Proc. Natl. Acad. Sci. USAVol. 85, pp. 4605-4609, July 1988Biochemistry

Human renal carcinoma expresses two messages encoding aparathyroid hormone-like peptide: Evidence for thealternative splicing of a single-copy gene

(hypercalcemia of malignancy factor/alternative RNA splicing)

MARK A. THIEDE*t, GORDON J. STREWLER*, ROBERT A. NISSENSON*, MICHAEL ROSENBLATT*,AND GIDEON A. RODAN**Department of Biological Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486; and Veterans Administration Medical Center andUniversity of California, San Francisco, CA 94121

Communicated by Alexander G. Bearn, February 22, 1988 (receivedfor review November 11, 1987)

ABSTRACT A peptide secreted by tumors associated withthe clinical syndrome ofhumoral hypercalcemia of malignancywas recently purified from human renal carcinoma cell line786-0. The N-terminal amino acid sequence of this peptide hasconsiderable similarity with those of parathyroid hormone(PTH) and of peptides isolated from human breast and lungcarcinoma (cell line BEN). In this study we obtained thenucleotide sequence of a 1595-base cDNA complementary tomRNA encoding the PTH-like peptide produced by 786-0 cells.The cDNA contains an open reading frame encoding a leadersequence of 36 amino acids and a 139-residue peptide, in which8 of the first 13 residues are identical to the N terminus ofPTH.Through the first 828 bases the sequence of this cDNA isidentical with one recently isolated from a BEN cell cDNAlibrary; however, beginning with base 829 the sequencesdiverge, shortening the open reading frame by 2 amino acids.Differential RNA blot analysis revealed that 786-0 cells expresstwo major PTH-like peptide mRNAs with different 3' untrans-lated sequences, one of which hybridizes with the presentlydescribed sequence and the other one with that reported for theBEN cell PTH-like peptide cDNA. Primer-extension analysis of786-0 poly(A)+ RNA together with Southern blot analysis ofhuman DNA confirmed the presence of a single-copy genecoding for multiple mRNAs through alternate splicing. Inaddition, the 3' untranslated sequence of the cDNA describedhere has significant similarity to the c-myc protooncogene.

The clinical syndrome of humorally mediated hypercalcemiaassociated with malignancy (HHM) closely resembles hyper-parathyroidism and occurs frequently in patients with certaincancers (1). In 1941, Albright (2) postulated that this syn-drome may result from ectopic biosynthesis and secretion ofparathyroid hormone (PTH) by tumors. Recent work hasrevealed that lung (cell line BEN) (3), breast (4), and renal(cell line 786-0) (5) carcinomas secrete peptides similar, butnot identical, to PTH. The molecular weight of the lung andbreast peptides is =17,000, and the sequence similarity withPTH was limited to the N terminus (3, 4). A cDNA comple-mentary to the PTH-like peptide (PTH-LP) from a lungcarcinoma (cell line BEN) has been cloned and sequenced(6), confirming structural data obtained by protein sequenc-ing. Recently a peptide corresponding to residues 1-34 of thePTH-LP was chemically synthesized and confirmed to pos-sess PTH activities on renal and osteoblast membranes invitro and on calcium and inorganic phosphate fluxes in vivo(7, 8). This structural and functional information has rein-forced the hypothesis that HHM is mediated by tumor-secreted factors with biological properties like PTH.

A peptide purified from the human renal carcinoma cell line786-0 was also found by peptide sequencing to have N-terminal similarity to PTH (5) and to be identical to theN-terminal region of the lung and breast peptides. Like theother PTH-LPs, the renal peptide produced in cell culturesalso possesses PTH-like activities. However, the molecularweight of this tumor product was only 6000.The aim of this study was to characterize the diversity of

the PTH-LPs by obtaining the complete deduced amino acidsequence of the renal carcinoma PTH-LP. Using recombi-nant DNA techniques, we found that the cDNA codingsequence of renal carcinoma PTH-LP5 was identical to thatof the lung-derived PTH-LP cDNA except that it lacks thelast two C-terminal residues due to the introduction of adifferent 3' untranslated sequence. Our studies indicate thata single gene codes for the PTH-LPs produced by varioustumors and that an alternate mRNA splicing mechanismgenerates multiple PTH-LP mRNAs in 786-0 cells. Further-more, one of these mRNAs contains similarities to anothertumor-associated gene product, the c-myc protooncogene.

MATERIALS AND METHODSReagents. Cultures of the human renal carcinoma cell line

786-0 (clone KEC) were maintained as described (9). AcDNA synthesis kit, Hybond-N, [a-32P]dCTP (3000Ci/mmol) (1 Ci = 37 GBq) and [y_32P] ATP (3000 Ci/mmol)were purchased from Amersham. Avian myeloblastosis virusreverse transcriptase was from Seikagaku, Kogyo (Tokyo).Human c-myc exon III DNA was purchased from Oncor(Gaithersburg, MD).

Construction and Screening of a Renal Carcinoma (Cell Line786-0) cDNA Library. A AgtlO cDNA library was constructedfrom poly(A) + RNA prepared from 786-0 cells as described(10). Plaque hybridization was done using a 5' end-labeledoligonucleotide (5' CTTCCCCTTGTCATGGAGGAGCT-GATGTTCAGACACAGCTC 3'). This sequence is comple-mentary to bases 107-147 of a PTH-LP cDNA reported bySuva et al. (6). Conditions for filter hybridization andwashing were 6 x SSC (1 x SSC = 0.15 M NaCl/0.015 Msodium citrate, pH 7) at 50°C and 2 x SSC/0.1% NaDodSO4at 60°C, respectively. Washed filters were exposed to KodakXAR film using DuPont Cronex intensifying screens.

Subcloning and Sequence Analysis of Clone 1OB5. TheEcoRI cDNA inserts from positive plaques were subclonedinto the EcoRI site of pBS/M13 + (Stratagene, San Diego,

Abbreviations: HHM, humoral hypercalcemia of malignancy; PTH,parathyroid hormone; PTH-LP, PTH-like peptide.TTo whom reprint requests should be addressed.§The sequence reported in this paper is being deposited in theEMBL/GenBank data base (IntelliGenetics, Mountain View, CA,and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03802).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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CA), and their sequences were determined by the method ofMaxam and Gilbert (11). Sequence analysis was done usingIntelliGenetics computer programs.RNA Blot Analysis. Poly(A) + RNA was prepared from cells

or tissue as described (10), electrophoresed in 1% agarosegels containing 2.2 M formaldehyde, and transferred toHybond-N (12). Blots were prehybridized overnight in asolution containing 50%o (vol/vol) formamide at 420C. Blotswere hybridized to cDNA probes at 420C and washed at 650Cin a solution of0.1 x SSC/0.1% NaDodSO4. Hybridization ofblots to oligonucleotides was done at 350C. The oligonucle-otide 1OB55' (5' CTGGCCAGCAGTTCTCCTGGGTTCGT-GGAACAGGGCTAACCGCCTCC 3') was derived from thecomplement of bases 49-95 of the clone lOB5 sequence. Theoligonucleotide AP7 (5' AGAATCCTGCAATATGTCCTT-GGAAGGTCTCTGCTGAAAATTTCAATGCCTC 3') iscomplementary to bases 525-576 of the sequence of Suva etal. (6) After hybridization, filters were washed at 60'C in 1 xSSC/0.1% NaDodSO4.

Primer-Extension Analysis of the 5' End of Cell Line 786-0PTH-LP mRNA. A 25-pmol aliquot of a 5' end-labeledoligonucleotide 1OB55' (specific activity 3 x 108 cpm/,ug)was hybridized to 2 ,ug of cell line 786-0 poly(A)+ RNA asdescribed (13). After hybridizing of primer to RNA overnightat 60°C, primer extension was done for 1 hr at 42°C using 15units of avian myeloblastosis virus reverse transcriptase. Theextension products were separated on an 8% denaturingpolyacrylamide gel. Autoradiography of the wet gel wascompleted with intensifying screens.

Southern Blot Analysis. The procedures used in the prep-aration of large-molecular weight DNA from human termplacenta and the subsequent Southern blots have beendescribed (10). The cDNA probes used for Southern analysisare outlined in Fig. 4. Hybridizations and washes were doneat 65°C in 6 x SSC and 0.1 x SSC, respectively. A blotprepared using the oligonucleotide AP7 was hybridized at50°C in 6x SSC and washed at 63°C in 3 x SSC/O.1%NaDodSO4.

RESULTSIsolation and Analysis of cDNAs Complementary to the

PTH-Like Factor mRNA Expressed in Human Renal Carci-noma 786-0 Cells. The partial N-terminal amino acid sequencefor the PTH-LP secreted by human renal carcinoma cell line786-0 (6) has at least 50% similarity with PTH and nearlycomplete similarity to a PTH-LP purified from human lungcarcinoma cell line BEN (3). An oligonucleotide encoding thePTH-like sequence of the BEN cell PTH-LP cDNA (seeMaterials and Methods) was used to screen a AgtlO cDNAlibrary constructed from 786-0 cell poly(A)+ RNA. Weisolated three clones varying in size from 625-1600 base pairsin length. The DNA and deduced amino acid sequences ofthenearly full-length clone lOB5 are presented in Fig. LA.A 303-base 5' untranslated sequence is followed by a

525-base open reading frame. The start codon ATG ispreceded by a sequence similar to the consensus sequence forthe initiation of translation of eukaryotic mRNA (14). ThePTH-like sequence begins at base 412; 8 of the first 13 aminoacids encoded are identical to PTH. The remainder of thepeptide, as shown by Suva et al. (6), shares only slightsimilarity to PTH. The nucleotide sequences and openreading frames of the cDNAs for the lung and renal PTH-LPsare identical through base 828, where they totally diverge. Inclone lOB5, there is a stop codon, which removes the last tworesidues (Arg-His) from the BEN cell-derived cDNA se-quence. The 3' untranslated sequence from clone lOB5 has767 bases and is punctuated by two polyadenylylation sites(AATAAA) followed by a short poly(A) stretch. In contrast,the 3' untranslated sequence of the BEN cell PTH-LP cDNA

is 550 nucleotides in length (6). Interestingly, both 3' se-quences are A+T rich and contain multiple copies of anATTTA motif that has been shown to play a role in mRNAturnover (15).Renal Carcinoma Cells Express Two Major PTH-LP

mRNAs with Different 3' Untranslated Sequences. The RNAblot seen in Fig. 2, lane 1, reveals the presence of two major(-1800 and 1550 nucleotides) and one minor (2400 nucleo-tide) species of PTH-LP mRNAs. This observation could beexplained by multiple PTH-LP genes, by the alternate use ofdifferent polyadenylylation sequences within the 3' untrans-lated region, or by alternate splicing of different 3' untrans-lated sequences (16).To investigate these possibilities, we hybridized 786-0

poly(A)+ RNA to probes derived from the 3' sequences ofeither lOB5 or the BEN cell cDNA. The lOB5 3'-specificcDNA probe hybridized to the 2400 and 1800 base mRNA(Fig. 2, lane 2), whereas an oligonucleotide (AP7) derivedfrom the 3' end of the BEN cell cDNA hybridized to the1550-base message (lane 4). Hybridization with both probes(lane 3) demonstrated that all mRNAs were present in thesame sample. The blots in lanes 2 and 4 were overexposed toreveal the absence of cross hybridization between the two 3'untranslated sequences. The 5' oligonucleotide 1OB55' hy-bridized to all three mRNAs, indicating that the 5' untrans-lated sequence of lOB5 is common to all three PTH-LPmRNAs expressed by these cells (lane 5).To further clarify the basis for multiple mRNAs we have

sequenced a cDNA, 1OC4, which appears to be derived froma partially processed PTH-LP mRNA. The sequence termi-nates with a short stretch of adenines, lacks an in-frame stopcodon and polyadenylylation sites, and diverges at the samepoint in the sequences as the cell line lOB5 and the BEN cellsequences (BRF.50) cDNAs (6). In Fig. 1B we present acomparison of the three known cDNA sequences derivedfrom this part of the message. From these data we postulatethat the point at which the three cDNA sequences diverge isa splice site (17) that can accept one of at least two different3' untranslated sequences and that the last 35 bases of thecDNA 1OC4 sequence are intron sequences.

Primer-Extension Analysis of the 5' End of PTH-LP mRNA.Primer-extension analysis was done to investigate whetherthe multiple PTH-LP mRNAs expressed by 786-0 cells resultsfrom initiation of transcription at different promoters (Fig. 3).A5' oligonucleotide, 1OB55', which hybridized to all PTH-LPmRNAs in 786-0 cells (Fig. 2, lane 5), was used as a primer.The two major extension products of 111 and 113 bases inlength (Fig. 1A), obtained by using a primer beginning at base% of the lOB5 sequence, indicate that the putative 5' cap sitesof the PTH-LP mRNAs in 786-0 cells are 15-17 basesupstream from the 5' end of sequence lOB5.The PTH-LPs Are Encoded by a Single-Copy Gene in

Humans. Southern blot analysis of human placental DNAwas done using the different 32P-labeled cDNA probesoutlined in Fig. 4. All probes derived from the lOB5 cDNA(blots A-C) hybridized to a single EcoRI fragment of -15kilobases (kb), whereas 5'- or 3'-specific probes hybridized toone of two different Pvu II fragments (blots A and C).To examine whether the two different 3' untranslated

sequences are found in the same gene, blot D in Fig. 4 washybridized to an oligonucleotide (AP7) complementary to the3' end of the BEN cell PTH-LP cDNA. This sequencehybridized to an EcoRI fragment common to the lOB5 probes(A-C), but hybridized to a unique Pvu II fragment (Fig. 4, blotD). These data indicate that both 3' untranslated sequencesare contained within the same 15-kb EcoRI fragment, but canbe mapped to two distinct Pvu II subfragments. To test forpossible related sequences within the human genome, anadditional blot was hybridized to probe A at lower stringency(550C). This resulted in a general increase in background

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A.CCGGTTCGCAAAGAAGCTGACTTCAGAGGGGGAAACTTTCTTCTTTTAGGAGGCGGTTAGCCCTGTTCCA 70

CGAACCCAGGAGAACTGCTGGCCAGATTAATTAGACATTGCTATGGGAGACGTGTAAACACACTACTTAT 140

CATTGATGCATATATAAAACCATTTTATTTTCGCTATTATTTCAGAGGAAGCGCCTCTGATTTGTTTCTT 210

TTTTCCCTTTTTGCTCTTTCTGGCTGTGTGGTTTGGAGAAAGCACAGTTGGAGTAGCCGGTTGCTAAATA 280

AGTCCCGAGCGCGAGCGGAGACG ATG CAG CGG AGA CTG GTT CAG CAG TGG AGC GTC GCG 339M Q R R L V Q Q W S V A -24

GTG TTC CTG CTG AGC TAC GCG GTG CCC TCC TGC GGG CGC TCG GTG GAG GGT CTC 393V F L L S Y A V P S L G R S V E G L -6

AGC CGC CGC CTC AAA AGA GCT GTG TCT GAA CAT CAG CTC CTC CAT GAC AAG GGG 447S R R L K R A V I j H I L L H D K Gj 12

AAG TCC ATC CAA GAT TTA CGG CGA CGA TTC TTC CTT CAC CAT CTG ATC GCA GAA 501K S I Q D L R R R F F L H H L I A E 30

ATC CAC ACA GCT GAA ATC AGA GCT ACC TCG GAG GTG TCC CCT AAC TCC AAG CCC 555I a T A E I R A T S E V S P N S K P 48

TCT CCC AAC ACA AAG AAC CAC CCC GTC CGA TTT GGG TCT GAT GAT GAG GGC AGA 609S y N T JL N H P y R F G

_D D E G R 66

TAC CTA ACT CAG GAA ACT AAC AAG GTG GAG ACG TAC AAA GAG CAG CCG CTC AAG 663Y L T Q E T N K V E T Y *, E Q P L K 84

ACA CCT GGG AAG AAA AAG AAA GGC AAG CCC GGG AAA CGC AAG GAG CAG GAA AAG 717T P G K K K K G K P G K R K E Q E K 102

AAA AAA CGG CGA ACT CGC TCT GCC TGG TTA GAC TCT GGA GTG ACT GGG AGT GGG 771K K R R T R S A W L D S G V T G S G 120

CTA GAA GGG GAC CAC CTG TCT GAC ACC TCC ACA ACG TCG CTG GAG CTC GAT TCA 825L E G D H L S D T S T T S L E L D S 138

CGG TAA CAGGCTTCTCTGGCCCGTAGCCTCAGCGGGGTGCTCTCAGCTGGGTTTTGGAGCCTCCCTTC 893R -- 139

TGCCTTGGCTTGGACAAACCTAGAATTTTCTCCCTTTATGTATCTCTATCGATTGTGTAGCAATTGACAG 963

AGAATAACTCAGAATATTGTCTGCCTTAAAGCAGTACCCCCCTACCACACACACCCCTGTCCTCCAGCAC 1033

CATAGAGAGGCGCTAGAGCCCATTCCTCTTTCTCCACCGTCACCCAACATCAATCCTTTACCACTCTACC 1103

AAATAATTTCATATTCAAGCTTCAGAAGCTAGTGACCATCTTCATAATTTGCTGGAGAAGTGTATTTCTT 1173

CCCCTTACTCTCACACCTGGGCAAACTTTCTTCAGTGTTTTTCATTTCTTACGTTCTTTCACTTCAAGGG 1243

AGAATATAGAAGCATTTGATATTATCTACAAACACTGCAGAACAGCATCATGTCATAAACGATTCTGAGC 1313

CATTCACACTTTTTATTTAATTAAATGTATTTAATTAAATCTCAAATTTATTTTAATGTAAAGAACTTAA 1383

ATTATGTTTTAAACACATGCCTTAAATTTGTTTAATTAAATTTAACTCTGGTTTCTACCAGCTCATACAA 1453

AATAAATGGTTTCTGAAAATGTTTAAGTATTAACTTACAAGGATATAGGTTTTTCTCATGTATCTTTTTG 1523

TTCATTGGCAAGATGAAATAATTTTTCTAGGGTAATGCCGTAGGAAAAATAAAACTTCACATTTAAAAAA 1593AA 1595

B.CTCGATTCAC GGTAACAGGCTTCTCGGCCCGTAGCCTCAGCGGGGTGCL D S R ---

CTCGATTCAC GGAGGCATTGAAATTTTCAGCAGAGACCTTCCAAGGACL D S R R H ---

CTCGATTCAC GTACAGCACTTCTGTGGGGTTTGAAAAAAAAAAAAAAAL D S splice

10B5

BRF.50

10C4end

FIG. 1. Nucleotide and deduced aminoacid sequences from clone 1OB5 cDNAand analysis of the presumptive site ofalternate splicing. (A) The open readingframe begins with the AUG at base 304.The amino acid sequence is presentedusing the single letter designation. Aminoacids - 1 to - 36 represent the presump-tive leader plus pro-specific sequence.The vertical arrow designates the start ofthe 139 residue PTH-LP. Those aminoacids shared by the PTH-LP and PTH areunderlined. Two polyadenylylation sites(AATAAA) at the end of the sequenceare boldly underlined. (B) These se-quences are taken from correspondingsequences of three different PTH-LPcDNAs. The nucleotide and deducedamino acid sequences begin with base817 of the 10B5 sequence or base 514 ofthe BRF.50 sequence (6). The arrowrepresents the point at which the threesequences diverge. In-frame stop codonspresent in the sequences of 10B5 orBRF.50 are underlined. In the 10C4 se-quence the presumptive intron consensusdinucleotide GT is underlined. The 10C4sequence terminates with a stretch ofadenines.

without the appearance of any new bands (data not shown),indicating the existence ofa single gene that encodes multiple3' untranslated mRNA sequences.Sequence Similarities Between the 3' Sequence of PTH-LP

and c-myc mRNAs. A computer search of the data base ofavailable DNA sequences revealed regions of considerablesimilarity between the 3' untranslated sequences of sequencelOB5 and the 3' untranslated sequence of the human c-mycprotooncogene (18). Several regions of similarity between thetwo sequences are shown in Fig. 5.

DISCUSSIONWe have isolated and sequenced a cDNA that is comple-mentary to the 1800-base mRNA encoding a PTH-LP ex-

pressed by the human renal carcinoma cell line 786-0. Thepredicted N-terminal sequence of this peptide is consistentwith the amino acid sequence of the peptide purified from786-0 cells, and 8 of the first 13 amino acids are similar toPTH. This cDNA encodes a molecule of 139 amino acids thatis identical to, but two amino acids shorter than, a peptidesequence deduced from a cDNA isolated from a human lungcarcinoma (cell line BEN). The lower apparent molecularweight ofthe peptide purified from the renal carcinoma (6000)compared with the molecular weight of 15,678 deduced fromthe cDNA may be due to proteolysis during purification.However, we cannot exclude the possibility of cell-specificprocessing.Our data clearly show that in the 786-0 cells there are two

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2 3 4 5

28S 1

18S *UI-iU........

FIG. 2. RNA blot analysis of 786-0 poly(A)+ RNA reveals thepresence of two different 3' untranslated sequences in PTH-LPmRNA. A 3-tig aliquot of cell line 786-0 poly(A)+ RNA wasseparated by agarose gel electrophoresis in the presence of 2.2 Mformaldehyde and blotted to nylon membranes. Individual blots werehybridized with the 1595-base-pair 10B5 cDNA (lane 1), bases872-1595 of clone 1OB5 sequence (lane 2), the oligonucleotide AP7derived from the 3' end of the BEN cell PTH-LP cDNA (6) (lane 4),or the 5' oligonucleotide 10B55' (lane 5). To generate the blot seenin lane 3, the blot seen in lane 4 was rehybridized to the probe usedto generate the blot seen in lane 2 without removing the first probe.RNA size markers are rat 18S and 28S rRNAs.

major PTH-LP mRNAs, of approximately equal abundance,generated by alternate splicing of sequences at the 3' end ofthe coding sequence. In addition, our data suggest that the1550-base mRNA may be the template for the BEN cell-derived PTH-LP cDNA (6), whereas sequence 1OB5 is a copyof the 1800-base message. This conclusion is based onequivalent 5' untranslated sequences and the contribution bythe poly(A) tail.

Alternate splicing of a single gene, a ubiquitous process ineukaryotic cells, can generate a diversity of protein productsand can serve as a potential mechanism for developmentaland tissue-specific gene regulation (16). One well-characterized example of this event is the splicing of thecalcitonin gene. Tissue-specific alternate splicing of 3' exonsof the calcitonin gene results in the synthesis of two struc-turally and functionally unrelated peptides, calcitonin (18)and calcitonin-gene-related peptide (19).

Breitbart and Nadal-Ginard (20) used a minigene of tropo-nin T to demonstrate that aberrant processing of troponin TmRNA occurs in cells that do not normally express thetroponin T message. Their findings suggest that there arecell-specific trans-acting elements responsible for the tissue-specific processing of precursor RNA. For PTH-LP, it is ofinterest that the human osteosarcoma cell line Saos-2 (data

113 '

FIG. 3. Primer-extension analysis of the 5' end of PTH-LPmRNA expressed by 786-0 cells. The end-labeled oligonucleotide10B55' (25 pmol) was hybridized to 2 ,g ofpoly(A)+ RNA from 786-0cells. The extension products were separated by electrophoresisthrough a 8% polyacrylamide gel containing 8.2 M urea. Autoradi-ography was done for 2 days. The DNA size markers were a Maxamand Gilbert sequencing ladder (ladder data not shown).

A B C DP E P E P E P E

23.1 --9.4--6.6 .f4.3

2.3 -2.0

AE A P

B E

C

D

1 OB5A PC H E 10B5

E 1OB5- AP 7

FIG. 4. Southern blot analysis of human placental DNA. Largemolecular weight DNA (10 ,ug) was digested overnight with eitherPvu II (P) or EcoRI (E). Digested DNA was electrophoresed through0.8% agarose gels and transferred to Hybond-N membranes. Indi-vidual blots A-D were hybridized to the corresponding DNA probesoutlined below. Thin and bold lines indicate untranslated and codingsequences, respectively. Other restriction sites are Ava I (A), Cla I(C), and HindIII (H). The 52-base oliogonucleotide AP7 is derivedfrom the BEN cell PTH-LP cDNA (6). The numbers at the leftrepresent the HindIII-digested ADNA size markers and are ex-pressed as the number of kilobase pairs.

not shown) also expresses approximately equal amounts ofthe two major PTH-LP messages and that 786-0 cells containa larger presumptive precursor RNA. Further studies areneeded to determine the normal tissue distribution of thePTH-LP messages and the physiological role of alternatesplicing.The mRNAs expressed by the renal carcinoma cells

potentially encode two PTH-LPs that would differ by twoamino acids at their C termini. Such a minor alteration in thestructure of a peptide as a result of alternate splicing is rare(21). The functional consequences in terms of biologicalactivity of such a change is unknown, but this change could

1330 13401OB5 CTTTTTATTTAATTAAATGTATTTAATT

***** *** **** * ** ***

C-MYC TTTTTTTTTTCTTTAACAGATTTGTATT940 950 960

1380 1390lOB5 TAATGTAAAGAACTTAAATTATGTTT-----TAAAC

*** * **** *** * ****** ****

C-MYC AAATTTTAAGAT-TTACACAATGTTTCTCTGTAAAT990 1000 1010

1410 1420 14301OB5 CCTTAAATTTGTTTAA-TTAAATTTAACTCTGGTTTCTAC

* ****** * *** ** *** *** **** **

C-MYC CATTAAATGTAAATAACTTTAATAAAAC ----GTTTATAG1030 1040 1050

1510 1520 15301OB5 GTTTTTCTCATGTATCTTTTTGTTCATTG

***** * * ***** * **

C-MYC TTTTTTATTTAAGTACATTTTGCTTTTTA1130 1140

1550 1560 15701OB5 ATTTTTCTAGGGTAATGCCGTAGGAAAAATAAAAC

******** ** * *** **********

C-MYC -TTTTTCTATTGTTTT----TAGAAAAAATAAAAT1170 1180

FIG. 5. Nucleotide sequence similarity between the 3' untrans-lated sequences of 1OB5 and the c-myc gene. DNA homology searchof the GenBank DNA sequence data base and subsequent sequencealignment was completed using the IFIND and ALIGN programs ofIntelliGenetics. The astericks (*) denote base matches between thetwo sequences, whereas dashes (-) represent gaps introduced tomaximize base matching.

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potentially have a profound influence on protein secretion.Recently it was reported that a C-terminal tetrapeptideLys-Asp-Glu-Leu was required for retention of certain pro-teins in the lumen of the endoplasmic reticulum; the additionof a single amino acid to the end of the protein caused theprotein to be secreted (22). Along similar lines, Collins et al.(23) have shown that tissue-specific alternate splicing ofplatelet-derived growth factor A chain mRNA removes abasic domain at the C terminus that results in a markeddecrease in the secretion of the molecule. Alternate splicingof the PTH-LP mRNA may also provide a 3' untranslatedsequence that could influence mRNA stability or translat-ability.

In contrast to earlier views that the different tumor-derivedPTH-LPs may be encoded by a family ofgenes, the Southernblot analysis indicates that a single-copy gene is responsiblefor the multiple PTH-LP mRNAs. Suva et al. (6) speculatedthat different sequences seen at the 5' end of the cDNA mayresult from alternate splicing. RNA blot and primer-extension analysis presented here show that in 7860 cells,the three PTH-LP mRNAs have common 5' untranslatedsequences. The close proximity of the two initiation sitessuggests that the two transcripts are initiated by the samepromoter. Although it seems unlikely, we cannot exclude thepossibility that the gene may encode two 5' noncodingregions of similar size or that alternate use of 3' untranslatedsequences is determined by the start site used.The significance of similarities in the 3' untranslated

sequence of PTH-LP and c-myc mRNAs is unknown at thistime. Elevated levels of c-myc have been correlated with celltransformation (24) and may play a role in the phenotype ofthese tumor cells. The A+ U rich sequences shared by thePTH-LP and c-myc messages may be a common signal formRNA turnover (15). These sequence similarities may becoincidental but are sufficiently interesting to warrant exam-ination of tumor cells that express PTH-LP for a possibleregulatory role of the 3' untranslated regions.

Little is known about the physiological regulation ofPTH-LP gene expression. PTH-LP may be the product of adevelopmentally regulated gene, the transcription of which ispromoted as a consequence of cell transformation. Such aphenomenon is exemplified by the ectopic expression of thedevelopmentally regulated a-fetoprotein gene by certainhepatomas and teratocarcinomas (25). On the other hand,PTH-LP may normally be expressed at lower levels innontumor cells, serving a physiological function yet to beelucidated. Further studies are needed to elucidate thepattern ofPTH-LP gene expression in normal tissues as wellas in tumors.

Note Added in Proof. After the submission of this manuscript a paperby Mangin et al. (26) appeared describing the cDNA of a PTH-likepeptide expressed in the human renal carcinoma cell line SKRC-1.The coding region of that cDNA sequence is identical to the onepresented here; however, the 3' untranslated sequence is differentfrom this one and is identical to that reported for the cDNA derivedfrom the cell line BEN. In addition, Mangin et al. (26) found that avariety of tumors as well as cultured keratinocytes express multiplePTH-LP mRNAs, indicating that alternative splicing may be acommon mechanism responsible for the generation of heterogenousPTH-LP mRNAs in tumor cells.

We thank Dr. Ellis Golub for the computer-assisted sequenceanalysis, Dr. T. J. Martin for helpful advice during the course of thiswork, and Drs. Robert Stein and Michael Caufield for discussionsduring the preparation of this manuscript.

1. Stewart, A. F. & Broadus, A. E. (1987) in Endocrinology andMetabolism, eds. Felig, P. F., Baxter, J., Broadus, A. E. &Frohman, L. A. (McGraw-Hill, New York), 2nd Ed., pp.1317-1453.

2. Case Records of Massachusetts General Hospital Case 27461(1941) N. Engl. J. Med. 225, 789-791.

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