Gene, 112 (1992) 247-250 © 1992 Elsevier Science Publishers B.V. AI! rights reserved. 0378-1119/92/$05.00

247

GENE 06348

The cDNA cloning and immunological characterization of hamster p53 (Recombinant DNA; tumor-suppressing gene; DNA sequencing; monoclonal antibodies)

Y. Legros a'b, P. Mclntyre c and T. Soussi a'b "Laboratoire de G~n&ique Mol~culaire, Ecole Normale Sup~rieure, 75230 Paris (France) Te!.(33)43291225; h hlstitut de G~n~tique Mol~culaire, U301 INSERM, 75010, Paris (France); and " Sandoz Institute for Medical Research, London WCIE 6BN (England) Tel. (44-1) 713874445

Received by H. van Ormondt: 19 August 1991 Revised/Accepted: 11 October/8 November 1991 Received at publishers: 7 January 1992

SUMMARY We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75 % homologous to human and mouse p53, respectively. Immunoprecipitations of the eDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.

INTRODUCTION Rodent cells have been very useful in the studies of different transforming processes. Studies of the mouse p53 gene have led to the classification of p53 as a tumor... suppressor gene which is mutated in murine and human tumors or in transformed cells (Lane and Benchimol, 1990; Levine, 1990). Several studies involved hamster transformed cells but so far, the hamster p53 sequence has not been available. Furthermore it is already known that the hamster p53 protein does not resemble other rodent p53 proteins in its interaction with mAbs. The aim ofthe present study was to clone and determine the sequence of hamster

Correspondence to: Dr. T. Soussi, U301 INSERM,27 rue J. Dodu, 75010 Paris (France) Tel. (33)42499269; Fax (33)42069531.

Abbreviations: aa, amino acid(s); bp, base pair(s); eDNA, DNA complementary to RNA; kb, kilobase(s) or 1000bp; mAb,monoclonal antibody; nt, nucleotide(s); ORF, open reading frame(s); p53, tumor suppressor protein encoded by the p53 gene(DNA); PAGE,polyacrylamidegel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; wt, wild type; X., Xenopus.

p53 cDNA and to establish its homology to other mammalian p53 proteins.

EXPERIMENTAL AND DISCUSSION

(a) Library screening A Syrian hamster 2gtl0 cDNA library was prepared from RNA extracted from MP1 cells which had been derived from baby hamster kidney (BHK) cells (Giulotto et al., 1987). The library was simultaneously probed with two 300-bp probes derived from the 5' and 3' ends of the mouse p53 eDNA (Jenkins et al., 1984). The positives clones were processed as described in the accompanying paper (Caron de Fromentel et al., 1992).

(b) Sequence of hamster p53 The complete nt sequence is shown in Fig. 1. The insert contains a single ORF extending from an ATG at nt 138 to a stop codon at nt 1328. The encoded protein is 396 aa long with a deduced Mr of 43 603. This size is significantly shorter than the apparent Mr of the protein produced by in vitro transcription/translation and analysed by P A G E SDS gel (Fig. 3, see accompanying paper of Caron de

248 10 20 30 GARTTCCCTACCATCCTGGCTGCAGGTAGC 40 50 60 70 60 90 100 110 120 130 137 TATTGGGRTCAGGGGGCACCTGGGCATCCGGGTCCGGARCCTACTTCCCRGCAGGGTARCACGCTTCCCTGAAGACCTGRAGCCTGGCTGACTTCCTGAGTGCTGCC 200 210 220 160 190 150 160 170 ATG GAG GAG CCA CAG TCA GAC CTC RGC ATC GAG CTC CCT CTG AGT CRG GRG ACA TTT TCA GAC CTG TGG AAA CTA CTT CCT CCA ARC RAT Met 91u glu pro 9In set Qsb leu set lie glu leu pro leu set gin 91u thr phe set asp leu trp lys leu leu pro pro ash ash 3O 20 10 290 300 310 270 260 230 240 250 260 GTT CTG TCC ACC TTG CCG TCC TCT GAT TCC ATT GAR GRA CTG TTC CTG TCC GAG ART GTT GCA GGC TGG CTA GAA GAC CCA GGT GAA GCT val leu sat thr leu pro set set asp set lie 91u 91u leu phe leu ser 91u ash val alo gly trp leu 91u asp pro gly 91u ola 60 5O 40 360 390 400 360 370 320 330 340 350 CTC CAR GGG TCG GCG GCT GCG GCA GCG CCG GCG GCT CCT GCA GCA GAG GAC CCT GTA GCT GAG ACT CCT GCA CCG GTG GCC TCT GCG CCA leu 91n 91y see alo ala ala alo ala pro ala ala pro ala alo 91u asp pro vQl alo glu thr pro ale pro val alo set ola pro 9O 80 70 4?0 460 490 450 460 410 420 430 440 6CC ACT CCC TGG CCC CTC TCA TCT TCT GTC CCA TCC TAT AAA ACC TAC CAG GGC GA¢ TAT GGT TTC CGT CTG GGC TTC CTG CAC TCG GGG ala the pro trp pro Isu set see see vol pro set tyr lys thr tyr gin 91y asp tyr 91y phe or9 leu 91y phe leu his see 91y 120 110 100 560 570 560 540 550 500 510 520 530 RCG GCC ARR TCT GTC ACA TGC ACG TAC TCA CCT TCC CTC AAT AAG CTG TTC TGC CAG CTG GCG AAA ACA TGC CCC GTG CRG CTG TGG GTC thr ala lys set val thr cue the tyr set pro set Isu ash lye leu phe ©ys gin leu ala lys thr cys peo val gln leu trp vol 150 140 130 650 560 670 530 640 590 600 610 520 RGC TCC ACA CCT CCA CCT GGC ACC CGT GTC CGT 6CC ATG GCC ATC TAC AAG AAG TTA CAA TAC RI"G RCG GAA GTT GTA AGR CGC TGT CCC set set thr pro pro pro 91y thr ar9 val or 9 ala met alo lie tyr lye lye leu gin fur mt:t thr glu val val or 0 or 9 ©ys pro 160 170 160 740 750 ?60 ?20 730 680 690 700 710 CAC CAC GAG CGC TCC TCC GAA GGC GAT GGT TTQ GCT CCT CCT CRG CAT CTT ATC CGA GTG GAA GGA RAT ATG CRT GCC GAR TRC CTG GAT hie his 91u ar9 set set 91u 91y asp gly leu ala pro pro gin his leu lie ar9 val 91u 91Y aan met his ala glu tyr leu asp 210 200 190 630 640 650 610 620 770 760 790 600 GAC RAG CAG ACT TTT CGG CAC AGT GTG GTG GTG CCC TAT GAG CCA CCT GAG GTT GGC TCT GAC TGT ACC ACC ATC CAC TAT ARC TAC ATG asp lys gin thr phe arg his set ual val val pro fur 91u pro pro 91u vol 91Y set asp cys thr the lie his tyr a~n tyr met 240 23O 220 900 910 920 930 940 660 670 600 090 TGT ART RGT TCC TGC ATG GG6 GGC ATG AAC CGG COG CCT RTC CTC flCC RTC RTC RCG CT~ 0~6 GAC ccc ROTTGpGRAC CTG C]G.,GGA CGG cys ash set set cys met 91y 91y met ash or 9 arg pro lie leu the lie lle thr leu glu asp pro set 91y ash leu leu 91y or 9 260 270 250 990 1000 1010 1020 1030 950 960 970 960 RAC AGC TTT GAG GTT CGT_.ATT TGT GCC TGC CCT GGG RGA G~ CGT C~T ACA GAG GAA AAA ART TTC CAR RAG RAG GGA Grin CCT TGC CCR aan set phe glu val or9 lie cys ala cys pro 91Y ar9 asp or 9 or 9 the 91u glu lys ash phe 91n lys lys 91Y 91u pro cys pro 290 300 200 1100 1110 !120 1040 1050 1060 1070 1080 1090 GAR CTR CCC CCR RAG RGT GCT ARA CGA GCA TT6 CCT ACC ARC ACA RGC TCC TCT CCC CAG CCA RAG AGA AAA ACA CTT GAC GGA GAA TAT 91u lau pro pro lys ser alo lys ar9 alo leu pro ~hr ash thr ~er get ger pro gin pro lys or 9 lya thr leu asp gly olu tyr 330 320 310 1130 1140 !150 1160 1190 1200 1210 1170 1160 TTC ACC CTT ARG RTC CGT GGT CRR GRA CGC TTC RnG ATG TTC CRR GAR TTG ART GAG GCC TTG GAA CTG RAG GAT GCR CRG GCT TTG RAG phe the leu lys lle or9 91y gin 91u org phe lys met phe gin 9lu leu aan 91u alo lau 91u leu lys osp olo gin aio leu lye 350 340 35O 1220 1230 1240 125C 1260 1270 1260 1290 1300 GCG TCR GAG GAC AGT 6GT GCT CRC TCC RGC TPC CTG RAG TCC ARG RAG 6GC CAG TCT GCC TCC CGT CTT AAA RAA CTR ATG ATC RAG AGA ola sop 91u asp see 91y alo his set see tdr leu lys set lye lys 919 gin ser ala set org leu lys lys leu met lie lys or 9 3?0 360 390 1310 1320 1330 1350 1350 1370 1360 1390 1400 1410 GAG GGG CCT GAC TCG GAC TGA CCTCCTTCCT ~CCTCCTGTC CCCCATCAGCRACCTCCCRCCCCCCTCCTTTCCTGCCATTTTGTCACTTGGGTGCTTGRRCT glu 91y pro osp set osp EHO 396 1420 1430 1140 1450 1460 1470 1460 1190 1500 1510 1520 CCCCRGCTTGGGTRGATCTGTCRTCTCTGTTATCTGGGGRGGGTGGTOGGRTACACCRGGACACCRGCTCAGCTTTTROGATTCTGACCATGAGGAAAGOARGAAAATTC 1530 1540 1550 1560 1570 1580 1590 1500 1610 1620 1630 TCAATRGGTGGTGTTAGARTTTACRRCTGGCCRTGTGCRGTGGTGAGGCCCCACECRRCTCTCTRAGRAAOCCATCTGAGTTTACTTTCTCCTGACTTGAATAGCCACAT 1640 1650 1660 1670 1680 1590 1700 1710 1720 1730 1740 CTGTAAAATG CAGRTGGGCATACCTGCTTCTCRGGGTTTGTTGTGRTGCT TGTCCCTTCCRCACCTTTTATTACCTGTGGCCTGGRCTCAGTCCCCTCTGTGAGTGCTGG 1T50 1760 1770 1760 1790 1600 1810 1620 1830 1840 1850 TTCCTGGCCCCTOTTGGGTGOTAGTTACCAGGTCTCTGCTGCTGCTGGCCCRGCGAARTCCTRTCCRGCCACTTGRTGGACCCTGGCACCTARAATGAAATTTCACCCCG 1660 1670 1660 1690 1900 1910 1920 1930 1940 1950 1960 CCCTACACCC TGTARGRTTCTATCTTGGGCTCTCATGGGGTCCATARCCR CCAAGACCCRTTTTCTTTCCACCCTGCRCTCGGGRTCAGTTTCTTTGAACTCTGCCCRGC 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 CTTTCTAGCC TTTCTGCATTTACCATCCCCCCCTCTCTTT TTATACCATG CCCCCTTTTATATACCGATTTCTTATTTTACARTAAAATT TTGTTATCAAAAAAARARAA 2060 2067 AAAARRAARR GGRRTTC

Fig. I.

249

SDLWKLLPPNNULSTLPSSDSIEELFLS ENVAGWL EDP 57

HAMSTER MEEPQSDLSI MOUSE

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52

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HAMSTER G EALQGSAAAARPARPAAEDPVRETPAPUASAPATPWPLSSSUPSYKTYQGDYGFRLGFLHS 119 -PS---RU-G-P--QDPVT ---G---P--F - - - Q . . . . . N - - - H . . . . Q- !10 MOUSE HUMAN

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175

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181

HAMSTER SSEGDOLAPPQHLIRUEONMHAEYLDDKQTFRHSVUUPYEPPEVGSDCTTIH 236 C-D LYP---E-R A--EY . . . . 227 MOUSE HUMAN

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233

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340 HAMSTER 6EPCPELPPKSAKRALPTNTSSSPQPKRKTLDGEYFTLKIR6QER EVL . . . . . . 6 C A P Q K P RK331 MOUSE HUMAN

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396

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Fig. 2. Comparison of the deduced aa sequences of human, mouse and hamster p53. The sequences were arranged to yield the maximum similaritybetween the proteins. The aa identical to those in the hamster p53 are indicated by dashes. The five black boxes, named I-V, represent the five evolutionarily conserved domains (Soussi et al., 1990). The open box delineates the PAb122 epitope defined by Wade-Evans and Jenkins (1985). The human p53 sequence was taken from Matlashewski et al. (1984)'and Zakut-Houri et al. (1985). The sequence of wt mouse p53 was taken from Finlay et al. (1989).

it is c o m p a r e d to h u m a n a n d m o u s e p53. The h a m s t e r p53 is 78% similar to h u m a n p53 a n d 75% h o m o l o g o u s to

(Soussi et al., 1990). These regions contain most of the p53 mutations found in human or mouse transformed cells and tumors (Nigro et al., 1989). Examination of the hamster sequence shows that aa 277 in domain V is lie whereas it is Val in all other p53s from rainbow trout to man (Soussi et al., 1990). Furthermore, this residue has already been shown to be mutated in a human tumor (Slingerland et al., 1991). In order to assess the correctness of the sequence,

m o u s e p53. C o m p a r i s o n of all p53 aa sequences available so far shows that five domains are conserved through evolution

we prepared e D N A from total cellular R N A purified from hamster embryo. The e D N A was then amplified by P C R with primers that flanked d o m a i n V and contained cloning

Fromentel et al., 1992, for discussion). The encoded product exhibits the characteristics found in other p53 proteins: (i) the five highly conserved domains, (ii)an acidic Nterminal aa sequence; (iii) an hydrophilic C-terminal aa sequence, and (iv)a penultimate Ser residue. The predicted aa sequence of the hamster p53 is shown in Fig. 2 where

Fig. 1. The nt sequence of hamster p53 eDNA and the deduced aa sequence. The putative polyadenylation signal is underlined (AATAAAA; nt 20422048). The doubly underlined segment corresponds to the sequence amplifiedfrom hamster embryo RNA, whereas the singly underlined segment corresponds to the primers used in the PCR rea:tion. The nt sequence data reported in this paper have been submitted to GenBank and were assigned the accession No. M75144.

250 sites (Fig. 1). The resulting c D N A / P C R products were cloned. Since the PCR reaction is known to be associated with a relatively high rate of nt misincorporation, we performed the experiment twice and several independent clones were sequenced. From this study, we can confirm that in the wt hamster p53, aa 277 is, indeed, Ile 277.

The identification and characterization of wt hamster

p53 c D N A is the first step of a study aimed at characterizing the mutations in the p53 gene of hamster transformed cells or tumors.

ACKNOWLEDGEMENTS

(c) Immunological characterization of hamster p53 Several mAbs specific for human or rodent p53 were tested for their recognition of hamster p53. PAbl801 is known to be human-specific (Banks et al., 1986). The mAbs HR231 and HT216 were produced by immunization of mice with human p53 (Y.L., M. Pla and T.S., unpublished). HR231 recognizes human, rat and mouse p53, whereas HT216 is a mAh which reacts also with X. laevis p53 (Y.L., M. Pla and T.S., unpublished). The 200.47 mAb is rodentspecific (Dippold et al., 1981). All these mAbs were tested by immunoprecipitation with hamster p53 produced via in vitro transcription/translation (Fig. 3). As expected, PAblS01 is human-specific and does not recognize hamster p53 (Fig. 3, lane 1). The mAb HR231 recognizes an epitope similar to PAbI22 in the C terminus of human p53 (Wade-Evans and Jenkins, 1985; Figs. 2 and 3). It does not react with hamster p53 (Fig. 3, lane 2) and this is certainly caused by the two aa substitutions found in the sequence of this epitope (Fig. 2). Hamster p53 is efficiently immunoprecipitated by HT216 (Fig. 3, lane 4). The mAb 200.47 was already known to react well with hamster and mouse p53, poorly with rat p53 and not at all with human p53 (Dippold et al., 1981) (Fig. 3, lane 3). Taken together, these data indicate that our c D N A clone encodes a full-length hamster p53.

1

2

3

4

Human ,~, ~ , ~ . . . . .

Mouse Rat Hamster

Fig. 3. Immunologicalcharacterization of mammalian p53. The different p53 proteins were synthesized by in vitro transcription/translation of the corresponding cDNAs. Immunoprecipitations were carried out with the following mAbs: PAblS01, lane 1; 231, lane 2; 200.47, lane 3 and 216, lane 4. Immunoprecipitates were subjected to 10% PAGE-0.1% SDS.

We thank M. Ernoult-Lange and M. Kress for their generous gift of hamster embryo RNA. This work was supported by grants from the Association pour la Recherche sur le Cancer. REFERENCES Banks, L., Matlashewski, G. and Crawford, L.: Isolation of human-p53 specific monoclonal antibodies and their use in the studies of human p53 expression. Eur. J. Biochem. 159 (1986) 529-534. Caron de Fromentel, C., Pakdel, F., Chapus, A., Baney, C., May, P. and Soussi, T.: Rainbow trout p53: eDNA cloning and biochemical characterization. Gene 112 (1992) 241-245. Dippold, W.G., .lay, G., DeLeo, A.B., Khoury, G. and Old, LJ.: p53 transformation-related protein: detection by monoclonal antibody in mouse and human cells. Proc. Natl. Acad. Sci. USA 78 (1981) 16951699. Finlay, C.A., Hinds, P.W., Tan, T.H., Eliyahu, D., Oren, M. and Levine, AJ,: Activatingmutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. J. Virol. 8 (1988) 531-539. Guilotto, E., Knight, K. and Stark, G.R.: Hamster cells with increased rates of DNA amplification, a new phenotype. Cell 48 (1987) 837845. Gurney, E.G., Harrison, R.O. and Fenno, J.: Monoclonal antibodies against simian virus40 T antigens:evidence for distinctsubclassesof largeT antigen and for similaritiesamong nonviralT antigens.J. Virol. 34 (1980) 752-763.

Jenkins, J.R., Rudge, K., Redmond, S. and Wade-Evans, A.: Cloningand expression analysis of full length mouse eDNA encoding the transformation associated protein p53. Nucleic Acids Res. 12 (1984) 56095626. Lane, D.P. and Benchimol, S.: p53: oncogene or anti-oncogene? Genes Develop. 4 (1990) 1-8. Levine, A.J.: Tumor suppressor genes. Bioassays 12 (1990) 60-66. Nigro, J.M., Baker, S.J., Preisinger, A.C., Jessup, J.M., Hostetter, R., Cleary, K., Bigner, S.H., Davidson, N., Baylin,S., Devilee,P., Glover, T., Collins, F.S., Weston, A., Modali, R., Harris, C.C. and Vogelstein, B.: Mutations in the p53 gene occur in diverse human tumour types. Nature 342 (1989) 705-708. Slingerland, J.M., Minden, M.D. and Benchimol, S.: Mutation of the p53 gene in human acute myelogenous leukemia. Blood (1991) 77 15001507. Soussi, T., Caron de Fromentel,C. and May, P.: Structural aspects of the p53 protein in relation to gene evolution. Oncogene 5 (1990) 945-952. Wade-Evans, A. and Jenkins, J.R.: Precise epitope mappingof the murine transformation-associated protein, p53. EMBO J. 4 (1985) 699-706. Zakut-Houri, R., Bienz-Tadmor, B., Givol, D. and Oren, M.: Human p53 cellular tumor antigen: eDNA sequence and expression in COS cells. EMBO J. 4 (1985) 1251-1255.