Identification of three transcriptional regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene

Gene. 167 (1995)255-260 © 1995ElsevierScienceB.V.All tights reserved.037g-1119/95/$09.50

255

GENE 09390

Identification of three transcriptional :regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene (Promoter deletion analysis; DNasc I protection; GC boxes; peripheral-type receptor)

Alessandra Oberto, Patrizia Longone and Karl E. Krueger Fidia-Georgetown Ir~titutefor the Neurosciences, and the Department of Cell Biology, Georgetown UniversitySchool of Medicine, Washington, DC 20007, USA

Receivedby C.M.Kane:9 November1994;Revised/Accepted:1 August/13August1995;Receivedat publisher;:25 September1995

SUMMARY The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene (MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, eleetrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations - 5 1 / - 33, - 2 6 7 / - 249 and - 5 5 5 / - 526. The most distal and proximal domains are positiveacting, whereas the element at - 2 6 7 / - 2 4 9 acts in a negative manner. The positive-acting - 5 1 / - 3 3 element contains the middle of three consensus Spl-recognition sequences found in this region of the gene. Binding of Y1 cell nuclear protein to a DNA fragment corresponding to this region of the gene is competed by a synthetic oligodeoxyribonucleotide bearing the.'consensus Spl-binding site sequence. These studies provide the first reported functional evidence localizing transcriptional elements of M B R .

INTRODUCTION The mitochondrial (mr) benzodiazepine receptor (MBR) is comprised in part by an 18-kDa protein specifying recognition sites having a rather broad but specific pharmacophore for numerous drugs (Bourguignon, 1993). This protein is chiefly present on outer mt membranes (Anholt et al., 1986) and is likely to associate with Correspondence to: Dr. K.E. Krueger, Department of Cell Biology, Georgetown University School of Medicine, 3900 Reservoir Rd., Washington, DC 20007, USA. Tel. (1-202) 687-1094; Fax (!-202) 687-1823;e-mail:[email protected]

Abbreviations:13Gal,[3-galactosidase;bp, basepair(s);CAT,Cm acetyltransferas¢; cat, gene encodingCAT; Cm, chlorampbenicol;EMSA, electrophoreti¢mobilityshiftassay;Exo,exonuclease;kb, kilobasc(s)or 1000bp; MBR, mitochondtial benzodiazepinereceptor; MBR, gene encoding MBR; MMEC, mouse mammary epithelialcells;rot, mitochondrial;oligo,oligodeoxytibonucleotide;Pollk, Klenow(large)fragment of E. coli DNA polymeraseI; tsp, transcriptionstart point(s). SSDI 0378-1119(95)00686-9

other proteins probably involved with mt transport processes (McEnery et ai., 1993). A definitive role of MBR has not been elucidated but strong evidence indicates that this protein plays an important role in intramitochondrial cholesterol transport during steroid biosynthesis (Krueger and Papadopoulos, 1990; 1992) and is coupled to mechanisms affecting mt respiration (Hirsch et al., 1989). The tissue distribution of MBR is very broad but shows a distinct cell.specific pattern of expression (Anholt et al., 1985; Moynagh et al., 1991). Receptor levels are also regulated in a tissue-dependent manner by a number of factors including hormonal regulationand stress (Gavish et al., 1992). The M B R genes from rat (Casalotti et al,, 1992) and human (Linet al., 1993) have recently been cloned. Both representations of this gene have equivalent exon/ intron organizations comprised of four exons where the first intron, which interrupts the 5' untranslated region, constitutes the majority of the primary transcript.

256 Noteworthy features o1 the putative promoter region in both species are the absence of a T A T A box and the conspicuous presence of multiple consensus sequences foi transcription factor Spl. To begin to understand the factors involved with regulating M B R expression and receptor levels, this paper reports the first functional study to identify transcriptional regulatory sequences in MBR.

EXPERIMENTALAND DISCUSSION (a) Promoter deletion analysis in pCAT constructs To determine whether the sequence upstream from the tsp in M B R (Casalotti et al., !992) is ab!c to promote transcription, this region was inserted upstream from the cat gone within the promoterless plasmid peAT-Basic (Promega, Madison, WI, USA). A fragment spanning from 36 bp into the first exon of M B R with about 4 kb of upstream genomic sequence was subcloned in both orientations. When mouse Y1 adrenocortical cells, a steroidogenic line that exhibits high MBR levels (Mukhin et al., 1989), were transfected with the construct containing the 4-kb M B R fragment inserted in the appropriate orientation relative to cat, an eightfold higher level of CAT activity was observed in comparison to cells transfected with either peAT-Basic or the 4-kb fragment inserted in the inappropriate orientation (Fig. 1A). These results verify that this region of the gone is able to direct transcriptional activity. When the more distal 3-kb extent of this region is removed essentially no significant change is found in its ability to drive cat expression (construct 1219/+36 in Fig. 1A). It should be pointed out that in these promoter constructs the association of the tsp with the first 36 bp of the first M B R exon is kept intact, therefore, transcription may proceed via fusion of this untranslated M B R sequence with a portion of noncoding polylinker (MCS) within pC'~tT-Basic before the open reading frame of cat is e n c o ~ ,~~u. No appreciable change in CAT activity was observed with further deletions to -674. As more deletion constructs were tested, three principal regions were found to result in pronounced changes in CAT activity of the transfected YI cells (Fig. IA). Deletion of the region between - 6 2 4 to - 4 2 0 reduced cat exprcssion by at least 50°/'0, whereas a deletion of - 2 5 7 to - 8 8 gave rise to an increase. At locations proximal to - 8 8 there are three GC boxes corresponding to consensus sequences for Spl transcription factor. To look at the potential roles of each of these units selective deletions were made to examine transcriptional activity as each individual GC box was removed. Removal of the most distal GC box exhibited no change in cat expression, although, a deletion that removed only -

part of this first GC box (construct --80/+36 in Fig. 1A) consistently showed a slightly lower activity than constructs with only two or all three GC boxes. Expression of cat dropped substantially upon deletion of the second GC box (construct - 3 4 / + 3 6 ) and a slightly further reduction to just above background levels was observed after the proximal GC box was removed (construct -14/+36). These results suggest that there are at least three transcriptional regulatory elements in the M B R promoter: one negative-acting element between - 2 5 7 to - 8 8 and two positive-acting elements, between - 6 2 4 to - 4 2 0 and - 5 1 to - 3 4 , the latter of which includes a Spl-binding site consensus. We then examined whether these pCATM B R promoter constructs showed a similar transcriptional profile in other mouse cell lines exhibiting lower MBR levels. One feature of MBRs is that virtually all rodent well lines express this receptor. Two other mouse cell lines used for cat transfection studies were 3T3 and MMEC (Telang et al., 1991) cells. Scatchard analyses of specific 13HIPKII195 binding (Le Fur et al., 1983) in cell suspensions indicated that these cells contain about 10 and 0.8 pmol of MBR/mg of protein, respectively. This is in comparison to the density of 50 pmol/mg of protein found in Y1 cells (Mukhin et al., 1989). Relative to the profile observed with YI cells, the effects of different M B R promoter deletions on cat expression showed similarities in 3T3 and MMEC cells (Fig. IB and C), although two differeJ~ce~ are apparent. Deletion of the positive-acting element in the region from - 6 2 4 to -513 did not result in a decrease of CAT activity in MMEC cells. Because this cell line expresses rather low levels of M B R , MMEC cells may lack a positive-acting transcriptional factor binding in this region. Similarly, deletion of the region between -1219 to - 6 2 4 resulted in an appreciable increase in cat expression with 3T3 and MMEC cells, whereas only a small increase was found with Y1 cells. This may be indicative of a negative-acting element within this region. The possibility that Y1 cells are insensitive to this element may account to some extent for the robust MBR levels they express. The data of Fig. 1 show that 3T3 cells produce higher levels of CAT compared to Y1 cells, due in part to their higher transfection efficiency. However, 3T3 cells also seem to exhibit a greater response dependent on upstream M B R sequence• In this respecL the first 8-kb intron may be of potential relevance in transcriptional regulation of M B R . Similar cat expression studies were performed using a construct which includes over 9 kb of M B R sequence starting at - 1219 and proceeding downstream to 3 bp within the second exon. With this pCATM B R plasmid, YI cells demonstrate nearly threefold more CAT activity than 3T3 cells (data not shown), more consistent with the relaUve MBR levels found m these •

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Fig. 1. Transfection with MBR-prnmoter/pCAT vectors. Extents of MBR ~equence spanned by each construct in pCAT-Basic are indicated schematically in panel A. Exon 1 is denoted by a large black box and locations of three consensus Spl-binding sites are depicted as smaller boxes. Note that the bottom construct is in the inappropriate orientation relative to cat. Transfection of YI (A), 3T3 (B) and MMEC (el cells with specific plasmids arc indicated. All data are expressed relative to results using promoterless pCAT-Basic vector. Results are from one representative experiment expressed as means_+SD of triplicate cultures. Methods: To subclone the promoter region of MBR into the polylinker of pCAT-Basic we first used a 2.2-kb Pstl MBR fragment in pBluescript II (Stratagene, La Jolla, CA, USA) spanning from 1219 bp of 5' flanking sequence continuing downstream through the first untranslated exon of 56 bp to 983 bp inside the first intrnn. This vector was cut in the polycloning region with Clal and partially digested with AIwNI, an enzyme that cut within the first exon. Protruding ends of the restricted plasmid were filled in with Pollk followed by blunt-end figation to circularize the plasmid containing only 36 bp of the first exon plus 1219 bp of upstream sequence. After cloning the appropriate recombinant plasmid, the insert of 1255 bp was cut out by PstI + Salt digestion. A second 3-kb Sall-Pstl fragment, derived from another MBR genomic clone, was combined with its contiguous 1255 bp Pstl-Sall fragment allowing them to be joined at their coinciding Pstl sites, subcloned in pBluescript at its Sail clqning site. This insert, represented as including 36 bp of the first exon with over 4 kb of upstream sequence, was isolated after Sail digestion and then subeloned in both orientations at the Sail site of pCAT-Basic. Different promoter deletions were then made by digesting the pCAT recombinant vector - 4 kb/+ 36 (of appropriate orientation) with various enzymes specifying restriction sites located within 1.3 kb of the first exon. After restriction cutting, Pollk fill-in was used to make blunt ends and the plasmids were circularized by ligation with "1"4DNA ligase. The restriction enzymes used to make these selective deletions are indicated as follows: Pstl, - 1219/+ 36; Smal, - 740/+ 36; Apal, -- 624/+ 36; Avrll, -- 513/+ 36: Ahv441, --420/+ 36; EcoRl, -- 317/+ 36; SacL - 257/+ 36: Stul, - 130/+ 36; EcoNL -- 88/+ 36 and BstEii, -- 14/+ 36. Other selective deletions were obtained by linearizing the pCAT vector - 1219/+36 with Stul at position - 130 and digesting with Exo Ill at 7°C. At several time points of digestion the DNA was recovered, digested with HindII! (cutting the pCAT polylinker 5' to the MBR promoter) and the plasmid was reciseularizcd by making blunt ends with Pollk follrawed by ligation. Four constructs were selected from this procedure corresponding to designations --80/+ 36, - 5 I / + 36. --34/+ 36 and - 4 / + 36 as determined by DNA sequencing of each plasmid. Transfections were performed by Ca.phosphate coprecipitation (Chen and Okayama, 1987). In each 100 mm culture dish 20 pg of plasmid DNA. 2.5 pg of the internal control plasmid pSV.13Gal (Promega) and 40 pg of sonicated salmon sperm DNA were used. The lacZ gen¢ was used as an inte:nal reference to monitor transfection efficiency. For constructs containing 4 kb of upstream sequence, 30 pg of plasmid DNA wa.s used to approximate the molar quantities used for the pCAT vectors with considerably smaller inserts. The precipitate was left on the cells for 14 h at 37°C under a 3% COz atmosphere. The cells were then washed twice with sterile Dulbecco's phosphate-buffered saline, refed with fresh medium and incubated an additional 24 h in a 6% CO2 atmosphere. Cells were scraped from the dishes and CAT activity was measured using a kit from Promega based on the acylation of [14C]Cm in the presence of n-botyryl coenzyme A. The reaction products were extracted with a small volume of xylenc and a portion of the organic phase was subjected to liquid scintillation counting. The same extracts of transfected cells were also used to measure ~Gal activity (Sambrook ct al., 1989). Relative units of CAT activity arc normalized to the internal control [3Gal. The specific activities of CAT from cells transfected with promotedess pCAT-Basic vector in these experiments, expressed in nmol/h per mg of protein, arc: Y1, 11; 3T3, 26; MMEC, 1.6.

cells. T h i s s u g g e s t s t h a t t h e first M B R i n t r o n m a y c o n t a i n additional transcriptional regulatory elements not cons i d e r e d b y t h e p r e s e n t studies.

n u c l e a r e x t r a c t s f r o m Y1 cells. F r a g m e n t s A, B a n d C , s h o w n in F i g . 2, w e r e f o u n d t o b e e l e c t r o p h o r e t i c a l l y r e t a r d e d w h e n p r e i n c u b a t e d w i t h Y1 n u c l e a r e x t r a c t s . I n c o n t r a s t , p r o b e F d i d n o t e x h i b i t a shift in m o b i l i t y . T h e s e

(b) EMSA analysis of upstream regions

results a r e in a g r e e m e n t w i t h t h e a f o r e m e n t i o n e d d e l e t i o n

T o d e t e r m i n e w h e t h e r p r o t e i n - b i n d i n g e l e m e n t s exist in t h e u p s t r e a m s e q u e n c e e x a m i n e d b y d e l e t i o n a n a l y s i s of MBR-pCAT constructs, EMSA was performed using

a n a l y s i s w h i c h s u g g e s t e d t h a t p r o m i n e n t r e g u l a t o r y elements should be present within the regions spanned by t h e first t h r e e p r o b e s .

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Fig. 2. EMSA analysis of upstream MBR sequence.The upper panel depicts the various MBR fragments used as probes and competitors. Consensus Spl-binding sites are indicated as triangles. Reference locations of designated restriction sites are indicated for clarity. The competitor signified as Spl is the double-stranded oligo $'-A'FI'CGATCGGGGCGGGGCGAGC (Promega). Metheds: Crude nuclear extracts were prepared from YI cells according to Dignan et al. (1983) with modifications(Costa et al., 1988). DNA fragments were prepared by digestion with the corresponding restriction eodonuclcases of the MBR promoter. After agarose gel separation and purification with Q1AEX resin (Qiagen~Chatswonh, CA, USA), DNA probes were labeled with [¥-s2P]ATP in the presence of T4 polynucleotide kinase. EMSA experiments were performed as described elsewhere (Grayson et al., 1988). Prutein-DNA binding reaction mixtures (20 Id), containing 1 ng (approx. 5 × 104 dpm) of 3zp_ end-labeled DNA probes and lO pg of crude nuclear extract, were incubated in the presence 4 pg of [poly(dI-dC).poly(dl-dC)] in 20 mM HEPES-NaOH (pH 7.6)/40mM KCI/2 mM MgOa/l mM dithiothreitol/4% Ficoll. Unlahelled competitors were added at 100-foldexcess over probes. Incubations were carded out at room temperature for 30 rain and ¢lectrophoresis was performed on non-denaturing 4% polyacrylamide gels at 4~C.

Specificities of the proteins binding to these probe sequences were demonstrated using different fragments as competitors. For example, probe A was competed by itself, by fragment D which still included two consensus S p l - b i n d i n g sites, and by a synthetic oligo containing a S p l consensus. Fragment B, which lacked the S p l consensus sequence but included the more distal extent of probe A, was not a n effective competitor. These results suggest that within sequence spanned by probe A one or more of the consensus Spl-binding sites appears to

account for a protein-binding e~ement. By the same reasoning, probe B was competed by itself but not by fragments A and E suggesting that a protein-binding d o m a i n is located between nt - 3 1 7 and - 2 5 7 . Similarly, probe C was competed by itself and by fragment G but not by fragment F implying that the region between nt - 6 2 4 and - 5 1 3 corresponds to another protein-binding element recognized by other positive-acting transcription factors different than those recognized in fragment A. The localization of these protein-binding regions is in good

259 coherence with the results obtained using pCAT constrncts in promoter deletion analysis. EMSA using nuclear extracts from MMEC cells gave similar results with the exceptions that a retardation ef probe C was more difficult to detect and one of the hands observed with probe A was absent (data not shown). (c) DNase I protection analysis To more definitively localize the protein-binding sites within the MBR promoter, DNase I protection assays were performed on the appropriate DNA fragments. Nuclear exiracts from YI and 3T3 cells gave similar, but not identical, protection patterns as shown in Fig. 3. The

Probe A

footprinting pattern within probe A was found to localize over about 19 bp ( - 51/-33) which surrounds the second consensus Spl-binding site distant from the tsp. Within probe B a region of about 18 bp ( - 2 6 7 / - 2 4 9 ) was protected. This region contains a SacI site and corresponds to a negative-acting element as determined by promoter deletion analysis. Concerning the most distal positiveacting clement, a region of about 30bp ( - 5 5 5 / - 5 2 6 ) exhibited protection with enhanced DNase I sensi:ivity at its boundaries. Comparing these two cell lines, there are several details of these fragmentation patterns where differences are observed within or flanking the major protected areas. Footprinting patterns observed from

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Fig. 3. DNase I footprinting analysis. Probes A, B and C from Fig. 2 ~vere tested in protection assays with nuclear extracts from YI and 3T3 cells. The same probes, subjected to standard chemical sequencing procedures (Sambrook et al., 1989), were co.electrophoresed to correlate the fragmentation pattern with known MBR sequence of the sense strand as indicated to the right of each panel. The ranges of upstream sequence covered in each image are given at the ends of each sequence. For the left panel, consensus Spl-binding sites are in lowercase letters. Regions showing either protection or hypersensitivity to DNase I are enclosed within a white outline with the respective DNA sequences inzluded within brackets. Methods: DNAbinding reactions were carried out in a 100 Itl volume containing about 5 fmol ofend.labded DNA fragment (approx. 2 x 104 dpm), 1.5 I~gof unlabeled carrier DNA [poly(dl-dC).poly(dl-dC)] and from 40 to 80 Ilg of crude nuclear extract in 20 mM HEPES-NaOH (pH 7.6)/40 mM NaCI/2 mM CaCl2/5 mM MgCIz/l mM dithiothreitol/10% glycerol. Samples were first incubated for 30 rain at room temperature. Then 1 Id of DNase I (50-100 pg/ml), freshly diluted in l0 mM HEPES-NaOH (pH 7.6)/25 mM CaCIjI00 pg bovine serum albumin per mL was added and digestion of the DNA-protein complexes was allowed to proceed for 1 4 rain at room temperature. The digestion reactions were stopped by the addition of 100 Ill of 2% sodium dodccy! sulfate]10 mM EDTA (pH 8)/250 pg per ml of tRNA. Samples were then extracted twice with phenol-chioroform-isoamyl alcohol 125:24;i), precipitated with ethanol and loaded onto a 7 M urea-6% polyacrylamide DNA sequencing gel for analysis.

260 M M E C n u c l e a r extracts, p a r t i c u l a r l y for p r o b e A, resembled m o r e closely those o f 3T3 cells (data n o t shown). These differences are likely to reflect, in part, the t r a n scriptional basis b y w h i c h these cell lines exhibit differing levels o f M B R . (d) Conclusions (1) T h e sequence s p a n n i n g 600 b p u p s t r e a m f r o m the first exon in rat MBR c o n t a i n s at least three elements ( - 5 1 / - 33, - 2 6 7 / - 249, - 5 5 5 / - 526) involved in t r a n scriptional r e g u l a t i o n o f this gene. T h e m o s t p r o x i m a l a n d distal elements act in a positive fashion whereas the t h i r d is a negative-acting element. (2) T h e positive-acting element closest to the tsp contains a c o n s e n s u s S p l - b i n d i n g site t h a t seems p a r a m o u n t for its interaction with the c o r r e s p o n d i n g D N A - b i n d i n g protein(s). T h e locations of the o t h e r t w o sites occupied b y o t h e r D N A - b i n d i n g proteins h a v e likewise been identified b y D N a s e I protection. Definitive identification of the transcriptional r e g u l a t o r y proteins binding to these three p r o m o t e r elements will require further investigation.

ACKNOWLEDGMENTS This w o r k was s u p p o r t e d by the Fidia Research F o u n d a t i o n a n d N I H g r a n t M H 4 4 2 8 4 to K.E.K.

REFERENCES Anholt, R.R.H., DeSouza, E.B., Oster-Granite. M.L. and Snyder, S.H.: Peripheral-type benzodiazepioc receptors: autoradiographic localization in whole-body sections of neonatal rats. J. Pharmacol. Exp. Ther. 233 (1985) 517-526. AnholL R.R.H., Pedersen, P.L., DeSouza, E.B. and Snyder, S.H.: The peripheral-type benzodiazepine receptor:. Localization to the mitocbondrial outer membrane. J. Biol Chem. 261 (1986) 576-583. Bourguignon, J.-J.: Endogenous and synthetic ligands of mitochondrial b~nzodiazepine receptors: structure-affinity relationships. In: Giesen.Crouse, E. (Ed.L Peripheral Iknzodiazepine Receptors. Academic Press, London, 1993, pp. 59-85.

Casalotti. S.O. Pclaia. O. Yakovlev, A.G. Csik6s. T~ Grayson, D.R. and Krucger, K.E.: Structure of the rat gene encoding the mitochondrial benzodiazepine receptor. Gone 121 (1992) 377-382. Cheu, C. and Okayama, H.: High-efficiencytransformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7 (1987} 2745-2752. Costa, R.H, Eseng, L., Grayson, D.R. and Damell, J.E.: The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specificfactor(s) at two other sites. Mol. Cell. Biol. 8 {1988) 81-90. Dignan, J.D., Lebovitz, R.M. and Roeder, R.G.: Accurate transcription initiation by RNA polymerase Ii in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11 (1983) 1475-1489. Garish, M., Katz, Y., Bar-Ami, S. and Weizman, R.: Biochemical, physiological, and pathological aspects of the peripheral bcnzodiazepine receptor. J. Neurochem. 58 (1992) 1589-1601. Grayson, D.R., Costa, R.H., Xanthopoulos, K.G. and Darnell, J-E.: A cell-specificenhancer of the mouse ~tl-antitrypsin gene has multiple functional regions and corresponding protein-binding sites. Mol. Cell. Biol. 8 (1988) 1055-1066. Hirseh, J.D., Beyer, C.F, Malkowiv~ L., Beer, B. and Blume, AJ.: Mitochondrial benzodiazepine receptors mediate inhibition of mitochondrial respiratory control. Mol. Pharmacol. 35 (1989) 157-163 Krueger, K.E. and Papadopoulos, V.: Peripheral-type benzodiazepine recepto~ mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J. Biol. Chem. 265 (1990) 15015-15022. Krueger, K.E. and Papadopoulos. V.: Mitochondrial benzodiazepine receptors and the regulation of steroid biosynthesis. Annu. Rev. PharmacoL Toxicol. 32 (1992) 211-237. Le Fur, G, Perrier, M.L., Vaucher, N., Imbault, F., Flamier, A., Uzan, A., Renault, C., Dubrocucq, M.C. and Gueremy. C.: Peripheral benzodiazepine binding sites: effect of PKI 1195 {142-chlorophenyl)-N-methyI-N-(l-methyl-propylb3-i soquinoline-carboxamide, 1. In vitro studies. Life Sci. 32 (1983) 1839-1847. Lin. D., Chang, YJ., Strauss IlL J.F. and Miller, W.L: The human peripheral benzodiazepine receptor gene: cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia. Genomics 18 (1993) 643-650. McEnery, M.W., Snowman, A.M., Trifiletti, R.R. and Snyder, S.H.: Isolation of the mitochondrial bcnzodiazepine receptor:,association with the voltage-dependent anion channel and the adenine dinucleotide carrier. Proc. Natl. Acad. Sci. USA 89 (1992) 3170-3174 Moynagh, P.N., Bailey, CJ.. Royce, SJ. and Williams, D.C.: hnmunological studies on the rat peripheral-type benzodiazepine acceptor. Bio:hem. J. 275 (1991) 419-425. Mukhin, A.G., Papadopoulos, V., Costa, E. and Krueger, K.E.: Mitochondrial benzodiazepine receptors regulate steroid biosynthesis. Proc. Natl. Acad. Sci USA 86 (1989) 9813-9816. Sambrook, J., Fritseh, E.F. and Maniatis. T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, 1989. Telang, N.T., Narayanan, R., Bradlow, H.L. and Osborne, M.P.: Coordinated expression of intermediate biomarkers for tumorigenic transformation in RAS-transfected mouse mammary epithelial cells. E'~-eastCancer Res. Treat. 18 (1991) 155-163.