Partial characterization of the mRNA that codes for enkephalins in ...

Proc. Natli Acad. Sci. USA Vol. 79, pp. 360-364, January 1982

Biochemistry

Partial characterization of the mRNA that codes for enkephalins in bovine adrenal medulla and human pheochromocytoma (hybridization/pro-enkephalin/oligodeoxynucleotide probe/cDNA)

MICHAEL COMB*, EDWARD HERBERT*, AND ROBERTO CREAt *Department of Chemistry, University of Oregon, Eugene, Oregon 97403; and tGenentech Inc., 460 Point San Bruno Blvd., South San Francisco,

California 94080

Communicated by George Streisinger, October 2, 1981

ABSTRACT [Met]enkephalin and [Leu]enkephalin are derived from a protein in bovine adrenal medulla that contains multiple copies of [Met]enkephalin [Kilpatrick, D. L., Taniguchi, T., Jones, B. N., Stern, A. S., Shively, J. E., Hullihan, J., Kimura, S., Stein, S. & Udenfriend, S. (1981) Proc. NatL Acad. Sci USA 78, 3265-3268.]. Here we characterize pro-enkephalin mRNA from bovine and human tissue by use of an oligodeoxynucleotide pentadecamer probe complementary to codons for Tyr-Gly-GlyPhe-Met ([Met]enkephalin). This probe hybridizes specifically to a species of poly(A)^RNA from adrenal medulla and human pheochromocytoma, (1400-1450 bases), and also to [Met]enkephalincontaining pro-opiomelanocortin mRNAs from bovine pituitary (1200 bases) and from mouse pituitary tumor cells (1100 bases). A cloned cDNA probe (144 bases) complementary to the region of pro-opiomelanocortin mRNA that codes for lipotropin does not hybridize to the RNA from bovine adrenal medulla, demonstrating that the latter RNA is not pro-opiomelanocortin mRNA. The pentadecamer probe was extended to make .cDNA with reverse transcriptase after hybridizing it to adrenal poly(A)-RNA. The sequence of an extended cDNA, 62 bases in length, was found to correspond exactly to that expected from the amino acid sequence of peptide E (a bovine adrenal peptide containing [Met]- and [Leu]enkephalin sequences). This cDNA also forms a specific hybrid with the RNA from bovine adrenal and human pheochromocytoma, confirming that these species of RNA are pro-enkephalin mRNA.

granules ofbovine adrenal medulla (10). These granules contain much higher levels of enkephalins than one finds in the brain. Several enkephalin-containing peptides have been purified from these granules and their sequences have been determined. One peptide, 34 amino acids in length, contains two [Met]enkephalin sequences, and another peptide, 39 amino acids in length, contains a [Leu]enkephalin and a [Metlenkephalin sequence (11, 12). From these studies it is concluded that the precursor protein contains multiple copies of [Met]enkephalin and at least one copy of [Leu]enkephalin. In this study, we have used synthetic oligodeoxynucleotide probes complementary to mRNA sequences that code for [Met]and [Leu]enkephalin to identify pro-enkephalin mRNA. Probes of this kind have been used previously for the detection and characterization of mRNA for several proteins, including interferons (13), gastrin (14), and relaxin (15), and for the detection of specific DNA fragments (16). We show here that this procedure allows the detection and partial sequence analysis of the enkephalin mRNA, which makes up only about 0.05-0.10% of the poly(A)-RNA in bovine adrenal medulla. MATERIALS AND METHODS Preparation of Poly(A)-RNA. Bovine adrenal glands were obtained within half an hour ofdeath ofthe animal. The adrenal medulla was dissected free ofcortical tissue and frozen in liquid nitrogen. Frozen human adrenal tumors were obtained from Lee Eiden of the National Institutes of Health. Frozen tissue was ground to a fine powder in liquid nitrogen. One gram of the frozen powder was homogenized with a Polytron (Brinkmann) in 8 ml of a solution containing 5 M guanidinium thiocyanate (Fluka), 100 mM Tris HCl (pH 7.6), 10 mm EDTA, and 5% (vol/vol) 2-mercaptoethanol. Total RNA was isolated from this homogenate as described (17). Poly(A)-RNA was recovered' after two cycles of chromatography on oligo(dT)-cellulose columns (T-2 from Collaborative Research, Waltham, MA). Synthesis of Oligonucleotides and Blot Hybridization. The pentadecamer C-A-T-G-A-A-G-C-C-G-C-C-G-T-A was synthesized by using the phosphotriester method in solution and three nucleoside diphosphates as building blocks (18). The pool of probes containing tetradecanucleotides T-C-C-A-T-C-T-T-c-TT-C-A-T was obtained as reported (13). The general procedures for purification and characterization of the synthetic oligonucleotides were similar to those described by Crea et al. (18). The 5''phosphates of oligonucleotides were labeled by using polynucleotide kinase (New England BioLabs) and [y-32P]ATP. The labeled oligonucleotides were separated from [y-32P]ATP as described by Noyes et aL (14). RNA preparations were denatured with glyoxal (19) and fractionated by electrophoresis on 1.75% agarose gels. The RNA was transferred to nitrocellulose by the method ofThomas (19). Blots were prehybridized at 90°C

In 1975 Hughes et al. (1) isolated two pentapeptides from brain tissue that exhibited opioid activity. These peptides are called [Leu]- and [Met]enkephalin. Since that time other opioid peptides have been discovered, including /&endorphin (2,. 3), aneo-endorphin, and dynorphin (4). The demonstration that ,Bendorphin and dynorphin are [Met]enkephalin and [Leu]enkephalin, respectively, with COOH-terminal extensions (2-4) suggested that these peptides might be precursors to the enkephalins. However, in the past 3 years biosynthetic studies have revealed that B3-endorphin is derived from a different gene product than the enkephalins are. ,3-Endorphin is synthesized from a precursor protein that also contains the sequence ofadrenocorticotropin and a-, A3-, and. y-melanocyte-stimulating hormones (5-7). This protein, which is called pro-opiomelanocortin, is present in a number of different tissues, including the pituitary and hypothalamus (8). Although pro-opiomelanocortin is processed efficiently to ,B-endorphin in these tissues, there is no evidence for conversion of, -endorphin to [Met]enkephalin. A separate origin for enkephalins, dynorphin, and endorphin is also indicated by differences in distribution ofthese peptides in pituitary and the brain (9). A source of [Met]- and [Leu]enkephalin has been elucidated recently by studies of the opioid peptide content of chromaffin The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: kb, kilobase(s). 360

Biochemistry: Comb et al. for 4 min in 0.6 M NaCI, 0.06 M sodium citrate, 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 10 mM EDTA (pH 7.4), yeast tRNA (50 ,ug/ml) (Sigma), and 0.1% Sarkosyl. The blots were allowed to cool to room temperature for 5 min. The incubation mixture was supplemented with 1-2 x 107 cpm of 32P-labeled oligonucleotide (4-8 X 106 cpm/ pmol) and again applied to the blot. The blot was heated to 900C for 5 min, cooled rapidly to 70'C, and then allowed to cool for 3 hr to 25-30'C. Hybridization with the pentadecamer probe was performed for 2-3 days at 25-300C. The blot was washed three or four times with a solution of 0.6 M NaCl and 0.06 M sodium citrate in 0.1% Sarkosyl at 40C, allowing 4 min for each wash. Autoradiography was carried out using Du Pont Lightning Plus intensifying screens at -700C for 15-48 hr. Hybridization with the 144-base cloned mouse cDNA probe ME-150 (20), which is complementary to the region of pro-opiomelanocortin mRNA that codes for -lipotropin, was by the method of Thomas (19). Oligonucleotide Primed cDNA Synthesis. Typically, 30 pmol of primer was incubated with 50 pmol of [y-32P]ATP (4500 Ci/ mmol; 1 Ci = 3.7 x 1010 becquerels) and 3 units of polynucleotide kinase (New England BioLabs) in a final volume of 10 ,ul as described by Houghton et aL (21). After 30 min at 37C, the reaction was terminated by addition of 2 vol of phenol/ chloroform (1:1, vol/vol). The aqueous phase was made 0.3 M in NaOAc (pH 5), 3 vol of 95% (vol/vol) ETOH was added, and the mixture was chilled at -70°C for 10 min. The precipitate was dissolved in 20 Al of a solution containing poly(A)-RNA (1 mg/ml), 5 mM EDTA, and 5 mM sodium phosphate (pH 7.7). The mixture was heated at 90°C for 4 min, then incubated at 50°C for 20 min in the presence of 100 mM KCl. Components were added to the mixture to give the following composition: poly(A)-RNA (50 ,g/ml); dATP, dCTP, dGTP, and dTTP (500 ,uM each); pH 8.3 Tris HCl (50 mM); KCl (50 mM); MgC12 (5 mM), and dithiothreitol (5 mM). After the temperature had been adjusted to 42°C, reverse transcriptase (RNA-dependent DNA polymerase) was added (lot no. 608979, from J. W. Beard) to a final concentration of 10 units/,ug of poly(A)-RNA and the reaction mixture was incubated at 42°C for 30 min. After the reaction had been terminated with phenoVchloroform, DNA was precipitated with 3 vol of 95% ethanol as above. The precipitate was resuspended in 0.1 M NaOH and incubated at 70°C for 20 min. An equal volume of a solution containing 95% (vol/ vol) formamide, 0.03% xylene cynanol, and 0.03% bromphenol blue was added and the mixture was incubated at 90°C for 1 min, cooled in an ice bath and layered on an 8% polyacrylamide/7 M urea gel (40 X 20 X 0.05 cm). After electrophoresis the band containing unextended primer was removed and the gel was autoradiographed for 1-3 hr at room temperature. DNA sequence analysis was carried out as described by Maxam and Gilbert (22).

RESULTS of Synthesis a Pentadecamer [Met]enkephalin Probe. Because the [Met]enkephalin sequence is reported to be present in the precursor in multiple copies, we felt that a probe to this sequence might provide a relatively high degree of sensitivity for the detection of enkephalin mRNA. However, in designing a DNA probe to this mRNA one has to contend with 64 possible coding sequences. We synthesized the pentadecamer probe shown in Fig. 1 on the basis of the observed frequency of codon usage in mammals (23), the sequence of mouse pro-opiomelanocortin mRNA as determined by Roberts et aL (20), and the effect of mismatching of bases on the stability of DNA-RNA hybrids

(24, 25). Hybridization of the [Met]enkephalin Probe to Mouse Proopiomelanocortin mRNA. Different degrees of mismatching of

Proc. Natl. Acad. Sci. USA 79 (1982) Protein NH2..

Tyr

Gly A

Gly Phe A

361

Met ... . COOH

mRNA 5'.... UACU GGCg GGC UUg AUG.... 3' U U 64 possible different coding sequences Mouse POMC mRNA 5'.... UAC GGU GGC UUC AUG.... 3' Met-enk pentodecomer. ATG CCG CCG AAG TAC. .. 5'

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FIG. 1. Hybridization of [Metlenkephalin pentadecamer probe to poly(A)-RNA from AtT-20 cells by the blot procedure. (Upper) Protein sequence of [Met]enkephalin, its possible mRNA coding sequences, the mouse pro-opiomelanocortin (POMC) mRNA sequence coding [Met]enkephalin (20), and the nucleotide sequence of the [Met]enkephalin probe that was chemically synthesized. (Lower) Hybridization of [32Plpentadecamer to decreasing amounts of poly(A) from AtT-20D16, cells was analyzed by blot procedures. The pro-opiomelanocortin mRNA levels in the AtT-20 poly(A)-RNA preparation were determined to be 1.5% of the total poly(A) by solution hybridization; fmol of proopiomelanocortin mRNA, applied to lane 1, 80; lane 2, 40; lane 3, 20; and lane 4, 10. The faint bands seen at 2200 and 5600 bases represent binding of the probe to a small amount of 18S and 28S ribosomal RNA. The length scale (number of nucleotides) was constructed by running glyoxal-denatured DNA fragments from Hae m digests of phage 4X174 replicative form DNA and HindA digests of phage 2 A DNA on adjacent lanes of the blot.

bases between the [Met]enkephalin probe and various of the [Met]enkephalin sequences in pro-enkephalin mRNA appeared likely. For this reason, we used pro-opiomelanocortin mRNA, which contains a single copy of the [Met]enkephalin sequence, to determine the effect of mismatching of bases on the stability and specificity of the hybrid formed between the probe and mRNA. As a source of pro-opiomelanocortin mRNA we used a poly(A)-RNA preparation extracted from the adrenocorticotropin-secreting cell line AtT-20-D16, (20). As shown in Fig. 1 Upper, there is one mismatch (d-U) between the sequence of the pentadecamer probe and the [Metlenkephalin sequence of pro-opiomelanocortin mRNA of the AtT-20-D16, cell line. To determine whether specific hybridization occurs and to evaluate the stability of the hybrid formed between the probe and AtT-20 mRNA, various amounts of poly(A)-RNA were fractionated by gel electrophoresis, blotted, and hybridized with the pentadecamer probe as described in Materials and Methods. The results in Fig 1. Lower show that the probe hybridizes efficiently to a species of RNA 1.1 kilobases (kb) in length. To determine whether the 1.1-kb RNA contains pro-opiomelanocortin sequences, hybridization of the AtT-20 poly(A)RNA was tested with the cloned mouse cDNA probe ME-150 (20). This probe hybridizes to RNA in the same region of the gel as the pentadecamer probe (Fig. 2, lane 1), suggesting that

362

Proc. Nad Acad. Sci. USA 79 (1982)

Biochemistry: Comb et aL

both probes hybridize to the same species of RNA, pro-opiomelanocortin mRNA. Furthermore, because only one band is seen upon hybridization of the pentadecamer probe with a complex mixture of mRNA extracted from the AtT-20 cells, we conclude that the pentadecamer probe is specific. The sensitivity of the probe for detecting mouse pro-opiomelanocortin mRNA is 2-5 fmol for an overnight exposure (Fig. 1). We have found that the pentadecamer probe forms a specific hybrid with bovine pro-opiomelanocortin mRNA, although at reduced efficiency (data not shown). Examination of the sequence of bovine pro-opiomelanocortin mRNA determined by Nakanishi et aL (26) indicates that there is a single mismatch (dG-G) between this mRNA and the pentadecamer probe. Identification of Enkephalin mRNA in Adrenal Medulla. In an attempt to identify the mRNA coding for enkephalin in the bovine adrenal medulla, poly(A)-RNA was isolated from that tissue and subjected to a blot analysis with the pentadecamer probe. The pentadecamer probe hybridizes to a species ofRNA approximately 1.45 kb in length (Fig. 2a, lane 2). To determine if this RNA contains pro-opiomelanocortin sequences, the blot was subjected to a second hybridization with the ME-150 cDNA probe, as described earlier for the AtT-20 RNA. In contrast to the earlier results with the mouse cell line RNA, the bovine adrenal medulla RNA that hybridizes to the [Met]enkephalin probe does not hybridize to the ME-150 cDNA (Fig. 2b, lane 2). Because ME-150 hybridizes efficiently to bovine pro-opiomelanocortin from RNA extracted from the pituitary intermediate lobe (Fig. 2b, lane 3), these results show that the 1.45-kb RNA in adrenal medulla is not pro-opiomelanocortin mRNA. To enrich for the hybridizing species of RNA in adrenal medulla, poly(A)-RNA from this tissue was centrifuged in a 5-29% sucrose gradient. Fractions from the gradient were analyzed by the blot procedure with the pentadecamer [Met]enkephalin probe as described above. The 17S fraction from the gradient was found to be enriched approximately 4-fold for the hybridizing species of RNA (results not shown). The amount of hybridizing RNA in other fractions from the gradient was much less than that in the 17S fraction. Detection of Pro-enkephalin mRNA with a Probe to a Nonenkephalin Sequence. One of the chromaffin granule peptides referred to earlier (11) contains a Met-Lys-Lys-Met-Asp sequence adjacent to a [Met]enkephalin sequence. We synthesized a pool offour probes complementary to this sequence, 14

22000 145Ow

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nucleotides in length; as shown in the upper part of Fig. 3. This pool of oligonucleotides is certain to contain one probe that is perfectly matched to the sequence ofinterest in the mRNA. The probe pool was labeled with 32P by using T4 polynucleotide kinase and hybridized to a blot of poly(A)-RNA from adrenal medulla and from AtT-20 cells. The pool of probes hybridized efficiently to a species of RNA (1.45 kb) (Fig. 3 Lower Right, lane 1) in poly(A)-RNA from adrenal medulla but not to any species of RNA in poly(A)-RNA from AtT-20 cells (Fig. 3 Lower Right, lane 2). The hybridization of this new set of probes and also the pentadecamer probe to 1.45-kb RNA in the adrenal medulla (Fig. 3) supports the conclusion that this mRNA codes for the enkephalin precursor. Oligonucleotide-Primed cDNA Synthesis. To further characterize the species of RNA that hybridizes to the probes, the [Met]enkephalin pentadecamer was used as a specific primer for the reverse transcription reaction. The probe was annealed with total poly(A)-RNA from adrenal medulla and with the partially purified 17S RNA fraction from the sucrose gradient and incubated with reverse transcriptase (24) as described in Materials andMethods. The cDNA products were resolved on urea/ polyacrylamide sequencing gels and visualized by autoradiography. Fig. 4a shows the cDNA products transcribed from total adrenal medulla poly(A)-RNA and the partially purified 17S RNA fraction. The [Met]enkephalin pentadecamer, like oligonucleotide probes used by other workers (27), primes the synthesis of a large number of different cDNA products from the total poly(A)-RNA. The amount and size ofcDNA products transcribed from bovine adrenal medulla total poly(A)-RNA was highly reproducible for three different RNA preparations. All of these preparations gave sharp bands on blots, providing evidence for intactness of the RNA.

2

1

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FIG. 2. Hybridization of [Met]enkephalin pentadecamer and /-lipotropin cDNA probes to poly(A)-RNA from pituitary and adrenal medulla tissue. The following RNAs were subjected to electrophoresis andblotted: 1.4 jg of AtT-20D16, poly(A)-RNA (lanes 1), 8 pg of bovine adrenal medulla poly(A)-RNA (lanes 2), and 17 pg of total RNA from neurointermediate lobe of bovine pituitary (lane 3). The blot was hybridized with: (a) 107 cpm of pentadecamer (4 x 106 cpm/pmol) or (b) 5 x 106 cpm of nick-translated mouse 150-base t3-lipotropin probe (20) at 5 x 108 cpm/pg as described (19).

2

FIG. 3. Hybridization of [Metlenkephalin and Met-Lys-Lys-MetAsp probes to adrenal medulla and AtT-20 poly(A)-RNA. (Upper) Schematic drawing of the multi-enkephalin-containing precursor protein. The oligonucleotide probe sequences complementary to two different regions of the enkephalin mRNA and the RNA species detected with the oligonucleotide probes are shown below the precursor. Placement of all the enkephalin sequences (open bars, [Metlenkephalin; open diamond, [Leulenkephalin) within the precursor is arbitrary. Hybridizations of the [Metlenkephalin pentadecamer (LowerLeft) and of the 32Plabeled Met-Lys-Lys-Met-Asp tetradecamer pool (Lower Right) are shown to poly(A)-RNA from bovine adrenal medulla and mouse AtT20-D16, tumor cells. Eight micrograms of bovine adrenal medulla poly(A)-RNA (lanes 1) and 1.4 pg of AtT-20 poly(A)-RNA (lanes 2) were treated as described for Fig. 1 and hybridized with 107 cpm of the Met-Lys-Lys-Met-Asp tetradecamer pool (4 x 107 cpm/pmol) at 30°C for 3 days. Autoradiography was performed for 16 hr.

Proc. Natl. Acad. Sci. USA 79 (1982)

Biochemistry: Comb et aL a

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FIG. 4. Analysis of cDNA transcripts primed with the [Met]enkephalin pentadecamer probe by using bovine adrenal medulla poly(A)-RNA as template RNA. (a) Poly(A)-RNA from adrenal medulla was fractionated in a 5-29% isokinetic sucrose gradient by centrifugation at 40C for 13 hr in a Beckman SW 41 rotor at 36,000 rpm. The 17S fraction of the gradient was 4- to 5-fold enriched for enkephalin mRNA as determined by blot analysis using the [Met]enkephalin probe. Two micrograms of RNA from the 17S fraction of the gradient (lane 1) and 2 ,ug of unfractionated poly(A)-RNA from bovine adrenal medulla (lane 2) were used as template RNA to synthesize cDNA. Six picomoles of 5'-end-labeled [Metlenkephalin pentadecamer (4 x 106 cpm/pmol) was used as primer. The conditions of priming and cDNA synthesis are described in the text. The cDNA products were purified by electrophoresis on polyacrylamide/urea sequencing gels. The size of cDNA transcripts was determined by sequence analysis. (b) Hybridization of [Metlenkephalin pentadecamer or cDNA to adrenal medulla poly(A)-RNA. Two micrograms of 17S poly(A)-RNA was hybridized with 107 cpm of the [Met]enkephalin pentadecamer (4 x 106 cpm/pmol) (lanes 1) and 2 x 104 cpm of cDNA62 (4 x 106 cpm/pmol) (lanes 2).

Comparison of the two lanes (Fig. 4a) reveals that the only cDNA transcript enriched in the partially purified 17S RNA fraction is cDNA62. To determine whether cDNA products transcribed from the 17S RNA fraction are synthesized as a result of specific priming on the polyenkephalin mRNA, the prominent bands were eluted from the gel and their sequences were determined by the Maxam-Gilbert method (22). The sequence ofcDNA62 is shown in Fig. 5. It codes for a potent opioid peptide from adrenal medulla known as peptide E (12), which contains both a [Met]enkephalin and a [Leu]enkephalin sequence. The priming event occurs at the [Leu]enkephalin sequence and terminates abruptly at the second base of the methionine codon of [Met]enkephalin. cDNA62 was also back hybridized to bovine adrenal medulla poly(A)-RNA. As shown in Fig. 4b cDNA62 Protein (NH2). mRNA

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hybridizes to a RNA species identical in size to that detected with the two synthetic oligonucleotide probes. This further demonstrates that the 1.45-kb mRNA contains a nucleotide sequence coding for both [Met]enkephalin and [Leu]enkephalin. To determine whether the various-sized cDNA fragments observed (Fig. 4) are due to nonspecific priming at many different places, the sequences of a number of the fragments have been determined. Several of the shorter fragments have been found to be premature termination products of longer ones. For example, cDNA55 (Fig. 4) is a shortened transcript of cDNA'25, which itself is a shortened transcript of cDNA2 (not shown). The complexity of the transcription products is thus due, at least in part, to the presence of discretely terminated fragments primed from the same initiation site. The sequences of these fragments differ from the sequence of cDNA62; thus priming is not from the same site as for the latter DNA. Detection of Enkephalin-Coding mRNA in Human Pheochromocytoma mRNA. Human pheochromocytomas have been reported to contain high levels of enkephalin-relatefd peptides (28). Hence we used this tissue as a source of RNA for characterization of human enkephalin mRNA. Poly(A)-RNA was isolated from the human pheochromocytomas and a blot was prepared as described in Materials and Methods. When the blot was probed with the enkephalin pentadecamer, a species of RNA, 1.4 kb, was detected as shown in Fig. 6a, lane 1. This species of RNA is slightly smaller than the bovine pro-enkephalin mRNA, which is shown in lane 2 of Fig. 6a. To determine whether the human-enkephalin-like mRNA contains sequences homologous to peptide E of bovine adrenal, the same blot was probed with cDNA62. The results in Fig. 6b indicate that there is sufficient homology between sequences in cDNA62 and human pro-enkephalin mRNA to form a specific hybrid, but that the signal is much weaker for this hybrid than for the hybrid formed with bovine pro-enkephalin mRNA. Because the blots indicate that pro-enkephalin mRNA is present at a higher level in the human tumor than in bovine adrenal, this result suggests that there are sequence differences in this mRNA from the two species in the region complementary to cDNA62.

DISCUSSION Both bovine and mouse pro-opiomelanocortin mRNA form a stable and specific hybrid with the pentadecamer [Met]enkephalin probe in spite of single-base mismatches. The pentadecamer probe is capable of detecting 2-5 fmol of mouse pro-opiomelanocortin mRNA in an overnight exposure to the autoradiogram (Fig. 1). The enkephalin probe hybridizes efficiently to a single size class (1.45 kb) of adrenal medulla poly(A)-RNA. This RNA does not code for pro-opiomelanocortin as shown by its failure to hybridize with a cDNA probe (ME-150) that is specific for the ,B-lipotropin sequence of pro-opiomelanocortin mRNA. Further evidence for the identity of the 1.45-kb RNA has been ob-

Gly - Gly - Phe- Met- Arg - Arg - Val- Gly - Arg - Pro- Glu - Trp - Trp- Met-

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FIG. 5. Nucleotide sequence of the

[Met]enkephalin pentadecamer-primed -Asp -Tyr - Gin - Lys - Arg -Tyr- Gly - Gly - Phe - Leu . U GGN G -GAC UAC CAG AAA AGG UAC GGN GGN UUC CUN ... -CTG ATG GTC TTT TCC ATG CCG CCG AAG TAC .

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62-base cDNA transcript. The amino acid sequence of bovine adrenal medulla opiate peptide E (12) is shown on the top line. The nucleotide sequence of cDNA62 and the mRNA sequence from which it was derived are shown on the next two lines. The nucleotide sequence of [Metlenkephalin primer is

shown on the bottom line.

364

Biochemistry: Comb et al.

Proc. Natl. Acad. Sci. USA 79 (1982)

RNA prepared from human pheochromocytoma, we have detected a RNA species (Fig. 6a) slightly smaller than the bovine pro-enkephalin mRNA.

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Note Added in Proof. Since this paper was submitted we have cloned

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and determined the sequence of a near-full-length cDNA of human

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We thank Dr. Lee Eiden of the National Institutes of Health for supplying human pheochromocytoma material. This work was supported by National Institutes of Health Grants 2 R01 AM 16879 and DA 02736.

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FIG. 6. Hybridization of poly(A)-RNA isolated from a human pheochromocytoma to [Metlenkephalin pentadecamer and cDNA62. Five micrograms of human adrenal poly(A)-RNA was applied to lanes 1 and 3 and 5 ug of bovine poly(A)-RNA was applied to lanes 2 and 4. The electrophoresis, blotting, and hybridization procedures were as described in the text. Blot a was probed with 107 cpm (3 x 106 cpm/ pmol) of the pentadecamer. Blot b was probed with 3 x 104 cpm (6 x 106 cpm/pmol) of cDNA62.

tained by hybridization to a probe that is complementary to the Met-Lys-Lys-Met-Asp sequence. Amino acid sequence analysis of a 3000-dalton peptide from adrenal medulla has revealed that the Met-Lys-Lys-Met-Asp sequence is adjacent to one of two enkephalin sequences in this peptide and hence should be coded for by the same mRNA as the enkephalins (11). This probe formed a stable hybrid with the same size mRNA as the enkephalin probe. Therefore, chemically synthesized probes complementary to nucleotide sequences in RNA that code for two different regions of the same peptide hybridize to the same size RNA from the adrenal medulla, providing strong evidence that this RNA is pro-enkephalin mRNA. The [Met]enkephalin probe hybridizes to a single class of poly(A)-RNA from bovine adrenal medulla, yet when used to prime cDNA synthesis from the same RNA preparation it results in the synthesis of a large number of different-sized cDNA products (Fig. 4). Similar size heterogeneity of cDNA transcripts has been reported for several other oligonucleotide primers (15, 27). We found that some of the complexity of the observed transcription products is due to the premature termination of products primed from the same initiation site. We do not yet know the reason for this premature termination. Efficient priming has been observed with probes as short as 11 bases (27) under conditions similar to those used here. It is possible that the specificity of priming with the pentadecamer probe is reduced to that of an undecamer in our priming experiments. To identify cDNA transcripts

derived from the enkephalin

mRNA, bovine adrenal medulla poly(A)-RNA

was

fractionated

gradient and a fraction enriched for the enkephalin mRNA was used to prime cDNA synthesis. A 62-base cDNA product was observed to be enriched severalfold over that produced from the total poly(A)-RNA. Back hybridization and DNA sequence analysis revealed that this transcript arose from the 1.45-kb RNA species, and that it codes for the bovine adrenal medulla peptide E (12). This extremely potent opioid peptide contains both a [Met]enkephalin and a [Leu]enkephalin sequence. Interestingly, this transcript arises from a priming event at the [Leu]enkephalin sequence, indicating that the 5'terminal nucleotides of the [Met]enkephalin probe do not contribute significantly to the specificity of priming under the conditions of annealing and transcription described. Recent studies with human adrenal medulla tumors (pheochromocytomas) (28) indicate that [Met]enkephalin is synthesized as a high molecular weight precursor in humans and that this precursor contains multiple copies of enkephalins. Using the [Met]enkephalin oligonucleotide to probe blots of poly(A)on a sucrose

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