analyzed at various time points following
the shifting of cells from low glucose- to oleic acid-containing media. The
results demonstrated that PIP2-lacZ expression in the
adr1
strain reached
50% of the wild-type level at all
time points measured (Table
III). Only minimal
-galactosidase activity was detected in
the corresponding pip2
mutant.
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The influence of Adr1p on the transcription of PIP2 was also
investigated under non-inducing conditions using the afore-mentioned
lacZ reporter gene
(1). Serial 10-fold dilutions
of wild-type and adr1 cells harboring the PIP2-lacZ
reporter gene were applied to YPD plates. The chromogenic substrate X-Gal was
added to the surface of the medium to assess the expression of the reporter
construct. As expected, Adr1p was not critical for the onset of growth on
rich-glucose medium, as verified by comparing colony sizes
(Fig. 3). The results
demonstrated that expression of
-galactosidase was reduced in the mutant
strain because, unlike the wild-type strain, which turned light blue, mutant
colonies remained white (Fig.
3). This experiment also revealed the importance of Adr1p for
PIP2 transcription in the absence of a fatty acid substrate,
i.e. in cells sensing the depletion of glucose.
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Adr1p Is Critical for Pip2p Expression on Oleic AcidTo
further substantiate the apparent requirement for Adr1p to express Pip2p,
protein levels were monitored using a plasmidborne gene that encodes a
Myc-tagged Pip2p expressed from the native promoter. This tag did not
interfere with the function of Pip2p, because Pip2p-Myc was as efficient as
the untagged protein in complementing the growth phenotype of a
pip2 strain (not shown). Immunoprecipitation was performed on
soluble protein extracts from wild-type, adr1
, or
pip2
oaf1
cells harboring this centromeric
plasmid. The results demonstrated that, under oleic acid-medium conditions,
Adr1p was important for obtaining wild-type levels of Pip2p
(Fig. 4, top panel).
The pattern of Cta1p signals (Fig.
4, middle panel) concurred with those of previous
experiments using immunoblotting as well as catalase assays, which
demonstrated a higher level of dependence on Pip2p-Oaf1p than on Adr1p for
expression under these conditions
(19). Kar2p served as an
internal loading control (Fig.
4, bottom lane), whereas the control lane on the far left
of Fig. 4 consisted of an
extract from a wild-type strain expressing an untagged Pip2p.
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Pip2p-Oaf1p Complexes Formed on OREs Are Less Abundant in Cells Devoid
of Adr1pThe above data indicated that, in the absence of Adr1p,
Pip2p expression was compromised. It followed, therefore, that the amount of
Pip2p-Oaf1p binding to target OREs might also be affected in the
adr1 mutant. Hence, EMSA was performed on an ORE obtained from
the promoter of the POT1/FOX3 gene, which, unlike the
corresponding element in the PIP2 promoter, does not overlap its
neighboring UAS1 (Fig.
5A). Formation of the Pip2p-Oaf1p complex was monitored
using soluble protein extracts derived from BJ1991 wild-type,
adr1
, or pip2
oaf1
strains that
were incubated with the labeled ORE fragment.
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The results demonstrated that the signal due to the Pip2p-Oaf1p complex was
appropriately missing from the lane containing the control
pip2oaf1
extract
(Fig. 5B, lane
4). In addition, this signal, using extracts obtained from oleic
acid-grown cells lacking Adr1p (lane 3), appeared to be less intense
compared with that using wild-type cells extracts (lane 2). This
situation seemed to be exacerbated using cells grown to a late exponential
phase on glucose or ethanol (Fig.
5C, lanes 3 and 6). Hence, in
accordance with abnormally low levels of Pip2p in oleic acid-grown
adr1
cells (Fig.
4), the amount of transcription factor that could bind OREs in
vitro was appropriately reduced.
Uncoupling PIP2 Transcription from Adr1p Control Partially Suppresses
the adr1 Mutant Phenotype on Oleic Acid
Overexpression in wild-type cells of just Pip2p (without also overproducing
Oaf1p) does not lead to an increase in ORE-dependent gene transcription
(8,
21). To determine the effect
of uncoupling PIP2 transcription from the control of Adr1p on the
expression of SPS19 and CTA1, PIP2 was placed behind the
constitutive TPI1 promoter in plasmid pTPI1-PIP2HA3
(Table II) that was used to
transform the adr1
or pip2
mutant cells that
additionally harbored an SPS19-LacZ reporter gene.
The result of propagating cells on oleic acid medium for 18 h demonstrated
that SPS19-lacZ reporter gene activities were 2.5-fold greater in
adr1 mutant cells ectopically expressing Pip2p compared with
those producing Pip2p exclusively from the native locus
(Table IV). This effect was
also manifested on catalase activity, which had increased 2-fold in the former
cell type. Complementation with the TPI1-driven PIP2 was
also monitored in a pip2
mutant, and, as anticipated, this
demonstrated a 28-fold increase in SPS19-lacZ expression as well as a
3.6-fold increase in catalase activity
(Table IV). Hence, it was
reasoned that levels of SPS19-lacZ and CTA1 expression were
elevated in the adr1
mutant because TPI1-PIP2 gave
rise to sufficient amounts of Pip2p. However, due to Adr1p also acting
directly at the SPS19 and CTA1 promoters, the observed
effects were only partial.
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As mentioned previously, cells devoid of Adr1p fail to degrade fatty acids
(15) because they do not
express Pox1p, which is necessary for -oxidation to occur
(19). To examine whether
ectopic expression of Pip2p could also re-establish fatty acid breakdown in
adr1
cells, the formation of clear zones was monitored on
solid oleic acid medium. This medium also contained Tween 80, which acted to
disperse the fatty acids but was also a poor carbon source, and, therefore,
mutant cells could grow to some extent on such plates, but the transparent
zones in the opaque medium around regions of cell growth indicated utilization
of the fatty acid substrate
(39). Three independent
adr1
mutant strains expressing a functional Pip2p from a
pTPI1-PIPHA3 construct were compared with a mutant harboring the YCplac22
plasmid vector for formation of clear zones. As seen in
Fig. 6A, all three
transformants overexpressing Pip2p could give rise to narrow zones of clearing
that were absent from the region surrounding the vector-containing
adr1
strain. Expression of the construct in the
pip2
mutant gave rise to more pronounced clear zones in the
medium, as could be expected from self-complementation
(Fig. 6B). Hence,
restoration of Pip2p levels in the adr1
mutant using the
TPI1-driven construct probably caused POX1 and possibly
other Adr1p-regulated genes to be expressed in amounts sufficient for moderate
levels of fatty acid degradation.
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DISCUSSION |
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The present data allowed us to conclude that Adr1p-dependent control over Pip2p affects ORE-dependent gene regulation. EMSA performed on cells lacking Adr1p demonstrated that such cells gave rise to a reduced Pip2p-Oaf1p signal. This indicated that Adr1p influenced the amount of the transcription factor being expressed, or alternatively, it modulated the factor's binding activity, e.g. by loss of synergy. Because the fragment used in these EMSAs consisted of an ORE in isolation, and immunoprecipitation revealed that less Pip2p was produced in cells devoid of Adr1p, we argue that the reason for the importance of Adr1p lies most probably in maintaining normal levels of Pip2p and not in increasing the binding efficiency of the transcription factor that subsequently forms with Oaf1p.
Adr1p-independent expression of PIP2 in adr1 cells
was sufficient to partially release these mutants from their oleic acid
induction deficiency. Analysis of the expression of two genes encoding
peroxisomal enzymes, Cta1p and Sps19p, showed that, as a result of the
uncoupling of PIP2 from Adr1p control, their respective levels
increased 2-fold, reaching between about a third (Sps19p-LacZ) to half (Cta1p)
of the potential of the wild-type expression levels, as measured by the
self-complemented BJ1991pip2
strain. Moreover, the capability
of this strain to degrade fatty acids improved significantly. Hence, it was
reasoned that the expression of additional ORE-regulated genes that are
critical for
-oxidation, e.g. POX1, was also likely to be
positively affected by such uncoupling. The extent of the transcriptional
cascade operating through Adr1p and Pip2p-Oaf1p is very significant for
metabolism because, by regulating Pip2p-Oaf1p, Adr1p could influence at least
24 additional genes involved in diverse functions, including those encoding
peroxisomal proteins but also a mitochondrial protein as well as proteins of
unknown function (2,
40,
41).
Cells shifted directly from fresh, rich glucose medium to that supplemented
with fatty acids as the sole carbon source without first undergoing
derepression fail to achieve the metabolic switch in a timely manner and do
not undergo efficient oleic acid induction. However, the molecular mechanism
ensuring the integrity of this link between the two phases of derepression and
induction has remained hitherto unclear. Adr1p might turn out to be important
for maintaining normal levels of Pip2p in non-induced cells in which
Pip2p-Oaf1p is transcriptionally inactive. For example, a PIP2-lacZ
reporter gene failed to turn blue in cells devoid of Adr1p that were grown on
glucose plates containing X-gal. In addition, adr1 mutants
grown to late exponential phase on glucose or ethanol media gave rise to
weaker signals representing Pip2p-Oaf1p binding to OREs than did wild-type
cells. Although non-induced wild-type cells contain only trace amounts of
Pip2p, it is attractive to speculate that these quantities nevertheless
support a faster response in derepressed cells whose growth medium was
supplemented with oleic acid.
There exists some evidence that Adr1p is additionally dependent on a signal emerging from the mitochondria. A temperature-sensitive mutation in the RML2 gene encoding a mitochondrial ribosomal protein disables the induction of peroxisomal catalase (42). Interestingly, this deficiency could be alleviated by overexpressing Adr1p (42). However, the fact that transcription of Adr1p-dependent POX1 and POT1/FOX3 was not affected by this mutation argued against a communication pathway appearing to course from the mitochondria to the peroxisomes via the nucleus. Nevertheless, such a retrograde pathway is clearly at work in respiratory-deficient [rho0] cells in which several ORE-regulated genes are induced despite the absence of fatty acids from the growth medium (43). A subsequent microarray-based study using cells devoid of Adr1p underscored the fact that, of the 43 genes that are responsive to changes in mitochondrial function, no less than 17 are regulated by Adr1p (44). Hence, it would be interesting to determine the extent of the sharing of transcription factors between the metabolic events triggered by oleic acid and respiratory deficiency. It is worth noting that, in such Adr1p-less cells, at least two ORE-regulated genes, DCI1 (2, 45, 46) and IDP3 (47, 48), are down-regulated (44) despite lacking obvious UAS1s in their promoters (18). It is not entirely impossible that this is due to a regulatory circuitry based on Adr1p controlling Pip2p abundance at their OREs.
In addition to acting on PIP2, Adr1p also regulates a number of ORE-regulated genes directly by binding to UAS1 elements that are situated in the vicinity of OREs. Experimental evidence for such a dual control exercised by Pip2p-Oaf1p and Adr1p was demonstrated previously for the promoters of CTA1, SPS19, POX1, and POT1/FOX3 (15, 18, 19, 20). This type of combinatorial control seems to be a more general feature of Adr1p, as it also acts in concert with the transcription factor Cat8p at promoters of genes encoding gluconeogenic and glyoxylate-cycle enzymes so as to allow their expression in glucose-exhausted media (4951). Our demonstration of PIP2 being regulated by Adr1p extends this concept of combinatorial control in that Adr1p acts not only together with Pip2p-Oaf1p under fatty acid medium conditions but also in the step preceding the oleic acid induction cascade.
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FOOTNOTES |
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¶ Current address: Division of Biochemistry and Molecular Biology, Brain
Research Inst., University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.
Tel.: 43-1-4277-629-53; Fax: 43-1-4277-629-60.
We dedicate this work to the memory of our co-author, Professor H. Ruis,
who died September 1, 2001.
|| To whom correspondence should be addressed. Tel. 43-1-4277-52815; Fax: 43-1-4277-9528; E-mail: AG{at}abc.univie.ac.at.
1 The abbreviations used are: ORE, oleate response element; UAS1, upstream
activation sequence type 1; X-Gal,
5-bromo-4-chloro-3-indolyl--D-galactopyranoside; EMSA,
electrophoretic mobility shift analysis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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