* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Nucleotide Sequence of the DNA Complementary to Avian (Chicken
Real-time polymerase chain reaction wikipedia , lookup
Peptide synthesis wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Multilocus sequence typing wikipedia , lookup
Non-coding DNA wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Biochemistry wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Gene expression wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Point mutation wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Polyadenylation wikipedia , lookup
Community fingerprinting wikipedia , lookup
Genetic code wikipedia , lookup
Biosynthesis wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Nucleotide Sequence of the DNA Complementary to Avian (Chicken) Preproparathyroid Hormone mRNA and the Deduced Sequence of the Hormone Precursor John Russell and Louis M. Sherwood Departments of Medicine (J.R., L.M.S.) and Biochemistry (L.M.S.) Albert Einstein College of Medicine Bronx, New York 10461 The nucleotide sequence of avian (chicken) preproPTH (prepro-PTH) mRNA was determined from a 2.3kilobase fragment of complementary chicken parathyroid DNA cloned in E. co/i MM 924. Northern blot analysis of chicken parathyroid mRNA, using both bovine and chicken cDNA probes, showed that the mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for mammalian prepro-PTH. Cleavage of the cloned DNA with restriction endonuclease Pst\ resulted in three fragments, each of which was subjected to sequence determination. The hormone sequence deduced from the DNA showed that chicken preproPTH mRNA encoded a 119-amino acid precursor which included a 25-amino acid signal sequence, a six-residue prohormone peptide, and an 88-amino acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs and included gene deletions and insertions. There was significant homology of sequence in the biologically active 1-34 region with mammalian hormones, but much less in the middle and carboxyl-terminal regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in studying evolution of the gene as well as structurefunction relationships of the hormone. (Molecular Endocrinology 3: 325-331, 1989) end of the hormone molecule (2-4). At present, the sequences of prepro-PTH from four different mammalian species (bovine, human, rat, and porcine) have been determined (5-8). All four share a high degree of homology and consist of a precursor hormone molecule of 115 amino acids, which includes a 25-member signal sequence, a hexapeptide prohormone region, and an 84-residue hormone sequence. To investigate PTH from nonmammalian species and to extend our studies on its biosynthesis (9-11), we have cloned the cDNA for chicken prepro-PTH mRNA. Northern blots of chicken prepro-PTH mRNA and subsequent analysis of the sequence of cloned cDNA demonstrated that it shared considerable homology with mammalian preproPTH mRNA, but it also contained some striking differences. Chicken prepro-PTH mRNA was almost 3 times larger than its mammalian counterparts (2.3 kilobases compared with 0.8-0.9 kilobases), and it encoded a larger hormone precursor containing 119 amino acids. The additional length could be accounted for by the hormone portion of the polypeptide, which contained 88 amino acids instead of 84. In this study we report the sequence of several cDNA fragments which has allowed us to deduce the primary structure of chicken prepro-PTH as well as the structure of its mRNA. RESULTS INTRODUCTION PTH is a major regulator of the serum calcium concentration. In conjunction with 1,25-dihydroxyvitamin D3, the active metabolite of vitamin D, PTH acts on bone and the kidney to mobilize calcium into the extracellular fluid (1). The biological activity of PTH on its target tissues has been shown to be present in a unique series of amino acids from residues 1 -34 at the amino-terminal 0888-8809/89/0325-0331 $02.00/0 Molecular Endocrinology Copyright © 1989 by The Endocrine Society In a wheat germ cell-free system, the poly(A+) RNA fraction of chicken parathyroid glands translated a major protein component which migrated slightly ahead of bovine prepro-PTH on sodium dodecyl sulfate (SDS)polyacrylamide gels (Fig. 1). This protein fraction could be immunoprecipitated by antibodies to the aminoterminal portion of bovine PTH; the addition of a microsomal membrane preparation to the translation mix converted it to a smaller protein, indicating the presence of a signal sequence. When total chicken parathyroid mRNA was analyzed by Northern blotting and hybridization with a cDNA probe containing the coding se325 Vol 3 No. 2 MOL ENDO-1989 326 kb -4.4 43- -2.37 -I.35 26- -0.33 18- 1 2 B C Fig. 1. Poly(A+) from Bovine (Lane B) and Chicken (Lane C) Parathyroid Glands Was Translated in a Wheat Germ System and Immunoprecipitated with Antibodies to the Amino-Terminal Portion of Bovine PTH Translation products were separated on 15% SDS-polyacrylamide gels along with mol wt markers which included ovalbumin and chymotrypsinogen, /Mactoglobulin, lysozyme, bovine trypsin inhibitor, and insulin (a- and /3-chains). quence for bovine PTH, a faint but distinct band was observed midway between 28S and 18S ribosomal RNA (Fig. 2). The size of the mRNA was calculated to be about 2.3 kilobases. A cDNA library was generated from the total poly(A+) fraction described, ligated into the Pst\ site of PBr322, and used to transform E. coli MM294 (12). Transformed colonies were screened with a cDNA probe for bovine PTH mRNA. After screening approximately 1000 colonies by in situ hybridization, we selected 22 for further characterization. Since the cross-hybridization between bovine and chicken PTH sequences was weak, we used the additional technique of hybridization selection (13) to eliminate any false positives from the first screening step. Of the 22 positive clones from in situ hybridization, five (Pc-4, Pc-7, Pc-9, Pc-17, and Pc-19) were also shown to be positive by this procedure. Further characterization of the plasmids from these colonies showed that the one with the largest cDNA insert produced three fragments when cut with Pst\ (PC-4). The fragments contained a total of Fig. 2. Northern Blot Analysis of Bovine and Chicken Parathyroid Gland Poly(A+) RNA Approximately 5 ng chicken (lane 1) and 1 fig bovine poly(A+) RNA (lane 2) were denatured in formaldehyde, separated on a 1.5% agarose gel, and transferred to a nylon filter by electroblotting. The filter was probed with nick-translated cDNA for the coding region of bovine PTH mRNA, and autoradiography was performed overnight at - 8 0 C using Kodak X-Omat AR film. 2.3 kilobases (see Fig. 3). When nick-translated and hybridized individually to a Northern blot of total chicken parathyroid mRNA, each one produced an intense band in the region of 2.3 kilobases where earlier we had seen cross-hybridization with the bovine cDNA probe. In contrast to the bovine probe, however, the chicken cDNA resulted in a signal that was approximately 100fold greater. Sequence analysis (together with overlaps provided by the other enzyme cleavages) showed that the smallest Pst\ fragment was at the 5' end, the intermediate fragment in the middle, and the largest fragment at the 3' end (Fig. 4). An initiator codon (ATG) was found in the middle of the smallest fragment, beginning an open reading frame which could be read downstream from this site for 357 bases, where it terminated in a stop codon (TGA). The last 181 bases of open reading frame were in the intermediate fragment. The longest fragment was placed at the 3' end and contained the polyadenylation signal (AATAAA) and the poly(A) tail. A second polyadenylation signal was identified in the intermediate fragment (see Fig. 4). The sequence of the three fragments was confirmed by overlapping the fragments obtained with the other endonucleases (Haelll, H/ndlll, and Taq\). Sequence of cDNA to Chicken Prepro-PTH mRNA 327 500 I HaeHI I Pc-4 Psll HindlH I Toql I I I HaeEI TaqI 2000 I 1500 I Hindi! Psll I 1 Pc-9 1000 Hoelll Toql Haeffi I I I I I Toql HaeUI Toql H Fig. 3. Partial Restriction Map of the cDNA Insert from Pc-4 Showing Fragments Used to Determine the Sequence Pc-9 is shown below which is identical to Pc-4, except that the 5' end is shortened by 156 bases. SEQUENCE OF cDNA CORRESPONDING TO AVIAN PRE-PRO-PTH mRNA 100 GATTTAAGGGATCCACTAAACCAATTCAGTAGTAGTCTTTAAATATACTTGACATAAGACACAGCCArCTGCTGACArACCCCAACCAGAAAACTGTTAAGGACAATATCTGATAAAA 200 ATG ACT TCT ACA AAA AAT CTG GCC AAG GCC ATA GTG ATT TTA TAT GCT ATA TGT TTT TTT ACA AAC TCT GAT GGA AGA CCA ATG ATG Met Thi Ser Thr Lys Asn Leu Ala Lys Ala lie Val lie Leu Tyr Ala le Cys Phe Phe Thr Asn Ser Asp Gly Atg Pro Met Met AAG AGA TCG GTG AGT Lys Arg Scr Val Ser 300 CTG CAG GAT GTG CAC Leu Gin Asp Val His GAG ATG CAA TTA ATG CAT AAC CTT GGA GAG CAT CGA CAC ACT GTG GAG AGA CAG GAC TGG CTT CAG ATG AAG Glu Met Gin Leu Met His Asn Leu Gly Glu His Arg His Thr Val Glu Arg Gh Asp Trp Leu Gin Met Lys AGT GCC CTT GAG GAT GCC AGG ACC CAG AGG CCT CGA AAC AAG GAG GAT ATT GTC CTG GGG GAG ATA AGA AAC Ser Ala Leu Glu Asp Ala Arg Thr Gin Arg Pro Arg Asn Lys Glu Asp le Val Leu Gly Glu le Atg Asn T 400 CGG AGG CTG CTC CCT GAG CAT TTG CGG GCA GCA GTG CAG AAG AAA TCC ATT GAC CTG GAC AAA GCT TAC ATG AAT GTA CTC TTT AAA Arg Arg Leu Leu Pro Glu His Leu Atg Ala Ala Val Gin Lys Lys Set le Asp Leu Asp Lys Ala Tyr Met Asn Val Leu Ptie Lys soo ACT AAG CCA TGA TGA AAAGACCAAGAGCATTATAACTGCCAAGTAAGCACATGTTGTAGATCACTGACCAGTTAGGGCATTTTATTTATT Thr Lys Pro MO ATTTTTTTTTATTTAACTCAAACTATGATAAGGATTAAAGGCTCCATGCCAGACTGTAGCCCCACTGAGATGGGTATTTCACAACTAAATA 700 CTATATGGAGAGCATTTGTCTGTAATCTTTAGACCTACTAGTACTGTAAACTAACAACGTAATATAGGCATAACTGCATTAGTCCTAGGGT 1(KK> ATAA^ATTGTGCATAAAAAGAAGAACAAGTTTTACATATACTGAAATGGAAGGGAGGTTTATTAACTTTCCCTCTTAATTATGAGCTGTCAC 1200 1 1300 GATAAAAACAAGACTTGTTTCAATTGTTATCATCTCTCCTTCAGTCAATAATCTATGAGTTTCTGTATATTGTGCTTAGGCCACATGGGTA AGTGGCTCACATAAAATTACTCATCTTCACATGTGCACTTATACAGAAATTGGGATTTCAGTTTGTTAAAACCCTGAAATTACAACCATTA AAATATAGAAATCAAAACCTGGGAACCATCAGTTAAAATATAAGCAGGATTCAGAAAGAATTTGACAGGAACATGGATGGGAGAAAATGAT ibOO GATTAATAATATAGAAAAGAAAGCAGCAAATATAAAATGATTTTGAATTGTATAGACAAGTAGTGCTTATGACCTCGACACCTTCTGAAT GCCAACACATCCTGTGTGGTCCCATACCACTTCCTTGTTTGTTTGATGTCCTCGACACATCTTGGGAACGTAATGAAAAACTCATACATAATT TACAAAATAAAGTGGACCAAAAGCTCTTGGACATTATTTTAAAGCATAAAAAAAAAAAAAAA Fig. 4. Base Sequence of DNA Encoding Chicken Prepro-PTH mRNA In the open reading frame (starting on line 2), the bases are separated as triplets, and the deduced amino acid sequence for the hormone precursor of 119 amino acids is indicated. The open reading frame extends for 357 bases, followed by two TGA stop codons. The Pst\ cleavage sites separating the three DNA fragments (Fig. 3) are indicated by arrows, and the two polyadenylation signals are underlined. Complete sequence analysis of the intermediate and large fragments allowed us to place almost 1700 additional basepairs in sequence beginning at the 3' end of the termination codon and ending at the poly(A) tail. Figure 4 shows the base sequence for cDNA strand corresponding to the sequence of chicken prepro-PTH mRNA together with the deduced amino acid sequence for the coding region. A comparison of the amino acid sequence of chicken prepro-PTH with those of bovine, human, and rat is shown in Table 1. All four precursor hormones contained a 25-member signal peptide which terminates in glycine. In this region the chicken sequence shared 44% homology with bovine, 48% with human, and 36% with rat peptides. The six residue pro sequence of the chicken precursor shared 50% homology with the rat propeptide, but only 33% with both the bovine and human sequences. The region that showed the most conservation of homology with the mammalian hormones consisted of residues 1-34 of the hormone sequence, which is the part of the molecule responsible for hormonal activity (2, 3). In this region the chicken sequence shared 59% homology with bovine, 65% with human, and 65% with rat amino-terminal peptides. Twenty residues in the 1 -34 region of all four hormone sequences were identical, and all residues were encoded by the same triplet or had only a single substitution. At the remaining 14 positions, all but two resi- Vol 3 No. 2 MOL ENDO-1989 328 Table 1. Comparison of Chicken Prepro and 1-34 Sequences with Mammalian Counterparts Conserved Amino Acid (%) Prepeptide Propeptide PTH-(1-34) Conserved Nucleotide (%) Bovine Human Rat Bovine Human Rat 11/25(44) 2/6 (33) 20/34 (59) 12/25(48) 2/6 (33) 22/34 (65) 9/25 (36) 3/6 (50) 22/34 (65) 47/75 (63) 11/18 (61) 70/102(69) 49/75 (65) 12/18 (67) 75/102(74) 42/75 (56) 13/18 (72) 65/102(64) -20 -10 T[S]T[K]N L A[K]A I V I L YA M[S]A[K]D M V[KJV H I V H L -5 I C F F T N S D GR P N N K R S V S E A I C F L A R S D GK S V K K R A V S E IT N H M H I|Q|F 20 30 [HIR H T RK [HJLSS 50 R P R N K E D I V L GITII 70 60 80 R N R R L L P E H L R A A V Q K K S I D L OKA R P R K K E D N V L V|E|S HQ K S L G E A Y N N D K ADVD 88 V L V L I[K Fig. 5. Comparison of Chicken and Bovine PTH Sequences with Regions of Homology Enclosed in Solid Black Lines The two proposed deletion/insertion events are indicated. dues (5 and 18) showed changes at two or three bases. As was true for the mammalian hormones, the first 14 residues of the chicken hormone also showed a high degree of homology (7 of 14) with the amino-terminus of the PTH-related peptide associated with humoral hypercalcemia of malignancy (14). Beyond the 1-34 region sequence homology between chicken and mammalian hormones was much less conserved. In fact, the chicken hormone sequence was longer by four amino acids, giving it a total length of 88 amino acids, compared with 84 for the mammalian sequences. The additional length and loss of homology could be explained on the basis of gene deletions and insertions, which will be discussed below (see Fig. 5). The larger size of the chicken hormone was somewhat surprising, since chicken prepro-PTH migrated faster than bovine prepro-PTH on SDS-polyacrylamide gels (Fig. 1). The bovine precursor hormone migrated with an apparent mol wt of 13,800, which was in good agreement with its actual mol wt of 13,000. In contrast, when SDS-gel electrophoresis was used to calculate the mol wt of chicken prepro-PTH, it was determined to be 11,250 compared with the actual mol wt of 13,920 based on the amino acid sequence. As yet, analysis of the primary structure has yielded no obvious clues that would explain the anomalous behavior of the peptide on SDS-gel. The 5' noncoding portion of chicken prepro-PTH mRNA contained 115 bases and was slightly larger than the 5' flanking sequences reported for mammalian PTH mRNA. The 3' noncoding region was quite large and consisted of almost 1700 basepairs, which was 1300 basepairs longer than the longest 3' noncoding sequence reported for mammalian PTH mRNAs. DISCUSSION A 2.3-kilobase mRNA was identified in extracts of avian (chicken) parathyroid glands which encoded a precursor prepro-PTH molecule of 119 amino acids. The mRNA was almost 3 times the size of its mammalian homologs and encoded a precursor molecule four residues longer than the mammalian precursors. In the deduced precursor peptide sequence, the signal and propeptides were identical in size with the mammalian proteins, but the hormonal peptide contained 88 residues rather than 84. Although the signal peptide of the chicken sequence was identical in length with that in the mammalian hormones, there was only moderate homology in this region. The main regions of homology were in the sequence around the cysteine at position 18 as well as the glycine at position 25 which serves as the site of cleavage of the leader sequence. The signal sequence had a high degree of hydrophobicity, however, which is characteristic of such sequences. In addition, the two positive charges contributed by lysines at positions - 2 7 and - 2 3 were preserved in the chicken sequence, which is in agreement with the concept that a net positive charge in this region is necessary for secretory function. The chicken hormone also contained a sixmember propeptide which ended in the dibasic sequence Lys-Arg, as is true for the mammalian homo- 329 Sequence of cDNA to Chicken Prepro-PTH mRNA logs. Like mammalian prohormone sequences, it was highly positively charged, with three instead of four residues being basic. The dibasic carboxyl-terminal sequence serves as a recognition point for proteolytic cleavage. It was not surprising to find that the greatest conservation in sequence homology was in the amino terminal 1-34 portion of the hormones, since this region has been shown to be responsible for the biological activity of PTH (2, 3). Within this region, the amino-terminal Ser-Val sequence is identical to that of human PTH, and from previous studies has been shown to be required for biological activity (2, 6). In addition, two domains have been delineated that bind the PTH receptor (15). These domains are located within residues 1 27 and 25-34, although the latter may be more important in binding activity. Although the 25-34 region of the chicken PTH sequence contains many features in common with the mammalian hormones, it lacks the tribasic sequence from residues 25-27, which is thought to be important for receptor binding (15). The Arg-Lys sequence from 25-27 present in the mammalian hormones corresponds to Gln-Met-Lys in the chicken sequence. In the region of the amino-terminal peptide from residue 1-14 there was 7 1 % homology (10 of 14 residues) between chicken and bovine peptides, with only 50% homology (10 of 20) between residues 15 and 34. The amino acid sequence of the tumoral peptide in humoral hypercalcemia of malignancy also shows striking similarity to mammalian PTH in the 1-13 region (8 of 13 residues) (14). It will be of interest to compare the biological activities of synthetic chicken and mammalian hormones to determine whether this change in structure affects its ability to bind PTH receptors and produce specific biological effects. In an initial study (16) we have compared the biological activities of synthetic avian PTH(1-34) and bovine PTH(1-34) in mammalian assays and showed a marked relative decrease in the potency of the avian peptide. A comparison of the amino acid hydropathy (17) in this region showed that the change in amino acid sequence resulted in a more hydrophobic environment around these residues in the chicken hormone, which could alter its ability to bind receptors. Beyond the 1-34 portion of the hormone, the homology was much less apparent. The additional length of the chicken hormone and the difference in sequence could be attributed to at least two deletion and insertion events. The first deletion involves residues 32-44 of the mammalian sequence, which is replaced by four amino acids in the chicken sequence (see Fig. 5). This occurs in the region where the mammalian hormone is cleaved to produce the biologically active amino-terminal fragment (15). It is not known at this time whether the chicken hormone undergoes similar proteolytic processing, but if it does, the difference in sequence in this region might imply that an enzyme(s) with a different specificity may be involved. This first deletion/insertion is followed by a region of strong homology from residues 37-53 of the chicken peptide, which corresponds to residues 45-61 of the mammalian sequence. This is followed by a second region of nonhomology resulting from a deletion of residues 62-70 of the mammalian sequence corresponding to an insertion of 22 amino acids in the chicken sequence. A high degree of homology between the avian and mammalian sequences is observed again in the COOH-terminal region corresponding to residues 76-88 in the chicken and residues 71-83 in the cow, human, and rat (see Fig. 5). The chicken hormone sequence ends with two in-phase termination codons (TGA TGA), which is similar, although not identical, to the human hormone sequence, which terminates in TGA AAA TGA. Downstream from the hormonal sequence was a 3' noncoding region of almost 1700 basepairs, far longer than that for any of the mammalian hormones. At least two polyadenylation signals were present in the sequence (see Fig. 4), but only one appears to be functional. In these studies we have determined the structure of the mRNA for avian prepro-PTH and the deduced hormone sequence. This is the first nonmammalian PTH sequence to be determined, and it shows some interesting differences from mammalian homologs. In the biologically active amino-terminal end there is significant homology in the 1-14 region, but some critical changes in the tribasic region (residues 25-27) thought to be important for receptor binding. Our findings provide new opportunities to study correlations of structure and function and regulation of avian PTH gene expression. MATERIALS AND METHODS Preparation of Poly(A+) RNA Newborn chicks were raised on a vitamin D3-deficient diet in the dark for 5-6 weeks. The glands were harvested, and total RNA prepared by the guanidinium isothiocyanate method (18). The poly(A+) fraction was then isolated by chromatography on oligo(dT) Sephadex (19). Approximately 1 ng poly(A+) RNA was translated in 25 fi\ cell-free wheat germ extract using labeled [35S]methionine (Amersham, Arlington Heights, IL). Incorporation of [35S]methionine was typically 7- to 10-fold compared with the blank reaction which contained no mRNA. Translation products were characterized by electrophoresis on 15% SDS-polyacrylamide gel electrophoresis. Immunoprecipitation of Translation Products Translation mixtures were diluted 10-fold with immunoprecipitation buffer (10 miui Tris, pH 7.4; 0.15 M NaCI; 10 ITIM EDTA; 0.5% Triton X-100; 0.5% sodium deoxycholate; and 3 mg "gelatin/ml), and 63 /*g guinea pig antiserum to synthetic aminoterminal (residues 1-34) bovine PTH (Beckman, Fullerton, CA) were added. The mixture was incubated on ice for 1 h, at which time 10 n\ goat antiguinea pig 7-globulin (Arnel, New York, NY) were added and incubated overnight at 4 C. The mixture was spun at 15,000 x g for 10 min, and the pellet was resuspended in SDS sample buffer containing 8 M urea, boiled for 2 min, and subjected to electrophoresis on 15% SDS-polyacrylamide gel electrophoresis. Northern Blot Analysis Approximately 2.5 ng total poly(A+) RNA were denatured by incubation for 15 min at 55 C in buffer containing 50% form- Vol 3 No. 2 MOL ENDO-1989 330 amide, 20 HIM (3-[N-Morpholine] propane-sulfonic acid) (pH 7.0), 6% formaldehyde, and 1 ITIM EDTA. The sample was loaded onto 1.5% agarose gels and separated by electrophoresis for 4 h at 75 V. The RNA was transferred from the gel to a 2 eta probe nylon membrane (Bio-Rad, Richmond, CA) in 10 nriM Tris acetate (pH 7.4) and 1 mM EDTA by electroblotting at 250 mamps overnight. The dried membrane was hybridized with a 650-basepair cDNA probe containing the entire coding sequence for bovine prepro-PTH (12). The probe was labeled by random primer extension (IBI) to a specific activity of 109 cpm/jtg DNA, and hybridization was carried out at 42 C overnight in buffer containing 4x FSPE (1x FSPE = 0.15 M NaCI, 10 mM Na2HPO4, and 1 mM EDTA, pH 7.4), 5x Denhardt's solution (1x Denhardt's = 0.01% Ficoll, 0.01% BSA, and 0.01% polyvinyl pyrollidone), 0.5% SDS, and 0.05 mg/ml salmon sperm DNA. After hybridization, the filters were washed twice with 2x SSC (1 x SSC = 0.5 M NaCI and 0.015 M Na3 citrate, pH 7.0) at room temperature and then three times in 0.1 x SSC at 55 C. Preparation of cDNA and Amplification in E. coli Single-stranded cDNA was prepared from the poly(A+) fraction using reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD) by procedures described previously (12). The reaction was terminated by extraction with phenol and ethanol precipitation. The cDNA:mRNA hybrid was converted to double-stranded DNA by the method of Gubler and Hoffman (20), and double-stranded cDNA was poly d(C)-tailed and annealed to Pst\ cut, poly d(G)-tailed PBr 322. The annealed circular DNA was used to transform E. coli strain MM 294 (Clontech, Palo Alto, CA) by a modification of the method of Hanahan (21). Colonies that contained plasmids with cDNA inserts were ampicillin sensitive and tetracycline resistant, and were selected using an AMP screen (Bethesda Research Laboratories). Identification of Chicken Prepro-PTH cDNA Colonies were initially identified by filter hybridization (22) using a radiolabeled cDNA probe for bovine prepro-PTH as described above. Weak hybridization between the bovine and chicken PTH sequences necessitated the use of an additional screening procedure, hybridization selection (13). Positive colonies were selected by their ability to hybridize selectively the mRNA for chicken prepro-PTH, which could be eluted and identified by translation in the wheat germ system. DNA inserts were cut out by digestion with Pst\ and analyzed on 2% agarose gels. Sequence Analysis cDNA fragments were subcloned into m13 (New England Biolab, Beverly, MA) and sequenced by the dideoxy method of Sanger (23). To provide overlapping sequences, additional fragments were created by digestion with the restriction endonucleases H/ndlll, Haelll, and 7agl. All fragments were purified by gel electrophoresis and cloned into m13 vectors. Ambiguities were resolved by sequencing both strands of DNA and, in some instances, by substituting ITP for GTP to eliminate compression. To verify the sequence, two independent clones were analyzed. Acknowledgments We wish to thank Ms. Deborah Lettieri for her excellent technical assistance in this project and Dr. S. Hurwitz of the Volcani Center, Israel for suppling the chicken parathyroid glands. Received October 19,1988. Accepted November 14,1988. Address requests for reprints to: Louis M. Sherwood, Department of Medical and Scientific Affairs, Merck, Sharp and Dohme International, P.O. Box 2000, Rahway, New Jersey 07075. These studies were supported in part by Grants NIDDK 34822 and 28556 from the USPHS. A preliminary account of the work was presented at the 10th Annual Meeting of the American Society of Bone and Mineral Research, June 6,1988, New Orleans, LA. REFERENCES 1. Feinfeld D, Sherwood LM 1988 Parathyroid hormone and 1,25(OH)2D3 in chronic renal failure. Kidney Int 33:10491058 2. Tregear GW, Van Rietschoten J, Greene E, Keutmann HT, Niall HD, Reit B, Parsons JA, Potts Jr JT 1973 Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity. Endocrinology 93:1349-1353 3. Potts JT, Tregear GW, Keutmann HT, Niall HD, Sauer RT, Deftos LJ, Dawson BF, Hogan ML, Aurbach GD 1971 Synthesis of a biologically active N-terminal tetratriacontapeptide of parathyroid hormone. Proc Natl Acad Sci USA 68:63-67 4. Rosenblatt M 1986 Peptide hormone antagonists that are effective in vivo. Lessons from parathyroid hormone. N EnglJMed 315:1004-1013 5. Weaver CA, Gordon DF, Kemper B 1982 Nucleotide sequence of bovine parathyroid hormone messenger RNA. Mol Cell Endocrinol 28:411-424 6. Hendy GN, Kronenberg HM, Potts Jr JT, Rich A 1981 Nucleotide sequence of cloned cDNAs encoding human pre-proparathyroid hormone. Proc Natl Acad Sci USA 78:7365-7369 7. Heinrich G, Kronenberg HM, Potts Jr JT, Habener JF 1984 Gene encoding parathyroid hormone. Nucleotide sequence of the rat gene and deduced amino acid sequence of rat pre-proparathyroid hormone. J Biol Chem 259:3320-3329 8. Sauer RT, Niall HD, Hogan ML, Keutmann HT, O'Riordan JL, Potts Jr JT 1974 The amino acid sequence of porcine parathyroid hormone. Biochemistry 13:1994-1999 9. Silver J, Russell J, Sherwood LM 1985 Regulation of vitamin D metabolites of messenger RNA for pre-proparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA 82:4270-4273 10. Cantley LK, Russell J, Lettieri D, Sherwood LM 1985 1,25-Dihydroxyvitamin D3 suppresses PTH secretion from bovine parathyroid cells in tissue culture. Endocrinology 117:2114-2119 11. Russell J, Lettieri D, Sherwood LM 1986 Suppression by 1,25(OH2)D3 of transcription of the parathyroid hormone gene. Endocrinology 119:2864-2866 12. Russell JR, Lettieri D, Sherwood LM 1983 Direct regulation by calcium of cytoplasmic mRNA coding for preproparathyroid hormone in isolated bovine parathyroid cells. J Clin Invest 72:1851-1855 13. Ricciardi RP, Miller JS, Roberts BE 1979 Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci USA 76:49274931 14. Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood Wl 1987 A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 237:893-896 15. Nussbaum SR, Rosenblatt M, Potts Jr JT 1980 Parathy- Sequence of cDNA to Chicken Prepro-PTH mRNA roid Hormone. Renal receptor interactions. Demonstration of two receptor-binding domains. J Biol Chem 255: 10183-10187 16. Caulfield MP, Levy JJ, McKee RL, Goldman ME, DeHaven PA, Reagan JE, Heaney L, Nutt RF, Winquist RJ, Russell J, Sherwood LM, Rosenblatt M, 1988 Avian (chicken) parathyroid hormone: synthesis and comparative biological evaluation of the 1-34 fragment. Endocrinology, 123:2949-2951 17. Kyte J, Doolittle RF 1982 A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-132 18. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294- 331 5299 19. Aviv H, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography in oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69:14081412 20. Gubler U, Hoffman BJ 1983 A simple and very efficient method for generating cDNA libraries. Gene 25:263-269 21. Hanahan D1983 Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557-580 22. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, pp 312-328 23. Sanger F, Nicklen S, Coulson AR 1977 DNA Sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467