(Received for publication, September 6, 1995; and in revised form, November 13, 1995)
From the
In Saccharomyces cerevisiae, unsaturated fatty acids
are formed from saturated acyl-CoA precursors by Ole1p, a -9 fatty
acid desaturase. OLE1 mRNA levels are differentially regulated
by the addition of saturated or unsaturated fatty acids to the growth
medium. One component of this regulation system involves the control of OLE1 transcription. Saturated fatty acids induce a 1.6-fold
increase in transcription activity, whereas a large family of
unsaturated fatty acids repress OLE1 transcription as much as
60-fold. A deletion analysis of OLE1 promoter::lacZ fusion reporter genes identified a 111-base pair (bp) fatty
acid-regulated (FAR) region approximately 580 bp upstream of the start
codon that is essential for transcription activation and unsaturated
fatty acid repression. Deletion of an 88-bp sequence within that region
resulted in a complete loss in transcription activation and unsaturated
fatty acid regulation. The 111-bp FAR element strongly activates
transcription and confers unsaturated fatty acid regulation on a
heterologous CYC1 promoter test plasmid. Essential elements
required for unsaturated fatty acid repression of OLE1 were
found in the 5` and 3` region of the 111-bp sequence. The FAR
element-mediated activation and fatty acid repression of transcription
was found to be closely tied to fatty acyl-CoA metabolism. Two fatty
acid activation genes, FAA1 and FAA4, were found to
be essential for unsaturated fatty acid repression of OLE1 through the FAR sequences. Disruption of either gene results in
reduced levels of unsaturated fatty acid repression; disruption of both
genes completely blocks the regulatory response. Acyl-CoA binding
protein (ACBP) plays a role in determining the level of FAR element
activated transcription. Disruption of the ACBP gene causes a
>5-fold activation of OLE1 transcription and a similar
increase in OLE1 mRNA levels. Unsaturated fatty acid
repression of OLE1 transcription, however, is not affected by
the disrupted ACBP gene. These studies show that promoter elements
responsible for unsaturated fatty acid-mediated transcription
repression are tightly linked to OLE1 activation sequences and
that OLE1 transcription levels are closely tied to acyl-CoA
metabolism.
Nutrient fatty acids can exert strong regulatory effects on a number of lipogenic enzymes in fungi. Medium and long chain fatty acids are readily internalized by fungi and incorporated into membrane and storage lipids. Saturated and unsaturated acids appear to differentially regulate the expression of a number of lipid biosynthetic genes, including those encoding acetyl-CoA carboxylase (2, 3, 4) , fatty-acid synthase(5, 6) , and fatty acid desaturase(1) . The mechanisms by which cells sense fatty acids, discriminate among molecular species, and modulate gene activity in different parts of the lipid metabolic web is unclear, although it appears that multiple systems have evolved to regulate different lipogenic functions.
To identify the mechanisms that control lipid
synthesis in response to extracellular acids, we examined the
regulation of the -9 fatty acid desaturase, an enzyme involved in
the formation of unsaturated fatty acids. The OLE1 gene, which
encodes that enzyme in Saccharomyces, is strongly regulated in
response to extracellular fatty acids(1) . This can be seen as
a rapid reduction in OLE1 mRNA levels when unsaturated acids
are added to the growth medium and an increase in enzyme activity when
cells are exposed to saturated fatty acids. The exogenous fatty acids
that trigger these responses are rapidly incorporated into membrane
lipids; thus it is reasonable to expect that the sensors and signal
transducers that regulate OLE1 in response to nutrient fatty
acids may also be a part of broader controls that regulate membrane
lipid composition in response to other stimuli.
Our recent studies
suggest that fatty acid regulation of OLE1 is under at least
two forms of control(7) . One component acts to repress
transcription of the OLE1 gene; a second acts by
post-transcriptional mechanisms to further modify OLE1 mRNA
levels(7) . In this paper, we examine the promoter of the
desaturase gene to identify transcriptional controlling elements that
respond to unsaturated fatty acids and to assess their contribution to OLE1 expression. An essential transcription activation region,
designated the FAR ()element, is identified that also
contains the elements required for unsaturated fatty acid-mediated
repression of transcription activity. There appears to be a close
connection between cellular acyl-CoA metabolism and regulators that act
on that region of the OLE1 promoter.
Plasmid p62::-93488 was
constructed by isolating a fragment consisting of OLE1 nucleotides -934 to -576 and fusing it to a fragment
containing bases -489 to +81. The resulting fragment was
ligated into the parent p62 plasmid, yielding an OLE1 insert
with the same ends as p62::-934 but lacking 88 bases between
-576 and -489. All fragments in plasmid p62 series had a
3`-end point at nucleotide +81. The promoter deletion constructs
were used to transform the phenotypically wild-type OLE1 strain, L8-25A, to uracil prototrophy.
Vectors
pCT714 and pCTm714 were constructed by inserting a 714-bp HindIII/HpaI fragment that extended between bases
-934 and -221 of the OLE1 promoter into the
pCT multiple cloning region. Vectors pCT111 and pCTm111 were
similarly constructed by inserting 111 bp of the OLE1 promoter
region that extends from bases -576 to -466. Vector
pCTm111E10 was made by inserting an EcoRI linker (sequence
CCGAATTCGG) into the SmaI restriction site at the 5`-end of
the 111-bp fragment of pCTm111. pCTm100 was derived from pCTm111E10 by EcoRI digestion to remove the 11 upstream base pairs of the OLE1 promoter sequence.
Vectors pCTm67 and pCTm40 were constructed using synthetic paired oligonucleotides. Vector pCTm67 contains bases -582 to -516, and plasmid pCTm40 includes bases -582 and -543 of the OLE1 promoter sequences.
Vector pCTm91 was constructed using a 91-bp fragment encompassing bases -556 to -466 of the OLE1 promoter (derived by PCR amplification of OLE1 promoter sequences). The base sequence of the PCR fragment was determined by DNA sequencing. Plasmid pCTm114 contains bases -579 to -466 of the OLE1 promoter. Vector pCTm25 was prepared by digestion of vector pCTm114 at the ApaI restriction site within the 114-bp OLE1 sequence and the vector XhoI site.
Integrating vectors pITm714 and pITm111 were constructed by removing the ARS and CEN sequences from their respective pCTm parents by HindIII digestion and religation.
Figure 1:
Deletions of the OLE1 promoter:lacZ fusion constructs and their activity in the
phenotypically wild-type strain L8-25A. The upper left scale represents DNA sequences upstream of the OLE1 protein coding region. The relative locations of two proposed TATA
boxes at positions -160 (TATATA) and -30 (TATAAA), and the
start codon are indicated. Restriction sites shown on the scale that
were used for constructions have the following designations: H, HindIII; S, SmaI; P, PstI. Deletion constructs are shown in the column below the
scale. The narrow line represents OLE1 promoter
sequences, the solid black bar represents the amino-terminal
27 amino acids of the OLE1 coding sequence fused to E.
coli lac Z (hatched bar). The number above each line
indicates the position of the deletion end point with respect to the
ATG start codon of the wild-type base sequence with the A of the codon
designated as +1. Deletion end points were verified by sequencing
as described under ``Materials and Methods.'' The
p62::-93488 construct represented by the bottom line contains an 88-bp deletion at the indicated position in the
promoter region. Bars to the right of each diagram
illustrate the
-galactosidase activity in Miller units (30) produced by each construct in cells grown without
unsaturated fatty acids (hatched bars) and with unsaturated
fatty acids (solid bars). Units of activity indicated are the
average of at least three independent experiments performed on two
separate transformants. Standard deviations of these values were less
than 20% of the mean.
Deletion of bases -934 to -576 caused an
approximate 3-fold loss of reporter gene activity. Removing bases
-576 through -488 caused a further 20-fold drop in reporter
gene activity, indicating the presence of an activating sequence in the
region near base -576. Deletions producing 5` ends between
-396 and -255 exhibited small (2-fold) increases in
expression corresponding to approximately one-thirtieth of the activity
seen with the entire 934-bp promoter fragment. Deletions beyond base
-255 produced reporter gene activities near the basal level
exhibited by the plasmid that contains no OLE1 promoter
sequences. An 88-bp deletion in the putative activation region between
bases -576 through -489 (Fig. 1) showed a 27-fold
reduction in -galactosidase activity, indicating that essential
transcription activation elements were located in that region.
The activity of the reporter containing the 88 bp deletion within the 934-bp fragment was reduced 2-fold in response to unsaturated fatty acids.
Tests performed on the effects of saturated fatty acids on strains containing the promoter deletion series produced a different pattern of regulation (data not shown). Supplementation of media with 0.5 mM 14:0 produced a 1.2-1.5-fold increase in activities of cells that contained plasmids p62::-934 and p62::-792. By contrast, reporter activity of the plasmid p62::-576 was decreased 5-fold by the addition of 14:0. Activities of plasmids containing more extensive 5` deletions in cells incubated with 14:0 did not differ significantly from those grown in media with no fatty acid supplements.
Figure 2:
Expression of reporter gene activity from
heterologous pCT vectors containing OLE1 promoter sequences.
Diagrams on the left compare the OLE1 promoter fragment in
construct p62::-934, with OLE1 fragments inserted into
heterologous constructions employing yeast CYC1:lacZ gene fusion plasmids. Plasmid pCT (20) contains a
polylinker upstream of the S. cerevisiae CYC1 promoter that
lacks the CYC1 UAS sequence. Fragments of the OLE1 promoter were inserted into the polylinker region to test for the
ability to activate transcription and confer fatty acid regulation on
this test plasmid, as described under ``Materials and
Methods.'' Plasmid pCT714 contains a 714-bp fragment extending
from bases -934 to a HpaI site at position -221,
which lies upstream of the OLE1 TATA sequences. OLE1 promoter sequences are indicated by the black line. The left-most box placed in the OLE1 promoter sequences
indicates the position of the 111-bp FAR region shown to be essential
for transcription activation and unsaturated fatty acid repression. Black bars represent CYC1 promoter sequences; hatched boxes indicate lacZ coding regions.
Activities of heterologous promoter test plasmids are shown to the right of the corresponding figure and are expressed in nmol of o-nitrophenyl--D-galactopyranoside (ONPG) hydrolyzed/min/mg protein. Activities indicated are the
averages of at least three independent experiments ± S.D. for
each vector. Hatched bars represent activities in cells grown
without added fatty acids (NFA), shaded bars represent activities of cells grown in the presence of 0.5 mM 16:1 and 0.5 mM 18:1 (UFA).
Plasmid pCT714 contains a 714-bp OLE1 promoter fragment that extends between bases -934 and
-221 (Fig. 2). Insertion of that fragment upstream of the CYC1 TATA region produced 66-fold higher levels of reporter
gene activity, compared with the parent vector that contains no insert.
Plasmid pCT71488 contains the same 88-bp deletion as that in
p62::-934
88. That vector showed only a 2-fold increase in
activity above the basal levels produced by the parent plasmid.
Reporter gene activity was also strongly activated in vector pCT111,
which contains a 111-bp fragment derived from bases -576 to
-466. The 5`-end of that fragment coincides with the 5`-end of
the 88 bases deleted from the pCT71488 vector that were found to
be essential for transcription activation. Plasmid pCT111 exhibited a
28-fold increase in activity over the control plasmids that contained
no OLE1 promoter sequences.
Vectors that included either
the 111-bp sequence or the intact 714-bp OLE1 promoter
sequence were repressed by the addition of unsaturated fatty acids to
the growth medium (Fig. 2). Vector pCT111 showed an approximate
2-fold repression of activity under conditions where the reporter that
contained the entire promoter was repressed 7-fold. The vector
containing the 88-bp deletion within the 714-bp promoter sequence was
not repressed by unsaturated fatty acid regulation. These data suggest
that primary elements essential for unsaturated fatty acid regulation
are contained within the 111-bp promoter sequence. A parallel set of
results was also obtained in tests of these fragments in the related
pCZ plasmid(20) . That vector differs from the plasmid pCT
in that it contains a shorter part of the CYC1 basal promoter
region that includes only one TATA sequence (data not shown). Given the
ability of the 111-bp DNA fragment to strongly activate transcription
and confer fatty acid-specific repression on heterologous vectors, it
was designated as the OLE1 FAR sequence. The nucleotide
sequence of this fragment and flanking sequences is shown in Fig. 3.
Figure 3: Nucleotide sequences associated with the FAR element responsible for transcription activation and unsaturated fatty acid regulation. Underlined sequences represent symmetric GC-rich sequences found within the 114-bp fragment of plasmid pCTm114. Bases in lower case and boldface represent the FAR region sequence required for unsaturated fatty acid-mediated repression of OLE1 transcription in plasmid pCTm100.
Figure 4:
-galactosidase activities of
L8-25A cells transformed with pCTm vectors that contain
derivatives of the 111-bp FAR region in the OLE1 promoter. The top left diagram illustrates the -934 to -221 OLE1 promoter fragment placed in vector pCTm714 and the
relative position of the FAR element with respect to the start codon.
The arrows indicate the relative size and position of the
111-base fragment in an exploded view. The hatched and black boxes on the left diagrams refer to the
positions of CCC and GGG bases within those fragments. Activities of
reporter plasmids indicated on the right are expressed in nmol of o-nitrophenyl-
-D-galactopyranoside (ONPG) hydrolyzed/min/mg of protein. Hatched bars refer to activities in cells grown under derepressed (no fatty
acid conditions). Solid bars refer to activities in cells
grown in presence of 1 mM 18:2. Cells were grown in 100 ml of
UDt medium to a density of 1.5
10
/ml with or
without 1 mM 18:2 for 10 h.
-Galactosidase activity was
measured using the cell disruption method of Buchman et al. (12) as described under ``Materials and Methods.''
Three independent transformants were tested for each vector. Error
bars in the figure represent standard deviations determined from a
minimum of five independent experiments for each
vector.
Because the previously identified 111-bp FAR region fragment contains only the GGG bases of the upstream CCCGGG sequence, vector pCTm114 was constructed to include both members of the symmetric pair. Compared with pCTm111, the activity of pCTm114 was slightly reduced, but there were no significant changes in the level of unsaturated fatty acid repression. Two additional vectors were constructed to test the effects of disrupting both CCCGGG sequences. A 10-bp linker was inserted into the remaining CCCGGG sequence of pCTm111 to produce pCTm111E10. That vector exhibited an approximate 50% decrease in reporter activity (compared with pCTm111) with no significant changes in the level of unsaturated fatty acid repression. Vector pCTm100 was constructed to contain only the GGG of the downstream CCCGGG site. It showed activities and level of fatty acid repression nearly identical to vector pCTm111E10. Deletion of an additional nine base pairs (pCTm91), however, resulted in a 50% reduction of derepressed activity and complete loss of fatty acid repression. While that observation indicates that the 9-bp fragment contains sequences essential for fatty acid-mediated repression, deletion of a 50-bp fragment from the distal end of the FAR region (pCTm67) indicates that those sequences are also essential for activation and repression. Shorter test fragments containing 40- and 25-bp sequences encompassing the distal part of the FAR region showed a complete loss of activation and repression. They exhibit levels of activity similar to that of the parent vector with no inserted promoter sequences. Vectors containing fragments shorter than the 100-bp fragment in pCTm100 also exhibit slightly increased levels of activation in the presence of unsaturated fatty acids, similar to that observed with the vector alone. Taken together, these data indicate that both the 5`- and 3`-ends of the pCT111 FAR region are required for maximal activation and unsaturated fatty acid-mediated repression.
To determine their response to saturated fatty acids, pCT plasmid-bearing strains were tested by supplementing cells with 14:0 (data not shown). Similar results were obtained to that seen with the 5` deletion series. Plasmid pCTm714 containing the 714-base promoter region exhibited a 1.6-fold increase in activity in cells grown in the presence of 0.5 mM 14:0. Vectors containing elements of the FAR region, including pCTm114, pCTm111, pCTm91, and pCTm67, did not show any significant increase in activity in cells incubated with the saturated fatty acid.
The presence of doubly disrupted faa1 and faa4
genes also appears to have a
strong effect on the activation levels of the reporter genes.
Derepressed activities of both pCTm714 and pCTm111 in the double
disruptant were increased 2-3-fold over the wild-type parent.
To assess the effects of this protein on OLE1 regulation, the gene was cloned by PCR and disrupted by replacing sequences that flank the coding region of the protein with the Saccharomyces LEU2 gene. Surprisingly, no visible phenotype other than a slight retardation in growth was observed in the disruptant strains. The effect of the disruption, however, was to produce an approximate 3-4-fold increase in transcription activity of reporter plasmids that contain the entire OLE1 upstream region (p62::-934 and pCTm 714) and an approximate 5-fold increase in transcription activity with the pCTm111 vector containing the 111-bp FAR region (Table 6).
The effect
of the ACBP gene disruption on transcription had a concomitant effect
on OLE1 mRNA levels (Fig. 5). Quantitative phosphor
image analysis, using the Saccharomyces PGK1 gene as an
internal standard, revealed that OLE1 mRNA levels in the
disruptant strain were 5.5-fold higher than in its wild-type parent
grown in fatty acid-deficient medium. Surprisingly, disruption of the
ACBP gene had no effect on relative levels of unsaturated fatty acid
repressed OLE1 transcription (Fig. 5). ACBP disruption
also appears to have profound effects on cellular fatty acid
composition. The increased levels of OLE1 transcript seemed to
result in a net increase in desaturase activity relative to saturated
fatty acid biosynthesis. Analysis of total cellular fatty acids
revealed a striking increase in 14-16 carbon species and an
increase in the ratio of unsaturated to saturated fatty acids. Due to
increased desaturation of 14:0 and 16:0, 14:1 levels increased to 5%
wild type from less than 1% and 16:1 levels increased from
41.7-59.2% in the acbp::LEU2 disruptant. Levels of
the 18 carbon species were reduced in the disruptant, primarily as a
function of the reduction in 18:1-21% (down from 29%) of the
total fatty acid mass (Table 7).
Figure 5:
Northern blot analysis of OLE1 expression in DTY10A (wild type) and JY001 (acbp::LEU2) under derepressed and 18:2 repressed
conditions. Repression of OLE1 mRNA following addition of
unsaturated fatty acids to 1) strain JY001, which contains a disrupted ACBP gene, and 2) wild type. Cells were grown to a density of
2
10
/ml at 30 °C in UDt medium, as described
under ``Materials and Methods.'' At time 0, 1 mM 18:2 was added to the growth medium. Total RNA was extracted by
the hot phenol extraction procedure as described at intervals indicated
in the figure. RNA blots were probed with an OLE1 specific
probe. After phosphor image analysis, blots were stripped and reprobed
with the Saccharomyces PGK1 gene as an internal standard.
Phosphor images of the resulting blots are shown in the
figure.
The absence of the
Hap1p transcription factor appeared to have no significant effect on
fatty acid regulation of the reporter gene. Under the standard assay
conditions, activity in the hap1::LEU2 disruptant strain
grown in the presence of unsaturated fatty acids was reduced to
approximately 20% of that found in cells grown in fatty acid-deficient
medium (data not shown). Given the effects of increased OLE1 transcription on fatty acyl composition seen in the
acbp::LEU2
disruption strain, it was thought that
complementary changes might be produced by the reduced level of OLE1 transcription in the hap1::LEU2 disrupted
strain. Analysis of the disruptant and its parent strain revealed no
significant differences in either the fatty acyl composition or the
total cellular fatty acid content of the two strains (data not shown).
Saccharomyces, like other eukaryotes, maintains a
balanced ratio of unsaturated and saturated fatty acids in its membrane
lipids under a wide range of physiological conditions. This requires
the coordinated regulation of fatty acid synthesis, which produces
saturated fatty acids, and fatty acid desaturation, which converts most
of the saturated acids to unsaturated species. The maintenance of fatty
acyl composition appears to be important in controlling the properties
of both cellular membranes and storage lipids. The -9 fatty acid
desaturase is a critical component of this system and is a highly
regulated activity that responds to both nutrient and physiological
controls(7) . A major question concerns how cells monitor the
availability of fatty acid precursors and the acyl composition of
glycerolipids to regulate the activities of the desaturase and other
lipogenic enzymes. Recent evidence from this laboratory suggests that
the regulation of unsaturated fatty acid formation involves a diverse
array of controls.
This analysis of the OLE1 promoter indicates that the formation of unsaturated fatty acids is strongly regulated at the level of transcription by nutrient fatty acids. This appears to be a major component of the previously observed elevation of OLE1 enzyme activity by saturated fatty acids and its repression in response to unsaturated acids(1) . Essential transcription activation and unsaturated FAR elements appear to be located in a short 111-bp region located approximately 500 bp upstream of the OLE1 transcribed region. That fragment is sufficient to activate and confer unsaturated fatty acid repression on an unrelated gene that contains only the basal promoter elements and no upstream transcription activating sequences. Saturated fatty acids, however, do not activate vectors containing only the FAR region elements, suggesting that sequences that respond to those stimuli lie in another region of the promoter. Taken together these observations suggest that there are at least two independent systems that regulate OLE1 transcription in response to fatty acids.
The identification of transcription factors that act through the FAR region is critical to our understanding of the mechanisms of fatty acid-mediated repression. The data presented here indicate that a specific 9-bp sequence at the distal end of the 100-bp FAR fragment is essential for fatty acid-mediated repression and plays some role in activation ( Fig. 3and Fig. 4). A region at the opposite end of that fragment is essential for activation. If the latter sequences are targets for transcription-activating DNA binding proteins, then they must also play a role in FAR region-mediated unsaturated fatty acid repression.
The data presented here are consistent with either of two models for control of OLE1 transcription(21) . One is that repression is mediated by an unsaturated fatty acid-responsive DNA binding protein. When activated, it competes with transcription factors for binding to FAR element sequences. A second model is that repression is triggered by an unsaturated fatty acid-activated auxiliary transcription factor that interacts with DNA binding proteins occupying the FAR region.
Associated with the essential regulatory sequence at the 5` boundary of the FAR region is a series of symmetric GC-rich elements. These include a pair of CCCGGG sequences followed by an inverse GGGGCCC sequence within a 30-bp region. A homologous pair of CG-rich sequences was found in the transcription-activating region of the promoter of the sterol biosynthetic gene, ERG11(22) . That gene encodes a microsomal cytochrome P-450 enzyme responsible for the demethylation of lanosterol. The significance of the CG-rich sequences is further reinforced by the occurrence of homologous paired sequences in the upstream noncoding region of the ERG3 gene, which encodes the sterol C-5 desaturase(23, 24) . All three genes encode intrinsic enzymes that act at the endoplasmic reticulum surface. The existence of homologues to the OLE1 sequences in another lipid biosynthetic pathway suggests they may be involved with a type of transcriptional control for lipogenesis that has yet to be identified.
The effect of the combined faa1, faa4 disruption in blocking repression of OLE1 transcription is intriguing and suggests that repression is related to the availability of acyl-CoA species formed from the exogenous fatty acids. The two genes that affect the repression of OLE1, (FAA1 and FAA4), account for approximately 99% of the cellular 14:0 CoA and 16:0 CoA synthetase activities in wild-type cells grown in glucose medium(29) . Furthermore, both genes are responsible for almost all of the activation of imported 14:0 and 16:0. It is not clear, however, whether they are also the primary activators for unsaturated species such as 16:1, 18:1, or 18:2. Disruption of FAA1 and FAA4 results in a striking reduction in the incorporation of these fatty acids into glycerolipids and a sharp increase in cellular fatty acids (27) . One possibility is that the OLE1 regulatory circuit responds to the size or the composition of the intracellular acyl-CoA pool generated by the two synthetases. Alternatively, Faa1p and Faa4p may be involved in the intracellular transport of exogenous fatty acids or in their partitioning to cellular locations that are accessible to the regulatory sensor.
The acyl-CoA binding protein appears to play a significant role in OLE1 expression. Disruption of this abundant and highly specific binding protein increases OLE1 transcription greater than 5-fold, which accurately correlates with a 5-fold increase in OLE1 mRNA levels. Although disruption of the ACBP gene does not produce a significant phenotype with respect to growth, it appears to cause an increase in the levels of unsaturated fatty acids relative to saturated species in total cellular lipids. One interpretation of this response might be that the regulatory sensor that detects available substrate for the desaturase responds to saturated ACBP-bound acyl-CoAs. The absence of ACBP-bound substrate may elicit a cellular response ordinarily used to monitor levels of available saturated substrates for the enzyme. In the absence of this signal, OLE1 expression is increased to compensate for the perceived reduction in substrate. If the actual acyl-CoA levels are not rate-limiting, this increase in OLE1 expression could have the concomitant result of increasing levels of cellular unsaturated fatty acids.
Hap1p (28) appears to be one of several transcription activators that recognize FAR region elements. Analysis of reporter gene activity in Hap1p-deficient cells indicates that it is responsible for more than half of the OLE1 transcription activity in wild-type cells grown under fermentative conditions. Disruption of HAP1, however, does not appear to affect either the balance of cellular saturated and unsaturated species or the relative levels of fatty acids found in those cells. This indicates that, in the absence of HAP1p, other transcription factors activate the OLE1 FAR element to produce sufficient mRNA to maintain normal membrane fatty acyl lipid composition. We are currently attempting to identify these unknown activation and repressor elements by the isolation of regulation defective mutants.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U42698[GenBank].