From the
The Drosophila glutathione S-transferase
(gstD) genes are a family of divergently transcribed,
intronless genes and pseudogenes. Under control conditions, the
steady-state level of gstD1 mRNA is 20-fold higher than that
of the gstD21 mRNA despite a lower transcription rate of the
gstD1 gene. The GST D1 protein level is four times as abundant
as the GST D21 protein. The gstD1 and gstD21 genes
responded rapidly to pentobarbital (PB) as changes in mRNA levels were
detectable within 30 min of treatment. Maximal induction of
gstD1 and gstD21 resulted in 3-fold and 20-fold
elevation of their respective mRNA levels. The major mechanism for the
increase in gstD1 mRNAs appears to be transcriptional
activation. The 2-fold increase in the rate of gstD21
transcription, however, cannot fully account for the 20-fold increase
in the steady-state level of gstD21 mRNA. Therefore,
post-transcriptional mechanism(s) should also be responsible for the
increase of gstD21 mRNA by PB. Because the gstD21
mRNA is relatively unstable under control conditions, induction of the
intronless gstD21 mRNA by PB occurs mainly at the level of
enhanced mRNA stability. The GST D1 protein level in adult
Drosophila was increased approximately 2-fold after PB
treatment, whereas the GST D21 level remained relatively the same.
Thus, an increase in gstD21 mRNA stability by PB treatment is
probably coupled to a regulatory effect at the translational level.
Glutathione S-transferases (GSTs, EC
2.5.1.18)
General anesthetics, such as barbiturates, are thought to exert
their effects on synaptic transmission by binding to the ion channel
proteins in the central nervous system
(15, 16) . The
GABA
Since little is known about the molecular mechanism(s) of PB
induction of the gst genes in general, the major objective of
this investigation was intended to understand the molecular
mechanism(s) of the responsiveness of gstD genes to PB. We
report that both the mRNA levels of gstD1 and gstD21
and the protein levels of GST D1 were induced by PB. We also report
that accumulation of the gstD21 mRNA in Drosophila upon PB treatment is mainly due to enhanced mRNA stability. A
model is proposed for the enhancement of gstD21 mRNA stability
and the apparent translational regulation of GST D21 synthesis in the
presence of PB.
The nuclear run-on data were calculated for folds of
induction relative to the internal references, the rp49 gene,
histone and actin genes. The actin and
histone genes are better reference markers for the
determination of transcription initiation rates because of their higher
transcription activities than that of the rp49 gene. The
induction ratios (in the presence/absence of PB) were calculated by
comparisons of the ratios of 1) the gstD signals to the
rp49 gene signals, 2) the gstD signals to the
histone gene signals, and 3) the gstD signals
registered on two duplicate filters hybridized separately with the
GST D1 and GST D21 are relatively abundant proteins, consisting of
approximately 1% and 0.25%, respectively, of the total soluble proteins
in Drosophila adults. These estimates were derived from
Western blots by a comparison of the histochemical or
The results from this investigation are relevant to several
observations in the literature. It was reported that DCNB conjugation
activity in house flies was elevated 2-4-fold after 4 days of
exposure to 2.5 mg/ml PhB and that treatment of house flies with 10
mg/ml PhB resulted in a high mortality rate
(43) . Another group
observed an increase of DCNB conjugation activity (2.8-fold) after 2
days of exposure to 10 mg/ml PhB in house flies and higher
concentrations of PhB caused high mortality
(44) . These
induction conditions were different from those used in this report
where Drosophila adults were exposed to various concentrations
of PB, including 1, 2, 5, 20, 50, 200, 500, and 1000 mg/ml PB on paper
strips, after starvation for 5-6 h. It took longer to observe
maximal induction as PB concentrations decreased. The flies responded
to PB treatment in a concentration-dependent manner probably because a
minimum dosage is required. The higher the PB concentration used to
treat the flies (e.g. OR strain), the shorter the time it took
for the flies to achieve maximal gstD gene induction
(Fig. 2, ). The induction time courses revealed that
the mRNA levels gradually decreased soon after peaking (Fig. 2,
).
Under normal conditions, the steady-state level of
the gstD1 mRNA was at least 20-fold more abundant than that of
the gstD21 mRNA (Fig. 1). The transcription rate of the
gstD1 gene, however, was lower than that of the
gstD21 gene (Fig. 4). This suggested that the
gstD21 mRNA is considerably less stable than the
gstD1 mRNA, resulting in a lower steady state level at
The differences in gstD mRNA
stability may be mediated through cis-acting sequence(s) which
could respond to changes in trans-acting factor(s) induced or
activated by PB treatment. For example, the gstD21 mRNA may
have at least one destabilizing sequence which can be recognized by
cellular RNase for rapid degradation under normal conditions (for
recent reviews on mRNA degradation in eucaryotes, see Refs.
46-49). These destabilizing elements could be protected against
degradation by the binding of trans-acting factor(s) induced
or activated by PB.
The relative instability of gstD21 mRNA
under control conditions was probably responsible for a 20-fold lower
steady-state level of gstD21 mRNA than gstD1 mRNA and
a 4-fold lower level of GST D21 than GST D1 in Drosophila adults. Considering the 20:1 ratio in favor of the steady-state
gstD1 mRNA, this 4:1 ratio in the corresponding protein levels
suggested that translation of gstD21 mRNA should be more
efficient than that of the gstD1 mRNA. As a matter of fact,
the sequence around the initiation codon (underlined) in
gstD21 mRNA (ATCCCCAACATGG) is a better fit for Kozak's
consensus sequence (GCCGCCPuCCATGG) than that of the gstD1
mRNA (TACAGTAAAATGG)
(50) . It is also a better fit for the
Drosophila translation initiation site
[(C/A)AA(A/C)ATG] proposed by Cavener
(51) . The
After a 5 h starvation, the control flies were fed with 5% sucrose
only and the PB-treated flies were fed with 5% sucrose containing 50
mg/ml or 200 mg/ml PB.
We thank Diana Cox-Foster for instruction in
Drosophila staging, Donna Carey for producing the polyclonal
antibodies against recombinant GST D1 and GST D21, Susan Abmayr for
providing the rp49 and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
are a family of multifunctional
dimeric proteins which are involved in many detoxification processes.
Abundant and ubiquitous in nature, they are products of a gene
superfamily with overlapping but different substrate specificities.
GSTs catalyze the conjugation of the thiol group of reduced glutathione
(GSH) to a variety of hydrophobic molecules possessing a reactive
electrophilic center (e.g. 1-chloro-2,4-dinitrobenzene,
1,2-dichloro-4-nitrobenzene). The GSH conjugation products, which are
generally less toxic and more hydrophilic, are further metabolized and
subsequently excreted from cells. Certain GSTs also possess
GSH-peroxidase activities capable of reducing organic hydroperoxides.
GSTs also bind a diverse group of endogenous and exogenous lipophilic
compounds with high affinity, reducing their intrinsic cytotoxicity and
facilitating their intracellular
export
(1, 2, 3, 4, 5) .
Drosophila melanogaster and Musca domestica have been
used extensively for studying insecticide metabolism. The best known
detoxification systems of insects are composed of cytochrome P450s,
GSTs, and esterases. Their expressions are inducible by many xenobiotic
compounds, and increased detoxification enzyme activities are important
for developing metabolic resistance in many
organisms
(3, 4, 5, 6, 7) .
Drosophila has at least two families of GSTs, the GST D
isozymes and GST-2, which are products of different genes. They are
immunologically distinct from each
other
(8, 9, 10, 11) . The GST-2 gene has little sequence homology to the gstD genes
(<8-15%), but has sequence homology to mammalian GST Pi genes
and nematode GST genes
(10) . The gstD gene family is
composed of at least eight closely linked intronless genes and
pseudogenes. The amino acid sequence identity of the GST D isozymes
ranges from 53% to 75% in pairwise
comparisons
(12, 13, 14) . They constitute a new
family of GSTs in the GST superfamily
(9, 14) .
receptor channel complex has been recognized as a
major target for barbiturates in their hypnotic effects. Prolonged
channel opening is probably the molecular mechanism for the
potentiation of
-aminobutyric acid action by
barbiturates
(15, 16, 17) . Besides their
neurochemical effects on the GABA
receptor, barbiturates
are also inducers for detoxification enzymes. Phenobarbital (PhB) and
pentobarbital (PB) are the most commonly used inducers for cytochrome
P450s and GSTs (for recent reviews, see Refs. 4 and 18). However,
evidence for direct metabolic conversions or binding of both compounds
by GSTs and/or specific cytochrome P450s is still incomplete. The
molecular mechanism of PhB induction of detoxification enzymes is best
characterized in the rat liver system
(18) . Certain cytosolic
GSTs, cytochromes P450 2B1 and 2B2 in rat liver are inducible by
PhB
(4, 19, 20) . The rapid increase in
cytochromes P450 2B1 (50- to 100-fold) and 2B2 mRNA (20-fold) levels
upon PhB induction was primarily due to increased transcription rates
of the corresponding CYP2B genes
(21, 22, 23, 24) . Although
there has been much investigation on the transcriptional regulation of
many PB-inducible genes, the putative PB receptor(s) and the subsequent
signaling pathway(s) remain poorly understood in eucaryotic organisms
(18).
Materials
Pentobarbital (PB), phenobarbital
(PhB), 1-chloro-2,4-dinitrobenzene (CDNB), and
1,2-dichloro-4-nitrobenzene (DCNB), reduced glutathione (GSH),
S-hexyl GSH-linked agarose, alkaline phosphatase-conjugated
goat anti-rabbit IgG, nitro blue tetrazolium, and
5-bromo-4-chloro-3-indolyl phosphate, were purchased from Sigma.
S-Hexyl GSH-linked Sepharose 6B was prepared according to the
published procedure
(25) . Restriction endonucleases, Random
Primed DNA Labeling Kit, T4 DNA ligase, and phenylmethylsulfonyl
fluoride were products of Boehringer Mannheim. RQ1 RNase-free DNase
(1,000 units/ml), RNase A, RNase T1, and RNasin ribonuclease inhibitor were purchased from Promega Corp. The
[
-
P]dCTP (3,000 Ci/mmol),
[
-
P]UTP (3,000 Ci/mmol), and
I-labeled goat anti-rabbit IgG were products of Amersham
Corp. or ICN. The bacterial expression vector pKK223-3 was purchased
from Pharmacia Biotech Inc. Bacteriological media were products of
Difco Laboratory. Biotrans
nylon membranes, slot-blot
nitrocellulose filters, and Immobilon-P transfer membranes were
obtained from ICN, Schleicher & Schuell, Inc., and Millipore Corp.,
respectively. The Drosophila strain Oregon-R (OR) was obtained
from Dr. Tao-shih Hsieh of Duke University. Flies were maintained under
the standard cornmeal agar medium in half-pint milk bottles at
22-23 °C.
Construction of gstD-specific DNA Probes
The
unique 3`-untranslated regions for gstD1 and gstD21
were subcloned from GTDm101 DNA into plasmid (pUC19) or phagemid
(pBluescript II KS
, Stratagene) and purified
by CsCl gradient centrifugation
(14, 26) . They are 403
bp (13855-14258, ScaI-HindIII) and 283 bp
(8914-9197, TaqI-TaqI) long, respectively. The
complete sequence of
GTDm101 genomic DNA clone (14707 nucleotides)
can be retrieved from the GenBank via accession number M97702. The
probes for the gstD23 and the gstD27 genes are 397 bp
(6631-7028, BamHI-StuI) and 132 bp
(992-1124, HindIII-PstI), respectively. The
pairwise comparison of sequence homology among these probes was
performed by the homology analysis in the DNA Inspector
IIe program (TEXTCO, West Lebanon, NH). No sequence homology was
found with a stringency of 3 mismatches out of a 12-nucleotide window.
The GST-2 probe
(10) was kindly provided by Dr. Eric
Fyrberg of The Johns Hopkins University.
Administration of Pentobarbital (PB) and Phenobarbital
(PhB)
Drosophila adults (3-4 days old) and the
third instar feeding larvae were used throughout this investigation.
Groups of 100 flies were starved for 5-6 h in separate milk
bottles and then fed with filter paper strips soaked in 5% sucrose
containing 0-200 mg/ml PB or PhB. The paper strips were removed
from the bottle right before harvest at various time points. The exact
amount of PB consumed by each fly, however, was not determined under
the experimental condition.
Extraction of Drosophila Total RNAs
RNA extraction
was carried out according to the published procedures with minor
modifications
(27, 28) . The extracted RNA was
resuspended in diethylpyrocarbonate-treated HO and stored
in aliquots at -20 °C. The RNA concentration was determined
by measuring absorbance at 260 nm. The fly materials for these RNA
isolations were processed through the same PB treatment conditions in
four separate milk bottles. The time course of gstD mRNA
induction by PB was carried out by RNA hybridization analyses, using
total RNAs extracted from PB-treated and control flies at various time
points, and as a function of PB dosage. Each data point of the PB
induction curve was the average of values from four independently
extracted total RNA samples.
RNA Blot Hybridizations
These were carried out on
Biotrans nylon membranes with
P-labeled
(>10
cpm/µg specific activity) DNA
probes
(26, 29) . To verify the uniformity of RNA
loadings, the ethidium bromide-stained intensity of rRNA bands were
photographed after electrophoresis. Also, the ribosomal protein
rp49, histone and actin genes
(30, 31, 32) were used as internal
references to normalize against minor variations in RNA loadings and in
subsequent hybridizations. The rp49 mRNA was consistently
present at high levels under all experimental conditions, including all
stages of Drosophila development, and in control or
chemical-treated samples. The actin and histone genes
are two other commonly used internal references for RNA blot
hybridization analyses. This process allowed normalization of the
hybridization signals among samples and calculation of the statistic
mean. Quantitation of the hybridized mRNA signals for each gene was
performed with a
etagen and Betascope 603
analyzer.
The relative abundance of the gstD1 and gstD21 mRNAs
to the rp49 mRNA was determined as the ratios of counts per
min of each band on Northern blot divided by each probe's
specific activity in counts/min/µg. For example, the mRNA ratio of
gstD1/rp49 is calculated as = (Betascope
counts of gstD1 band/counting time/specific
activity)/(Betascope counts of rp49 band/counting
time/specific activity).
Nuclear Run-on Assay
The protocol for isolation of
nuclei was a modification of the published
procedure
(33, 34, 35) . The freshly isolated
nuclei were checked under a microscope for intactness and used
immediately in nuclear run-on assays. An aliquot of isolated nuclei
(2
10
nuclei) was used to start each reaction
at room temperature for 30-40 min in the presence of
[
-
P]UTP (100 µCi at 3,000 Ci/mmol). The
residual DNA in the labeled RNA samples was removed by digestion with 1
unit of RQ1 RNase-free DNase for 20 min at 37 °C followed by phenol
extraction and ethanol precipitation. The radioactive RNA pellets were
resuspended in diethylpyrocarbonate-H
O and used for
hybridization to the same gstD-specific DNA fragments as those
used in Northern analyses. The denatured DNAs (2-5 µg) were
immobilized on nitrocellulose filters with a Minifold II Slot Blot
apparatus (Schleicher & Schuell). After transfer, the DNAs were
UV-cross-linked to the filters, followed by baking in an 80 °C
vacuum oven for 2 h. Hybridization was performed according to the
procedures of McKnight and Palmiter
(36) at 45 °C for 3 days
(1 to 6
10
cpm of
P-labeled RNA) with
gentle shaking. After hybridization, the DNA filters were washed in
buffer E (10 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 2
mM EDTA) containing 0.1% SDS at 45 °C for 2 h and again
with buffer E only at 45 °C for 30 min. The filters were then
incubated with DNase-free RNase A (20 µg/ml) and RNase T1 (1
µg/ml) in buffer E at 37 °C for 30 min to remove unhybridized
RNAs. This is followed by washing of the filters in buffer E containing
0.1% SDS at 45 °C for 1 h. The filters were dried in air before
autoradiography, and the radioactive signals were quantitated by a
Betascope.
P-labeled RNAs isolated from the nuclei of PB-treated and
control flies. The gstD signal ratios from the third
calculations above were normalized against the input (total counts) of
each
P-labeled nuclear RNAs. The three comparisons gave
similar PB induction ratios of the gstD genes. The induction
results calculated from the last method are presented in
I.
GST Activity Assays
Two hundred control or
PB-treated flies were homogenized (20-30 times) in 4 ml of 25
mM Tris-HCl buffer (pH 8.0) containing 1 mM
phenylmethylsulfonyl fluoride at 4 °C. Homogenates were centrifuged
twice at 10,000 g at 4 °C for 30 min. The
supernatant fractions were carefully transferred to new Eppendorf tubes
as the crude extracts. Total protein concentrations were determined by
the bincinchonic acid (BCA) protein assay (Pierce Chemical Co.) using
bovine serum albumin as standard. The GST conjugation activity in crude
extracts was determined with CDNB and DCNB as substrates according to
Habig and Jakoby
(37) at 340 nm and 344 nm, respectively. A
molar extinction coefficient 9,600 M
cm
was used in the enzyme unit calculations.
One unit of conjugation activity is defined as 1 µmol of GSH
conjugate formed per min. Each sample assay was repeated 3-4
times.
Purification of Recombinant GST D1 and GST D21 and
Western Blot Analyses
The GST D1 and GST D21 expression
constructs in pKK223-3 were made by polymerase chain reaction
amplification. The GST D proteins were purified from Escherichia
coli according to previously published
procedures
(38, 39) . Polyclonal antibodies against
recombinant GST D1 and GST D21 were prepared in rabbits by Donna Carey
at The Pennsylvania State University. The different electrophoretic
mobilities of the two GST D proteins and the lack of immunological
cross-reactivities of the two antisera ensured the unequivocal
detection of GST D1 and GST D21 in Drosophila crude extracts.
Levels of the GST D proteins in fly crude extracts were quantitated by
Western blot analyses relative to predetermined amounts of purified
recombinant GST D proteins
(26, 39, 40) .
Induction of gstD Gene Expression by PB
The
steady-state gstD mRNA levels in control and chemical-treated
Drosophila RNA samples were analyzed with the gene-specific
probes by RNA blot hybridization analyses. Among the chemicals tested,
water-soluble sodium pentobarbital (PB, anesthetic barbiturate, t = 15-48 h) and sodium phenobarbital (PhB,
anticonvulsant barbiturate, t = 80-120 h)
(41) were the two most effective inducers for gstD1 and
gstD21 gene expression. They exhibited similar
sedative/hypnotic effects on Drosophila (data not shown) and
induced gstD1 and gstD21 gene expression
(Fig. 1). Since PB is more potent than
PhB
(41, 42) , it was chosen for studying the mechanism
of gstD gene induction in subsequent experiments.
Figure 1:
Induction of the
gstD genes and the GST-2 gene by pentobarbital
(PB) or phenobarbital (PhB) treatment in adult
Drosophila. Total RNAs were isolated from adult Drosophila fed after a 5-h starvation for 2 h on filter paper strips soaked
in 5% sucrose solution (control), 5% sucrose solution containing 200
mg/ml PB, or 5% sucrose solution containing 200 mg/ml PhB. Equal
amounts of total RNAs (50 µg/lane) from four independently
extracted RNA samples were fractionated by gel electrophoresis in 1.5%
agarose in the presence of 2.2 M formaldehyde. Four
identically prepared RNA filters were hybridized separately to the
P-labeled probes for the gstD1, gstD21,
GST-2, histone (not shown),
-tubulin (not shown), and
rp49 genes. The specific activities of these probes were 1.1
10
cpm/µg for the histone probes and 4-6
10
cpm/µg for the other probes. Conditions for
hybridization were described under ``Experimental
Procedures.'' Exposure time was 1 h for the histone probe, 2 h for
the rp49 probe, 5 h for the GST-2 probe, 10 h for the
gstD1 and gstD21 probes, and 4 days for the
-tubulin probe.
Induction
of the gstD1 and gstD21 mRNAs was quantitated after
Northern analyses as a function of PB dosage and duration of PB
treatment. A series of preliminary studies on the time course of
gstD mRNA induction by different concentrations of PB were
carried out. The results indicated that high concentrations of PB
(e.g. 200 mg/ml) were much faster in inducing the
gstD gene expression than low concentrations of PB (e.g. 10 mg/ml) (data not shown). This suggested that the responses were
dosage-dependent. The results of mRNA induction by 200 mg/ml PB and 200
mg/ml PhB after 2-h treatments are shown in Fig. 1. The maximal
increase of the mRNA levels by both compounds was 3-fold for the
gstD1 mRNA and
20-fold for the gstD21 mRNA. On
the other hand, the GST-2 mRNA level was not affected under
the same conditions. After PB treatment, the gstD21 mRNA,
being a minor species under the normal condition, became a major
gstD mRNA species comparable to the uninduced gstD1
mRNA level. Their abundance relative to rp49 mRNA is presented
in . After PB induction, the level of gstD1 mRNA
became 7.6% of the rp49 mRNA whose level is comparable to the
levels of total histone mRNAs. Regarding other gstD mRNAs, the
expression levels of the gstD23 and gstD27 genes were
moderately induced (2-3-fold) by PB (Fig. 3).
Figure 3:
Induction patterns of the gstD
genes in response to PB treatment at the adult and the third instar
feeding larval stages. Northern blot analysis of the total RNAs
isolated from adults and third instar feeding larvae under control or
PB-treated conditions has been described under ``Experimental
Procedures.'' The adults were starved for 5 h before the PB
treatment, while the third instar feeding larvae were treated with PB
without starvation. Equal amounts of total RNAs (50 µg/lane) were
fractionated by gel electrophoresis in 1.5% agarose in the presence of
2.2 M formaldehyde. Identically prepared RNA filters were
hybridized separately to P-labeled probes for the
gstDs, the complete gstD1 cDNA, GST-2,
rp49, and
-tubulin genes. The specific
activities of these gene-specific probes were 2-8
10
cpm/µg. Quantitation of the hybridized mRNA signals
for each gene was performed by a
etagen and Betascope 603
analyzer. The relative mRNA ratios of
gstD1/rp49 were elevated 3-fold for the adults and
1.5-fold for the third instar feeding larvae after PB treatment, while
those of gstD21/rp49 were elevated 20-fold for both
the adults and the third instar feeding
larvae.
The
induction time courses of the gstD1 and gstD21 genes
by PB were analyzed by Northern blotting analyses. One representative
result of PB induction is shown in Fig. 2. The gstD1 and
gstD21 mRNAs were elevated to their respective maximal levels
2 h after PB (200 mg/ml) treatment. At the peak of induction, the
gstD1 mRNA was elevated 3-fold and the gstD21 mRNA
20-fold over their respective levels in control flies. The maximal
levels of induction shown in Fig. 2were nearly identical with
those shown in Fig. 1, demonstrating reproducibility of the
gstD mRNA induction by PB. The gstD1 cDNA probe
hybridized with mRNAs from other members of the gstD multigene
family through homology in their coding regions. Therefore, the
hybridization signals were stronger (lower left panel,
Fig. 2
) than those from the D1 specific probe (upper right
panel, Fig. 2). The differences between the two (lower
right panel, Fig. 2) reflected mainly contributions from
hybridizations to gstD21 mRNAs. Contributions from other
gstD mRNAs should be negligible due to their very low
abundance in adults (Fig. 3). Similar time courses of induction
were observed with 10 or 50 mg/ml PB treatment, except that maximal
induction occurred at 4 h (). The gstD genes were
also inducible by PB at the developmental stage of the third instar
larvae. Their levels of induction were similar to those observed at the
adult stage (Fig. 3), but the mechanism (i.e. transcription or post-transcription) of induction at the larval
stage was not investigated. The basal level of gstD mRNA
expression was generally higher at the larval stage than in adults
(Fig. 3). Nevertheless, Drosophila adults were the
preferred subjects in this study because they were easier to manipulate
in quantity than larvae.
Figure 2:
Induction time course of the
gstD1 and gstD21 genes in response to PB treatment by
RNA blot hybridization analyses. Northern blot analyses were carried
out as those described in the legend to Fig. 1. The specific activities
of the gene-specific probes were 2-8 10
cpm/µg. After counting each band by a Betascope analyzer, the
ratios of gstD1 mRNA/rp49 mRNA and gstD21
mRNA/rp49 mRNA from four independent groups of PB-treated
flies were compared with those of the control flies at each time point.
The normalized induction ratios were plotted against time in
minutes.
Mechanisms of gstD Induction by PB
For intronless
genes, an increase in steady-state mRNAs can be due to an increase in
transcription initiation, increased mRNA stability, or a combination of
the two. To differentiate between transcription and post-transcription
mechanisms, nuclear run-on assays were conducted to measure changes in
transcription rates of the gstD genes in response to PB
treatment. The conditions for PB treatment in nuclear run-on assays
were 200 mg/ml PB for 1.5 h or 50 mg/ml PB for 4 h according to
``Experimental Procedures.'' These conditions were adapted
from the results of Northern analyses on PB induction kinetics with 200
mg/ml PB (Fig. 2) or with 50 mg/ml PB (). The
duration of PB treatment in nuclear run-on assays was, however,
slightly shorter than that for the maximal induction of the
gstD genes in Northern analyses. Results in
Fig. 2
indicated that the rate of increase of gstD mRNA
accumulation in the presence of PB was linear at the 1.5-h time point.
Therefore, it was chosen to be the best time point for preparation of
nuclear run-on assays because the induction would have peaked out at 2
h in the presence of 200 mg/ml PB. Control flies received 5% sucrose
solution only. Results shown in Fig. 4and I
indicate that PB enhanced the rates of transcription initiation of both
gstD1 and gstD21 genes to a moderate extent. Although
the transcription rates of the gstD genes were lower than
those for the actin and histone genes in the control
flies, the gstD21 gene consistently showed a higher
transcription rate than that of the gstD1 gene. However, the
steady-state level of the gstD1 mRNA was at least 20-fold
higher than that of the gstD21 mRNA, which was barely
detectable by Northern analysis (Fig. 1). These results indicate
that the gstD21 mRNA is relatively unstable under control
conditions ( Fig. 1and Fig. 4).
Figure 4:
Effect of PB on transcription initiation
rates of the gstD genes in adult Drosophila. The
P-labeled nascent RNAs were used as probes for the
following gene-specific DNA fragments, gstD1, gstD21,
gstD1 cDNA, rp49, histones, actins,
and the pBluescript plasmid DNA (negative control). The DNAs (5 µg
of each) were immobilized on two identically prepared nitrocellulose
filters. The lengths of the gene-specific DNA fragments were:
gstD1, 403 bp, containing the 3`-untranslated region of
gstD1 gene; gstD21, 283 bp, containing the
3`-untranslated region of gstD21 gene; gstD1 cDNA,
770 bp, containing the entire gstD1 coding region;
rp49, 640 bp, containing the entire ribosomal
protein-49 coding region; histones, 4.8 kb, containing
all five histone genes; actins, 0.94 kb, containing
part of the coding region of an actin gene. Hybridization was
carried out at 40 °C in the presence of 50% formamide with
10
cpm probes for 72 h. The washed filters were dried
in air and exposed for 3 days with an intensifying screen. Calculation
of results is presented in Table III.
The increase in the rate
of gstD1 transcription paralleled the increase in the
steady-state gstD1 mRNA level by PB treatment (
Fig. 1
and Fig. 4, Tables I and III). The rate increase was
sufficient to account for the 3-fold increase of the gstD1
mRNA level. However, a 2-fold increase in the transcription rate of
gstD21 by PB treatment cannot fully account for the 20-fold
elevation in the steady-state gstD21 mRNA. Therefore, the
increase of the gstD21 mRNA was mainly due to an enhanced RNA
stability. Thus, distinct molecular mechanisms are responsible for the
PB induction of Drosophila gstD mRNAs.
Changes in GST D Protein Expression in Response to
PB
Changes in GST D1 and GST D21 protein levels in response to
PB were determined in Drosophila crude extracts using GST D
isozyme-specific antisera by immunoblotting analyses. Although GST D1
and GST D21 share 70% amino acid identities
(14) , the GST D1
antiserum exhibited only weak cross-reactivity with GST D21 on Western
blots. The GST D21 antiserum, on the other hand, exhibited no
detectable cross-reactivity with GST D1 by Western blot analyses.
Furthermore, GST D1 (209 amino acids per subunit) and GST D21 (215
amino acids per subunit) can be separated from each other on SDS-PAGE.
These differences allowed the unequivocal analyses of the GST D1 and
GST D21 proteins in fly crude extracts by immunoblotting procedures.
I
signals at the respective GST D protein bands in 250-µg fly
extracts to the reference signals from the purified recombinant GST D1
and D21 (data not shown). The increase in GST D1 after PB treatment was
consistently 1.5-2-fold over control (Fig. 5). Since GST D1
is by far the most active Drosophila isozyme toward CDNB
conjugation, a comparison of CDNB conjugation specific activity in
adult fly crude extract (0.16 µmol/min/mg) and that of the purified
recombinant GST D1 (58.1 µmol/min/mg in Ref. 39) provided an
independent estimation of its abundance (0.28%). This estimation is
reasonably consistent with the result from immunoblotting (1%). The
CDNB conjugation activity in the crude extracts of PB-treated flies
increased 1.5-fold over that of the control flies (data not shown).
Since the specific activity of GST D1 in CDNB conjugation is 300-fold
higher than that of GST D21
(39) , the 1.5-fold increase of CDNB
conjugation activity in PB-treated extracts mainly reflected an
increase in GST D1 protein. This is consistent with the Western blot
analyses (Fig. 5) where no detectable change in the GST D21
protein level in PB-treated flies relative to controls was observed for
up to 6 h (data not shown). The DCNB activity in the same extract has
no detectable changes.
Figure 5:
Western blot analyses of GST D1 and GST
D21 protein expression in response to PB treatment. A,
SDS-PAGE analyses. Drosophila protein crude extracts were
prepared at different time points from control and PB-treated flies as
described under ``Experimental Procedures.'' Equal amounts of
total protein extract (250 µg/lane) were fractionated by 12%
SDS-PAGE and stained with 0.25% Coomassie Brilliant Blue R-250 to check
for the equal loading of the protein samples. Another two protein gels
were then prepared for electrophoresis under the same conditions. After
electrophoresis, the proteins were transferred onto Immobilon-P
transfer membranes. Affinity-purified recombinant GST D21 (3 µg on
the left and 6 µg on the right sides of the gel) were used to mark
the positions of GST D proteins in the crude extracts. Protein
standards (SDS-7) were included on both sides of the gel and were
marked in kilodalton units. B, Western blot analyses. The
Immobilon-P transfer membranes in A were probed separately
with antibodies against GST D1 and recombinant GST D21 as described
under ``Experimental Procedures.'' The amounts of GST D1
proteins expressed in control and PB-treated crude extracts were
quantitated by incubating the first membrane, previously reacted with
anti-GST D1 antibodies, with the I-labeled goat
anti-rabbit IgG secondary antibodies. The signals on GST D1 proteins
were analyzed by autoradiography. Then, the corresponding GST D1
protein bands were excised and quantitated using a Beckman Gamma 700
counter. The amounts of GST D21 proteins were detected by incubating
the second membrane, previously reacted with anti-GST D21 antibodies,
with alkaline phosphatase-conjugated goat anti-rabbit IgG secondary
antibodies. GST D21 protein bands were visualized by color formation
using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate.
5%
of the gstD1 mRNA. Under PB induction (200 mg/ml, 2 h), the
gstD21 mRNA was elevated 20-fold ( Fig. 1and
Fig. 2
) to become a second major gstD mRNA species. The
ratio of gstD1 mRNA to gstD21 mRNA became close to
3:1 in contrast to 20:1 in control flies. PB apparently induced
gstD gene expression at the levels of both transcription
initiation and mRNA stability. The increase of gstD1
transcription rate after PB treatment was sufficient to account for the
increase of the relatively stable gstD1 mRNA. In contrast, the
20-fold increase in the steady state gstD21 mRNA after PB
treatment cannot be fully accounted for by the 2-fold increase in the
rate of gstD21 gene transcription. Therefore, PB enhanced the
expression of the gstD1 gene primarily at the level of
transcription initiation, but the gstD21 gene activation by PB
was primarily at the level of enhanced mRNA stability. Among 13 other
Drosophila strains tested including a
1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane-resistant
Drosophila strain PSU-R, the gstD21 mRNA was induced
10- to 20-fold with no clearly detectable increase in the GST D21
protein level
(45) .
3-fold induction of the gstD1 mRNA by PB resulted in a
readily detectable increase of GST D1 (Fig. 5) above the
pre-existing control level at 1% of the soluble Drosophila proteins. There was also a corresponding increase of GST
D1-associated CDNB conjugation activity. The presumed better efficiency
of gstD21 mRNA translation and the 4-fold less abundance of
GST D21 than GST D1 under normal conditions would predict that any
increase from newly synthesized GST D21 due to a 20-fold increase of
the gstD21 mRNA be detectable by Western analysis in
Fig. 5
. Therefore, the lack of increase in GST D21 by PB
treatment suggests a mechanism for translational regulation, although
theoretically protein turnover could also contribute to the steady
state level of GST D21. A simple mechanism for simultaneously achieving
gstD21 mRNA stabilization and regulating GST D21 protein
synthesis is by binding of trans-acting factor(s) to the
gstD21 mRNA. Such binding at destabilizing cis-acting
element(s) could not only protect the gstD21 mRNA from
degradation but also prevent its efficient translation (Fig. 6).
Such a mechanism could be an effective mechanism for regulating the
expression of GST D21 in the presence of a 20-fold increase in mRNA. A
second independent stimulus could then relieve the translation
regulation and allow a brief period of increased GST D21 synthesis.
Other models involving compartmentalization of gstD21 mRNA are
also possible mechanisms for consideration.
Figure 6:
Schematic summary of gstD mRNA
and GST D protein expression in response to PB treatment. Bold
arrows indicated relative position and direction of transcription
of each Drosophila gstD gene. The open reading frames were
shown by the shadowed boxes. The complete nucleotide sequence
(14,707 nucleotides) can be retrieved from the GenBank via
accession number M97702 (14). The expression levels of the
gstD mRNAs were indicated by the wiggled lines, and
those of the GST D proteins were indicated by two hemispheres.
The relative number of the corresponding symbols represents the
relative expression levels of the gstD mRNAs and GST D
proteins. The elevations in the expression levels of the gstD
mRNAs and GST D proteins by PB were illustrated quantitatively by the
increase in the number of their corresponding
symbols.
The proposed mechanism
for gstD21 mRNA stabilization by PB and the associated
translation regulation can be tested by identification and
characterization of the putative destabilizing sequence(s) on the
gstD21 mRNA in vivo and its interactions with the
putative PB-induced or activated factor(s) in vitro. The
transcription activation of PB-responsive gstD genes by PB may
be due to certain conserved regulatory strategies employed across
species by way of a conserved trans-acting factor(s) or
conserved ``PB receptor'' itself. The string of 17 base pairs
[ATA(T/G)CNAAAGC(T/A)GG(T/A)GG] in the 5`-flanking regions of
genes encoding barbiturate-inducible cytochromes P450 and P450
from Bacillus megaterium and of
the 5` sequences of genes for barbiturate-inducible cytochromes P450b
and P450e of the rat
(52) are not found in the intergenic region
of gstD1 and gstD21, however. Changes of mRNA
stability of mouse P
450 and P
450 by
tetrachlorodibenzo-p-dioxin in a tissue-specific manner were
reported earlier by Kimura et al.
(53) . Nevertheless,
the Drosophila gstD21 gene provides a much simpler system for
investigating mechanisms on the effect of chemicals (e.g. PB)
on mRNA stability. Drosophila is also likely to be a useful
system for studying the mechanism of gene regulation by PB.
Table:
Relative abundance of the gstD1 and gstD21 mRNAs
Table:
Time course of PB induction of gstD
mRNAs
Table:
Induction of gstD genes by pentobarbital
-aminobutyric acid; PB, pentobarbital; PhB, phenobarbital;
bp, base pair(s).
-tubulin gene probes, Eric Fyrberg
for providing the GST-2 gene probe, and David Gilmour for
providing the actin and histone gene probes. Amy Tang
especially thanks Michael Sypes and Ross Whitwam for editing of her
early versions of this manuscript. We also thank Eileen McConnell for
secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.