THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 5, Issue of March 10, pp. 3320-3329, 1984 Printed in U.S.A.

Gene Encoding Parathyroid Hormone NUCLEOTIDE SEQUENCE OF THE RAT GENE AND DEDUCED AMINO ACID SEQUENCE OF RAT PREPROPARATHYROID HORMONE*

(Received for publication, July 11, 1983)

Gerhard Heinrich$, Henry M. Kronenbergs, John T. Potts, Jr.7, and Joel F. HabenerSJI From the $Laboratory of Molecular Endocrinology and §Endocrine Genetics Unit, YDepartment of Medicine, Massachusetts General Hospital, Harvard Medical School and the /(Howard Hughes Medical Institute Laboratories, Harvard Medical School. Boston. Massachusetts 021 14

The nucleotide sequence of the rat parathyroid hor- mone (PTH) gene was established from a 14.5-kilobase pair fragment of rat liver DNA cloned in bacteriophage Charon 4A. The transcriptional unit of the gene of 2.5 kilobase pairs is interrupted by two introns (1600 and 111 base pairs). Blot hybridization of restriction en- zyme digests of rat spleen DNA using 32P-labeled frag- ments of the cloned PTH gene suggests that the gene is unique and present in a single copy in the genome. A promoter sequence (Goldberg-Hogness or TATA box) is situated 28 base pairs upstream from the point of initiation of transcription which was found by S1 nu- clease mapping and by oligonucleotide-primed reverse transcription of rat PTH mRNA. The gene is flanked on its 5’ side by repetitive DNA and contains a differ- ent, more abundant repetitive DNA on its 3‘ side. The mRNA encoded by the parathyroid hormone gene con- sists of 800 2 50 nucleotides as determined by electro- phoresis on agarose gels. The 5’ untranslated region of the mRNA contains three AUG triplets. Only one AUG triplet initiates biosynthesis of preproparathyroid hor- mone (prepro-PTH). The 3’ untranslated segment of the mRNA contains two AATAAA sequences charac- teristic of polyadenylation signals. The mRNA encodes prepro-PTH, a precursor of PTH of 115 amino acids. When the amino acid sequence of the rat precursor is compared with the analagous bovine and human pre- cursors, it becomes evident that the hormone sequences are highly conserved in two regions of known function near the NH2 terminus and in a third region near the carboxyl terminus whose biologic function, if any, has not yet been defined.

PTH’ acts directly on bone and kidney to mobilize and conserve calcium and indirectly on the intestine to promote calcium absorption; the hormone is therefore an important determinant of the concentration of ionized calcium in the extracellular fluid (1).

The PTH gene encodes a 115 amino acid polypeptide in mammals (2). Proteolytic site specific cleavage during post- translational processing of this polypeptide, prepro-PTH, gives rise to PTH, a peptide of 84 amino acids secreted from the parathyroid glands ( 3 ) . The secretion and production of

* This work was supported by New Investigator Research Award Grant AM29669 and Grant AM11794 from National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicat,e this fact.

The abbreviations used are: PTH, parathyroid hormone, prepro- PTH, preproparathyroid hormone; kb, kilobase; bp, base pair.

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PTH are inversely regulated by the concentrations of calcium in the extracellular fluid (4, 5 ) .

We previously studied the short term (several hours) expression of the PTH gene at the levels of PTH secretion ( 6 ) , the translation and cellular content of the mRNA encod- ing prepro-PTH ( 7 ) , and the proteolytic processing of prepro- PTH (8). Regulatory studies at each of these levels of PTH gene expression showed that the concentration of extracellu- lar calcium is the principal regulator of parathyroid gland activity (1). Regulation at the level of PTH secretion occurs through negative feedback; elevation of extracellular ionized calcium above the physiologic level inhibits, and a fall below this level dramatically stimulates, PTH secretion both in vitro (7) and in vivo (9). Regulation of the production of PTH a t the levels of proteolytic processing and synthesis of prepro- PTH and pro-PTH is modest and has been more difficult to demonstrate ( 5 ) . Recently, we showed that cellular levels of PTH mRNA are perturbed little, if at all, under conditions of short term stimulation and suppression of PTH secretion in vitro ( 7 ) .

Compared with knowledge of short term regulation, little is known about the molecular mechanisms of long term regula- tion of PTH gene expression. Chronic stimulation of PTH secretion (for days and weeks) consistently induces enlarge- ment and proliferation of parathyroid secretory cells (10, 11). This hypertrophic and proliferative response must involve regulation of a complex array of genes whose coordinated expression ultimately raises the capacity of the parathyroid gland for the production and secretion of PTH. To analyze reguIation of PTH gene expression within the array of regu- lated genes in the chronically stimulated parathyroid gland, we wish to study the biosynthesis of parathyroid hormone in vivo using a convenient laboratory animal, the rat. We deter- mined the structure of the rat PTH gene to provide knowledge of the primary nucleotide sequence of the gene and of the deduced amino acid sequence of the encoded rat PTH precursor and to provide complementary DNA probes for hybridization analyses of PTH gene expression at the nucleic acid level.

EXPERIMENTAL PROCEDURES

Screening of Rat Genomic Library-A library of rat liver DNA partially digested with EcoRI and cloned in the A-phage derivative Charon 4A (12) was screened by filter hybridization at a density of 3,000 plaques/l5-cm plate (13). The hybridization probe was a cDNA to bovine PTH mRNA previously cloned in the plasmid pBR322 and designated pPTHml (14). The cDNA insert containing >80% of the PTH mRNA sequence was nick-translated in the presence of [a-”P] deoxycytosine to a specific activity of 10’ cprnlpg of DNA (15). Filter hybridizations were performed in 6 X SSC (1 X SSC = 0.15 M NaC1, 0.015 M Na, citrate) at 65 “C, and the filters were washed in 2 X SSC

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Preproparathyroid Hormone Gene 3321

at room temperature. Two of the approximately 200,000 Plaques, representing approximately one genome, hybridized to the cDNA.

Characterization of Recombinant Cloned DNAs-The two clones of recombinant Charon 4A phages hybridizing to the bovine cDNA were isolated, and DNA was prepared from the purified recombinant phages. Restriction enzymes were obtained from New England Bio- labs, Beverly, MA, or Bethesda Research Laboratories, Bethesda, MD, and used according to the manufacturer's recommendations.

Restriction fragments hybridizing to the cDNA were identified by the blot-hybridization technique of Southern (16). Hybridization Conditions were identical with those used for library screening. How- ever, a cloned cDNA to human PTH mRNA, designated pPTHm122, was used as a 32P-labeled hybridization probe (17).

Subcloning of Phage Genomic DNA into Plasmid pBR322"Inas- much as the two rat genomic DNAs inserted in the Charon 4A phages were shown to share several identical restriction fragments (see below), subsequent analyses and sequencing were performed on the larger clone, rPTHg-1 (Fig. 1111). One pg of rPTHg-1 was alterna- tively digested with the restriction endonucleases BamHI or HindIII, and the individual mixtures of restriction fragments was ligated into the corresponding restriction site of pBR322 using T4 DNA ligse (P- L Biochemicals). Aliquots of the ligation mixtures were used to transform Escherichia coli strain C600. Single bacterial colonies con- taining BarnHI fragments of PTH-related sequences (rPTHs-I, Fig. 1IV) were selected by colony hybridization (18) using the pPTHml insert as a 32P-labeled hybridization probe. A subclone (rPTHs-2; Fig. 111) adjacent to the 5' end of PTHs-1 was identified in the HindIII subclone library by colony hybridization using the 32P-labeled 1.9- and 2.0-kb EcoRl fragments of rPTHg-1 (Fig. 11ZI) as hybridization probes. From this subclone (rPTHs-Z), a 1.4-kb EcoRIjHindIII frag- ment representing the 5' end of the 8.5-kb EcoRI fragment of rPTHg- 1 was further subcloned as rPTHs-3 (Fig. 11). To accomplish this, the orientation of the rPTHs-2 insert with respect to pBR322 was first determined by restriction enzyme analyses. These analyses in- dicated that the desired EcoRIjHindIII fragment was located toward the BamHI site of pBR322 (Fig. 111). Therefore, rPTHs-3 was con- structed from rPTHs-2 by digestion of rPTHs-2 with EcoRI and religation with T4 DNA ligase. Ligation conditions were adjusted by dilution of the rPTHs-2 digest to favor intramolecular ligation. The resulting construct, rPTHs-3, is shown in Fig. 11.

Sequencing of Genomic Insert of rPTHg-I-AI1 DNA sequencing was performed by the chemical method of Maxam and Gilbert (19). Restriction fragments labeled at the 5' end were prepared by a 32P- exchange reaction with [Y-~'P]ATP catalyzed by polynucleotide ki- nase (P-L Biochemicals), and 3' labeling was accomplished with the Klenow fragment of E. coli DNA polymerase (Bethesda Research Laboratories) (20), or by transferring [32P]cordycepin (Bethesda Re- search Laboratories) to the 3' OH end using terminal transferase (Bethesda Research Laboratories) (21).

Determination of the Size of Rat PTH mRNA-Hyperplastic para- thyroid glands were obtained from rats rendered hypocalcemic for 6 weeks by feeding them a diet deficient in vitamin D and calcium (22). The glands were immediately frozen on dry ice. Twenty-four glands, weighing approximately 0.6 mg each, were homogenized in 0.5 ml of 4 M guanidine thiocyanate, 0.1 M (3-mercaptoethanol, and 0.02 M sodium citrate (pH 7) using a Brinkman Polytron homogenizer. 0.08- ml aliquots of the homogenate were layered over 0.05 ml of a 5.7 M CsCl cushion, and centrifuged for 16 h a t 100,000 X g in a Beckman Airfuge. The supernatant was discarded, and the pellet was taken up in 0.1 ml of 0.2 M Na acetate (pH 5), and immediately precipitated by addition of 2.5 volumes of 95% ethanol. The precipitate was dissolved in 50 p1 of HzO, and stored a t -70 "C.

For the determination of the size of PTH mRNA, an aliquot of 1 p1 of the RNA was denatured in an aqueous solution consisting of 50% formamide (v/v) and 6% formaldehyde (v/v) and separated by electrophoresis through a 1.4% agarose gel containing 6% formalde- hyde. Size markers were human and bovine PTH mRNA, 28 S, 18 S calf liver RNA (P-L Biochemicals), and yeast tRNA (Bethesda Re- search Laboratories). The fractionated RNAs were transferred to Gene Screen (New England Nuclear) by electrophoresis. The blot was hybridized with the nick-translated 450-bp NcoIIXbaI fragment of rPTHs-1, a subclone of rPTHg-1, encompassing >90% of the protein coding sequence of rPTH mRNA, and intervening sequence B (111 base pairs long, see below). Hybridization was performed in 6 X SSC and 50% formamide at 42 "C. The blot was washed three times in 2 x SSC, 0.1% sodium dodecyl sulfate (wjv) for 30 min a t room temperature. Inasmuch as the rat NcoIjXbaI fragment hybridized at

a nearly undetectable level to human and at an undetectable level to bovine PTH mRNA under these conditions, the blot was rehybridized with 3ZP-labeled human cDNA (pPTHm122) and bovine cDNA (pPTHml) and washed again under these conditions.

Localization of the 5' Untranslated Sequence of PTH mRNA in the Cloned Genomic DNA-An intervening sequence (intron A or Ivs A) was found to interrupt the 5' untranslated sequence of PTH mRNA, 5 nucleotides upstream from the initiation codon ATG. The cloned human/bovine cDNAs (pPTHm122, pPTHml) could not be cross- hybridized to the rat 5' untranslated sequences even under minimally stringent hybridization conditions (40 "C, 2 X SSC). Therefore, the untranslated sequence in the genomic DNA was localized by hybrid- izing various nick-translated fragments of a genomic subclone to a rPTH mRNA blot. A 1.4-kb EcoRljHindIII fragment of the subclone rPTHs-2, located 1.6-kb upstream from the 3' end of intron A, hybridized to the immobilized rPTH mRNA. This fragment of DNA was subcloned (rPTHs-3) and sequenced as described above.

Identification of the Site of Initiation of Transcription and Location of Intron A in the PTH Gene-We used the methods of specific primed cDNA synthesis and S1 nuclease mapping to determine the sequence of the 5' untranslated region of rPTH mRNA, the point of transcriptional initiation, and the location of intron A. The phospho- triester method (23) was used to synthesize a pentadecanucleotide complementary to codons 5-10 of rPTH mRNA as encoded in the cloned genomic DNA and located 19 nucleotides downstream from intron A. The pentadecanucleotide was labeled with 32P at the 5' end and used to prime the reverse transcription of rPTH mRNA using reverse transcriptase (Life Sciences). The resulting 5"labeled cDNAs were separated by electrophoresis through an 8% polyacrylamide gel containing 8 M urea. The largest cDNA transcript was eluted from the gel and sequenced according to the method of Maxam and Gilbert (19). Comparison of the cDNA sequence with that of the 5' genomic subclone hybridizing to rPTH mRNA revealed the 5' end of intron A and indicated the nucleotide in the rPTH gene near which tran- scription begins. To further substantiate the transcriptional start positions, an AvaIIjBamHI fragment of 730 base pairs spanning the putative site of initiation and the Goldberg-Hogness sequence (TATA box) and beginning just upstream from intron A (see Fig. l), was labeled at the downstream 5' end with [-y3'P]ATP, and hybridized to rPTH mRNA. The hybrid was digested with S1 nuclease (Bethesda Research Laboratories) using the method of Weaver and Weissmann (24). The DNA fragment was then chemically sequenced (19) and analyzed along with the S1-digested DNA by electrophoresis through an 8% polyacrylamide gel containing 8 M urea.

Identification of Repetitive Sequence DNA Flanking the rPTH Gene-Restriction digests of rPTHg-1 (2 pg) were blotted onto Gene Screen (New England Nuclear) by electrophoresis and hybridized with 1 pg of genomic rat liver DNA that was nick-translated with 32P to lo' cpmjpg of DNA. Hybridization was performed in 6 X SSC at 65 "C for 16 h. The blot was washed three times in 2 x SSC a t room temperature, and exposed for 4 h at -70 "C using a Cronex Lightning Plus intensifying screen. The relation of the repetitive sequences flanking the PTH gene to each other was further examined by blotting of restriction fragments of rPTHs-1 onto Gene Screen and hybridiz- ing the blot with rPTHs-2 labeled with 32P by nick translation (15).

RESULTS

Restriction Mapping of Genomic Clones-Two plaques of recombinant Charon 4A phages hybridized to the 32P-labeled bovine PTH cDNA. The hybridizing phages were isolated and repeatedly plaque purified. Inasmuch as the recombinant phages were constructed from rat liver DNA partially digested with restriction endonuclease EcoRl and, therefore, the entire genomic inserts can be released and distinguished from phage Charon 4A DNA by digestion with this enzyme, the relation- ship between the two recombinant phages was first examined by EcoRl digestion and agarose gel electrophoresis of the purified and digested phage DNAs. The smaller recombinant phage, rPTHg-2, contained only two genomic EcoRl frag- ments of 0.6 and 8.5 kb, respectively. The 5' limit of rPTHg- 2 is denoted by an open arrow in Fig. l Z U . The larger recom- binant phage, rPTHg-1, contained two additional EcoRl frag- ments of 1.9 and 2 kb in length, respectively, indicating that

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3322

I

II

m

m

rPTHs-3 (pBR322)

rPTHs-2 (pBR322)

rPTHg- 1 rPTHg- 2

(Charon 4 A )

rPTHs- I (pBR322)

1 CII , ‘ Exon11 ExonII t ,

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\ Exon111 \

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P

”- -” -“

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FIG. 1. Recombinant X-phage containing the rPTH gene and three subclones of the genomic insert in plasmid pBR322. A recombinant bacteriophage (rPTHg-1) containing rPTH gene sequences was isolated by plaque hybridization from a rat liver DNA library using a bovine PTH cDNA as a hybridization probe. Restriction fragments of rPTHg-1 (ZZZ) were subcloned into plasmid pBR322. (Z, ZZ, and ZV). The nucleotide sequences of selected subclone segments were determined according to the strategy indicated by the arrows in structures Z and V. Orientation of the rPTH gene (5’ to 3‘) is from left to right. Open boxes represent DNA of the cloning vehicles, A-phage Caron 4A, and plasmid pBR322. Shaded and closed boxes indicate exons of the rPTH gene in which shaded boxes are untranslated regions of PTH mRNA and closed boxes represent prepro-PTH coding sequences. Lines represent remaining rat DNA, including rPTH gene introns A and B. The site of priming of rat PTH mRNA with a synthetic pentadecamer to determine the insertion site of intron A is indicated by a wavy arrow on rPTHs-1. Regions of repetitive sequence DNA are labeled (R. DNA), and a region of nine pairs of alternating thymine and guanine bases is designated by TpG,. 0 , 5 ’ end of rPTHg-2; 0, DNA labeled at the 3’ end; 0, DNA labeled at the 5’ end.

rPTHg-1 and rPTHg-2 shared sequences in common. More detailed restriction analysis was performed and yielded the map of rPTHg-1 shown in Fig. 1. A single, centrally located XbaI site was found in both rPTHg-1 and rPTHg-8. Single and double digests of both recombinant phages with the restriction endonucleases NcoI and XbaI and hybridization blotting of the digests (16) revealed a single band of 450 bp that hybridized to the human PTH cDNA. Thus, sequences that hybridized to PTH cDNA were found close to the XbaI site in both recombinant phages. The site of the hybridizable NcoI/XbaI fragment of the two recombinant phages was com- pared more precisely by 5‘ labeling of the phage DNAs at the XbaI sites with 32P, and digesting with NcoI. The digests were then analyzed by electrophoresis through a 5% polyacrylam- ide gel. Autoradiography revealed that the XbaIINcol bands were of the same length (data not shown).

Moreover, both recombinant phages contained all se- quences hybridizable with human cDNA (essentially full

length) in a single 5-kb pair NcoI fragment. This NcoI frag- ment was isolated from rPTHg-1 and rPTHg-2, and the fragments were digested with the restriction endonuclease AuaII. The digests were then compared by electrophoresis through a 5% polyacrylamide gel. The mobilities of seven discrete AvaII fragments ranging from 160 to 2000 base pairs were identical in both recombinant phages (data not shown). Therefore, by restriction enzyme mapping, the two phages rPTHg-1 and rPTHg-2 are closely related, and are indistin- guishable in the sequences that hybridize to human PTH cDNA. However, single digests of each with HindII1, and double digests with HindIII/EcoRl revealed differences in the electrophoretic pattern of bands that could not be accounted for by opposite orientations of the genomic inserts in the cloning vector Charon 4A. These differences are discussed further below.

Sequence and Organization of the rPTH Gene-The NcoI restriction fragment of rPTHg-1, which hybridized to human

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Preproparathyroid Hormone Gene 3323

RAT PARATHYROID HORMONE GENE

5 '

ccagattaat gtgtgcaatt aagaaatgta gttagtgttt attgaatgct attacgatac ctcaaaaacc caagttc;at gaatttaaaa ataatttctc tttatttgg -1050 ttccataact tccacattta caagcataag atgcttgtgt aaaaataaag atatcacatt cactgaagaa ttactgtaat aattaccaga ataccagaat taccagaata -3160

caaggccggg tatcttcctc tagttttgct ctcttccttg tttttgcata tggtacctca ctgaacctaa ggcttgctgt ttaitGac tggctagcct gccctgggga - 830 tacttcaaat ctgtatcaag cactctgaat attttcttat gtccattgtt ttcctctgtg tgcatgagtg tgagagagag agagtgtgtg tpt@Lgtg taggcaaggc - g40 tttgtccata tccaattcct tcccccaagc actagtctta cagtgctccc caccatggtt ggcttgtaga tggcttctag gaatggaact caggtcctca tatttgcact - 720 ttactgactg agacatctgc cagctctcca gccttatacg cattaaacat atatacaata tgtgttaaac tgatcaaagg tgataagagt tggttggaaa tggcttataa - 610 gaaaaatcat gcaacatgat tccttaagaa tacaaagtct tataatataa ggcttataat aaggctgtgc aatgcatata ctcggctaat tatactgtgc ttcataattg - 500 tcact ....... 80 base pairs......g aaagtgcagg cccagtggca atgtctttaa ctttgcactc atgaatagag gcaagtggat cttgagtttt gggtcagcct - 316 gatctacata ttgagttcta ggccagccag agttcatagt gagacgctat ctcaaactgt tttaaatata aaatcagtat catggattac gtcagatttt tctaagtggt - 206 tacttaagca tttgctgcaa cttcttttgc agattctttg ccagcacctt gctctttttg aatccattat ctaagtatct gaaactttag aggagtgggc accgcccgat - 96 gagggtaggt ggctgttctg attcctatga ttgagaacca gagaaccagg catgacatca tccttcccba taaaat+tc ctcttggtga gcaaaAGGCC TGCATATGAA 15 ACTCAGGCTT GAAGAACTGC AGTCCAGTTC ATCACCTGTC TGGCTTACTC CAGCTTAATA CAGGGTCACT CCTGAAGGAT CCTCTCTGAG AGTCATTGTAGgtaaggaa 125 tctctcaatt gcccttttaa attccgtgag atttagaaaa ttgtgctagt ttttaataca taccattttc tatcaatatg tgccattttt taaaattgga aggtaggggt 235 gaatttgcca aaatggaagg atatggcaat aaaataattt agggtaggaa attcggactc atctatgtat atggttaa .. IVS A (1600 base pairs) ., gttc 1433 tatactaaag tatctgtcct ataaagatcc cacagaccca tgaaaagtgg catcagctaa tgactgtttc cttgggtggt tatgaataac agctactggg actgtgttta 1543

-31 -30 -20

cacactttag agcactgaca gtgtcttaaa atatctctgt ctctccttgt agTTAAG ATG ATG TCT GCA AGC ACC ATG GCT AAG GTC ATG ATC CTC ATG 1642

Leu Ala Val Cys Leu Leu Thr Gln Ala Asp Gly Lys Pro Val Ly CTG GCA GTT TGT CTC CTT ACC CAG GCA GAT GGG AAA CCC GTT AAgtaagtgc tgcagcccgt cgtcccaggg aagtcggaca tgaggctctg taggtttta 1742

Met Met Ser Ala Ser Thr Met Ala Lys Val Met Ile Leu Met

-10 -6

s Lys Arg Ala Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys -1 + 1 10

atgttgtggg CatggggagC taatggagtg gtCCtCtCtt tctgttctctctagG AAG AGA GCT GTC ACT GAA ATA CAG CTT ATG CAC AAC CTG GGC A A A 1842 20 30

His Leu Ala Ser Val Glu Arg Met Gln Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe Val Ser Leu Gly Val Gln Met Ala Ala CAC CTG GCC TCT G T G GAG AGG ATG CAA TGG CTG AGA AAA AAG CTG CAA GAT GTA CAC AAT TTT GTT ACT CTT MA GTC CAA ATG GCT GCC 1932

40

50 60 Arg Glu GlY Ser Tyr Gln Arg Pro Thr LYS LYs Glu ASP Asn Val Leu Val Asp Gly Asn Ser Lys Ser Leu Gly Glu Gly ASP LYS Ala

70

AGA GAA GGC AGT TAC CAG AGG CCC ACC AAG AAG GAG GAA AAT GTC CTT GTT GAT GGC AAT TCA AAA ACT CTT GGC GAG NG GAC AAA GCT 2022 80 84

Asp Val ASP Val Leu Val Lys Ala Lys Ser Gln END CAT GTG GAT GTA TTA ca."c AAG GCT AAA TCT CAG TAA ATGCT GACGTATTCT AGACCGTGCT GACCAATAAC ATATGCTGCT ATCCTTTCAA GCTCCACGAA 2123 GATCACCAGT GCTAATTCTT C T A C T G T W A G T T T G A AATTTCATTC CACTTTTGCT CTTTAACGTC TCTTCCAATG ATTCCATTTC AATATATTCT TCTTTTTAAA 2233 GTATTACACA TTTCCACTTC TCTCCTTAAA TATAAATAAA GTTTAATGAT CATGAACCAA Ataagcagtg tttcttactt gttaaaactt ttgtctcagt gttggagggc 2343 tggctcagaa gttaagagtg catactgctt c c t c a z cccgagtttg cttctcgg 240 1

3'

FIG. 2. Nucleotide sequence of the rat PTH gene. The nucleotide sequence was determined from the subcloned DNAs shown in Fig. 1. Exons of the rPTH gene are shown in capital letters and introns and flanking DNA in lower case letters. The promoter (Goldberg-Hogness) sequence is boned, and the AATAAA polyadenylation signals are indicated by wavy lines. ATG triplets in the 5' untranslated region of rPTH mRNA are underlined, and stop codons in the same reading frame with the ATG codons are ouerlined. The dashed lines indicate two sequences of alternating purines and pyrimidines which are possible sites of formation of Z-DNA. Nucleotides are numbered on the right of the figure beginning with +1 at the putative transcriptional initiation site. Nucleotides on the 5' and 3' sides of the point of initiation are given negative and positive numbers, respectively, beginning at the initiation site. The protein coding sequence of the gene is keyed by numbers above the assigned amino acids, +l (alanine) begins the sequence of PTH; the minus numbers designate the signal (leader) and propeptide sequences.

and bovine cDNAs and contains the unique XbaI site, was first used to obtain sequence directly from isolated recombi- nant phage DNA. Sequencing from the NcoI and XbaI sites established that this fragment contained rat PTH coding sequences. Phage rPTHg-1 was therefore subcloned as de- scribed above to yield the subclones indicated in lines I, 11, and IV of Fig. 1. Sequencing of subcloned recombinant plas- mids, rPTHs-1 and rPTHs-2, revealed the presence of three exons interrupted by two introns of 1600 (intron A) and 111 base pairs (intron B) (Figs. 1 and 2). The location of intron B in the propeptide segment of the rat PTH precursor was inferred from comparison with the sequence of the human PTH gene (25). The human gene contains an intron of 103 base pairs in the homologous location. Inasmuch as there is complete homology surrounding the insertion site of the in- tron, and both human and rat introns conform to the consen- sus splice site sequences (see Fig. 3), there is little doubt that the assignment of the site of intron B is correct.

Further comparison of the rat gene sequences with the human and bovine cDNA sequences and with the human gene sequence (14, 25) indicated the presence of another interven- ing sequence (intron A) 89 nucleotides upstream from intron B in the 5' untranslated region of rPTH mRNA and 5 nucleotides upstream from the initiator codon of rat prepro-

PTH. Inasmuch as the human and bovine cDNAs did not hybridize to the rat 5' untranslated region even under condi- tions of low hybridization stringency, rPTH mRNA was used in two ways to determine the location and sequence of the 5' untranslated region of rat PTH mRNA encoded in the rPTH gene. First, rPTH mRNA was hybridized to various fragments prepared from the 5' regions of the inserted DNA in the subclones of the recombinant phages. A 1.4-kb EcoRl/HindIII fragment of rPTHs-2 hybridized to rPTH mRNA immobilized on a filter which indicates that the remaining sequences corresponding to the 5' untranslated region must be contained therein. Second, rPTH mRNA was reverse transcribed start- ing 20 bp downstream from the insertion site of intron A, by using a synthetic pentadecanucleotide complementary to co-

- 5 Pro Val Ly

-1 S Lys A r q

R a t

Human T C T G T T AAetaaqt ... IVSB ... c c a q G AAG AGA Ser V a l Ly S L y s Arq

5 ' CCC G T T A A s a a g t . . . IVSB.. . c t a q G AAG AGA

3 '

FIG. 3. Comparison of the nucleotide sequences flanking the insertion sites of intron B in rat and human PTH genes. Exon and intron nucleotides are written in upper and lower case letters, respectively. The intron splice donor and acceptor consensus se- quences are underlined (27). Amino acids of the propeptide region are numbered -5 to -1 from NH, to COOH termini.

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3324 Preproparathyroid Hormone Gene

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IVSA 1.6 kb

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/ /

/ /

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Gl FIG. 4. Localization of intron A (ZVS A ) and site of tran-

scription of the rat PTH gene by reverse transcription of mRNA and S1 mapping of a DNA-RNA hybrid. mRNA prepared from rat parathyroid glands was hybridized to a synthetic penta- decamer labeled at the 5' end with "P, and then transcribed from the primed site with reverse transcriptase. The longest labeled tran- script was chemically sequenced. The sequencing ladders are dis- played on an 8% polyacrylamide gel containing 8 M urea. The auto- radiogram of the gel is shown on the right. The bases a t which aliquots of the labeled DNA were chemically cleaved are indicated abooe each lane. G, guanine; C, cytosine; A, adenine; T, thymine. The location of intron ( I V S ) A on the reverse transcript is indicated by a bracket. The basis denoted by asterisks at the stem of the converging dashed lines indicate the position of intron A on the genomic clone rPTHg-1 depicted in structure 111 of Fig. 1. T o determine the site of transcriptional initiation, an AuaII/RarnHl fragment of rPTHs-3 containing the promoter (see Fig. 1) was labeled at the 5' end of the

dons 10-15 of the signal peptide region of rat prepro-PTH prepared according to the sequence obtained from the recom- binant phage rPTHg-1 (see Figs. 1 and 2). The largest primed cDNA was sequenced, and the sequence was compared with the sequences of rPTHs-1 and rPTHs-3. The latter is a subclone of rPTHs-2 and contains the 1.4-kb EcoRl/HindIII fragment that hybridized to the 5' untranslated sequences of rPTH mRNA. The primed cDNA sequence (Fig. 4) was pre- cisely colinear with the genomic sequence of rPTHs-1 up to the predicted insertion of intron A (see Figs. 2 and 4), and continued in rPTHs-3 near the unique RamHl site. Hence, intron A was precisely localized to nucleotides 118-1595 of the rPTH gene. There exists perfect homology between the human and rat genes around and including the insertion site of intron A. However, the rat intron consists of 1.6 kb when compared with a corresponding intron of 3.5 kb pairs in the human PTH gene.

The unique XbaI site of the rPTH gene was located just downstream from the TAA stop codon of the genomic rPTH mRNA sequences (Fig. l ) , a location similar to that in human gene and cDNA. Sequencing downstream from this XbaI site revealed homology with both human and bovine cDNAs, including the presence of two potential polyadenylation signal sites, AATAAA (Fig. 2). We believe that this region contains no additional introns and that the second AATAAA sequence is probably recognized as a polyadenylation signal for the following reasons: (i) no intron is found in the human gene in this region; ( i i ) the size of 800 f 50 bp of rPTH mRNA as determined by agarose gel electrophoresis (Fig. 5) would allow only a small, if any, intron; and (iii) the presence of a single PTH mRNA band argues against alternate recognition of the two AATAAA signals (Fig. 5). Therefore, the rPTH gene whose sequence is shown in Fig. 2 is organized into three exons and two introns. Exon I consists of the sequence encod- ing the 5' untranslated region of rPTH mRNA and is followed by intron A of 1.6-kb length. Exon I1 encodes the prepeptide, or leader, sequence of rat prepro-PTH, and a part of the propeptide. Intron B is short (1 11 bp) and is followed by exon 111, which contains the remaining sequences of rPTH mRNA.

Comparison of Recombinant Phage DNA with Genomic DNA-To show that the cloned recombinant DNAs are rep- resentative of genomic DNA, restriction enzyme fragments prepared from rat spleen DNA and a mixture of rPTHg-1 and rPTHg-2 were compared by electrophoresis on a single gel, and by subsequent blotting and hybridization with ""P-labeled rPTHs-I. Both cloned and genomic DNA gave identical re- striction fragments, demonstrating that the recombinant phages contain DNA sequences that are representative of those in genomic DNA (Fig. 6). The presence of a single band in each of the genomic digests indicates that the differences observed in the HindIII digests of rPTHg-2 and rPTH g-1 must be located upstream or downstream from the central HindIII fragment of the rPTHg-1 genomic insert (the 5' end of the genomic fragment abuts the left arm of Charon 4A). Thus, the differences in restriction patterns observed between the two recombinant phages involve only one of several re- striction sites examined. Inasmuch as both the genomic li- brary and the spleen DNA were prepared from single animals,

RarnHI site and hybridized to rPTH mRNA. The hybrid was digested with S1 nuclease, and the products were separated by electrophoresis on an 8% polyacrylamide/urea gel (lane labeled SI) along with the products of the identical AoaII/RarnHI fragment prepared by chem- ical sequencing. The two autoradiographic sequence ladders were aligned, and the corresponding nucleotides were connected by a line. The sequence given in the upper right margin was determined from the ladder shown on the upper left. Note that the sequence is the complement of the coding (sense) strand of the gene shown in Fig. 2.

~~~~ ~~ ~~~~ ~ ~ ~ ~ ~. ~

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Preproparathyroid Hormone Gene 3325

these results suggest the presence of a HindIII site polymorph- ism closely linked to the PTH gene. Furthermore, the pres- ence in digested spleen DNA of only the DNA fragments predicted by the map of rPTHg-1 and rPTHg-2 shows that unique BamHI, HindIII, and EcoRI sites have been preserved 1-6 kb downstream from the PTH gene. The absence of heterogeneity in these sites argues for the existence of only one PTH gene/rat haploid genome. However, our data do not exclude the possibilities of the existence of two or more PTH genes/haploid genome or cloning artifacts that have occurred near the ligation site of the genomic fragment into the cloning vector, or both.

rPTH Gene Promoter and Site of Initiation of Transcrip- tion-The nucleotide sequence of the cDNA obtained from reverse transcription of rPTH mRNA using the synthetic pentadecanucleotide suggested that the promoter of the rPTH gene is located at position -24. To further substantiate this assumption, a genomic AuaIIIBamHI fragment, labeled a t the 5' end spanning the putative cap site, was hybridized to rPTH mRNA, and the heteroduplex was digested with S1 nuclease (24). The size of the protected fragment was then determined by co-electrophoresis with a sequencing ladder prepared from the same fragment (Fig. 4). Two clusters of bands are seen on the autoradiogram of the gel. The more prominent cluster contains five bands, of which two are strong and three are lighter, and the minor cluster contains two bands. The het- erogeneity of the major cluster may be due to true heteroge- neity of the transcription start site or the rather stringent hybridization conditions (50 "C, 80% formamide) and the presence of the mRNA cap that may prevent complete het- eroduplex formation at the 5' end of the mRNA (26), or may be due to partial denaturation of the heteroduplex by homo- duplex formation (both strands of the genomic DNA fragment were present during DNA-RNA hybridization). Inasmuch as reverse transcription of rPTH mRNA yielded only a single cDNA corresponding in size to the major cluster of S1 bands

I -

3-

A B C FIG. 5. Autoradiogram of blot of RNA from rat parathyroid

gland, human parathyroid adenomas and bovine parathyroid glands. RNA prepared from hyperplastic rat parathyroid glands, human parathyroid adenoma, and bovine parathyroid glands was subjected to electrophoresis through a 1.4% agarose gel containing 6% formaldehyde, and blotted onto Gene Screen. The blot was hybridized with the XbaI/NcoI fragment of rPTH S1 (Fig. I - I V ) , pPTHml, and pPTHm122, labeled with 32P by nick translation (10' cpm/pg). Several autoradiograms of the blot were obtained and su- perimposed to give comparable hybridization signals: A, human para- thyroid adenoma RNA; E , rat parathyroid gland RNA; C, bovine parathyroid gland RNA. Dots on the left margin numbered 1,2, and 3 mark the positions of the 910-, 659-, and 521-bp fragments, respec- tively, of an AluI digest of pBR322.

r ' Pvu II

r W "I

(L3 I

Ji

Barn H I 1

I.

Eco R I

Hind IJI

Pvu 71

Barn H I

Eco R I

Hind III

FIG. 6. Autoradiogram of genomic (Southern) blot of re- striction enzyme digests of rat spleen DNA and cloned DNA, phage rPTHg-1. One-pg aliquot of rat spleen DNA and 100-ng aliquots of rPTHg-1 DNA were digested with one of several restriction enzymes, subjected to electrophoresis through an 0.8% agarose gel and translcrred onto nitrocellulose. The blot was hybridized with a cloned DNA fragment (rPTHs-I), labeled with 32P by nick translation (10' cpm/pg), and autoradiographed. The sizes in kilobase pairs of the hybridizing restriction fragments are indicated by arrows on the right-hand margin.

and no cDNA corresponding to the minor S1 bands, the minor cluster may represent S1 nuclease overdigestion of the DNA- RNA hybrid, perhaps as a result of the relative abundance of adenine and thymine residues in this region (+lo to +15). Although the precise initiation point is not defined by this S1 mapping, comparison with other cap sites makes it likely that one of the four A's of the sequence is the cap acceptor (27). Therefore, the sequence AATAAAATA a t positions -27 to -18 is likely to be the TATA box equivalent of the rPTH gene (28).

Size and Complexity of rPTH mRNA-The (Southern) blot and the analyses of the restriction digests of the recombinant phages suggested the presence of a single type of PTH gene, making it unlikely that, at the RNA level, more than one cellular transcript would be found, and, in conjunction with the results of the primed reverse transcription of parathyroid gland RNA, further suggested that the recombinant phages contain sequences identical with those that are expressed in the parathyroid gland. For additional verification of this supposition and determination of the size of rPTH mRNA, RNA prepared from rat hyperplastic parathyroid glands, hu- man parathyroid adenoma, and normal bovine parathyroid glands was subjected to electrophoresis on agarose gels, trans- ferred to Gene Screen membrane filters, and hybridized to the labeled 450-bp NcoIIXbaI fragment containing most of the rat PTH gene coding sequences and intron B (Fig. 2), and

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3326 Preproparathyroid Hormone Gene

further hybridized to labeled human cDNA (pPTHm122) and bovine cDNA (pPTHm1). Even after long exposure (3 days) only a single discrete band hybridizing to the rat RNA was seen (Fig. 5). The fuzziness of the lower portion of the rPTH mRNA band may be due to heterogeneity of the poly(A) extension of rPTH mRNA ( 7 ) , or to minor degradation of rPTH mRNA, or to both. Thus, a single mRNA appears to be derived from the rPTH gene and is expressed in the parathyroid gland, and its size is intermediate between the known sizes of bovine PTH mRNA of 750 f 50 bases (14) and of human PTH mRNA of 850 k 50 bases (17).

Hepdtiue Sequence DNA-Two regions of rPTHg-1 were found to contain repetitive sequence DNA by the technique of hybridization of total genomic DNA labeled with :j2P by nick translation to rPTHg-1 restriction fragments (Fig. 7 ) . The small EcoRI fragments that map to the left of and 1.4 kb upstream from the PTH gene give a hybridization signal. The I.:coRI/XbaI fragment (Fig. 7 ) , which is on the 3' side of the PTH gene, gives a much stronger signal. The 4.3-kb BamHl band containing all of the PTH gene except the 5' un- translated region and promotor, hybridizes very weakly (Fig. '7, open arrow). Hence, the two introns of the PTH gene apparently do not contain highly repeated DNA sequences but, rather are surrounded by such sequences. More detailed mapping of the 3' repetitive sequences indicated that they are located a t least 4 kilobases downstream from the PTH gene.

The relation between the 5' and the 3' flanking repetitive DNA sequences was examined in greater detail by hybridiza-

FIG. 7. Repetitive DNA sequences flanking the rat PTH gene demonstrated by autoradiogram of a blot of restriction fragments prepared from rPTHg-1 and hybridized with la- beled rat spleen DNA. Two pg of rPTHg-1 were digested with the indicated restriction enzymes, the fragments were separated by elec- trophoresis through a 0.890 agarose gel, and transferred to a Gene Screen filter membrane. The filter was hybridized for 16 h with 1 pg of rat spleen DNA labeled by nick translation (10' cpm/mg). Auto- radiography was performed for 4 h at -70 "C using an intensifying screen (Cronex Lightning Plus). The open arrou! indicates the posi- tion of the RarnHl restriction fragment containing exons I1 and 111, introns H, and more than 8 0 4 of intron A of the rPTH gene. Up and down orrows denote repetitive DNA in the 5' and 3' flanking regions, respectively.

tion. Under stringent conditions (6 X SSC, 65 "C) that re- vealed hybridization between the 220-bp overlapping seg- ments of subclone rPTHs-1 and rPTHs-2, no hybridization occurred between the flanking repetitive sequences (data not shown). Thus, at least two different types of repetitive se- quences are present on rPTHg-1.

DISCUSSION

We isolated and determined the structure and organization of the rat PTH gene. From the complete nucleotide sequence of the PTH mRNA encoded by the rPTH gene, we deduced the previously unknown amino acid sequences of rat PTH and prepro-PTH, the biosynthetic precursor of PTH.

Our data indicate that the transcriptional unit of the rat PTH gene is located on an EcoRl restriction fragment of 8.5 kilobase pairs and contains three exons and two introns that span 2.5 kilobase pairs of DNA. The 5' flanking region of the rPTH gene contains a promoter sequence that resembles the Goldberg-Hogness or TATA box, a short sequence believed to be involved in initiation of transcription (28). The 3' untranslated region of the gene contains the sequence AA- TAAA that serves as the recognition signal in the polyaden- ylation of mRNAs (29).

Our analyses of restriction enzyme digests of rat genomic DNA by blot hybridization suggest that the rPTH gene is unique and is not a member of a multigenic family. In addi- tion, the rPTH gene appears to contain unique sequence DNA in both its flanking regions and its introns, inasmuch as blot hybridization studies and DNA sequencing did not reveal any repetitive DNA sequences in a 1.4-kilobase pair restriction fragment flanking the rPTH gene on its 5' side, in a 4.0- kilobase pair region on its 3' side, or in the two introns. Repetitive DNA was found, however, in more distant regions flanking the PTH gene.

The pattern of interruption of the rat PTH gene by introns is consistent with the proposal that functional domains of proteins are encoded in individual exons of the corresponding gene (30). Thus, exon I1 of the rPTH gene encodes the signal sequence of rat prepro-PTH (Fig. 9). Such hydrophobic signal sequences are found at the NH2 termini of most secreted proteins, have an important function in the secretory process, and are frequently isolated within exons of the genes encoding secretory proteins (31). Exon 111 of the rPTH gene encodes the biologically active and secreted PTH peptide of 84 amino acids. Precisely the same interruption of functional domains is found in the human PTH gene (25).

Reverse transcription of cDNA from the 5' region of rat PTH mRNA as well as S1 nuclease mapping indicate that

1

I 1 I

t FIG. 8. Comparisons of the nucleotide sequences of the rat

bovine and human PTH gene promoter regions. The nucleotide sequences of the promoter regions of rat and human PTH genes were aligned with each other and with the nucleotide sequence of a cDNA to the minor bovine PTH mRNA species (32). Gaps chosen by visual inspection were introduced into the sequences to maximize homology. The rat promoter and the major bovine promoter are boxed. Sites of transcriptional initiation are indicated by arroms (32). Homology between nucleotides is indicated by dots between the homologous pairs.

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rorc lne ~ o v 1 n r l i u r a n

8a t

Human B o v l n e

5 '

p o r c i n e B o v i n e Human

R a t

H u n a n B o v l n e

P o r c 1 r . e B o v i n e Human

R a t

Human B o v l n e

P o r c l n e B o v l n e Human

R a t

H u m a n B o v l n e

- 3 1 - 3 7

Preproparathyroid Hormone Gene - 2 0

3327 1 ' I

-6 -1 + 1

20

S e r - L e u - - A s n - Met - - S e r - Met - -

A l a Ser V a l G l u A r g

GCC TCT GTG GAG AGG

AG- --C - -A - -A M- --G A" _ _ _ --A

4 0

A l a Ser I l e - T y r - A l a Ser I l e V a l H ~ s -

A l a P r o L e u - Pro - V a l G l n Met A l a A l a A r 3

GTC CAA ATG GCT GCC AG?

-CT TCT --A --- -CT -CT C-A __- C-- _ _ _

TA- _ _ _

50

A s p - G l y Ser - - A s p - - Ser - - A s p A l a G l y Ser - - G l u G l y Ser T y r G l n A r c GAA GGC AGT TAC CAG AGG --T -CT G-- -C- _ _ _ _ _ _ --T --T -c- _" --A

7 0 8 0

Porcine Ser HIS G l n - - - - - A l a - - - la - - - - - _ - B o v l n e Ser H l s G l n - - - - - A l a - - - - - - - H u m a n Ser Ris G l u - - - - - A l a - - - - - I l e - - - - As?, - - T h r - - - R a t

G ~ Y A s n Ser LYS Ser L e u G l y G l u G l v A s p ~ y s la ASP V a l ASP v a l L e u V a l LYS A l a LYS

GGC AAT TCA AAA AGT C T r GGC GAG GGG GAC AAA GCT GAT GTG GAT GTA TTA GTT M G GCT H u m a n C-- GA- _-- _ _ _ _ _ _ --A _ _ _ B017 lne A" C-- CAG --- _-_ _ -_ -CA _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

--A --A -CA --- _-- _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ A - - - - - _ - AC- - -A _ _ _ _ _ _ A-- - - A _ _ _ _ _ _

FIG. 9. Nucleotide and deduced amino acid sequence of rat preproparathyroid hormone-compari- son with corresponding sequences of other species. The amino acids of the signal and propeptide region of prepro-PTH were assigned negative numbers, and the secreted (hormone) form was assigned positiue numbers starting at the NH, terminus. Identity of the nucleotide and amino acid sequences with the deduced rat sequences is indicated by dashes. Porcine hormone amino acid sequences (34) and bovine (33) and human cDNA structures

8 4

P r o - P r o -

S e r Cl? T C T ChG ' ' "C "- c-c " _

"

(25) have been reported.

transcription of the rat PTH gene is initiated from a single promoter. In contrast, the results of reverse transcription of bovine P T H mRNA performed by Weaver et al. (32) suggest that transcription may be initiated from two promoters about 25 nucleotides apart in the bovine PTH gene. In the bovine gene, transcription is initiated less frequently from the more upstream promoter. The resultant minor PTH mRNA is 25 nucleotides longer than the major species of bovine PTH mRNA. To detect differences in nucleotide sequences between the rat and bovine PTH genes which may account for the different utilization of the promoter regions of the rat and bovine genes, the sequence of the rat gene was aligned with the sequence of reverse transcripts (cDNAs) of the minor and major bovine PTH mRNAs (Fig. 8). The major site of initia- tion of the synthesis of the bovine transcripts is located 25 nucleotides downstream from a TATA sequence at the 5' end of the minor bovine cDNA (Fig. 8). This TATA sequence is also found in the corresponding region in the human gene. Comparison of this promoter with the corresponding region of the rat PTH gene shows three nucleotide substitutions, ie. the major bovine promoter sequence TATATATA has become

TGCATATG in the rat gene. It is not clear whether these nucleotide sGbstitutions, or sequence differences elsewhere or other factors contribute to the observed differences in utili- zation of this region for the initiation of mRNA synthesis.

The nucleotide sequence of the bovine PTH gene upstream from the 5' end of the cDNA whose sequence is shown in Fig. 8 is presently unknown and therefore cannot be compared directly with the sequences of the human and rat PTH genes in this region. If one assumes, however, that the promoter that directs the initiation of the minor bovine PTH mRNA is located 28 bases upstream from the point of initiation, this promoter can be aligned with the rat gene promoter. There- fore, the major rat PTH gene promoter probably corresponds to the minor, more upstream promoter of the bovine PTH gene. The location of the rat promoter in the more upstream position creates a longer 5' translated region in rat, in com- parison with the major bovine PTH mRNA, and also intro- duces at nucleotide position +15 an AUG triplet into the 5' untranslated region of the rat PTH mRNa (see below).

Multiple AUG sequences are present in the 5' region of the mRNA encoded by the rPTH gene, i.e. in positions 11, 115,

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3328 Preproparathyroid Hormone Gene

1600, and 1603 (see Fig. 2). The AUG codon at position 11 of rPTH mRNA is followed by an in frame stop codon (UAA) that is 19 codons downstream in the mRNA. AUG 115 and AUG 1600 are in positions precisely homologous to the AUG codons of human PTH mRNA, as is the stop codon UGA immediately following AUG 115. The two contiguous AUG codons at positions 1600 and 1603 of rPTH mRNA are homologous to those in bovine PTH mRNA (33). Although it is uncertain to what extent each of the four initiator codons of rPTH mRNA is used to initiate protein synthesis, several considerations suggest that AUG 1600 (or AUG 1603) is the preferred site of initiation. First, sodium dodecyl sulfate- polyacrylamide gel electrophoresis of the products of trans- lation of bovine and rat parathyroid gland mRNAs in the wheat germ cell-free system showed only a minor difference in the migration of bovine prepro-PTH and a major transla- tion product of rat parathyroid gland mRNA (data not shown). We previously showed that translation of bovine PTH mRNA in vitro is initiated from the sequence analogous to rat AUG 1600 (33). Second, the homology in the coding regions of rPTH mRNA with human and bovine PTH mRNAs indicates that the primary translation products de- rived from the mRNAs are analogous precursors of PTH (Fig. 9). We therefore conclude that rPTH mRNA translation is initiated primarily at AUG 1600.

We aligned (Fig. 9) the nucleotide sequence of the coding region of rat PTH mRNA and the deduced amino acid se- quence of rat prepro-PTH with the amino acid and nucleotide sequences of human and bovine prepro-PTH and the amino acid sequence of porcine P T H (34). A partial sequence of chick PTH was reported (35), and is currently being extended and revised.' Inspection of the sequences in Fig. 9 shows three distinct regions of conservation of the nucleotide and amino acid sequences among the various species.

The first region of conservation extends from amino acid -6 through +15, and comprises the propeptide (-6 to -1) and the NH, terminus of PTH (+1 to +15). Conservation in this region is consistent with analyses of structure and bioac- tivity of synthetic fragments and analogues of PTH that have shown that, for full biologic activity, PTH must begin pre- cisely at position +1 (36). Synthetic analogues lacking residue +1 (36) or containing glycine at +1 (37) or tyrosine at -1 (38) have little bioactivity. Moreover, pro-PTH is biologically inactive (39). A second region of conservation includes amino acids 23-38. Recent studies indicate that this region is involved in the binding of PTH to its receptor (40). In addition, proteolytic cleavage of PTH after its secretion from the parathyroid gland occurs at several sites between amino acids 32 and 38, generating the COOH-terminal and probably NH,-terminal fragments of PTH (41). Although the significance of proteolytic cleavage is as yet unsettled, there are reports that NH'-terminal fragments rather than PTH (1-84) are biologically active in bone (42). The third area of conservation of amino acid and nucleotide sequence extends from amino acid 49 to the COOH terminus of PTH a t amino acid 84. The significance of conservation in the COOH- terminal fragment is obscure, but raises the intriguing possi- bility that this fragment of P T H may have some functional role. By contrast, the region located between amino acids 39 and 49 shows considerable variation of amino acid sequence among the hormones of the four species. The lack of amino acid conservation may well correlate with heightened antige- nicity of this region. Immunization of animals with synthetic peptides corresponding to this region of rat prepro-PTH, may

R. R. MacGregor, personal communication.

allow us to develop high affinity anti-rat PTH antibodies useful in physiologic studies.

Knowledge of the structure of the cloned rat PTH gene will now enable us to study in vivo the regulation of its expression, and to examine the functional significance, if any, of the unique promoter and 5' untranslated regions of the rat PTH gene.

Acknowledgments-We thank Thomas Heinrich, David H. Drey- fus, Robert L. Buchanan, Phillip C. Dee, and Charles D. Brackett for excellent technical help, and Esther G. Hoomis and Louise Fred for preparation of the manuscript.

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28. 29.

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G Heinrich, H M Kronenberg, J T Potts, Jr and J F Habenerdeduced amino acid sequence of rat preproparathyroid hormone.

Gene encoding parathyroid hormone. Nucleotide sequence of the rat gene and

1984, 259:3320-3329.J. Biol. Chem. 

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