©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pentobarbital-induced Changes in Drosophila Glutathione S-Transferase D21 mRNA Stability (*)

Amy Hong Tang (§) , Chen-Pei D. Tu (¶)

From the (1) Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Glutathione S-transferases (GSTs, EC 2.5.1.18)() 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) .

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 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).

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.


EXPERIMENTAL PROCEDURES

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-HO 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.

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 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) .


RESULTS

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.

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 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.




DISCUSSION

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 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) .

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 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 P450 and P450 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

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.



FOOTNOTES

*
The project described was supported by Biomedical Research Support Grant 2S07 RR07082 and in part by Grant ES 05661 from NIEHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: 539 Life Science Addition Bldg., Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200.

To whom correspondence and reprint requests should be addressed: 401 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802. Tel.: 814-863-2096; Fax: 814-863-7024.

The abbreviations used are: GSTs, glutathione S-transferases; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; GSH, reduced glutathione; PAGE, polyacrylamide gel electrophoresis; GABA, -aminobutyric acid; PB, pentobarbital; PhB, phenobarbital; bp, base pair(s).


ACKNOWLEDGEMENTS

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 -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.


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