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Sequence of an exon of tumour suppressor p53 gene—A comparative study in domestic animals: Mutation in a Feline solid mammary carcinoma
13)"vet.J. (1995). 151,325
S E Q U E N C E OF AN E X O N OF T U M O U R S U P P R E S S O R p53 G E N E - - A COMPARATIVE S T U D Y IN D O M E S T I C ANIMALS: M U T A T I O N IN A FELINE SOLID MAMMARY CARCINOMA
B. MAYR,* G. SCHAFFNER,t R. KURZBAUER,t M. REIFINGER+ + and K. SCHELLANDER* *Institute for Animal Breeding and Genetics and $Institute for Pathology, Vete){na)y University, Linke Bahngasse 1 I, A-1030 Vienna and t lnstitute for Molecular Patholo~, Dr. Bohrgasse 7, A-1030 Vienna, Aust){a
SUMMARY Partial sequence determinations were performed on exon 8 of tumour suppressor gene p53 of cattle, sheep, goat, horse and pig. High sequence homology, between these species and other species including dog, cat, chicken and man is demonstrated. A mutation CGG--->TGG (arginine-->tryptophan) was detected in a feline solid carcinoma of the mammary gland. KE~WORDS: Gene sequence; p53; exon 8; domestic animals; feline mammary carcinoma.
INTRODUCTION Phylogenetic studies have demonstrated that tumour suppressor p53 contains five domains (I-V) highly conserved during the course of evolution (Soussi et al., 1987, 1990; Caron de Frementel & Soussi, 1992). Mutations in these five highly conserved regions are frequently involved in human tumours as so called hot spot regions (Nigro et al., 1989; Caron de Frementel & Soussi, 1992). The gene contains 11 exons interrupted by 10 introns; it codes for 393 amino acids. The major goal of our study was a comparative investigation of tumour suppressor p53 in several domestic animal species. Exon 8 (amino acids 262 to 308) harbours the highly conserved domain V (amino acids 270-286). Within this hot spot region, amino acids Arg 273 a n d Arg 282 are principle targets of mutation in man (Caron de Frementel & Soussi, 1992). Therefore, we included a panel of feline tumours in our present investigation 0007-1935/95/030325-05/$08.00/0
© 1995 Bailli6reTindall
326
BRITISH VETERINARY JOURNAL, 151, 3
both for comparative reasons and in order to extend our earlier data (Mayr el al., 1993).
MATERIALS A N D METHODS
Genomic DNA was extracted fl'om tile peripheral blood of healthy animals including five cattle (Bos taurus), five sheep (Ow;es ades), five goats (Capra hircus), five horses (Equus caball'us), five pigs (Sus scrofa) and 60 cats (Fells catus) according to standard techniques. DNA was also harvested fi'om neoplastic tissue of 17 tUlnOUrbearing cats. Pathohistologically the tumours were classifed as five adenocarcinomas (mamnla D, gland), one adenonaa (lllallllllal-}, gland), one haelnangioendotheliolna (mannnary gland), one haemangioma (mammalT gland), one solid carcinoma (lnanmla D, gland), one basosquamous cell carcinoma (thigh), one basalioma (thigh), one nwxosarkoma (oral), one adenoma of sweat glands (underarms) and four fibrosarcomas (head, bell),, jaw and shoulder). All these 17 feline ttnnours are different fi'om the eight previously reported (Mayr el al., 1993). Exon 8 of the p53 gene was amplified by a polynlerase chain reaction (PCR) using a 25 bp (5'TGGTAATCTACTGGGACGGAACAGC3') and a 20bp (5'TTACCTCGCTTACTGCTCCC3') primer. Thirty-five amplification cycles were performed. Template denaturation was 2 min at 97°C, primer annealing 1 rain at 57°C and extension 1 rain at 73°C. Four percent NuSieve/agarose gel electrophoresis was used for analysis. Elution of the 141 bp product used a Quiaex Kit (Quiagen, Chatsworth, CA) procedure. Sequencing was performed on the Automatic sequencer ABI 373 A applying the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).
RESULTS
Fig. 1 provides a comparison of the partial sequences in exon 8 of several domestic animal species (cattle, sheep, goat, horse, pig, cat and dog). Moreover, data from two primates (man and monkey), two rodents (mouse and rat) and tile domestic chicken were taken fl'om the literature and included for comparative pnrposes. Full identity (100%) at the amino acid level was present from codon no.s 970-'288 in all lnamnlalian species. The interspecific differences were restricted to several silent third base substitutions only. Even the avian species (chicken) is ve W similar in this region showing two amino acid changes (codon nos. 983 and 284) only. Between codon nos. 289 and 300, the conservation is remarkable too, both at amino acid and nucleotide level. For example, one amino acid difference separates cattle fi'om sheep or goat (no. -989) and dog from cat (no. 290) only (holnology 91.8%). The similarity between the bovidae cattle, sheep and goat is especially spectacular at the nucleotide level (homology 100% between sheep and goat and 97._9% to cattle, i.e. 35 of 36 nucleotides). Nonetheless, four amino acid changes separate cattle fl-om horses (codon nos. 989, -993, -994 and 995), dogs (codon nos. 289, -990, and 295) and five humans (codon nos. 989, -994, 295, 296 and 997), thus restricting homologies to 66.6 and 58.3%, respectively. At nucleo-
EXON 8 OF p53 IN DOMESTIC KNIMALS
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. . . . . .
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Fig. I ('Oml)arison of tlae nucleotide and predicted anfino acid sequences of codon nos. 270-300 of exon 8 of p53 from different species. Dots mean identity with the corresponding bovine nucleotide. The p53 sequences used fi'om literature are: dog (Mayr el aL, 19q4), human (Matlashewski et al., 1984), monkey (Rigaudy & Eckhart, 1989), mouse (Benchimol el al., 1984), rat (Soussi et aL, 1988b) and chicken (Soussi et al., 1988a). The chicken has been aligned and n u m b e r e d relative to mammals (Soussi et al., 1990) in order Io obtain homology'. Note also the deletion in the chicken f r o m codon nos. 294-298.
328
BRITISH VETERINARYJOURNAL, 151, 3
Fig. 2 Solid carcinoma of flae mammary gland of a cat; high mitotic activity. Bar represents 50 ~tm. tide level, the c o r r e s p o n d i n g homologies were 75% ( 2 7 / 3 6 c a t t l e - h u m a n and cattle-horse) and 80.5% ( 2 9 / 3 6 cattle-dog), respectively. All five individuals within the species cattle, sheep, goat, horse and pig had the same sequence. All o f the 50 health), and 16 o f the 17 t u m o u r - b e a r i n g cats possessed the n o r m a l feline 'wild type' s e q u e n c e o f e x o n 8. However, o n e p o i n t mutation o f this n o r m a l s e q u e n c e was d e t e c t e d in a solid c a r c i n o m a o f the m a m m a r y gland (Fig. 2) in o n e cat. T h e m u t a t i o n hit c o d o n no. 282 (CGG---)TGG, arginine--~tryptophan). Unfortunately, no n o n - t u m o u r tissue f r o m this animal was available for examination.
DISCUSSION N o n e o f the predicted a m i n o acid differences d e t e c t e d in o u r investigated domestic animals c o n c e r n e d the highly conserved d o m a i n V (Soussi et al., 1990) observed in o t h e r vertebrates. Thus, o u r present results on domestic animals supp o r t and e x t e n d earlier data (Soussi et al., 1990) suggesting the high evolutionary conservation o f p53. T h e h o m o l o g i e s in the less conserved region between c o d o n nos. 289 and 300 were considerable too, especially within families. Such comparative data are a worthwhile c o n t r i b u t i o n for a b e t t e r u n d e r s t a n d i n g o f the essential functional role suggested by its conservation. Exon 8 represents a p o r t i o n of the molecule which is crucial to its turnout suppressor function. CpG dinucleotides are prefentially involved in s p o n t a n e o u s mutations. T h e c o d o n for Arg 282 (CGG) is often hit in m u t a t i o n in m a n and can be c o n s i d e r e d as a h o t spot codon. It is remarkable that the same c o d o n was hit in
EXON 8 OF p53 IN DOMESTIC ANhMALS
329
the solid m a m m a r y c a r c i n o m a o f o u r p r e s e n t investigation a n d in a lymphosarc o m a investigated earlier (Mayr et al., 1993). Given the i m p o r t a n c e o f p53 in t u m o u r i g e n e s i s o f m a n a n d e x p e r i m e n t a l animals a n d the high f r e q u e n c y o f m u t a t i o n s f o u n d in h u m a n t u m o u r s its i n v o l v e m e n t in the t u m o u r s o f domestic animals is to be expected. Clearly, the search for nucleotide a n d a m i n o acid s e q u e n c e s o f this g e n e region a n d their alterations in domestic animals are o f g r e a t i m p o r t a n c e for veterinary a n d c o m p a r a t i v e medicine. T o f u r t h e r o u r u n d e r s t a n d i n g o f the c o n n e c t i o n between such p53 a b n o r m a l i ties a n d animal tumourigenesis, m o r e i n f o r m a t i o n is n e e d e d r e g a r d i n g the occurr e n c e a n d f r e q u e n c y o f m u t a t i o n s at known h o t spots as well as at new locations in different species a n d t u m o u r types.
REFERENCES BENCIIIMOL, S.,JENK1NS,J. R., C~XWFO~D,L. V. et al. (1984). Molecular analysis of tile gene for the p53 cellular tulnor antigen. Ill Cancer Celh. 2. Oncogenes and Virus Genes, eds G. F. Van
de Wonde, A.J. Levine, W. C. Topp & J. D. Watson, pp. 383-91. Cold Spring Harbor, New York: Cold Spring Harbor Laboratories. CARON DI- FREMENTEI,, C. ~¢: SoussI, T. (1992). Tp53 tumor suppressor gene: a model for investigating human mutagenesis. Genes, Chromosomes and Cancer4, 1-15. MATI~\SHEWSKI,G., L\~xn~, P., PI,xx, D., P.c.xcoc~,j., c~x~,~oRo, L. & BENCHIMOL,S. (1984). Isolation and characterization of a human p53 cDNA clone: expression of the human gene. EMBOJournal 3, 3257-62. Ma~a, B., HECZKO,U., SCHEI.taNI~ER,K., SCHL~:¢.~n,W. & R~.'mNGER,M. (1993). Sequence of an exon of tile feline p53 gene-nmtation in a lylnphosarcolna. British VeteTinmyJouma1149, 387-90. Mau~, B., SCHELL~NDER,K., SCHLE¢,ER,W. & R~IWNCE~,M. (1994). Sequence of an exon of the canine p53 gene-nmtation in a papilloma. British VeterinaryJournal 150, 81--4. Nl¢,Ro, J. M., B~x~R, S.J., PP,EISIN(;~,R,A. C. et al. (1989). Mutations in the p53 gene occur in diverse human tunlour types. Nature 342, 705-8. RIcauDv, P. & ECKI-L~RI",W. (1989). Nucleotide sequence of a cDNA encoding the monkey cellular phosphoprotein p53. Nucleic Acids Research 17, 8375. Sovssl, T., C,.XRONDE FRO~XlEN'rEt.,C., MECHaH, M., IVl..w,P. & KRESS, M. (1987). Cloning and characterization of a cDNA from Xenopus laevis coding for a protein homologous to human and murine p53. Oncogene 1, 71-8. Soussl, T., B~cw, A., I~Ess, M., S'rEHELIN,D. & MA~, P. (1988a). Nucleotide sequence of a cDNA encoding the chicken p53 oncoprotein. Nuc~'ic Acids Research 16, 11383. Soussl, T., Cakoy DE Fm:.,~IF~YTEL,C., Bastsc~o-r, C. & IVLw,E. (1988b). Nucleotide sequence of a cDNA encoding the rat p53 nuclear oncoprotein. Nucleic Acids Research 16, 11384. Sduss~, T., Carboy ~ F~OM~YTr~,C. & M.w, P. (1990). Structural aspects of the p53 protein ill relation to gene evolution. Oncogene 5, 945-52. (Acceptedfor publication 21 March 1994)
S E Q U E N C E OF AN E X O N OF T U M O U R S U P P R E S S O R p53 G E N E - - A COMPARATIVE S T U D Y IN D O M E S T I C ANIMALS: M U T A T I O N IN A FELINE SOLID MAMMARY CARCINOMA
B. MAYR,* G. SCHAFFNER,t R. KURZBAUER,t M. REIFINGER+ + and K. SCHELLANDER* *Institute for Animal Breeding and Genetics and $Institute for Pathology, Vete){na)y University, Linke Bahngasse 1 I, A-1030 Vienna and t lnstitute for Molecular Patholo~, Dr. Bohrgasse 7, A-1030 Vienna, Aust){a
SUMMARY Partial sequence determinations were performed on exon 8 of tumour suppressor gene p53 of cattle, sheep, goat, horse and pig. High sequence homology, between these species and other species including dog, cat, chicken and man is demonstrated. A mutation CGG--->TGG (arginine-->tryptophan) was detected in a feline solid carcinoma of the mammary gland. KE~WORDS: Gene sequence; p53; exon 8; domestic animals; feline mammary carcinoma.
INTRODUCTION Phylogenetic studies have demonstrated that tumour suppressor p53 contains five domains (I-V) highly conserved during the course of evolution (Soussi et al., 1987, 1990; Caron de Frementel & Soussi, 1992). Mutations in these five highly conserved regions are frequently involved in human tumours as so called hot spot regions (Nigro et al., 1989; Caron de Frementel & Soussi, 1992). The gene contains 11 exons interrupted by 10 introns; it codes for 393 amino acids. The major goal of our study was a comparative investigation of tumour suppressor p53 in several domestic animal species. Exon 8 (amino acids 262 to 308) harbours the highly conserved domain V (amino acids 270-286). Within this hot spot region, amino acids Arg 273 a n d Arg 282 are principle targets of mutation in man (Caron de Frementel & Soussi, 1992). Therefore, we included a panel of feline tumours in our present investigation 0007-1935/95/030325-05/$08.00/0
© 1995 Bailli6reTindall
326
BRITISH VETERINARY JOURNAL, 151, 3
both for comparative reasons and in order to extend our earlier data (Mayr el al., 1993).
MATERIALS A N D METHODS
Genomic DNA was extracted fl'om tile peripheral blood of healthy animals including five cattle (Bos taurus), five sheep (Ow;es ades), five goats (Capra hircus), five horses (Equus caball'us), five pigs (Sus scrofa) and 60 cats (Fells catus) according to standard techniques. DNA was also harvested fi'om neoplastic tissue of 17 tUlnOUrbearing cats. Pathohistologically the tumours were classifed as five adenocarcinomas (mamnla D, gland), one adenonaa (lllallllllal-}, gland), one haelnangioendotheliolna (mannnary gland), one haemangioma (mammalT gland), one solid carcinoma (lnanmla D, gland), one basosquamous cell carcinoma (thigh), one basalioma (thigh), one nwxosarkoma (oral), one adenoma of sweat glands (underarms) and four fibrosarcomas (head, bell),, jaw and shoulder). All these 17 feline ttnnours are different fi'om the eight previously reported (Mayr el al., 1993). Exon 8 of the p53 gene was amplified by a polynlerase chain reaction (PCR) using a 25 bp (5'TGGTAATCTACTGGGACGGAACAGC3') and a 20bp (5'TTACCTCGCTTACTGCTCCC3') primer. Thirty-five amplification cycles were performed. Template denaturation was 2 min at 97°C, primer annealing 1 rain at 57°C and extension 1 rain at 73°C. Four percent NuSieve/agarose gel electrophoresis was used for analysis. Elution of the 141 bp product used a Quiaex Kit (Quiagen, Chatsworth, CA) procedure. Sequencing was performed on the Automatic sequencer ABI 373 A applying the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).
RESULTS
Fig. 1 provides a comparison of the partial sequences in exon 8 of several domestic animal species (cattle, sheep, goat, horse, pig, cat and dog). Moreover, data from two primates (man and monkey), two rodents (mouse and rat) and tile domestic chicken were taken fl'om the literature and included for comparative pnrposes. Full identity (100%) at the amino acid level was present from codon no.s 970-'288 in all lnamnlalian species. The interspecific differences were restricted to several silent third base substitutions only. Even the avian species (chicken) is ve W similar in this region showing two amino acid changes (codon nos. 983 and 284) only. Between codon nos. 289 and 300, the conservation is remarkable too, both at amino acid and nucleotide level. For example, one amino acid difference separates cattle fi'om sheep or goat (no. -989) and dog from cat (no. 290) only (holnology 91.8%). The similarity between the bovidae cattle, sheep and goat is especially spectacular at the nucleotide level (homology 100% between sheep and goat and 97._9% to cattle, i.e. 35 of 36 nucleotides). Nonetheless, four amino acid changes separate cattle fl-om horses (codon nos. 989, -993, -994 and 995), dogs (codon nos. 289, -990, and 295) and five humans (codon nos. 989, -994, 295, 296 and 997), thus restricting homologies to 66.6 and 58.3%, respectively. At nucleo-
EXON 8 OF p53 IN DOMESTIC KNIMALS
('atth" Slu,ep Goal
270F 7"T'l" . . . .
271E GAG . . . . . .
Horse
. . . . . .
Dog Cat
II'C
Man
.
M o n k O'
.
Mo,,,~
. . . . . .
. .
272 V GTG . . . . . .
. .
.
.
.
.
.
.
.
.
.
.
.
.
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.
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277C TGT
. . . . . .
281D GAC . . . . . . . . .
.
.
iii
286 E Cattle Sheep
. .
276 A GCC
...
Rat
(:hi...,,
.
. .
.. . . . . . . .
280R AGA . . . . . .
.
.
A
ifc,
.
.
.
. .
275 C TGT . .
T
.
.
. .
. .
.
.
. . . . . . 279G GGG . . . . .
274 V G77" . . . . . .
. .A ..C
3"
Rat
(:hi~k~,,
. .
A A
. . . . . . . . . . .
. .
273 R CGT" . . . . . .
327
.
.
.
.
. . . .A . . . .
.G
.
7"(;
...
TG . TG TG GGG
... ... ... G..
Fig. I ('Oml)arison of tlae nucleotide and predicted anfino acid sequences of codon nos. 270-300 of exon 8 of p53 from different species. Dots mean identity with the corresponding bovine nucleotide. The p53 sequences used fi'om literature are: dog (Mayr el aL, 19q4), human (Matlashewski et al., 1984), monkey (Rigaudy & Eckhart, 1989), mouse (Benchimol el al., 1984), rat (Soussi et aL, 1988b) and chicken (Soussi et al., 1988a). The chicken has been aligned and n u m b e r e d relative to mammals (Soussi et al., 1990) in order Io obtain homology'. Note also the deletion in the chicken f r o m codon nos. 294-298.
328
BRITISH VETERINARYJOURNAL, 151, 3
Fig. 2 Solid carcinoma of flae mammary gland of a cat; high mitotic activity. Bar represents 50 ~tm. tide level, the c o r r e s p o n d i n g homologies were 75% ( 2 7 / 3 6 c a t t l e - h u m a n and cattle-horse) and 80.5% ( 2 9 / 3 6 cattle-dog), respectively. All five individuals within the species cattle, sheep, goat, horse and pig had the same sequence. All o f the 50 health), and 16 o f the 17 t u m o u r - b e a r i n g cats possessed the n o r m a l feline 'wild type' s e q u e n c e o f e x o n 8. However, o n e p o i n t mutation o f this n o r m a l s e q u e n c e was d e t e c t e d in a solid c a r c i n o m a o f the m a m m a r y gland (Fig. 2) in o n e cat. T h e m u t a t i o n hit c o d o n no. 282 (CGG---)TGG, arginine--~tryptophan). Unfortunately, no n o n - t u m o u r tissue f r o m this animal was available for examination.
DISCUSSION N o n e o f the predicted a m i n o acid differences d e t e c t e d in o u r investigated domestic animals c o n c e r n e d the highly conserved d o m a i n V (Soussi et al., 1990) observed in o t h e r vertebrates. Thus, o u r present results on domestic animals supp o r t and e x t e n d earlier data (Soussi et al., 1990) suggesting the high evolutionary conservation o f p53. T h e h o m o l o g i e s in the less conserved region between c o d o n nos. 289 and 300 were considerable too, especially within families. Such comparative data are a worthwhile c o n t r i b u t i o n for a b e t t e r u n d e r s t a n d i n g o f the essential functional role suggested by its conservation. Exon 8 represents a p o r t i o n of the molecule which is crucial to its turnout suppressor function. CpG dinucleotides are prefentially involved in s p o n t a n e o u s mutations. T h e c o d o n for Arg 282 (CGG) is often hit in m u t a t i o n in m a n and can be c o n s i d e r e d as a h o t spot codon. It is remarkable that the same c o d o n was hit in
EXON 8 OF p53 IN DOMESTIC ANhMALS
329
the solid m a m m a r y c a r c i n o m a o f o u r p r e s e n t investigation a n d in a lymphosarc o m a investigated earlier (Mayr et al., 1993). Given the i m p o r t a n c e o f p53 in t u m o u r i g e n e s i s o f m a n a n d e x p e r i m e n t a l animals a n d the high f r e q u e n c y o f m u t a t i o n s f o u n d in h u m a n t u m o u r s its i n v o l v e m e n t in the t u m o u r s o f domestic animals is to be expected. Clearly, the search for nucleotide a n d a m i n o acid s e q u e n c e s o f this g e n e region a n d their alterations in domestic animals are o f g r e a t i m p o r t a n c e for veterinary a n d c o m p a r a t i v e medicine. T o f u r t h e r o u r u n d e r s t a n d i n g o f the c o n n e c t i o n between such p53 a b n o r m a l i ties a n d animal tumourigenesis, m o r e i n f o r m a t i o n is n e e d e d r e g a r d i n g the occurr e n c e a n d f r e q u e n c y o f m u t a t i o n s at known h o t spots as well as at new locations in different species a n d t u m o u r types.
REFERENCES BENCIIIMOL, S.,JENK1NS,J. R., C~XWFO~D,L. V. et al. (1984). Molecular analysis of tile gene for the p53 cellular tulnor antigen. Ill Cancer Celh. 2. Oncogenes and Virus Genes, eds G. F. Van
de Wonde, A.J. Levine, W. C. Topp & J. D. Watson, pp. 383-91. Cold Spring Harbor, New York: Cold Spring Harbor Laboratories. CARON DI- FREMENTEI,, C. ~¢: SoussI, T. (1992). Tp53 tumor suppressor gene: a model for investigating human mutagenesis. Genes, Chromosomes and Cancer4, 1-15. MATI~\SHEWSKI,G., L\~xn~, P., PI,xx, D., P.c.xcoc~,j., c~x~,~oRo, L. & BENCHIMOL,S. (1984). Isolation and characterization of a human p53 cDNA clone: expression of the human gene. EMBOJournal 3, 3257-62. Ma~a, B., HECZKO,U., SCHEI.taNI~ER,K., SCHL~:¢.~n,W. & R~.'mNGER,M. (1993). Sequence of an exon of tile feline p53 gene-nmtation in a lylnphosarcolna. British VeteTinmyJouma1149, 387-90. Mau~, B., SCHELL~NDER,K., SCHLE¢,ER,W. & R~IWNCE~,M. (1994). Sequence of an exon of the canine p53 gene-nmtation in a papilloma. British VeterinaryJournal 150, 81--4. Nl¢,Ro, J. M., B~x~R, S.J., PP,EISIN(;~,R,A. C. et al. (1989). Mutations in the p53 gene occur in diverse human tunlour types. Nature 342, 705-8. RIcauDv, P. & ECKI-L~RI",W. (1989). Nucleotide sequence of a cDNA encoding the monkey cellular phosphoprotein p53. Nucleic Acids Research 17, 8375. Sovssl, T., C,.XRONDE FRO~XlEN'rEt.,C., MECHaH, M., IVl..w,P. & KRESS, M. (1987). Cloning and characterization of a cDNA from Xenopus laevis coding for a protein homologous to human and murine p53. Oncogene 1, 71-8. Soussl, T., B~cw, A., I~Ess, M., S'rEHELIN,D. & MA~, P. (1988a). Nucleotide sequence of a cDNA encoding the chicken p53 oncoprotein. Nuc~'ic Acids Research 16, 11383. Soussl, T., Cakoy DE Fm:.,~IF~YTEL,C., Bastsc~o-r, C. & IVLw,E. (1988b). Nucleotide sequence of a cDNA encoding the rat p53 nuclear oncoprotein. Nucleic Acids Research 16, 11384. Sduss~, T., Carboy ~ F~OM~YTr~,C. & M.w, P. (1990). Structural aspects of the p53 protein ill relation to gene evolution. Oncogene 5, 945-52. (Acceptedfor publication 21 March 1994)