©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Androgen Modulation of Multiple Transcription Start Sites of the Mitochondrial Aspartate Aminotransferase Gene in Rat Prostate (*)

Horng H. Juang (§) , Leslie C. Costello , Renty B. Franklin (¶)

From the (1) Department of Physiology, Dental School, University of Maryland at Baltimore, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitochondrial aspartate aminotransferase (mAAT) is one of two key enzymes in the pathway of citrate production in prostate. Expression of mAAT is modulated by testosterone and prolactin in prostate. We cloned the promoter and 5`-flanking region of the rat mAAT gene and sequenced 2.0 kilobases of the DNA. This fragment contains the 5`-regulatory promoter region that lacks a TATA and a CCAAT box but is G+C rich. The 5`-upstream flanking region contains sequences that have high homology with the consensus glucocorticoid response element/androgen response element (ARE) and a reported ARE sequence that is different from the consensus sequence. Functional transcription studies showed that a 481-base region containing the two ARE sequences was sufficient for androgen-regulated gene expression. There are multiple transcription start sites that are regulated by testosterone in prostate. In liver, on the other hand, castration did not affect transcription from any of the start sites. Therefore, these data provide evidence that transcriptional regulation of the rat pmAAT gene occurs through an ARE located in the 5`-region. In addition, not only is gene expression modulated by testosterone, but the effect of testosterone on transcription is cell specific.


INTRODUCTION

An extraordinarily high concentration of citric acid is characteristic of the prostate gland in many mammals. Our studies using rat ventral prostate and pig prostate epithelial cells have shown that aspartate via mitochondrial aspartate aminotransferase (mAAT)() serves as the major source of oxaloacetate for citrate synthesis in prostate. Therefore, mAAT is a key enzyme in the pathway for citrate production by prostate epithelial cells and contributes to the accumulation of citrate in the glandular lumen. The expression of mAAT is regulated by testosterone and prolactin in prostate. We have reported that prostate mAAT activity (1) and biosynthesis (2) are stimulated by testosterone. In addition, we showed that the effects of testosterone (3, 4) and prolactin (5) on mAAT were at the level of gene expression. mAAT is expressed in all tissues, however, it is specifically regulated by testosterone in prostate epithelium but not in other tissues (1) . Therefore, although mAAT is expressed in all cells, its hormonal regulation is cell specific.

To further investigate regulation of mAAT expression and the tissue-specific effects of hormonal stimulation, we cloned the promoter and 5`-untranslated region of the rat mAAT gene. Sequence analysis revealed that the general characteristics (absence of TATA box, G+C-rich content, and the presence of Sp1 sites) of housekeeping gene promoters were present. In addition, the gene contains multiple transcription start sites that are regulated by testosterone in prostate but not in liver. We speculate that the initiation of transcription from multiple start sites contributes to the mechanism of testosterone modulation of expression of mAAT and to the tissue-specific regulation of this housekeeping gene. To identify functional androgen response elements, transient expression experiments were performed using an androgen receptor vector and reporter genes. The results provide evidence that transcriptional regulation of the rat pmAAT gene occurs through an ARE located in the 5`-region.


MATERIALS AND METHODS

Animals and Animal Treatment

Male Wistar rats weighing 250-300 g were used as the source of prostate and liver mRNA. The animals were maintained and sacrificed in accordance with the National Institutes of Health guidelines. The procedures for castration and sham operation were conducted as previously described (6) .

RNA Preparation

Total RNA was isolated from rat prostate and liver using the guanidium thiocyanate extraction method described by Chomczynski and Sacchi (7) . Poly(A) RNA was isolated by oligo(dT)-cellulose chromatography (8) .

DNA Used as Hybridization Probes

A full-length rat precursor mAAT (pmAAT) cDNA cloned into the EcoRI site of the BlueScript II KS phagemid (9) was used. The full-length pmAAT cDNA was digested with PvuII, and the three resulting fragments were separated by agarose gel electrophoresis. A 560-bp EcoRI-PvuII fragment containing the 5`-end of the pmAAT cDNA, a synthetic oligonucleotide representing the first 22 bp of the signal peptide of pmAAT, and the full-length cDNA were used as probes.

Isolation and Sequencing of the 5`-Region of the pmAAT Gene

A SalI partially digested rat liver genomic library in the DASH vector from Stratagene was screened by the plaque hybridization technique (8) . Plaques (8 10) were screened according to the procedures recommended by the manufacturer using the total pmAAT cDNA labeled by random priming. Seven positive clones were isolated by three rounds of screening, and DNA was prepared and characterized by restriction endonuclease mapping. EcoRI fragments of the phage clone inserts were screened by hybridization using the probes described above. Four of these clones contained EcoRI fragments that hybridized with all three probes and probably represent pseudogenes. Only one of the three remaining clones contained an EcoRI fragment that hybridized to the synthetic signal peptide oligonucleotide probe. This 6.4-kb EcoRI fragment was digested using HindIII. The four resulting HindIII fragments were subcloned into BlueScript II KS, and the two 5`-subclones were sequenced according to Sanger et al.(10) .

Primer Extension Analysis

The primer extension reaction was carried out according to a slight modification of the method described by Modaressi et al.(11) . Two 18-base synthetic oligonucleotides (5`-GAAACCGAGGCGCGGGCTCC-3` and 5`-ACCGGAGTGCAGGAGGGC-3`) complementary to positions -80 to -62 and +4 to +22, respectively, were 5`-end labeled with [P]ATP (6000 Ci/mmol) and T4 polynucleotide kinase. The oligonucleotide at position -80 was used to ensure that the start sites identified with the oligonucleotide at position +4 included the most 5`-site. 4 µg of poly(A) RNA were mixed with 0.5 pmol of end-labeled primer in 10 µl of a solution containing 0.4 M NaCl and 10 mM Pipes (pH 6.4) and incubated for 10 min at 75 °C. After addition of 10 units of RNase inhibitor, the hybridization reaction was carried out for 2 h at 56 °C. 20 µl of 5 reverse transcriptase buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl), 20 µl of 10 mM dithiothreitol, 20 µl of 10 mM dNTP mixture, and 10 µl of 1 mM MgCl were added to the reaction solution. The final volume was adjusted to 100 µl by addition of nuclease-free water. The reaction was incubated at 42 °C for 2 h after adding 1200 units of Moloney murine leukemia virus (MMLV) reverse transcriptase. The reaction was stopped by addition of 5 µl of 0.5 M EDTA. The extended DNA was extracted with phenol chloroform and ethanol precipitated. The size of the extended DNA was analyzed by electrophoresis on a 7% polyacrylamide sequencing gel. Aliquots of a sequencing reaction using genomic DNA and the same oligonucleotides as primers were separated on the same gel.

Construction of Transfection Vectors

The 6.4-kb EcoRI fragment containing the 5`-flanking region was digested to remove the 5`-EcoRI-HindIII fragment (Fig. 1). This fragment was then subcloned into the BlueScript II SK phagemid. The resulting recombinant plasmid (pj226H2-1) was digested with PstI and AccI. A pmAAT-CAT chimeric gene (ARE(500)-CAT) was constructed by inserting the 481-bp PstI-AccI fragment of pj226H2-1 into the PstI-AccI sites of the pCAT-promoter plasmid from Promega. A second pmAAT-CAT chimeric gene (ARE(1400)-CAT) was constructed by inserting the PstI-HindIII fragment of pj226H2-1 into the PstI-HindIII sites of the pCAT-promoter plasmid. The rat androgen receptor expression vector (pSV40rAR) was a gift from Dr. Robert Matusik. Construction of this vector has been previously described (12) .


Figure 1: 5`-Region of the rat mitochondrial aspartate aminotransferase gene. A restriction map of the EcoRI fragment of the phage clone containing the 5`-region of the pmAAT gene is shown. The first exon is boxed; A, AccI; B, BstEII; H, HindIII; P, PstI; S, StuI; V, AvaI.



Cell Culture and Transfection

The PC-3 cell line was obtained from the American Type Culture Collection. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4,500 mg/liter D-glucose, 25 mM HEPES, 584 mg/liter L-glutamine, 44 mM NaHCO, and 10% FBS. The medium was changed twice per week. After transfection, the medium was replaced with DMEM containing 2% charcoal-dextran-stripped FBS and either no added steroid or 10-10M 5-DHT. The charcoal-dextran-stripped FBS was prepared as follows. The charcoal-dextran mixture (0.125 g of activated charcoal, 0.125 g of dextran T-70, 0.7 ml of 5 M NaCl, and 225 ml of HO) was vortexed for 5 min and then centrifuged for 10 min at 1000 g. The pellet was transferred to 500 ml of freshly thawed FBS, and the mixture was stirred with a magnetic stirrer at room temperature for 30 min; the solution was then heat inactivated at 56 °C for 30 min. The charcoal-dextran was removed from the FBS by centrifugation at 3000 g for 10 min, and the supernatant was filtered through a 0.22-µm pore filter. The charcoal-dextran-stripped FBS was divided into aliquots and stored -20 °C.

Before transient transfection, PC-3 cells were plated at 1 10 cells per 25-cm culture flask and grown in DMEM with 10% FBS until they reached 60-70% confluence. 1-3 h prior to DNA addition, the medium was replenished with fresh complete medium. The reporter vector and androgen receptor vector were introduced into PC-3 cells by the calcium phosphate-DNA coprecipitation method. A mixture of 50 µl of 2.5 M CaCl, 10 µg of ARE(500)-CAT or ARE(1400)-CAT reporter plasmid, 10 µg of pSV40rAR vector, and 20 µg of salmon sperm DNA in a final volume of 500 µl was prepared. An equal volume of 2 HBS (50 mM HEPES, 0.28 M NaCl, 1.6 mM NaHPO, pH 7.1) was slowly added to the DNA mixture using a 1-ml pipette with vortexing. The mixture was incubated 30-50 min at room temperature and added to the dishes. After 6 h at 37 °C, the DNA-containing medium was removed, and the cells were washed once with phosphate-buffered saline (2.7 mM KCl, 1.5 mM KHPO, 137 mM NaCl, and 16 mM NaHPO, pH 7.3), followed by addition of 15% glycerol in HBS and incubated for 2 min at room temperature. The cells were then washed twice with phosphate-buffered saline and placed in DMEM supplemented with 2% charcoal-dextran-stripped FBS with or without various concentrations of DHT. The Ethanol vehicle was added to the medium of control groups. In each transfection experiment, the treatment and control groups contained three replicate flasks of transfected cells. The cells were incubated for 48 h, after which they were harvested in TEN buffer (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 15 mM NaCl) and assayed for CAT activity.

CAT Activity Assay

The cell extracts were prepared by three cycles of freezing and thawing. Each cycle consisted of 5 min in an ethanol/dry ice bath and 5 min in a 37 °C water bath. CAT activity was determined using the CAT enzyme assay system from Promega. The reaction mixture was composed of 50 µl of supernatant cell extract, 65 µl of 0.25 M Tris-HCl (pH 8.0), 5 µl of [C]chloramphenicol (25 µCi/ml, Amersham), and 5 µl of n-butyryl coenzyme A (5 mg/ml, Pharmacia Biotech Inc.). After incubation at 37 °C overnight, the reaction was terminated by addition of 300 µl of mixed xylenes and vigorous vortexing. The xylene organic phase was extracted twice with 100 µl of TE buffer (10 mM Tris-HCl, pH 7.5, and 1 mM EDTA). 200 µl of the final extracted organic phase were counted in a liquid scintillation counter. CAT activity is expressed as the mean ± S.E. of n-butyryl [C]chloramphenicol dpm for the three replicates in each group.


RESULTS

The 5`-flanking region of the pmAAT gene was isolated by screening a rat genomic library with a 500-bp fragment of the rat pmAAT cDNA (Fig. 1). Fig. 2shows the sequence of the HindIII fragments that comprise the first exon, part of the first intron, and the 5`-regulatory region. The exon and 90 bp of the 5`-flanking sequence are 100% homologous to the 5`-region of the rat pmAAT cDNA. A 200-bp region from -1 to -202 (+1 is A of the ATG codon used for translation) is G+C rich (67% GC) and is characteristic of the promoter in housekeeping genes. This region does not contain a TATA-like element nor a CCAAT box in the G+C rich area but does contain three putative Sp1 sites (Fig. 1). Two sequences that are homologous to the ARE or to the ARE half-site are located at -1460 and -1439. The sequence at location -1439 (GGAAAAGACTGTTCT) differs from the consensus (GGAACANNNTGTTCT) ARE (13) by only one base in the left half-sequence. The other sequence at location -1460 (ATCTTGTTCTGTAG) is very similar to the ARE-2 reported by Rennie et al.(12) for the probasin gene. Thus, the sequences at location -1439 and -1460 could participate in testosterone modulation of pmAAT expression.


Figure 2: Sequence of the 5`-region of the rat mitochondrial aspartate aminotransferase gene. Transcription start sites are indicated by downarrows; Sp1 sites are doubleunderlined; exon 1 (signal peptide) is singleunderlined; putative AREs are boxed. +1 corresponds to the A of the first ATG used for translation. Asterisks are above bases that are different from the cDNA sequence reported by Mattingly et al. (9). All bases that are different are at the end of the cDNA and may be artifacts of cloning the cDNA.



We determined the 5`-boundary of the gene by identifying the transcription initiation sites using primer extension analysis. The primers were labeled at the 5`-end, hybridized with rat ventral prostate poly(A) RNA, and extended by MMLV reverse transcriptase. Primer extension analysis showed the presence of multiple transcription initiation sites. The sizes of the extended products were determined by comparison with the sequencing products of a DNA genomic fragment that was sequenced using the same primer. Seven transcription initiation sites were identified (Fig. 3). The presence of multiple transcription initiation sites has been reported for other housekeeping genes including rat cAAT (14) and mouse mAAT (15) . Consequently, this may be a general characteristic of genes that lack a TATA box in the promoter.


Figure 3: Transcription initiation sites of rat mitochondrial aspartate aminotransferase gene. Poly(A) RNA (4 µg) extracted from rat ventral prostate was extended using MMLV reverse transcriptase. The primer was complementary to the gene sequence at +4 to +22 and was end labeled with P. Arrows indicate locations of transcription initiation sites. Lanes1-4 are sequencing reactions of corresponding DNA (lanes are ACGT, respectively).



Testosterone stimulates the transcription of the mAAT gene (4) . Therefore, we wanted to determine the affect of testosterone on transcription from the multiple initiation sites. Poly(A) RNA was extracted from the ventral prostate and liver of sham-operated, castrated, and castrated + testosterone-treated rats. Fig. 4shows the affect of castration alone and with testosterone replacement on transcription from all of the pmAAT start sites. Castration reduced transcription from all start sites in prostate but did not affect any start site in liver. Transcription from start sites at locations -112, -102, -87, and -60 in ventral prostate were reduced below the level of detection. Transcription from locations -25, -38, and -42, on the other hand, were significantly decreased but were clearly evident. Testosterone replacement restored transcription from all sites to near the sham level.


Figure 4: Effect of testosterone on transcription from multiple transcription initiation sites. Poly(A) RNA extracted from ventral prostate and liver of sham-operated (S), castrated (C), or castrated plus testosterone-treated (T) rats. RNA was extended using MMLV reverse transcriptase and a P end-labeled primer complementary to the gene sequence at position +4 to +22. Arrows indicate transcription initiation sites; PS, prostate sham; PC, prostate castrate; LS, liver sham; LC, liver castrate.



Transcription enhancer activity of the 5`-flanking region of the rat mAAT gene was tested in PC-3 cells by cotransfection with rat AR expression vector and with CAT reporter plasmids containing a 481-bp PstI-AccI fragment or a 1400-bp PstI-HindIII fragment subcloned into the pCAT promoter vector from Promega. To test the AR expression vector, PC-3 cells transfected with AR were treated with DHT, and the mAAT activity was measured. Fig. 5shows that 10 nM DHT significantly increased mAAT activity in AR-transfected cells. Fig. 6presents the dose response of CAT activity to DHT in transiently transfected PC-3 cells. The results demonstrate that DHT in the range of 10-10M caused a 3-4-fold increase in CAT expression. PC-3 cells transfected with the control pCAT promoter (no insert) vector did not respond to DHT at a concentration of 10M (data not shown).


Figure 5: Effect of DHT on mAAT activity of PC-3 cells transfected with rat androgen receptor expression vector. PC-3 cells were transfected with pSV40rAR expression vector and incubated with or without 10M DHT for 24 h. mAAT activity of mitochondrial extracts was determined by activity staining in a 1% agarose gel. Activity is expressed as relative absorbance units determined by densitometer scanning. CONT., control.




Figure 6: Androgen-dependent transcriptional enhancer activities of 5`-flanking region fragments of the pmAAT gene. DHT induction of CAT activity by pmAAT-CAT constructs in PC-3 cells is shown. Fragments (1400 or 481 bp) containing putative AREs were cloned upstream of the CAT reporter in the pCAT vector from Promega. Nucleotide numbering corresponds to Fig. 1. Constructs were cotransfected with pSV40rAR into PC-3 cells. Cells were then grown in the absence or presence of the indicated concentrations of DHT. Brokenline indicates deletion construct.




DISCUSSION

We have isolated the rat pmAAT gene from a phage genomic library. The gene is 30 kb long and contains 11 exons. The first exon is the signal peptide and is separated from the second exon by a large (greater than 10 kb) intron. In this report, we describe a 2.0-kb fragment of the gene that contains the 5`-untranslated region, the first exon, and part of the first intron. The sequence that we determined for this fragment is 100% homologous with the reported sequence for the rat pmAAT cDNA (9) . The sequence of the promoter region is G+C rich (67% from base -1 to -202) and contains numerous CG or GC dinucleotide sequences. Similar CG islands have been reported in the promoter region of the mouse cAAT (16) and mAAT (15) genes, the rat cAAT gene (14) , and other mammalian housekeeping genes (16) . The promoter does not include a TATA box but does contain three putative Sp1 binding sites. Although we did find four CCAAT boxes in the reverse orientation, these were all far upstream of the G+C-rich area and are probably not a part of the promoter. Therefore, we do not believe that these CCAAT elements function in transcription regulation of the rat pmAAT gene.

The sequence of the pmAAT gene is highly conserved between the rat and mouse. In the region from -1 to -226, the two sequences are 90% identical. The Sp1 binding sites at -9 and -88 in the rat gene are at the same positions in the mouse gene. However, in both sequences there is a single base change between the rat and mouse sequences (CCGCCC to CCACCC and GAGGCGTCGT to GAGGTGTCGT). In each case, the change is a purine to purine or a pyrimidine to pyrimidine change. In addition to these two Sp1 sites, another sequence with high homology to the consensus Sp1 sequence GCCCCGCCCA is located at -115. The sequence in the pmAAT gene is GCCACGCCCA. The same sequence is present at the same location in the mouse gene; however, Tsuzuki et al. (15) did not report this sequence as an Sp1 site. Since the change here is purine to pyrimidine, this sequence might not bind Sp1 with high affinity. Additional binding studies are required to determine whether this sequence can function as an Sp1 binding site.

Seven transcription start sites were identified for the rat mAAT gene using primer extension analysis. Multiple start sites have also been reported for other housekeeping genes lacking a TATA box. Pave-Preux et al.(14) identified 5 start sites for the rat cAAT gene, and Tsuzuki et al.(15) identified four sites for the mouse mAAT gene. Therefore, our results are consistent with these other studies. In addition, Schatt et al.(17) reported that the presence of upstream enhancers in the absence of a TATA box resulted in initiation from sites scattered around the promoter region. Consequently, multiple transcription initiation sites may be a characteristic of genes that lack a TATA in the promoter.

We previously reported that testosterone stabilizes mAAT mRNA in rat ventral prostate and pig prostate (4) . This stabilization effect contributes to the testosterone-stimulated increase in the steady-state level of pmAAT mRNA. In addition, the kinetics of mRNA disappearance were not first order and suggested the presence of two populations of pmAAT mRNA. In the present study, castration and testosterone replacement resulted in differential stimulation of transcription from these multiple start sites in prostate. However, castration did not affect expression from any start site in liver. Discrimination among start sites by testosterone could account for a mixed population of pmAAT mRNAs. Moreover, if the half-life of the various pmAAT mRNA species were different due to structural differences, this might account for the lack of first order kinetics that we observed in the disappearance of pmAAT mRNA reported earlier. The intensity of the transcription start sites closest to the translation start codon was greater than the intensity of the most 5`-upstream start site. This difference in intensity was greater in the liver than in the prostate. In addition, the most 5`-upstream start site seemed to be affected by castration to a greater extent than the more 3`-sites.

Utilization of multiple transcription start sites has been reported for other genes. In some cases, transcription initiation from different start sites is tissue specific and may contribute to tissue-specific gene expression (18, 19, 20) . Our observations, however, are different since we observed the same multiple start sites in both prostate and liver in intact rats. In castrated rats, on the other hand, the more distal transcription start sites are not used in prostate but are used in liver. Thus, in this case it is the hormone induction that is tissue specific. Glucocorticoid regulation of multiple transcription start sites for the cAAT gene (14) and hypoglycemic modulation of transcriptional start sites in the preproenkephalin (19) gene have been reported. In the case of cAAT, dexamethasone increased transcription from two of the five transcription start sites 15-18-fold, while transcription from the three other start sites was only increased 3-fold. In addition, dexamethasone also induced transcription from three new start sites that were below the level of detection in the controls. Here, testosterone treatment of castrated rats resulted in induction of transcription from four start sites that were below the level of detection. Thus, if castration represents a condition in which there is a basal level of gene expression in androgen-responsive tissues, then these results suggest that the 5`-distal start sites might be the testosterone-induced sites while the other sites are constitutive. Multiple transcription initiation sites have been proposed as characteristic of ``housekeeping'' genes that show little or no tissue specificity. These results support the suggestion that hormonal regulation of multiple transcription start site usage may provide a mechanism to achieve tissue-specific hormonal responses in so-called ``housekeeping'' genes.

Sequence analysis of the 2.0-kb DNA fragment was carried out to determine if conserved sequence motifs might be involved in androgen induction of pmAAT expression. Steroid regulation of gene expression involves binding of activated steroid receptor to a specific DNA sequence motif (the steroid response element) that enhances transcription of the gene. Among the most studied and best characterized response elements is the GRE that contains the consensus sequence GGTACAnnnTGTTCT. Several studies (12, 21, 22, 23, 24, 25) have revealed that the consensus GRE can also mediate transcription activation by androgen. Studies with androgen-responsive genes (13, 26, 27) have revealed the presence of an ARE sequence that has a high homology with the consensus GRE sequence. Consequently, evidence is mounting to support the concept that the GRE might be characterized as a GRE/ARE. However, studies with the androgen-regulated probasin gene (12) revealed the presence of two putative AREs. Both AREs are required for functional activity and are unresponsive to glucocorticoid.

Two ARE-like sequences were identified in the 1.7-kb 5`-region of the pmAAT gene. One sequence is homologous to the consensus ARE sequence reported by Roche et al.(13) , and the second sequence is similar to ARE-2 in the probasin gene (12) and contains the consensus GRE/ARE right half-site. To determine whether the putative ARE sequences in the pmAAT gene are functional, a 1.4-kb and a 481-bp fragment were tested in transfection studies. The pmAAT-CAT constructs were cotransfected with a rat androgen receptor expression vector. We tested functional androgen receptor expression by determining mAAT activity in response to DHT treatment in PC-3 cells. PC-3 cells have a significantly lower mAAT activity level than LNCaP cells,() and since they do not contain functional androgen receptor, they do not respond to DHT treatment. However, after transfection with the androgen expression vector, mAAT activity was inducible by androgen. In addition, PC-3 cells, which do not accumulate citrate in the culture medium, produced significant levels of medium citrate after transfection (data not shown). Therefore, is it clear that the androgen receptor was functional in the transfected cells.

The pmAAT DNA fragments were cloned into the CAT vector downstream of the SV40 promoter. CAT activity with the larger 1.4-kb construct was increased 3-fold by treatment with androgen with the maximum response seen at 10M DHT. Deletion of this 1.4-kb fragment showed that the 481-bp region containing the putative ARE region was sufficient for androgen-responsive CAT activity. Furthermore, 10M DHT caused CAT induction with the 481-bp construct, while this level of DHT was not effective with the larger 1.4-kb construct. Thus, the functional transcription studies demonstrated that DNA fragments containing the two ARE sequences were capable of enhancing transcription of the SV40 promoter. Moreover, sequence analysis of the 481-bp fragment revealed no other sequences in this fragment with homology to any reported sequence for the GRE/ARE. We conclude that the putative ARE sequences identified in this fragment of the pmAAT gene function as an ARE. Moreover, these results are consistent with cooperativity among AREs as has been described in other genes regulated by androgen (12, 28) . Mutation studies must be carried out to determine whether both ARE sequences are required. Most importantly the current studies continue to support our proposal concerning the mechanism of testosterone regulation of citrate production by prostate epithelial cells. These studies link the important relationship of hormonal regulation of gene expression to a major function (i.e. citrate production) of prostate as described in our recent review (29) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK28015 and DK42839. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21158.

§
Current address: Dept. of Anatomy, Chan Gung College of Medicine and Technology, Tao-Yun, Taiwan, Republic of China.

To whom correspondence and reprint requests should be addressed: Dept. of Physiology, University of Maryland, Dental School, 666 W. Baltimore St., Baltimore, MD 21201.

The abbreviations used are: mAAT, mitochondrial aspartate aminotransferase; pmAAT, precursor mitochondrial aspartate aminotransferase; cAAT, cytosolic aspartate aminotransferase; kb, kilobase pair(s); bp, base pair(s); MMLV, Moloney murine leukemia virus; Pipes, 1,4-piperazinediethanesulfonic acid; ARE, androgen response element; GRE, glucocorticoid response element; DHT, dihydrotestosterone; CAT, chloramphenicol acetyltransferase; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium.

H. H. Juang, L. C. Costello, and R. B. Franklin, personal observations.


ACKNOWLEDGEMENTS

We express sincere appreciation to Dr. S. Biswas and E. Biswas (University of Maryland at Baltimore) for valuable suggestions and assistance. We are indebted to Dr. R. J. Matusik (University of Manitoba) for the androgen receptor expression vector and Dr. J. R. Mattingly (University of Missouri-Kansas City) for the pmAAT cDNA.


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