Department of Microbiology and Immunology,
University of Tennessee, Memphis, Tennessee 38163
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INTRODUCTION |
Nitrogen-catabolic gene expression
in Saccharomyces cerevisiae depends on the GATA family
transcriptional activators Gln3p and Gat1/Nil1p (for comprehensive
reviews of the GATA transcription factor literature, see references
7 and 26). In the presence of a
rich nitrogen supply, Gln3p and Gat1p form a complex with Ure2p (a
prion precursor and negative regulator of nitrogen catabolite repression (NCR)-sensitive gene expression) and are thereby excluded from the nucleus (2, 3, 9, 10, 14-17, 19, 27). Consistent with this exclusion, when CAN1 expression is repressed by
growth with glutamine as a nitrogen source, the GATA sequences in its promoter are unoccupied and hence are able to serve as surrogate TATA
elements (9).
NCR-sensitive gene expression is also subject to negative regulation by
two transcriptional repressors, Dal80p/Uga43p and Deh1p/Gzf3p (7,
26); mutation of DAL80 increases expression of the
regulated genes 10- to 20-fold (4, 12). Dal80p, Deh1p, Gln3p, and Gat1p are all GATA family DNA-binding proteins (7, 26), and Dal80p has been shown to bind to the same GATA elements as Gln3p (11). The opposing regulation mediated by the
GATA-family activators and repressors led us to propose that they might
work by competing with one another for binding to their target GATA sequences (Fig. 1) (1, 6, 11, 13,
23, 24, 26). The hypothesis of competitive control not only
applies to genes encoding transport and enzyme proteins; three of the
four GATA-factor genes (GAT1, DAL80, and
DEH1) contain multiple GATA sequences in their promoters,
and genetic data argue that they are subject to cross and autogenous
regulation (Fig. 1). Although genetic data overall support the model,
an important aspect of it has been questioned because it has not been
subjected to experimental scrutiny. Genetic data predict that
GAT1 and DAL80 expression should be tightly
coupled to one another (5, 6). In experiments in which
GAT1 was placed under GAL1,10 control,
DAL80 expression was shown to increase in parallel with that
of GAT1 (10).
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FIG. 1.
Proposed regulatory circuit of GATA-factor-dependent
transcription in S. cerevisiae. Open arrows and closed bars
indicate positive and negative regulation, respectively.
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The purpose of the work reported here was to test whether graded
DAL80 expression regulates GAT1 transcription in
particular and overall NCR-sensitive expression in general. The data
obtained support such a competitive model and suggest that the value of this complex regulation most likely accrues during the transition from
a good to a poor nitrogen supply.
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MATERIALS AND METHODS |
Strains, plasmids, and media.
We used strains TCY5
(MAT
lys2 ura3 trp1::hisG), TCY29
(Mat lys2 ura3 trp1::hisG
dal80::hisG), TCY36 (MAT lys2 ura3
trp1::hisG leu2::hisG),
TCY38 (MAT lys2 ura3 trp1::hisG
leu2::hisG
dal80::hisG), TCY46 (MAT lys2
ura3 trp1::hisG GAL1,10-GAT1),
TCY53 (Mat lys2 ura3 trp1::hisG
dal80::hisG GAL1,10-GAT1), TCY55
(MAT lys2 ura3 trp1::hisG
dal80::hisG
gln3
::hisG), TCY64 (MAT lys2 ura3
trp1::hisG dal80::hisG
gln3::hisG GAL1,10-GAT1), TSC65
(MAT lys2 ura3 trp1::hisG leu2::hisG his3::hisG
GAL1,10-DAL80).
We used plasmids pTSC417 (pT7-7-DAL80) (13),
pTSC491 (UGA4GATA-lacZ) (11), pTSC560
(DAL80-lacZ) (this work), pTSC565 (CUP1-DAL80) (this work), pTSC572 (DAL80-lacZ) (6),
pTSC624 (GAT1-lacZ) (6), pTSC666
(GAL1,10-GAT1-lacZ) (10),
pTSC674 (GAL1,10-DAL80) (this work), pTSC678 (see
below), pTSC679 (GAL1,10-DAL80-lacZ) (10), and pJD5-2 (DAL5-lacZ) (6).
To construct the GAL1,10-DAL80 fusion, the 0.9-kb
Asp-718-PvuII fragment from pTSC674 was cloned
into Litmus 28 digested with the same enzymes to yield pTSC678. pTSC678
was then digested with PvuII, a BamHI
oligonucleotide (10-mer) was ligated onto the blunted ends, and the
resulting plasmid was digested with BamHI and
XbaI. The 0.9-kb XbaI-BamHI fragment,
containing the 3' end of HIS3, the GAL1,10
UAS, and DAL80 5' end (to position +46) was isolated and ligated into plasmid pHP41 digested with XbaI and
BamHI, yielding pTSC679 (Fig.
2B). All fusion junctions were sequenced
to ensure that the fusions were in frame. Methods similar to those
above were used to construct CUP1-DAL80 pTSC565, except that
the plasmid backbone was derived from pSc3415 (20).
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FIG. 2.
Constructs for the production and assay of Dal80p and
Gat1p from the GAL1,10 promoter. Genomic loci are
shown prior to (A) and following (B) integration of a construct in
which a GAL1,10 promoter fragment (derived from
pTSC645 or pTSC674) replaces the native promoter of the gene; open
bars, yeast DNA; grey bars, GAT1 or DAL80 open
reading frame (ORF); black bars, yeast selectable marker; stippled
bars, GAL1,10 promoter DNA; dashed lines, vector
sequences. Strain numbers of the resulting strains are also indicated.
lacZ fusion plasmids were derived from
GAL1,10-GAT1 (C) and
GAL1,10-DAL80 (D), pTSC666 and pTSC679,
respectively. Symbols are as indicated for panels A and B above except
vertical lines, indicating lacZ DNA. (E)
CUP1-DAL80 plasmid.
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Pertinent structures of GAL1,10-GAT1 and
GAL1,10-DAL80, that replaced the wild-type
alleles in the genome, and GAT1-lacZ, DAL80-lacZ,
and CUP1-DAL80 plasmids are shown (Fig. 2). All
lacZ fusion constructs were carried in CEN vectors.
Minimal medium was 0.17% yeast nitrogen base (YNB) without aa or
(NH4)2SO4 (Difco) with indicated
carbon and nitrogen sources and auxotrophic requirements. Cultures for
experiments in which various amounts of glucose were added to the
cultures (Fig. 3, 4A and B, 5, and 7) were prepared in the following
manner. Standard overnight cultures were grown to log phase
(A600 = 0.6 to 0.8) in minimal 2% glucose
medium. Therefore, all GAL1,10-regulated gene
expression was heavily repressed at the onset of the experiment. A
sample of the culture was taken, washed with water, and inoculated into
fresh minimal galactose-proline medium containing the indicated amounts
of glucose. The inocula were such that the cultures reached cell
densities (A600) of 0.3 to 0.8 after overnight
growth. The cultures were then harvested and assayed for
-galactosidase. As the amount of glucose added to the culture was
increased, the cumulative amounts of either
GAL1,10-GAT1 or
GAL1,10-DAL80 expression decreased (Fig.
3). The premise upon which these
experiments were based is that the cumulative amount of GAT1
and DAL80 expression during growth of a culture can be
varied in a reproducible manner and that the effects of those
variations can then be measured. It must also be noted, however, that
while the method we used will test and correlate whether graded changes
in DAL80 expression elicit similarly graded changes in
GAT1 expression and vice-versa, they do not permit absolute
quantitation of the relationships between intracellular Gat1p and
Dal80p levels, subcellular distributions of the proteins, and the
levels of DAL80 and GAT1 expression.
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FIG. 3.
GAT1-lacZ expression driven from the
GAL1,10 promoter in cultures to which increasing
amounts of glucose were added. The detailed protocol for the experiment
is described in Materials and Methods. Data in Fig. 3B are those from
Fig. 3A plotted as the reciprocal of the glucose concentration to
facilitate understanding of the results.
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Asparagine to proline shift.
Strain TCY36 (wild type [WT])
or TCY38 (dal80), transformed with plasmids indicated in the
figures, was grown in 2% glucose-0.1% asparagine-YNB minimal medium
at 22°C to an A600 of 0.4. Each culture was
then harvested by filtration, washed with 100 ml of minimal medium
lacking a nitrogen source, and resuspended in the same volume (as the
original culture) of minimal glucose proline medium. The cultures were
sampled as indicated and assayed for -galactosidase as previously
described (11). The results were reported in Miller units,
which are based on 25 ml of culture.
Northern blot hybridization and -galactosidase assays were performed
as previously described (13).
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RESULTS |
Heterologous DAL80 and GAT1 expression
under GAL1,10 control.
If Dal80p
down-regulates transcription by competing with the GATA family
transcription activators for DNA binding, one would expect Gln3p- and
Gat1p-dependent transcription to vary inversely with DAL80
expression. To test this, we placed GAT1 or DAL80
expression under control of the GAL1,10 promoter
to remove it from GATA factor regulation and NCR sensitivity.
Variations in the amount of GAT1 or DAL80
expression were then generated by changing the levels of glucose (and
hence the level of carbon catabolite repression) in the medium. These
results are demonstrated in Fig. 3A, in which lacZ was fused
to the GAL1,10-GAT1 promoter. Finally, the
effects elicited by changes in GAT1 or DAL80
expression were measured using DAL80-lacZ or
GAT1-lacZ reporter genes containing native promoter regions
of the fused genes. This experimental strategy was previously used to
analyze the regulatory relationships between Gat1p and Ure2p
(10). Throughout this work, we have plotted the data as the
inverse of the glucose concentration (Fig. 3B) to facilitate
visualizing the experimental results; i.e., expression of the gene we
are changing experimentally (GAL1,10-GAT1, or
GAL1,10-DAL80) increases from left to right on
the abscissa, and the effects elicited by the experimental pertubation
are recorded on the ordinate as DAL80-lacZ or
GAT1-lacZ expression (-galactosidase production), respectively. Dal80p was chosen for these experiments rather than Deh1p
because (i) Dal80p is the best characterized GATAA-binding repressor
and (ii) the increasing suspicion that the Deh1p-regulated genes
reported so far may not be Deh1p's most physiologically relevant
targets (6, 23, 24).
To ensure the GAL1,10-DAL80 construct responded
appropriately to various levels of glucose, we measured steady-state
DAL80 mRNA levels and
GAL1,10-DAL80-lacZ reporter gene expression. As increasing amounts of glucose were added to the medium, steady-state DAL80 mRNA (Fig. 4A) and
-galactosidase production from
GAL1,10-DAL80-lacZ (Fig. 4B) decreased, arguing
that we can predictably regulate DAL80 expression. To
evaluate the possibility that growth on galactose vs glucose or WT vs
dal80::hisG strains might compromise
our experimental approach, we measured DAL80 expression
under these conditions. DAL80-lacZ expression increased
twofold in a dal80 mutant vs the WT and decreased less than
twofold in glucose vs galactose medium (Fig. 4C). Therefore, the
experimental system was not compromised by the experimental conditions
we used.
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FIG. 4.
Characterization of the Dal80p-production system. (A)
Northern blot analysis of DAL80 mRNA driven by the
GAL1,10 promoter. Total RNA (10 µg/lane) was
extracted from TCY65. The experimental protocol was as described in
Materials and Methods. The 32P-dCTP-labeled
DAL80 probe was synthesized from the 0.8-kb
NdeI-EcoRI fragment of pTSC417. HHT1
and HHT2 standards were probed with a radioactive
complementary oligonucleotide. (B) Effect of increasing glucose
addition on lacZ expression in strain TCY36 transformed with
GAL1,10-DAL80-lacZ pTSC679. (C) Expression of
DAL80-lacZ from pTSC572 in WT (TSC46) and dal80
(TCY53) strains growing in YNB-2% glucose-2% galactose-0.1%
proline medium. Cells were grown under standard conditions and
harvested for -galactosidase assays at a cell density
A600 of 0.4 to 0.8.
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Inverse regulation of GAT1 and DAL80 expression.
GAT1-lacZ pTSC624, in which lacZ is expressed
from the native GAT1 promoter, was used to transform
GAL1,10-DAL80 strain TCY65. As DAL80
expression increased, GAT1-lacZ expression decreased (Fig.
5A). In the reciprocal control
experiment, DAL80-lacZ expression from pTSC572 increased in
parallel with that of GAT1 expressed from a
GAL1,10 promoter in both WT TCY46 and
dal80 mutant TCY53 (Fig. 5B) as reported earlier
(10). Mutating the genomic copy of DAL80 further
increased DAL80-lacZ expression at each level of
GAT1 expression (Fig. 5A). We conclude that (i) DAL80
expression parallels that of GAT1 and (ii) GAT1
expression is inversely related to that of DAL80.
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FIG. 5.
Inverse regulation of GAL80 and
GAT1 expression. (A) GAT1 expression at different
levels of DAL80 expression from pTSC624 in strain TCY65. (B)
DAL80 expression at different levels of GAT1
expression from pTSC572 in strains TCY46 (WT) or TCY53
(dal80). Growth and assay conditions were as described in
Fig. 3. Steady-state -galactosidase production derived from
DAL80-lacZ and GAT1-lacZ fusion genes in WT,
dal80, and gat1 mutant backgrounds have been
reported (6). However, the values obtained in those
experiments should be only generally compared with the values obtained
here because of the differences in the experimental conditions used.
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The next experiment had two objectives: (i) to ensure that the Dal80p
regulation of gene expression we observed did not aberrantly derive
from the heterologous GAL1,10 promoter we used
and (ii) to ascertain whether the characteristics of Dal80p regulation described above extended to nonregulatory genes whose expression depended primarily upon Gln3p. We constructed CUP1-DAL80
fusion pTSC565. CUP1 is an inducible gene whose expression
is regulated by the levels of copper in the medium (18). We
found that copper concentrations up to 0.1 mM were not toxic to cell
growth and hence provided a useful range of induction capabilities
within which to work (data not shown). To meet the second objective, we
used a DAL3-lacZ reporter to monitor Dal80p regulation of
GATA-factor-dependent transcription. Electrophoretic-mobility-shift
assays have shown that Dal80p and Gln3p bind to the same GATA elements
in the DAL3 promoter (11) and further that
DAL3 transcription is largely Gln3p dependent and Gat1p
independent (6, 11). -Galactosidase production, supported
by DAL3-lacZ pTSC560, is approximately 14-fold higher in
dal80 strain TCY29 than in isogenic WT TCY5, irrespective of
the amount of copper added to the medium (Fig.
6). In strain dal80 TCY29,
transformed with CUP1-DAL80 pTSC565, DAL3-lacZ
expression depended on copper additions to the medium (Fig. 6). At 0.1 mM copper, DAL3-lacZ expression was close to the low level
observed in WT strain TCY5, in which the genomic copy of
DAL80 is expressed from its native promoter. These data
suggest that the inverse functional relationship between
DAL80 and GAT1 expression extends to
Gln3p-mediated DAL3-lacZ expression as well.
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FIG. 6.
DAL3 expression at various levels of
CUP1-DAL80 expression. Strains TCY5 (WT) and TCY29
(dal80::hisG) were transformed with
reporter pTSC560 alone or together with CUP1-DAL80 pTSC565.
Cultures were grown overnight in 2% glucose-0.1% proline medium,
containing the indicated concentrations of copper sulfate, to a cell
density (A600) of 0.4 to 1.0, at which time
samples were removed for the assay of -galactosidase, using standard
assay methods.
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Gat1p can partially replace Gln3p in transcription of
DAL80.
Varying GLN3 expression, as we did with
DAL80 and GAT1, was not possible because excess
Gln3p is toxic to the cell (21). Although we did not vary
GLN3 expression, we assessed Gat1p's ability to replace
Gln3p in activating DAL80 transcription. As GAT1
expression increased in gln3 TCY55 grown in minimal
proline medium, a small but measurable amount of -galactosidase was
produced from pTSC572 (Fig. 7 [compare
with Fig. 5]). When the additional variable of genomically derived
Dal80p was removed, by performing the experiment in a gln3
dal80::hisG double mutant (TCY64),
-galactosidase production increased to about 25% of the level seen
in a GLN3 strain (Fig. 7). This expression, however, was NCR
sensitive.
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FIG. 7.
The effect of GAT1 expression on
DAL80 expression in gln3 strains.
-Galactosidase production was supported by a pTSC572 transformant of
strain TCY55 (gln3 GAL1,10-GAT1) or TCY64
(gln3 dal80 GAL1,10-GAT1) with proline or
ammonium sulfate as the nitrogen source and 2% galactose plus the
indicated concentration of glucose as the carbon source as per Fig. 3
and Materials and Methods.
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Dal80p functions during the transition from good to poor nitrogen
sources.
The proposed GATA factor regulatory circuit consists of
multiple feedback loops (Fig. 1), and this prompts the question of what
use such complex control would be to the cell. When a good nitrogen
source is provided, there is little DAL80 expression, reasonably low levels of GAT1 expression, and little
functional Gln3p; nitrogen-catabolic gene expression is repressed to
very low levels. Alternatively, when a poor nitrogen source is
provided, GAT1 and DAL80 expression is high, and
Gln3p functions well; nitrogen-catabolic gene expression is high and in
some cases increases further via induction (7, 22). In these
steady-state situations, we see little need for such a complex and
dynamic regulatory circuit. Therefore, we imagined that Dal80p
functions during transition from good to poor nitrogen sources to allow
NCR-sensitive gene expression in general and GAT1 expression in
particular to occur as a rich nitrogen supply is depleted but only at
low levels.
We tracked reporter gene expression supported by four fusion plasmids:
pJD5-2, pTSC491, pTSC572, and pTSC624, following a rapid shift of WT
and dal80 mutant cultures from minimal-asparagine to
minimal-proline medium. DAL5-lacZ expression served as a
control, because Dal80p regulation of this gene is minimal
(13). The course of -galactosidase production driven by
the DAL5 promoter was the same in WT and dal80
cultures following the shift, increasing to high levels (Fig.
8A). A very different result was seen
when expression supported by the UGA4 GATA cluster was
followed. The overall kinetics of -galactosidase production in
dal80 strain TCY38 were the same as in Fig. 7A; i.e.,
production continuously increased following a lag of 20 to 30 min. In
contrast, -galactosidase production in the WT (TCY36) first
increased as seen in the dal80 mutant but then plateaued at
a low level (Fig. 7B). The same pattern was observed when
DAL80-lacZ and GAT1-lacZ expression was followed through a transition from a good to poor nitrogen source (Fig. 9).
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FIG. 8.
Expression of DAL5-lacZ and a UGA4
promoter fragment in a heterologous expression vector following a shift
of cultures from YNB glucose-asparagine to glucose-proline medium.
Strains TCY36 (WT) and TCY38 (dal80) were transformed with
DAL5-lacZ pJD52 (A) or UGA4GATA
pTSC491 (B).
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FIG. 9.
-Galactosidase production from DAL80-lacZ
pTSC572 (A) and GAT1-lacZ pTSC624 (B) transformed into
strains TCY36 (WT) and TCY38 (dal80). Conditions were as
described in the legend to Fig. 8.
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DISCUSSION |
Our data demonstrate that GAT1 expression is inversely
regulated by DAL80 expression. A similar relationship
appears to exist between DAL80 expression and Gln3p-mediated
transcription as well. DAL80 expression, in turn, is
regulated by Gat1p expression, Gln3p operation, and autogenous
repression by Dal80p (26). These results are expected if
Dal80p represses Gln3p- and Gat1p-dependent transcriptional activation
by competing with Gln3p and Gat1p for binding to their target GATA sequences.
The results also provide insight into the relationship of Gln3p- and
Gat1p-binding sites and functions. If the two transcriptional activators were identical in DNA binding and function, one would expect
Gat1p overproduction to more effectively suppress the gln3 deletion phenotype. Although some suppression was observed, it was
limited, arguing that Gln3p- and Gat1p-binding-site specificity or
their functions in transcription do not fully overlap. Alternatively, it is possible that differences in the relative strength of Gln3p and
Gat1p as transcriptional activators could account for the differences
observed (5, 12). This possibility is particularly important
since three additional GATA family members in S. cerevisiae, GAT2, GAT3, and GAT4, do not appear to
function in nitrogen-catabolic gene expression (8;
R. Rai and T. G. Cooper, unpublished observations).
When we consider the present data in light of recent reports that Gln3p
and Gat1p form complexes with Ure2p and that these complexes are
excluded from the nucleus (2, 3, 9, 10, 14-17, 19, 27), we
suggest that GATA factor transcriptional regulation consists of two
complementing facets. NCR preeminently controls the GATA factor
regulatory circuit, by dictating the amount of Gln3p and Gat1p that
gains access to the nucleus and hence their target binding sites. Finer
control of GATA-factor-mediated expression is then achieved by the
competition of Gln3p and Gat1p with Dal80p for binding to the DNA. The
fine tuning that this second level control can attain depends on the
autogenous and cross regulation of GATA transcription factor production
and competition. The overall outcome is a highly responsive but equally
highly buffered control circuit not unrelated to the feedback loops
known to electrical engineers.
We further suggest that such complex, interrelated control does not
afford its greatest advantage when the cell finds itself in rich or
poor nitrogen supplies but rather during the transition from the former
to the latter. At times of excess nitrogen, there is little need for
such finely tuned control, and little is available at the site of
transcription since, due to NCR, the GATA-transcriptional activators
Gln3p and Gat1p are excluded from the nucleus by Ure2p, and the
repressor, Dal80p, is not produced at significant levels (6). During continuous nitrogen limitation, the system is
again at steady state. Simple transcriptional regulation at the level of NCR would be quite adequate. However, as rich nitrogen sources begin
to dwindle, the cell must cast about for alternative sources. To do so,
only low levels of proteins required to transport and degrade
alternative nitrogen sources are needed. To produce more of these
proteins would be wasteful at a time when the cell can ill afford it.
Therefore, nitrogen limitation results in the release of Gln3p from the
Ure2p-Gln3p complex and its subsequent entry into the nucleus. There it
mediates transcription of GAT1, and the Gat1p produced can
then enter the nucleus and function as a transcriptional activator as
well. In this regard, it is probably significant that expression of
most NCR-sensitive genes requires both Gln3p and Gat1p (5, 13,
25). Therefore, when both Gln3p and Gat1p are available in the
nucleus, the conditions for nitrogen-catabolic gene expression to occur
have been met. However, these are also precisely the conditions that
activate DAL80 expression. As soon as Dal80p is produced, it begins
down-regulating not only nitrogen-catabolic gene expression per se, but
also production of Gat1p, the activator required for this expression.
In other words, there is only a short time during the transition when
nitrogen-catabolic gene expression occurs at an unrestricted rate,
i.e., the time needed for DAL80 to be expressed and
DAL80 mRNA to be translated. Thereafter, gene expression
moves toward the Dal80p-regulated level until a new steady-state level
is attained. It is important to note that we are unable to find any
evidence that Dal80p is subjected to the subcellular localization
control seen with Gln3p and Gat1p (M. Distler and T. G. Cooper,
unpublished observations). Although much remains to be learned about
the details of this complex regulatory circuit, the mechanistic
outlines through which it regulates a broad array of gene expression in
S. cerevisiae (8; K. Cox, R. Andhare, and T. G. Cooper, unpublished observations) are becoming
more clear.
We thank the UT Yeast Group for suggested improvements to the
manuscript and Tim Higgins for preparing the figures.
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Abstract
Introduction
