Rat Brain Preproenkephalin mrna

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1 THE JO~JRNAL OF BOLOGCAL CHEMSTRY Vol. 259, No. 22. ssue of November 25, pp ,1984 Printed in U.S.A. Rat Brain Preproenkephalin mrna cdna CLONNG, PRMARY STRUCTURE, AND DSTRBUTON N THE CENTRAL NERVOUS SYSTEM* (Received for publication, April 25, 1984) Kazuaki Yoshikawa, Christianna Williams, and Steven L. Sabol From the Laboratory of Biochemical Genetics, National Heart, Lung, and Blood nstitute, National nstitutes of Health, Bethesda, Maryland 225 A cdna-clone library was constructed from polyadenylated RNA of Fischer rat striatum and screened for inserts coding for the enkephalin precursor pre- proenkephalin. The insert of one positive clone, prpe2, wasequenced and found to contain coding the sequence (81 bases), as well as 316 and 155 bases of the 3 and 5 untranslated regions, respectively, of rat preproenkephalin mrna. The primary structure of rat preproenkephalin (269 amino acids, M, 3,932) is similar to those of previously sequenced bovine and human preproenkephalins (78 and 82% matched residues, respectively), and contains four copies of Met-enkephalin, one of Leu-enkephalin, one of Met-enkephalin- Arge-Gly7-Leus, and one of Met-enkephalin-Arge- Phe. Cell-free translation of rat striatal mrna selected by hybridization with prpe2 DNA resulted in the synthesis of a 31,-Da protein. Southern analysis of rat genomic DNA with 32P-labeled prpe2 frag- ments is consistent with a single preproenkephalin gene. A 32P-labeled prpe2 fragment hybridized specifically with preproenkephalin mrna (approximately 15 bases) on Northern blots of polyadenylated or total RNA of all brain regions but not liver. Relative abundances of preproenkephalin mrna in total RNA of specific regions of the rat central nervous system, determined by a sensitive dot-blot hybridization assay, had the following order: striatum >> hypothalamus, pons-medulla, spinal cord > cerebellum, midbrain, frontal cortex > hippocampus, thalamus. The prpe2 probe is useful for the analysis of the dynamics of preproenkephalin mrna in rat neurons. The search for endogenous opiate-receptor ligands in the brain led to the characterization in 1975 of the pentapeptides methionine- (Met5-) and leucine- (Leu5-) enkephalin (), which subsequently have been detected throughout the nervous system and in the adrenal medulla (reviewed in Refs. 2 and 3). The enkephalins are now known to be part of a family of structurally related peptides derived from a precursor protein proenkephalin (or proenkephalin A). From the characterization of these peptides from bovine adrenal medulla (3), as well as sequencing of cloned cdna for bovine and human adrenomedullary preproenkephalins (4-7), it is apparent that each precursor molecule can be processed to generate four copies of Met-enkephalin (Tyr-Gly-Gly-Phe-Met), one of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu), one of Met-enke- phalin-arg6-phe7, and one of Met-enkephalin-Ar$-Gly7-Leu8. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked oduertisernent in accordance with 18 U.S.C. Section 1734 solely to indicate this fact Extended enkephalin-like peptides, such as peptides E and F (3) and Met-enkephalin-Arg6-Arg7-Va18-NH2 (8, 9), are also derived from this precursor by incomplete or alternative processing. Because bovine and rat brain contain these peptides, other proenkephalin fragments (1-12), and mrna similar to adrenal preproenkephalin mrna (13-15), it has been assumed that the same preproenkephalin gene is expressed in the nervous system and the adrenal medulla. However, preproenkephalin mrna from nervous tissueper se has not been previously sequenced. Little is known about the regulation of preproenkephalin gene expression in the nervous system. The rat brain and spinal cord are important tissues for studies addressed to this problem, because most information about the localization and physiological functions of enkephalins, other opioid peptides, and opiate receptors is being obtained in the rat (reviewed in 2, 16). Because of the evolutionary distances separating the rat, human, and bovine species, the structure of rat preproenkephalin and characteristics of its gene expression may differ from those of preproenkephalins of other species. Thus, there is a need for information concerning the sequences of rat neuronal preproenkephalin mrna and its translation product, the number of gene(s), and the distribution of the mrna in the rat nervous system. Furthermore, a sensitive and specific assay for preproenkephalin mrna in small amounts of rat brain tissue is required. n an effort to provide necessary information as well as ideal probes for the study of the regulation of rat brain preproenkephalin gene expression, we report the molecular cloning and sequencing of rat brain preproenkephalin cdna. We also report the use of fragments of the clone as hybridization probes to determine the number of preproenkephalin genes in rat and to determine relative amounts of the mrna in various regions of the rat central nervous system by blothybridization analysis. EXPERMENTAL PROCEDURES Materials-Ribonuclease inhibitor (RNasin) was obtained from JEM Research, nc., Kensington, MD. Terminal deoxynucleotidyl transferase and T4 polynucleotide kinase were from P-L Biochemicals. Avian myeloblastosis virus reverse transcriptase was from Dr. J. Beard, Life Sciences, nc., St. Petersburg, FL. Oligo(dG)-tailed Pst-cut plasmid pbr322 was from Bethesda Research Laboratories. Restriction endonucleases were from New England Biolabs or nternational Biotechnologies, nc., except for Cpf from Worthington Biochemicals. [a-32p]dctp(28 Ci/mmol), [a-32p]cordycepin 5 - triphosphate (6 Ci/mmol), [T-~*P]ATP (29 Ci/mmol), and [35S] methionine (11 Ci/mmol) were from New England Nuclear. Escherichia coli 294 cells harboring plasmid phpe-9, which con- tains a human pheochromocytoma preproenkephalin cdna insert (17) was a generous gift of Drs. M. Comb and E. Herbert (University of Oregon, Eugene, OR). Plasmid was purified from bacterial lysates by CsC1-ethidium bromide density-gradient centrifugation. Tissue Preparation-Adult male Fischer-344 rats (2-25 g) were

2 1432 Preproenkephalin Rat Brain mrna killed in a saturated-c2 chamber. Brains were immediately dissected by the method of Glowinski and versen (18) with the following modifications: The thalamus (plus subthalamus) was separated from the midbrain by a transverse section between the posterior hypothalamus and mammillary bodies. The striatum was divided into two parts at the level of the optic chiasma, and only the anterior part was used, unless otherwise stated. The entire spinal cord was also isolated. The tissues were quickly frozen on dry ice and stored at -18 "C. Preparation of RNA-Total RNA was extracted from frozen tissue by homogenization with 4 M guanidinium thiocyanate and purified by selective ethanol precipitation from 8 M guanidinium chloride (19). Poly(A)+ RNA' was purified by two cycles of affinity chromatography on oligo(dt)-cellulose (Type 7, P-L Biochemicals). Preparation of Rat Striatum cdna Library-Double-stranded rat striatum cdna was prepared by the method of Land et al. (2). The reaction mixture for first-strand cdna synthesis was modified by the inclusion of 5 units/ml of ribonuclease A inhibitor (RNasin) and extension of the incubation time to 9 min. A portion of the final product, oligo(dc)-tailed double-stranded cdna,was annealed to oligo(dg)-tailed Pst-cut pbr322 DNA and used to transform E. coli C6 cells according to a procedure of D. Hanahan (21). Transformants were selected on LB agar plates containing 12.5 pg/ml tetracycline. All experiments with recombinant DNA were conducted with P1 containment according to the National nstitutes of Health guidelines for recombinant DNA research. dentification of Rat Preproenkephnlin cdna-containing Clones- Tetracycline-resistant transformants were screened by colony hybridization (22, 23). Filters were incubated for 16 h at 3 'C in a solution containing 5% formamide, 5 X SSPE (1 X SSPE =.18 M NaC1,lO mm sodium phosphate, 1 mm EDTA, ph 7.7), 7.5% dextran sulfate, 2 X Denhardt's solution (1 X Denhardt's =.2% each of bovine serum albumin, Ficoll-4, and polyvinylpyrrolidone), 1 pg/ml herring sperm DNA,.1% sarcosyl, and 1.1 ng/ml 918-bp Hinc fragment of human preproenkephalin plasmid phpe-9 (1.1 X 1' cpmlpg), labeled by nick translation with [CX-~~PJ~CTP. Putative positive clones were picked and purified by recloning. Plasmids were purified from bacterial lysates by CsC1-ethidium bromide equilibrium density-gradient centrifugation. Plasmid DNA Restriction Analysis and Sequencing-Plasmid DNA was digested with restriction endonucleases according to conditions specified by the manufacturer(s). Restriction fragments were visualized by ultraviolet light after electrophoresis in % agarose gels containing.89 M Tris-borate, ph 8.3,.1 M EDTA,.5 pg/ml ethidium bromide. Fragments were isolated by agarose gel electrophoresis, eluted from gels by either electroelution or diffusion from crushed gel pieces, and usually purified on NACS-Prepac columns (Bethesda Research Laboratories). DNA sequencing was performed according to the procedures (reactions R5-R9) of Maxam and Gilbert (24). Protruding 3' ends were labeled with [c~-~'p]cordycepin 5"triphosphate and terminal deoxynucleotidyl transferase as described (25); 5' ends were labeled with [Y-~'P]ATP and T4 polynucleotide kinase (24). Sequences were analyzed by the SEQ program (Stanford Molgen Group) and the NU- CALN and PRTALN programs (26) on the DEC-1 computer at the National nstitutes of Health Computer Center. Hybridization Selection and Translation of mrna-equimolar amounts, 1 pg and 7.7 pg, of EcoR-linearized prpe2 and pbr322 plasmids, respectively, were bound to 3 X 3 mm nitrocellulose membranes as described (27). Each filter was incubated for 17 h at 5 "C with.2 ml of a solution containing 2 pg of rat striatum poly(a)+ RNA, 65% formamide,.2 M 1,4-piperazinediethanesulfonic acid (ph 6.4),.2% sodium dodecyl sulfate,.4 M NaC1, 1 pg/ml calf liver trna. Each filter was washed, and RNA was eluted and purified as described (27). The RNA was translated in the rabbit reticulocyte lysate system as described previously (14). Translation products were subjected to immunoprecipitation with anti-met-enkephalin-arg'- Phe' serum, as described previously (14), except that 5 pl of a different antiserum (MEAP-, a gift of Dr. J. S. Hong, National nstitute of Environmental Health Sciences, Research Triangle Park, NC) was employed. n radioimmunoassays performed by us, this antiserum, tested at 1/5 dilution, was found to have a high affinity for Metenkephalin-Arg6-Phe7 with <.18% cross-reactivity with Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg', Met-enkephalin- Arg6-Gly7-LeuS, &endorphin, P-lipotropin, or dynorphin-[l-13]. m- The abbreviations used are: Poly(A)+ RNA, polyadenlyated RNA bp, base pair. munoprecipitates were characterized by electrophoresis on a.75-mm thick sodium dodecyl sulfate-11.3% polyacrylamide slab gel (28) followed by fluorography as described (14). Southern Blot Analysis-DNA was purified from the cerebral cortex (2.7 g) of Fischer-344 rats according to the method of Davis et al. (29) with the following modification: DNA was treated with 1 pg/ ml DNase-free RNase A (Millipore) for 2 h at 37 "C in 1 mm Tris- HCl (ph 7.51, 1 mm EDTA, and 1 mm NaC1.DNA was phenol extracted, ethanol precipitated, and finally dissolved in 1 mm Tris- HC1 (ph 7.51, 1 mm EDTA. The yield was.26 mg/g of tissue. The DNA (1 pg) was digested with restriction endonucleases (2-4 units) for 17 h at 37 "C, and electrophoresed on a 1.% agarose gel for 16 h at 2 V/cm).The DNA was subsequently partially depurinated in situ with.25 M HC1 for 1 min at 2 "C, denatured, and transferred to a nylon membrane (Genescreen, New England Nuclear) by the capillary blot method (3) with 1 X SSC buffer (1 X SSC =.15 M NaC,.15 M sodium citrate, ph 7). The membrane was incubated for 7 h at 36 "C in 1 ml of buffer H (buffer H = 5% formamide, 1 X Denhardt's solution,.5 M Tris-HC1 (ph 7.5), 1 M NaC1, 3.75 mm N~PzO~, 1% sarcosyl, 1% dextran sulfate, and 1 pg/ml denatured herring sperm DNA). The probe DNA, 513- and 417-bp Pst fragments of prpe2 labeled by nick translation with [cx-~'p]~ctp, was then added to a final concentration of 1 ng/ml, and the membrane was hybridized for 17 h at either 36 or 3 C. The membrane was washed twice with 1 ml of 2 X SSPE buffer,.1% sarcosyl, and twice with 1 ml of.1 X SSPE,.1% sarcosyl for 1 min at 2 "C, then autoradiographed with Kodak XAR-5 film at -85 "C in the presence of an intensifying screen. Northern Blot Preparation-RNA was denatured in 5% formamide, 6% formaldehyde for 15 min at 55 "C and electrophoresed in a 1.3% agarose gel containing 6% formaldehyde (31). The gel was soaked in 1 mm sodium phosphate buffer (ph 7), and the RNA was transferred to a Genescreen membrane by the capillary blot procedure with 1 X SSC buffer. The membrane was washed with 1 X SSC for 3 min, air dried, and baked at 8 "C for 2 h. Dot-blot Preparation-Total RNA or poly(a)+ RNA was serially diluted, lyophilized, and denatured in 2 p1 of 7.4% formaldehyde, 6 X SSC for 15 min at 6 "C(32), then diluted with 8 pl of 1 X SSC and filtered over a 2-h period by gravity flow through a Genescreen membrane placed in a filtration manifold (Bethesda Research Laboratories). The membrane was washed with 2 X SSC at 22 "C for 3 min, air dried, and baked at 8 "C for 2 h. Hybridization ofrnablots-a 941-bp Bsp1286 fragment was isolated from the prpe2 plasmid and nick translated with [cy-~'p] dctp to specific radioactivities indicated in the figure legends. Each blot was incubated for 7-19 h at 42 "C with 1 ml of buffer H, then incubated with 1 ng/ml probe DNA in buffer H for h at 42 "C. The membrane was washed twice with 1 ml of 2 X SSC,.1% sarcosyl, and twice with 1 ml of.1 X SSC,.1% sarcosyl for 1 min each time at 2 "C, and finally once with 2 ml of.1 X SSC,.1% sarcosyl for 3 min at 5 "C. The membrane was then subjected to autoradiography with Kodak SB-5 film at -85 "C with an intensifying screen. RESULTS solation and Characterization of Rat Brain Preproenkephalin Clones-Poly(A)' RNA from rat striatum (caudate, putamen, and globus pallidus) was used to construct a cdna library in the Pst restriction site of the plasmid pbr322. A clone library of over 11, tetracycline-resistant E. coli transformants was screened for the presence of preproenkephalin inserts by colony hybridization at moderate stringency with a nick-translated 918-bp Hinc fragment of the human preproenkephalin cdna clone phpe9 (6, 17). This probe was previously determined by us and others (15) to hybridize adequately, albeit weakly, with rat preproenkephalin mrna on Northern blots. The larger of two positive clones, designated prpe2, possessed an approximately 132-bp insert consisting of four excisable Pst fragments of chain lengths approximately 52,42,26, and 13 bp. A detailed restriction map of the prpe2 insert was deduced (Fig. 1) and DNA sequencing of the insert was performed according to the strategy diagrammed in Fig. 1. Sequencing was performed on

3 Rat Brain Preproenkephalin mrna 1433 both strands for 89% of the insert. For the remaining 11%, located in noncoding regions, sequencing of one strand was performed at least twice. Excluding the oligo(dc):oligo(dg) tails, the insert was found to consist of 1275 bp plus 4 da.dt bp that would be derived from the poly(a) tail of the mrna. Primary Structure of Rat Preproenkephalin mrna and Protein-The nucleotide sequence of rat preproenkephalin mrna is shown in Fig. 2. The reading frame and translation start codon were deduced by comparison with previously q FG. 1. Restriction map of prpe2 cdna insert and sequencing strategy. Numbers along the axis refer to distance in nucleotides from beginning of insert. Dotted lines extending from the axis represent oligo(dg):oligo(dc) tails. Experimentally determined relevant restriction sites are indicated by the number of the 5' nucleotide generated by the cleavage; in this case, the numbers correspond to those in Fig. 2, with nucleotide 1 at the start codon. Numerous other restriction sites are not shown. Arrows at the bottom indicate direction and extent of sequence determinations of fragments labeled at either the 5' end () or the 3' end (1, as described under "Experimental Procedures." characterized human and bovine preproenkephalins (4-7). The assigned start codon is also the first AUG triplet in the mrna sequence. The distance from this codon to the end of the first in-phase termination codon (UGA) is 81 nucleotides. Nine in-phase termination codons follow the initial one. The clone contains 155 nucleotides on the 5' side of the start codon and 312 nucleotides on the 3' side of the termination codon. The putative transcription-termination signal AAU- AAA is present twice, at 17 and 44 nucleotides upstream from the poly(a) tail. The mrna codes for a polypeptide chain of 269 amino acids (M, 3,982) that is structurally similar to bovine and human preproenkephalins (4-7). The amino-terminal region possesses characteristics of a signal peptide, which in the case of bovine preproenkephalin was inferred to be residues 1-24 (4, 5). The rat precursor contains four copies of Met-enkephalin and one copy each of Leu-enkephalin, Met-enkephalin- Arg6-Gly7-Leus, and Met-enkephalin-Arg6-Phe7. Each of these opioid peptides is separated fromneighboringresidues by pairs of basic amino acid residues. A sequence that may give rise to Met-enkephalin-ArgG-Arg7-Vala-NH2 (metorphamide or adrenorphin), isolated from human pheochromocytoma (8) and bovine brain (9), is also present in rat preproenkephalin (residues ). One site of potential asparagine-linked glycosylation, Asn-Ser-Ser (positions ), is present. Translcrtwn of Hybridization-selected Preproenkephalin mrna-our previous work (33) demonstrated that cell-free FG. 2. Primary structures of rat preproenkephalin mrna and polypeptide. The nucleotide sequence was derived from sequencing of the prpe2 cdna as indicated in Fig. 1. Nucleotides are numbered in the 5' to 3' direction, with nucleotide 1 at the beginning of the AUG start codon. Deduced amino acids are numbered starting with the initiating Met. A descending arrow indicates the putative site of signal peptide cleavage. Met-enkephalin, Leu-enkephalin, Metenkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe sequences are shown in boxes; the dotted box indicates amino acids contributing to the C-terminal structure of metorphamide (adrenorphin). Putative AAUAAA transcriptiontermination signals are underlined, and 4 A residues at the 3' end of the insert are shown.

4 Rat Brain Preproenkephalin mrna 1434 translation of rat striatum mrna results in the synthesisof up to four proteins that are specifically immunoprecipitated by antisera recognizing the carboxyl-terminus of preproenkephalin: a prominent oneof apparent M, 3,-31, and three less-conspicuous ones of apparent M, 31,-32,, 21,5-22,, and 2,. T o determinethe relationship between the prpe2 sequence and mrna(s) for these four proteins, linearized prpe2 DNA was bound to a nitrocellulose filter and allowed to hybridize with rat striatum poly(a)+ RNA understringent conditions. Hybridization-selected mrna was translated in a cell-free system, and translation products were immunoprecipitated with a Met-enkephalinAr$-Phe7 antiserum that recognizes preproenkephalin. As shown in Fig. 3, the same specifically immunoprecipitated proteins, i.e. those completely displaced by 5 p~ Met-enkephalin-Ar$-Phe', were obtained by translation of prpe2selected mrna and by translation of unselected rat striatum poly(a)+ RNA a major 31-kDa band, in agreement with the predicted molecular weight, a faint kDa shoulder, and Hybridization Selected mrna Unselected pbr322 prpe2 rnrna Met-Enk-Arg-Phe: M, X 1-3: 97- " and 2-kDa minor bands. Analysis of nonimmunoprecipitated translation products indicated that these were the only detected proteins dependent upon mrna selected by prpe2 DNA (not shown). These results indicate that these proteins are encoded by identical or similar mrna species that arehomologous to the prpe2dna insert. The variable abundance of the two smaller forms suggests that their production is related to mrna or polypeptide degradation. Southern Blot Analysis of Rat DNA-To determine the number of rat preproenkephalin gene(s), brain DNAwas digested with restriction endonucleases that do not cut the prpe2 insert between nucleotides 21 and Southern blots of restricted and electrophoresed DNA were hybridized under moderately stringent conditions with 32P-labeled prpe2 Pst fragment , or,in some experiments, with an equal mixture of 32P-labeledPst fragment and Pstfragment As shown in Fig. 4, the probe(s) hybridized with an apparently single band of DNA for each restriction digest. This result is consistent with a single preproenkephalin gene per rat haploid genome, although the existence of distantly related genes cannot be ruled out. Blot-hybridization Analysis of Rat RNA with a Rat Preproenkephalin Probe-To assess the sensitivity and specificity of the prpe2probe for the quantitationof preproenkephalin mrna, Northern blots of poly(a)+rna or total(unfractionated) RNA from several regions of the rat central nervous system were hybridized with a 32P-labeled 941-bp Bsp 1286 fragment of prpe2. This probe contains DNA corresponding 68" " m - - " _ m u " -- \ rp < g G $ $ 4332 L _ l BasePairs x 1-~ FG. 3. Analysis of gene products of mrna selected by hybridization with prpe2 DNA. Nitrocellulose membranes containing equimolar amountsof linearized prpe2 or control pbr322 DNA were hybridized with rat striatum poly(a)+ RNA asdescribed under "Experimental Procedures." Each eluted RNA samplewas translated in a cell-free system (.15 ml) containing [%]methionine. One-fifth of each reaction mixture was analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography (not shown because of interference by reticulocyte-mrna translation products). Four-fifthswere subjected to immunoprecipitation with an antiserum recognizing the COOH terminusof preproenkephalin, twofifths in the absence and two-fifths in the presence of 5 p~ Metenkephalin-Arg-Phe. n addition,.11 pg of rat striatum poly(a)+ RNA was translated in a.1-ml reaction mixture and immunoprecipitation was carried out as above. mmunoprecipitates were electrophoresed and autoradiographed with Kodak SB-5 film for 12 days. Numbers at the left refer to M,values and migrationsof "C-labeled protein markers (straight lines, Bethesda Research Laboratories) or interpolated apparentm,values of immunoreactive proteins(arrows). 1- FG.4. Southern blot analysis of preproenkephalin gene(s) in rat genomic DNA. Rat brain DNA (1 pg) was digested with the restriction endonuclease shown at the top of each lane and electrophoresed, transferred to a nylon membrane, and hybridized a t 36 "C with nick-translated513-bp Pst fragment of prpe2 cdna (2.9 X 1s cpm/pg), as described under "Experimental Procedures." Numbers at the left indicate the chain length (base pairs) estimated from DNA markers X-Hind111 digest and 4x174 RF-Hoe-11 digest. An autoradiogram of 18-h exposure is shown. dentical band patterns were obtained after hybridization of the same blot a t 3 "C with a mixture of two labeled Pst fragments ( and ) (not shown).

5 Rat Brain Preproenkephulin mrna B. Total RNA A. Poly (ATRNA Bovine Rat FG.5. Northern blot analysis of preproenkephalin mrna from various regions of rat central nervous system. A, poly(a)+ RNA (4 pg/sample) wasdenatured and electrophoresed on a 1.2% agarosegel containing 6% formaldehyde, transferred to a nylon membrane, and hybridized with a nick-translated Bsp 1286 fragment of prpe2 plasmid (2.4 X 1' cpm/pg), as described under "Experimental Procedures." The poly(a)+ RNA samples analyzed and the yields that were obtained during their purification (pg/g of tissue) are as follows: liver (LV) 9.5; striatum (ST), 26; cerebellum (CB), 21.8; spinal cord ( X ), 9.1; frontal cortex (FC), 17.7; bovine adrenal medulla (AM), 9.; and bovine striatum (ST), 8.. An autoradiogram of 36-h exposure is shown. Numbers at the left are the chain length (bases) of E. coli and calf liver rrna markers.b, total unfractionated cellular RNA (5 pglsample) was analyzed as described for p a n e l A. The RNA samples analyzed and their yields (mg/g of tissue) were as follows: liver (LV),.85; frontal cortex (FC), 1.97; striatum (ST), 2.15; hypothalamus (HT), 1.11; midbrain ( M B ), 1.83; cerebellum (CB), 1.; pons + medulla oblongata (PM), 1.19; spinal cord (SC), 1.4. An autoradiogram of 16-h exposure is shown. The broadness of the bands is a consequence of loading a large amount of RNA onto each lane. to the entire coding sequence of preproenkephalin mrna of hybridization to rat liver and small intestine RNA were (nucleotides , see Figs. 1 and 2). As shown in the negligible. As withthenorthernblots (Fig. 5, panel A ), autoradiograms of Fig. 5, theratpreproenkephalinprobe hybridization of the rat probe to bovine striatal poly(a)+ RNA hybridized with a single major band of poly(a)+ RNA (panel was weak in comparison to rat striatal poly(a)+ RNA. The A ) ortotalrna (panel B ) of lengthapproximately 15 signals of bovine frontal cortex and cerebellum were hardly nucleotides from central nervous system regions, but notfrom detected (Fig. 6, panel A ). n order to estimate relative abundances of preproenkeliver, a tissue essentially devoid of proenkephalin peptides. This size is consistent with that previously determined for phalin mrna ( i e. relative amounts per pg of RNA) in brain bovine and rat striatal preproenkephalin mrna (13, 15). For regions, the radioactivity of the probe hybridized to the dot the striatum, the region with the greatest abundance of this blots of total or poly(a)+ RNA was quantitated by excision mrna, a minor band of approximately 6 bases, possibly and liquid scintillation counting of the spots (Table ). n analyses of both total and poly(a)+ RNA, the striatum was a nuclear precursor of preproenkephalin mrna, was noted. Hybridization to RNA smaller than 15 bases was noted, found to have the highest abundance of the regions tested. fell into three groups: a high but the variability of the patterns among RNA preparations Regions other than the striatum group (hypothalamus, pons + medulla oblongata, and spinal indicates that the smaller RNA represents degraded material. a middle group T o examine the selectivity of the probe under stringent hy- cord) having 1-2% of the striatal abundance; bridization conditions, it was tested also with poly(a)+rna (cerebellum, midbrain, and frontal cortex) having 4-1% of from bovine adrenal medulla and striatum, tissues with pre- the striatal abundance, and a low group (hippocampus and proenkephalin mrna abundances presumably higher than or thalamus) having 1-2% of the striatal abundance. comparable to that of rat striatum. The amount of probe DSCUSSON hybridizedwas low compared to that for rat striatum, as expected from the mismatching (26%) of the rat and bovine Molecular cloning and sequencing of rat brain preproensequences. kephalin cdna was undertaken toprovide basic information Because the Northern blot patterns appeared free from necessary to study regulation of enkephalin biosynthesis in nonspecific hybridization, asimple dot-blot assay was adopted the nervous system of rat. The striatum was chosen as the for quantitation of relative abundances of preproenkephalin source of mrna for cloning because this region contains a mrna. As shown in Fig. 6, varied amounts of the probe relatively highconcentration of enkephalinergic neuronshavhybridized with poly(a)+rna (panel A ) or total RNA (panel ing cell bodies in the caudate-putamen and fibers terminating B ) from eight brainregions plus the spinal cord. The amounts in the globus pallidus. The prpe2 clone contains an insert

6 Rat Brain Preproenkephalin mrna ;is , ST- Rat # (, 1 2 vg. Lv- e o FC - FC - *' ST- (-- e. HT - sc - TH - :1 Bovine, HT - [AM - TABLE B. Total RNA A. Poly (A)*RNA HC - *. MB-. CB - c PM- sc - orno FG. 6. Dot-blot analysis of preproenkephalin mrna from regions of rat central nervous system. ndicatedamounts of poly(a)' RNA (panel A ) or total unfractionated cellular RNA (panel R) from various brain regions or tissues were denatured and blotted onto nylon membranes,then hybridized withnick-translated Bsp 1286 fragment of prpe2 plasmid (2.4 X l@ cpmlpg) as described under"experimental Procedures." A, poly(a)+rnasamplesand their yields (pg/g of tissue), where different from samples used for 1.; frontal Fig. 5, are asfollows: striatum (ST); hypothalamus (HT), cortex (FC); cerebellum (CB); spinalcord (SC);liver (LV); small intestine ( S o, 36.4; bovine adrenal medulla (AM); bovine striatum (ST); bovine frontal cortex (FC), 9.8; bovine cerebellum (CB),3.8. An autoradiogram of 8-h exposure is shown. E, t o t a l RNA samples and their yields (mg/g of tissue), where different from samples used for striatum (ST); Fig. 5, are as follows: liver (LV);frontal cortex (FC); hypothalamus (HT); thalamus (TH),.92; hippocampus (HC), 1.67; midbrain ( M E ) ;cerebellum (CB); pons + medulla oblongata (PM); and spinal cord (SC). An autoradiogram of 4.5-h exposure is shown. Relative abundance of preproenkephalin mrna inrat central nervous system Total or poly(a)+ RNA from various brain regions or tissues was adsorbed to nylon membranes and hybridized with the 32P-labeled Bsp 1286 fragment of prpe2. After autoradiography, shown in Fig. 6, 28-mm2 circles includingeach dotof bound RNA were excised from the membranes, and radioactivity was determined by liquid scintillation spectrophotometry. Preproenkephalin mrna-dependent radioactivity was calculated by subtracting the radioactivity of circles with equal amounts of liver RNA. The relative abundance of preproenkephalin mrna was calculated by dividing the preproenkephalin mrna-dependent radioactivity of a given amount of RNA with that of the corresponding amount of striatal RNA. For the analysis of total RNA, the preproenkephalin mrna-dependent radioactivities obtained for 1.25, 2.5, 5, 1, and 2 pg of striatal RNA were 49, 17, 25,446, and 616 cpm, respectively. Values listed are averages of three amounts of RNA (5,1, and 2 r g of total RNA or.5, 1, and 2 pg of poly(a)' RNA). Relative abundanceof preproenkephalinmrna Region Total RNA Poly(A)+RNA analysis analysis 9% of striatum (mean f S.E.) 1 1 Striatum 1.3 f f.6 Hypothalamus 1.8 f.1 ND" Pons + medulla oblongata f.8 Spinal cord 6.1 f f.7 Cerebellum ND 5.9 f.4 Midbrain 6.5 f f.5 Frontal cortex 2. f.2 ND Hippocampus ND 1.6 &.2 Thalamus * ND, not determined. thatrepresentstheentiretranslatedand 3' untranslated sequences, as well as mostor all of the 5' untranslated sequence of preproenkephalin mrna. Rat brain preproenkephalin structurallyresembles the preproenkephalins of bovine adrenal medulla and human pheochromocytoma. From computer-derived alignments (not shown) of the rat mrna sequence with the partial bovine sequence (4) or the complete human sequence (17, 34), the following percentages of nucleotide-sequence conservation were calculated: humanlrat, 75.8%; and bovinelrat, 71.2%. Sequence conservation asa function of position along the rat C Human vs Rat mrna sequence is plotted in Fig. 7. Three major domains of OBovtne Rat relatively high conservation are evident within the translated V U ', 1 ~_ il ~. region: ( a ) nucleotides 1-21, coding for rat amino acids w1 NTAL BASE OF RAT PREPROENKEPHALN mrna 2 BASE SEGMENT 7, a region devoid of opioid sequences but containing the FG. 7. Similarities of rat, human, and bovine preproenkesignal peptide and all 6 Cys residues of proenkephalin; (b) nucleotides , coding for rat amino acids , a phalin mrna sequences as a function of position. The rat sequence (Fig. 2) was aligned with either the complete human seregion containing three enkephalin sequences and including quence or the partial bovine sequence using thenucaln computer the peptidef sequence (amino acids 17-14); and ( c ) nucleo- program, which was instructed tominimize gaps between nucleotides tides , coding for amino acids , a region con- in the alignment (gap penalty = 7) (26). The alignments were divided taining four enkephalin sequences and including the peptide into 2 base segments, and the numbersof matched nucleotides were counted. A gap was considered to be one mismatch per nucleotide. E sequence (amino acids ). Short regionsbetween 2-base segment is plottedversus the these three domains,as well as the5' untranslated region, are The per cent similarity in each nucleotide at the beginning of each segment of the rat preproenkenotably divergent. The 3' untranslated region itself appears phalin sequence. Top, schematic diagram of the preproenkephalin to have possibly three well-conserved domains separated by coding region. Abbreviations, Sig, signal peptide; C,cysteine residues short divergent zones. in the proenkephalin portion; M, Met-enkephalin or extended MetConsiderable similarity among the three known preproen- enkephalin sequences; and L, Leu-enkephalin. kephalia amino acid sequences is evident from the computerderived alignments shown in Fig. 8. Rat preproenkephalin similarities between the precursors are indicated by the fol(269 amino acids, M,3,932) is slightly larger than the human lowing percentages of identical aminoacids: humanlrat, 82%; preproenkephalin (267 aminoacids, M,3,785 (6, 7), and the bovinelrat, 78%; and bovinelhuman,85%. The sequences and bovine precursor (263 amino acids, M,29,786) (4). The overall relative positions of the enkephalins are identical among the YS

7 Rat Brain Preproenkephalin mrna 1437 Human 6 Rat 1 6 Bovtne 1 6 FG. 8. Comparison of rat, human, and bovine preproenkephalin amino acid sequences. The sequences, indicated by standard one-letter amino acid abbreviations, were aligned with the assistance of the PRTALN computer program. Colons between aligned sequences indicate matched amino acids. The enkephalin or enkephalin-like sequences are shown in solid-line boxes. The putative signal peptide, the cysteine residues, and the amino acids that produce the COOH terminus of metorphamide (adrenorphin) are shown in broken- Line boxes. Human 61 Rat 61 BovLne hl Human 121 Rat 121 Bovine 118 Human 1R1 Rat 181 Bovine 17R....(.,......l....,., n un 1Rn R Human 239 Rat 241 Bovine 236 three preproenkephalins. The sequences of peptides E and F, the carboxyl-terminal 27 amino acid residues, the pairs of basic residues that are sites of proteolytic processing, and the potential glycosylation site are all highly conserved. Although the rat precursor contains an additional Cys residue in the signal peptide, the positions of the 6 Cys residues of the proenkephalin portion are entirely conserved. As proposed by others (6), these residues may form disulfide bridges important for the proper folding of the prohormone. The rat precursor contains 7 Ser or Thr residues (positions 27, 49, 153, 163,245,251, and 253) that may fulfill proposed requirements (Ser/Thr-X-acidic) for phosphorylation by one class of casein kinases (35). Four of these sites (Ser at 27,245,251, and 253) are conserved in the three species. Numerous regions of nucleotide-sequence similarity and dyad symmetry within the rat preproenkephalin mrna seqeunce were detected by a computer search. n addition to sequences coding for the enkephalins, 17 pairs of sequences of 218 bases were found to be over 75% similar. Two pairs of sequences ((-35)-(-18) and ; and and 76-78) were over 8% similar. These similarities suggest that the evolutionary history of the preproenkephalin gene includes duplication of non-enkephalin as well as enkephalin elements. This study demonstrates that large restriction fragments of prpe2 are specific and sensitive probes for preproenkephalin mrna of rat brain. Under stringent hybridization conditions, nonspecific binding of the probe is minimized, allowing hybridization experiments to be performed with unfractionated total RNA rather than purified poly(a)+ RNA. An advantage of probing blots of unselected RNA rather than poly(a)+ RNA from brain is that mrna molecules with small or absent poly(a) tails are included in the analysis. The extent of polyadenylation of brain preproenkephalin mrna has not been rigorously tested. The appearance of large amounts of poly(a)- mrna in developing postnatal mouse brain has been reported by Chaudhari and Hahn (36). Thus, a study of any neural mrna should not initially be restricted to poly(a)+ RNA. The relative abundances of preproenkephalin mrna in discrete regions of the rat central nervous system (Table ) are consistent with the regional distribution of Met-enkephalin-Arg-Phe-containing cell bodies detected by immunohistochemistry (16, 37) and are partially consistent with the relative tissue contents of Met-enkephalin and Met-enkephalin-Arg-Phe (38, 39, 15, and earlier studies). The relative preproenkephalin mrna abundance probably is a good indication of the average rate of enkephalin biosynthesis in a particular region, while the enkephalin concentration is dependent upon many factors, such as the rates of precursor biosynthesis and processing, transport to other regions, release, and degradation. The partial concordance between mrna and peptide abundances reflects the fact that many enkephalinergic neurons are interneurons possessing cell bodies (the sites of mrna) and nerve terminals (the sites of storage and release of enkephalins) in the same gross brain region (16, 37). The prpe2 probe described here should be a valuable tool for the localization and quantitation of preproenkephalin mrna in individual nuclei of the rat central nervous system by means of micro-punch dissection combined with dot-blot analysis of total RNA, as well as by in situ hybridization histochemistry. n initial attempts to study the regulation of enkephalin biosynthesis in the brain, we and others have demonstrated selective increases in the preproenkephalin mrna abundances in the striatum elicited by chronic haloperidol treatment (15, 33) and in the hypothalamus elicited by repeated electroconvulsive shock (4). More precise mapping of these and other changes in preproenkephalin mrna levels should now be possible. Acknowledgments-We are deeply indebted to Dr. Satya Dandekar for initiating recombinant DNA experiments in our laboratory. We are grateful to Drs. E. Herbert and M. Comb for providing the human preproenkephalin phpe9 clone, to Steven Lee for the preparation of several poly(a)+ RNA samples, and to Drs. H. Ueyama, M. Schubert, and K. Krueger for helpful advice. Addendum-Rosen et al. (41) have reported the sequence of most of a rat preproenkephalin genomic clone. The sequence of the exon portions of this gene are in good agreement with the sequence of our prpe2 cdna clone; the few discrepancies are in untranslated regions and might reflect strain differences.

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