- Email: [email protected]
p53 Pseudogene dating: Identification of the origin of laboratory mice
Gene 270 (2001) 153±159
www.elsevier.com/locate/gene
p53 Pseudogene dating: Identi®cation of the origin of laboratory mice Hiroshi Tanooka a,*, Hiroki Sasaki a, Toshihiko Shiroishi b, Kazuo Moriwaki c a
b
Genetics Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Laboratory of Mammalian Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-0806, Japan c Graduate University for Advanced Studies, Hayama, Kanagawa-ken 240-0193, Japan Received 17 January 2001; received in revised form 22 March 2001; accepted 5 April 2001 Received by T. Sekiya
Abstract Mutations were accumulated with a wide variety in the p53 pseudogene of various wild mouse species and subspecies captured at different localities, as extensively observed in the exon 4 ± exon 5 region. The rate of mutation accumulation in the mouse p53 pseudogene was estimated to be 1.4±2.1 £ 10 28 mutations/bp/year, which is 20±30 times faster than that of the functional p53 and makes the dating possible for the time range of 10 6 years or more. From comparison of the mutation spectrum, the origin of laboratory mice was identi®ed to one of two M. m. domesticus groups. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Murine genetics; Mus musculus; Tumor suppresser gene; Mutation; Evolution
1. Introduction Previously, we showed distribution of p53 pseudogogenes (Trp53-ps) with various mutations in the exon 4 ± exon 5 region and its absence in wild mice (Tanooka et al., 1995). Such p53 pseudogene variation was found in other part of exons (Ohtsuka et al., 1996) and extensively pursued in the 128-bp segment including the exon 4±5 junction and the second p53 pseudogene in rare subspecies (Prager et al., 1998). In this report, we add further information on the p53 pseudogene mutations in wild mice and will show the origin of laboratory mice from analysis of the p53 pseudogene structure. The gene p53 (Trp53) is important for cell-cycle control and tumor suppression (Levine et al., 1991). The p53 pseudogene of mice (Zakut-Houri et al., 1983) resides on chromosome 14 (Czosnek et al., 1984), while the functional p53 gene (Zakut-Houri et al., 1983; Bienz et al., 1984) is located on chromosome 11. The origin of a processed pseudogene is a reverse-transcribed mRNA of the functional gene integrated into the chromosome apart from the original gene. Functional genes are well conserved and their evolution rate is very slow, possibly due to preferential DNA repair of actively transcribed genes (Mellon et al., 1987) and elimination of un®tted mutation by selective constraint. Abbreviation: Trp53-ps, p53 pseudogogenes * Corresponding author. Tel.: 181-3-3542-2511; fax: 181-3-3541-2685. E-mail address: [email protected] (H. Tanooka).
However, once a functional gene is converted to a pseudogene, it becomes free from constraint and starts to accumulate mutations freely, which are then spread within a group of population (Kimura, 1983; Nei, 1987). This situation provides an opportunity to examine the timing of its conversion, which might be associated with separation of mouse species and subspecies. Various biological markers have been applied for the study of diversi®cation of wild mouse species and subspecies, such as mitochondrial DNA structure (Yonekawa et al., 1981; Ferris et al., 1983; Prager et al., 1998), Y chromosome structure (Nagamine et al., 1992; Prager et al., 1998), biochemical markers (Bonhomme et al., 1984), chromosome C-band pattern (Moriwaki et al., 1986), the 28S ribosomal gene (Suzuki et al., 1986), the major histocompatibility complex (Moriwaki et al., 1990), and the Sycp 1 pseudogene structure (Sage et al., 1997). The polyphiletic origin of laboratory mice was indicated from biochemical and genetic markers (Bonhomme et al., 1987). To pursue this problem further, more useful markers are needed. We demonstrate here that the p53 pseudogene structure adds useful information. 2. Materials and methods 2.1. DNA preparation Wild mouse DNAs were obtained from the stock at the
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00480-2
X X X X
5179 b 5126 b
5061 b 0533 b
X
0305 b
Type IIIA±C M. m. castaneus (A) Kualanpul, Malaysia Bundar Lanpung, Sumatra, Indonesia (B) Hemei, Taiwan (C) Guiling, China
X X X X
0299 b 0379 0395 5001
Type IIB M. m. domesticus Pomolie, Bulgaria Montpellier, France Binasco, Italy Pomolie, Bulgaria M. m. breviostris France
X X X X X X
X
0365 b 0375 0380 0388 5092
5051 b
Type I M. spicilegus Kranevo, Bulgaria
Exon 5
X
X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X
X X
X X
X X
X X
X
X X X X
X X X X X X
X
X X
X
X
X X
X X
X
X X X X
X X X X X X
X X
X
X
X
X X X X
X X X X X X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X
X X X X
X X X X X X
X
X
X
X
X
X X
X X
X X
X
X X X X
X X X X X X
Codon a 62 64 67 74 85 90 106 (106) 110 120±122 (122) 137 139 143 157 160 165 166 167 (167) 171 174 175 178 CGA TCA CTT ACC GCC GTG TTC TTC ATGTGCACG ACG ACG CCT TGG ATG TAC CAC ATG ACG AGA CCC CAC CGC CAA TTA CCT ACT GCT CCG TTG TTT TTT ACTG ACA ACA CTC TGA ACG TTC CAT ATT CTG ATG AGG CCT TAC TGC
Exon 4
Type IIA M. m. domesticus Ontario, Canada Afula, Israel Minorca, Spain Odis, Denmark Ontario, Canada Wild mice, Mozi das Cruzes, Brazil
MG-stock number
Pseudogeme type Mice (locality)
Table 1 Mutations in exon 4±5 of the p53 pseudogene of wild and laboratory mice
154 H. Tanooka et al. / Gene 270 (2001) 153±159
b
a
5103 b
0280 b 5046 b
0080 b 0293 b 0511 b 0649 b 0221 b 0233 b 0240 b 5039 b
0309 b 0338 b 5129 b
X X X X
X X X X
X X X X
X X X X
X X X X
X X X X
Upper sequences: codons of the functional p53; Lower sequences: mutated codons. Mutated bases are underlined, except for codon 120±122. Mouse DNAs in the previous study (Tanooka et al., 1995) were re-examined.
p53 Pseudogene absent: M. m. musculus Tosheivo, Bulgaria Klank, Denmark Bialowieza, Poland M. m. molossinus Beijing, China Oomuluk, China Langzou, China Shinto, China Shizuoka, Japan Niigata, Japan Osaka, Japan Koujuri, Korea M. m. bactrianus Lahore, Pakistan Mashhad, Iran M. spretus Ibiza, Spain
Laboratory mice C57BL C3H/He BALB/c ICR X X X X
X X X X
X X X X
X X X X
X X X X
H. Tanooka et al. / Gene 270 (2001) 153±159 155
156
H. Tanooka et al. / Gene 270 (2001) 153±159
National Institute of Genetics, Japan (Table 1, af®xed by MG-stock numbers). Brazilian wild mice were caught in an orchard in the suburbs of San Paulo. Laboratory mice; C57BL/6, C3H/He, BALB/c, and ICR, were commercially available (Charles River Japan). Mice were treated according the guidelines of National Cancer Center Research Institute. DNA extraction and other molecular methods followed Sambrook et al. (1989). 2.2. Sequencing methods The exon 4±5 regions of the p53 functional gene and pseudogene were obtained by PCR ampli®cation with the b1 primer at the 5 0 end of exon 4 (5 0 -CCATCACCTCACTGCATGGA-3 0 ) and the d4 primer at the 5 0 end of exon 6 (5 0 -TAAGATGCTGGGGAGGAGCC-3 0 ), and separated by 1% gel-electrophoresis as a 1329-bp band of the functional p53 with intron 4 (780-bp) and intron 5 (78-bp) and a 466±471 bp band of the p53 pseudogene without introns (Tanooka et al., 1995). Other primers (Ootsuyama et al., 1994) were used to con®rm the absence of the p53 pseudogene. DNA was recovered from gel by a glass milkbinding system (ISC BioExpress), and the nucleotide sequence of the exon 4±5 region (423-bp from codon 42 to codon 184) was analyzed from the 5 0 -3 0 and 3 0 ±5 0 directions using the b1 and d4 primers with an analyzer system (ABI-310, PE Applied Biosystems). Nucleotide sequences of p53 pseudogenes were compared with that of the functional p53 by a Genetyx-Mac 8.0 program. 3. Results and discussion 3.1. Distribution of p53 pseudogene in wild mice Table 1 shows a list of mutations found in the exon 4 ± exon 5 region of the p53 pseudogene of wild mice captured at different localities together with those found in laboratory mice. It includes 24 different mutations in the 423-bp region, including 5 mutations reported in a previous study (Tanooka et al., 1995) and 19 newly found mutations. The mice were divided in two groups, one without the p53 pseudogene and the other with the p53 pseudogene as previously reported. M. spicilegus is distinct from M. musclus in that it carries four unique mutations and in the absence of mutation at codon 120±122 which is characteristic of the p53 pseudogene of M. musculus. It is not yet con®rmed whether the p53 pseudogene of spicilegus, like that of musculus, resides on chromosome 14. However, common mutations were found at codons 62, 74, 85, 90, and 165, indicating that the common ancestor of M. spicilegus and M. musculus already possessed the p53 pseudogene with these ®ve mutations before the start of their diversi®cation. However, this result is dif®cult to reconcile with already accepted evolutionary data. A plausible explanation is that this p53 pseudogene is of domesticus origin. The subspecies of M. musculus tested had four common
mutations at codons 120±122, 143, 166, and 178. The mutation at codon 120±122 is unique in having multiple changes, 9 2 6 1 1, as previously noted (Tanooka et al., 1995). M. m. domesticus was divided into two groups based on the additional mutation at codon 64. M. m. castaneus showed a variety in the p53 pseudogene structure, which is divided into subtypes Type III-A, B, and C based on additional mutations at codons 106, 110, 137, 157, 167, 174, and 175. Fig. 1 shows the geographical distribution of wild mice with various p53 pseudogene types. Wild mice without a p53 pseudogene are widely distributed from Asia to Europe along the Silk Road. Bulgaria is, interestingly, a meeting point of wild mice with different types of p53 pseudogene. The mouse of Malaysia was distinguished from the mouse of Sumatra, probably because the two areas are separated by the narrow Malacca channel. A similar situation can also be seen between the mouse of southern China and the mouse of Taiwan. 3.2. Origin of laboratory mice determined from p53 pseudogene type The laboratory mouse strains tested were C57BL, C3H/ He, BALB/c, and ICR. All of them showed an identical sequence of the p53 pseudogene at exons 4±5, Type IIA, which coincides with the sequence of one of two M. m. domesticus groups and is distinct from that of other European domesticus with a type IIB pseudogene at codon 64 (Table 1). M. m. domesticus with the Type IIA p53 pseudogene from Denmark, Minorca, Israel, Canada, and Brazil are thought to be related to each other and to be possibly of north European origin, migrating later with humans to various localities. Analysis of mitochondrial DNA indicated the origin of laboratory mice is domesticus (Yonekawa et al., 1982; Ferris et al., 1983), while the polyphiletic origin of laboratory mice was shown with biochemical and genetic markers (Bonhomme et al., 1987). More recently, the presence of the pseudogene, Sycp 1 ps2, in laboratory mice was shown by Sage et al. (1997), while it is absent in wild domesticus, indicating new acquisition of Sycp 1 ps2 in laboratory mice. In our case, the Type IIA p53 pseudogene was already present in one of the two domesticus subspecies and was a useful marker for determining the origin of the laboratory mice. 3.3. Examination of mutation hot-spot at the exon 4±5 junction There was a unique change at codon 120±122 neighboring the exon 4 ± exon 5 junction site in the murine p53 pseudogene. Prager et al. (1998) pointed out that the 128bp sequence at this region is particularly variable. This hotspot characteristic has been recognized for mutations of the functional p53 in radiation-induced mouse tumors (Ootsuyama et al., 1994). However, the present study did not show a particularly high mutation rate at this region: 5
Fig. 1. Geographical distribution of wild mice with various p53 pseudogene mutations. Mice without p53 pseudogene (X), with p53 pseudogene Type I (W), Type IIA (O), Type IIB (K), Type IIIA (B), Type IIIB (A), and Type IIIC (S).
H. Tanooka et al. / Gene 270 (2001) 153±159 157
158
H. Tanooka et al. / Gene 270 (2001) 153±159
variable sites in the 128-bp region (0.039/bp) versus 15 variable sites in the 295-bp region outside the 128-bp region (0.051/bp). For the 128-bp segment, Prager et al. (1998) found 12 variable sites in mice captured at 68 different localities, while we found 5 at 16 localities (0.18 versus 0.31). In terms of mutation type, this ratio was 0.26 (18 types/ 68 localities) versus 0.38 (6 types/ 16 localities). Therefore, the 128-bp region is not particularly hyper-mutable, although it is a useful marker. 3.4. Mutation accumulation rate The p53 pseudogene accumulates mutations more rapidly than the p53 functional gene does. Its speed was estimated to be 1.39±2.08 £ 10 28 mutations/bp/year from the present results, assuming that diversi®cation of mouse subspecies occurred 10 6 years ago (Moriwaki et al., 1990). On the other hand, there was no change in the functional p53 structure in different species and subspecies tested in this study (data not shown). However, its evolutionary speed was estimated to be 0.7 £ 10 29 mutations/bp/year from amino acid data on mice and humans (Soussi et al., 1990), assuming that mice were separated from humans 8 £ 10 7 years ago. Therefore, the p53 pseudogene change is approximately 20±30 times faster than that of the functional p53. Furthermore, the effect of fast-breeding of laboratory mice was not found in the p53 pseudogene structure of laboratory mice compared with that of wild domesticus. Drake et al. (1998) reported the spontaneous mutation rate in mice as 0.9 mutations/effective genome/sexual generation, which represents 2.2 £ 10 28 mutations /bp/ year, assuming 8.0 £ 10 7 bp for the mouse effective genome size and 0.5 year for sexual generation. This is a good agreement with our estimation. Furthermore, the time of formation of the p53 pseudogene in the ancestral mice is estimated to be 0.53 £ 10 6 years before the start of diversi®cation of mouse subspecies. 3.5. Conclusions The present study showed the usefulness of the p53 pseudogene variation in the study of mouse evolution. The mutations accumulate 20±30 times faster than those of the functional p53 and can be applicable to dating of evolutionary events of mice. Acknowledgements We thank Sohei Kondo, Kinki University, and Takashi Gojobori, National Institute of Genetics, for critical reading of the manuscript, Masaaki Terada, President of the National Cancer Center and Teruhiko Yoshida, Chief of Genetics Division, for supporting this work. This work was supported by a grant-in-aid from the Ministry of Education and Culture and the Ministry of Health and Welfare, Japan, and by the Central Research Institute of Electric Power Industry.
References Bienz, B., Zakut-Houri, R., Givol, D., Oren, M., 1984. Analysis of the gene coding for the murine cellular tumor antigen p53. EMBO J. 3, 2179± 2183. Bonhomme, F., Catalan, J., Britton-Davidian, J., Chapman, V.M., Moriwaki, K., Nevo, E., Thaler, L., 1984. Biochemical diversity and evolution in the genus Mus. Biochem. Genet. 22, 275±303. Bonhomme, F., Guenet, J.-L., Dod, B., Moriwaki, K., Bul®eld, G., 1987. The polyphyletic origin of laboratory inbred mice and their rate of evolution. Biol. J. Linnean Soc. 30, 51±58. Czosnek, H.H., Bienz, B., Givol, D., Zakut-Houri, R., Pravtcheva, D.D., Ruddle, F.H., Oren, M., 1984. The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes. Mol. Cell. Biol. 4, 1638±1640. Drake, J.W., Charlesworth, B., Charlesworth, D., Crow, J.F., 1998. Rates of spontaneous mutation. Genetics 148, 1667±1686. Ferris, S.D., Sager, R.D., Prager, E.M., Ritte, U., Wilson, A.C., 1983. Mitochondrial DNA evolution in mice. Genetics 105, 681±721. Kimura, M., 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. Levine, A.J., Momard, J., Finlay, C.A., 1991. The p53 tumor suppresser gene. Nature 351, 453±456. Mellon, I., Spivak, G., Hanawalt, P.C., 1987. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51, 241±249. Moriwaki, K., Miyashita, N., Suzuki, H., Kurihara, Y., Yonekawa, H., 1986. Genetic features of major geographical isolates of Mus musculus. In: Potter, M., Nadeau, H., Cancro, M.P. (Eds.), Current Topics in Microbiology and Immunology, Vol. 127. Springer-Verlag, BerlinHeidelberg, pp. 55±61. Moriwaki, K., Sagai, T., Shiroishi, T., Bonhomme, F., Wang, C.G., He, X.Q., Jin, M.L., Wu, Z.G., 1990. Mouse subspecies differentiation and H-2 polymorphism. Biol. J. Linnean Soc. 41, 125±139. Nagamine, C.M., Nishioka, Y., Moriwaki, K., Boursot, F., Bonhomme, F., Lau, Y.F., 1992. The musculus-type Y chromosome of the laboratory mouse is of Asian origin. Mamm. Genome 3, 84±91. Nei, M., 1987. Molecular Evolution Genetics. Columbia University Press, New York. Ohtsuka, H., Oyanagi, M., Mafune, Y., Miyashita, N., Shiroishi, T., Moriwaki, K., Kominami, R., Saitou, N., 1996. The presence/absence polymorphism and evolution of the p53 pseudogene in the genus Mus. Mol. Phylo. Evol 5, 548±556. Ootsuyama, A., Makino, H., Nagao, M., Ochiai, A., Yamagishi, Y., Tanooka, H., 1994. Frequent p53 mutation in mouse tumors induced by repeated beta radiation. Mol. Carcinog. 11, 236±242. Prager, E.M., Orrego, C., Sage, R.D., 1998. Genetic variation and phylogeography of central Asian and other house mice, including a major new mitochondrial lineage in Yemen. Genetics 150, 835±861. Sage, J., Yuan, L., Martin, L., Mattei, M.-G., Guenet, J.-L., Liu, J.-G., Hoog, C., Rassoulzadegan, M., Cuzin, F., 1997. The Sycp 1 loci of the mouse genome: successive retropositions of a meiotic gene during the recent evolution of the genus. Genomics 44, 118±126. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Soussi, T., Caron de Fromentel, C., May, P., 1990. Structural aspects of the p53 protein in relation to gene evolution. Oncogene 5, 945±952. Suzuki, H., Miyashita, N., Moriwaki, K., Kominami, K., Muramatsu, M., Kanehisa, T., Bonhomme, F., Petras, M.L., Yu, Z.C., Lu, D.Y., 1986. Evolutionary implications of the non-transcribed spacer region of ribosomal DNA repeating units in various subspecies of Mus musculus. Mol. Biol. Evol. 3, 126±137. Tanooka, H., Ootsuyama, A., Shiroishi, T., Moriwaki, K., 1995. Distribution of the p53 pseudogene among mouse species and subspecies. Mammal. Genome 6, 360±362.
H. Tanooka et al. / Gene 270 (2001) 153±159 Yonekawa, H., Gotoh, O., Tagashira, Y., Matsushima, Y., Shi, L.I., Cho, W.S., Miyashita, N., Moriwaki, K., 1981. Evolutionary relationships among ®ve subspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA. Genetics 98, 801± 816. Yonekawa, H., Moriwaki, K., Gotoh, O., Miyashita, N., Migita, S.,
159
Bonhomme, F., Hiorth, J.P., Petras, M.L., Tagashira, Y., 1982. Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA. Differentiation 22, 222±226. Zakut-Houri, R., Oren, M., Bienz, B., Lavie, V., Hazum, S., Givol, D., 1983. A single gene and a pseudogene for the cellular tumour antigen p53. Nature 306, 594±597.
www.elsevier.com/locate/gene
p53 Pseudogene dating: Identi®cation of the origin of laboratory mice Hiroshi Tanooka a,*, Hiroki Sasaki a, Toshihiko Shiroishi b, Kazuo Moriwaki c a
b
Genetics Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Laboratory of Mammalian Genetics, National Institute of Genetics, Mishima, Shizuoka-ken 411-0806, Japan c Graduate University for Advanced Studies, Hayama, Kanagawa-ken 240-0193, Japan Received 17 January 2001; received in revised form 22 March 2001; accepted 5 April 2001 Received by T. Sekiya
Abstract Mutations were accumulated with a wide variety in the p53 pseudogene of various wild mouse species and subspecies captured at different localities, as extensively observed in the exon 4 ± exon 5 region. The rate of mutation accumulation in the mouse p53 pseudogene was estimated to be 1.4±2.1 £ 10 28 mutations/bp/year, which is 20±30 times faster than that of the functional p53 and makes the dating possible for the time range of 10 6 years or more. From comparison of the mutation spectrum, the origin of laboratory mice was identi®ed to one of two M. m. domesticus groups. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Murine genetics; Mus musculus; Tumor suppresser gene; Mutation; Evolution
1. Introduction Previously, we showed distribution of p53 pseudogogenes (Trp53-ps) with various mutations in the exon 4 ± exon 5 region and its absence in wild mice (Tanooka et al., 1995). Such p53 pseudogene variation was found in other part of exons (Ohtsuka et al., 1996) and extensively pursued in the 128-bp segment including the exon 4±5 junction and the second p53 pseudogene in rare subspecies (Prager et al., 1998). In this report, we add further information on the p53 pseudogene mutations in wild mice and will show the origin of laboratory mice from analysis of the p53 pseudogene structure. The gene p53 (Trp53) is important for cell-cycle control and tumor suppression (Levine et al., 1991). The p53 pseudogene of mice (Zakut-Houri et al., 1983) resides on chromosome 14 (Czosnek et al., 1984), while the functional p53 gene (Zakut-Houri et al., 1983; Bienz et al., 1984) is located on chromosome 11. The origin of a processed pseudogene is a reverse-transcribed mRNA of the functional gene integrated into the chromosome apart from the original gene. Functional genes are well conserved and their evolution rate is very slow, possibly due to preferential DNA repair of actively transcribed genes (Mellon et al., 1987) and elimination of un®tted mutation by selective constraint. Abbreviation: Trp53-ps, p53 pseudogogenes * Corresponding author. Tel.: 181-3-3542-2511; fax: 181-3-3541-2685. E-mail address: [email protected] (H. Tanooka).
However, once a functional gene is converted to a pseudogene, it becomes free from constraint and starts to accumulate mutations freely, which are then spread within a group of population (Kimura, 1983; Nei, 1987). This situation provides an opportunity to examine the timing of its conversion, which might be associated with separation of mouse species and subspecies. Various biological markers have been applied for the study of diversi®cation of wild mouse species and subspecies, such as mitochondrial DNA structure (Yonekawa et al., 1981; Ferris et al., 1983; Prager et al., 1998), Y chromosome structure (Nagamine et al., 1992; Prager et al., 1998), biochemical markers (Bonhomme et al., 1984), chromosome C-band pattern (Moriwaki et al., 1986), the 28S ribosomal gene (Suzuki et al., 1986), the major histocompatibility complex (Moriwaki et al., 1990), and the Sycp 1 pseudogene structure (Sage et al., 1997). The polyphiletic origin of laboratory mice was indicated from biochemical and genetic markers (Bonhomme et al., 1987). To pursue this problem further, more useful markers are needed. We demonstrate here that the p53 pseudogene structure adds useful information. 2. Materials and methods 2.1. DNA preparation Wild mouse DNAs were obtained from the stock at the
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00480-2
X X X X
5179 b 5126 b
5061 b 0533 b
X
0305 b
Type IIIA±C M. m. castaneus (A) Kualanpul, Malaysia Bundar Lanpung, Sumatra, Indonesia (B) Hemei, Taiwan (C) Guiling, China
X X X X
0299 b 0379 0395 5001
Type IIB M. m. domesticus Pomolie, Bulgaria Montpellier, France Binasco, Italy Pomolie, Bulgaria M. m. breviostris France
X X X X X X
X
0365 b 0375 0380 0388 5092
5051 b
Type I M. spicilegus Kranevo, Bulgaria
Exon 5
X
X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X
X X
X X
X X
X X
X
X X X X
X X X X X X
X
X X
X
X
X X
X X
X
X X X X
X X X X X X
X X
X
X
X
X X X X
X X X X X X
X X
X X
X
X X X X
X X X X X X
X
X X
X X
X
X X X X
X X X X X X
X
X
X X X X
X X X X X X
X
X
X
X
X
X X
X X
X X
X
X X X X
X X X X X X
Codon a 62 64 67 74 85 90 106 (106) 110 120±122 (122) 137 139 143 157 160 165 166 167 (167) 171 174 175 178 CGA TCA CTT ACC GCC GTG TTC TTC ATGTGCACG ACG ACG CCT TGG ATG TAC CAC ATG ACG AGA CCC CAC CGC CAA TTA CCT ACT GCT CCG TTG TTT TTT ACTG ACA ACA CTC TGA ACG TTC CAT ATT CTG ATG AGG CCT TAC TGC
Exon 4
Type IIA M. m. domesticus Ontario, Canada Afula, Israel Minorca, Spain Odis, Denmark Ontario, Canada Wild mice, Mozi das Cruzes, Brazil
MG-stock number
Pseudogeme type Mice (locality)
Table 1 Mutations in exon 4±5 of the p53 pseudogene of wild and laboratory mice
154 H. Tanooka et al. / Gene 270 (2001) 153±159
b
a
5103 b
0280 b 5046 b
0080 b 0293 b 0511 b 0649 b 0221 b 0233 b 0240 b 5039 b
0309 b 0338 b 5129 b
X X X X
X X X X
X X X X
X X X X
X X X X
X X X X
Upper sequences: codons of the functional p53; Lower sequences: mutated codons. Mutated bases are underlined, except for codon 120±122. Mouse DNAs in the previous study (Tanooka et al., 1995) were re-examined.
p53 Pseudogene absent: M. m. musculus Tosheivo, Bulgaria Klank, Denmark Bialowieza, Poland M. m. molossinus Beijing, China Oomuluk, China Langzou, China Shinto, China Shizuoka, Japan Niigata, Japan Osaka, Japan Koujuri, Korea M. m. bactrianus Lahore, Pakistan Mashhad, Iran M. spretus Ibiza, Spain
Laboratory mice C57BL C3H/He BALB/c ICR X X X X
X X X X
X X X X
X X X X
X X X X
H. Tanooka et al. / Gene 270 (2001) 153±159 155
156
H. Tanooka et al. / Gene 270 (2001) 153±159
National Institute of Genetics, Japan (Table 1, af®xed by MG-stock numbers). Brazilian wild mice were caught in an orchard in the suburbs of San Paulo. Laboratory mice; C57BL/6, C3H/He, BALB/c, and ICR, were commercially available (Charles River Japan). Mice were treated according the guidelines of National Cancer Center Research Institute. DNA extraction and other molecular methods followed Sambrook et al. (1989). 2.2. Sequencing methods The exon 4±5 regions of the p53 functional gene and pseudogene were obtained by PCR ampli®cation with the b1 primer at the 5 0 end of exon 4 (5 0 -CCATCACCTCACTGCATGGA-3 0 ) and the d4 primer at the 5 0 end of exon 6 (5 0 -TAAGATGCTGGGGAGGAGCC-3 0 ), and separated by 1% gel-electrophoresis as a 1329-bp band of the functional p53 with intron 4 (780-bp) and intron 5 (78-bp) and a 466±471 bp band of the p53 pseudogene without introns (Tanooka et al., 1995). Other primers (Ootsuyama et al., 1994) were used to con®rm the absence of the p53 pseudogene. DNA was recovered from gel by a glass milkbinding system (ISC BioExpress), and the nucleotide sequence of the exon 4±5 region (423-bp from codon 42 to codon 184) was analyzed from the 5 0 -3 0 and 3 0 ±5 0 directions using the b1 and d4 primers with an analyzer system (ABI-310, PE Applied Biosystems). Nucleotide sequences of p53 pseudogenes were compared with that of the functional p53 by a Genetyx-Mac 8.0 program. 3. Results and discussion 3.1. Distribution of p53 pseudogene in wild mice Table 1 shows a list of mutations found in the exon 4 ± exon 5 region of the p53 pseudogene of wild mice captured at different localities together with those found in laboratory mice. It includes 24 different mutations in the 423-bp region, including 5 mutations reported in a previous study (Tanooka et al., 1995) and 19 newly found mutations. The mice were divided in two groups, one without the p53 pseudogene and the other with the p53 pseudogene as previously reported. M. spicilegus is distinct from M. musclus in that it carries four unique mutations and in the absence of mutation at codon 120±122 which is characteristic of the p53 pseudogene of M. musculus. It is not yet con®rmed whether the p53 pseudogene of spicilegus, like that of musculus, resides on chromosome 14. However, common mutations were found at codons 62, 74, 85, 90, and 165, indicating that the common ancestor of M. spicilegus and M. musculus already possessed the p53 pseudogene with these ®ve mutations before the start of their diversi®cation. However, this result is dif®cult to reconcile with already accepted evolutionary data. A plausible explanation is that this p53 pseudogene is of domesticus origin. The subspecies of M. musculus tested had four common
mutations at codons 120±122, 143, 166, and 178. The mutation at codon 120±122 is unique in having multiple changes, 9 2 6 1 1, as previously noted (Tanooka et al., 1995). M. m. domesticus was divided into two groups based on the additional mutation at codon 64. M. m. castaneus showed a variety in the p53 pseudogene structure, which is divided into subtypes Type III-A, B, and C based on additional mutations at codons 106, 110, 137, 157, 167, 174, and 175. Fig. 1 shows the geographical distribution of wild mice with various p53 pseudogene types. Wild mice without a p53 pseudogene are widely distributed from Asia to Europe along the Silk Road. Bulgaria is, interestingly, a meeting point of wild mice with different types of p53 pseudogene. The mouse of Malaysia was distinguished from the mouse of Sumatra, probably because the two areas are separated by the narrow Malacca channel. A similar situation can also be seen between the mouse of southern China and the mouse of Taiwan. 3.2. Origin of laboratory mice determined from p53 pseudogene type The laboratory mouse strains tested were C57BL, C3H/ He, BALB/c, and ICR. All of them showed an identical sequence of the p53 pseudogene at exons 4±5, Type IIA, which coincides with the sequence of one of two M. m. domesticus groups and is distinct from that of other European domesticus with a type IIB pseudogene at codon 64 (Table 1). M. m. domesticus with the Type IIA p53 pseudogene from Denmark, Minorca, Israel, Canada, and Brazil are thought to be related to each other and to be possibly of north European origin, migrating later with humans to various localities. Analysis of mitochondrial DNA indicated the origin of laboratory mice is domesticus (Yonekawa et al., 1982; Ferris et al., 1983), while the polyphiletic origin of laboratory mice was shown with biochemical and genetic markers (Bonhomme et al., 1987). More recently, the presence of the pseudogene, Sycp 1 ps2, in laboratory mice was shown by Sage et al. (1997), while it is absent in wild domesticus, indicating new acquisition of Sycp 1 ps2 in laboratory mice. In our case, the Type IIA p53 pseudogene was already present in one of the two domesticus subspecies and was a useful marker for determining the origin of the laboratory mice. 3.3. Examination of mutation hot-spot at the exon 4±5 junction There was a unique change at codon 120±122 neighboring the exon 4 ± exon 5 junction site in the murine p53 pseudogene. Prager et al. (1998) pointed out that the 128bp sequence at this region is particularly variable. This hotspot characteristic has been recognized for mutations of the functional p53 in radiation-induced mouse tumors (Ootsuyama et al., 1994). However, the present study did not show a particularly high mutation rate at this region: 5
Fig. 1. Geographical distribution of wild mice with various p53 pseudogene mutations. Mice without p53 pseudogene (X), with p53 pseudogene Type I (W), Type IIA (O), Type IIB (K), Type IIIA (B), Type IIIB (A), and Type IIIC (S).
H. Tanooka et al. / Gene 270 (2001) 153±159 157
158
H. Tanooka et al. / Gene 270 (2001) 153±159
variable sites in the 128-bp region (0.039/bp) versus 15 variable sites in the 295-bp region outside the 128-bp region (0.051/bp). For the 128-bp segment, Prager et al. (1998) found 12 variable sites in mice captured at 68 different localities, while we found 5 at 16 localities (0.18 versus 0.31). In terms of mutation type, this ratio was 0.26 (18 types/ 68 localities) versus 0.38 (6 types/ 16 localities). Therefore, the 128-bp region is not particularly hyper-mutable, although it is a useful marker. 3.4. Mutation accumulation rate The p53 pseudogene accumulates mutations more rapidly than the p53 functional gene does. Its speed was estimated to be 1.39±2.08 £ 10 28 mutations/bp/year from the present results, assuming that diversi®cation of mouse subspecies occurred 10 6 years ago (Moriwaki et al., 1990). On the other hand, there was no change in the functional p53 structure in different species and subspecies tested in this study (data not shown). However, its evolutionary speed was estimated to be 0.7 £ 10 29 mutations/bp/year from amino acid data on mice and humans (Soussi et al., 1990), assuming that mice were separated from humans 8 £ 10 7 years ago. Therefore, the p53 pseudogene change is approximately 20±30 times faster than that of the functional p53. Furthermore, the effect of fast-breeding of laboratory mice was not found in the p53 pseudogene structure of laboratory mice compared with that of wild domesticus. Drake et al. (1998) reported the spontaneous mutation rate in mice as 0.9 mutations/effective genome/sexual generation, which represents 2.2 £ 10 28 mutations /bp/ year, assuming 8.0 £ 10 7 bp for the mouse effective genome size and 0.5 year for sexual generation. This is a good agreement with our estimation. Furthermore, the time of formation of the p53 pseudogene in the ancestral mice is estimated to be 0.53 £ 10 6 years before the start of diversi®cation of mouse subspecies. 3.5. Conclusions The present study showed the usefulness of the p53 pseudogene variation in the study of mouse evolution. The mutations accumulate 20±30 times faster than those of the functional p53 and can be applicable to dating of evolutionary events of mice. Acknowledgements We thank Sohei Kondo, Kinki University, and Takashi Gojobori, National Institute of Genetics, for critical reading of the manuscript, Masaaki Terada, President of the National Cancer Center and Teruhiko Yoshida, Chief of Genetics Division, for supporting this work. This work was supported by a grant-in-aid from the Ministry of Education and Culture and the Ministry of Health and Welfare, Japan, and by the Central Research Institute of Electric Power Industry.
References Bienz, B., Zakut-Houri, R., Givol, D., Oren, M., 1984. Analysis of the gene coding for the murine cellular tumor antigen p53. EMBO J. 3, 2179± 2183. Bonhomme, F., Catalan, J., Britton-Davidian, J., Chapman, V.M., Moriwaki, K., Nevo, E., Thaler, L., 1984. Biochemical diversity and evolution in the genus Mus. Biochem. Genet. 22, 275±303. Bonhomme, F., Guenet, J.-L., Dod, B., Moriwaki, K., Bul®eld, G., 1987. The polyphyletic origin of laboratory inbred mice and their rate of evolution. Biol. J. Linnean Soc. 30, 51±58. Czosnek, H.H., Bienz, B., Givol, D., Zakut-Houri, R., Pravtcheva, D.D., Ruddle, F.H., Oren, M., 1984. The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes. Mol. Cell. Biol. 4, 1638±1640. Drake, J.W., Charlesworth, B., Charlesworth, D., Crow, J.F., 1998. Rates of spontaneous mutation. Genetics 148, 1667±1686. Ferris, S.D., Sager, R.D., Prager, E.M., Ritte, U., Wilson, A.C., 1983. Mitochondrial DNA evolution in mice. Genetics 105, 681±721. Kimura, M., 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. Levine, A.J., Momard, J., Finlay, C.A., 1991. The p53 tumor suppresser gene. Nature 351, 453±456. Mellon, I., Spivak, G., Hanawalt, P.C., 1987. Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51, 241±249. Moriwaki, K., Miyashita, N., Suzuki, H., Kurihara, Y., Yonekawa, H., 1986. Genetic features of major geographical isolates of Mus musculus. In: Potter, M., Nadeau, H., Cancro, M.P. (Eds.), Current Topics in Microbiology and Immunology, Vol. 127. Springer-Verlag, BerlinHeidelberg, pp. 55±61. Moriwaki, K., Sagai, T., Shiroishi, T., Bonhomme, F., Wang, C.G., He, X.Q., Jin, M.L., Wu, Z.G., 1990. Mouse subspecies differentiation and H-2 polymorphism. Biol. J. Linnean Soc. 41, 125±139. Nagamine, C.M., Nishioka, Y., Moriwaki, K., Boursot, F., Bonhomme, F., Lau, Y.F., 1992. The musculus-type Y chromosome of the laboratory mouse is of Asian origin. Mamm. Genome 3, 84±91. Nei, M., 1987. Molecular Evolution Genetics. Columbia University Press, New York. Ohtsuka, H., Oyanagi, M., Mafune, Y., Miyashita, N., Shiroishi, T., Moriwaki, K., Kominami, R., Saitou, N., 1996. The presence/absence polymorphism and evolution of the p53 pseudogene in the genus Mus. Mol. Phylo. Evol 5, 548±556. Ootsuyama, A., Makino, H., Nagao, M., Ochiai, A., Yamagishi, Y., Tanooka, H., 1994. Frequent p53 mutation in mouse tumors induced by repeated beta radiation. Mol. Carcinog. 11, 236±242. Prager, E.M., Orrego, C., Sage, R.D., 1998. Genetic variation and phylogeography of central Asian and other house mice, including a major new mitochondrial lineage in Yemen. Genetics 150, 835±861. Sage, J., Yuan, L., Martin, L., Mattei, M.-G., Guenet, J.-L., Liu, J.-G., Hoog, C., Rassoulzadegan, M., Cuzin, F., 1997. The Sycp 1 loci of the mouse genome: successive retropositions of a meiotic gene during the recent evolution of the genus. Genomics 44, 118±126. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Soussi, T., Caron de Fromentel, C., May, P., 1990. Structural aspects of the p53 protein in relation to gene evolution. Oncogene 5, 945±952. Suzuki, H., Miyashita, N., Moriwaki, K., Kominami, K., Muramatsu, M., Kanehisa, T., Bonhomme, F., Petras, M.L., Yu, Z.C., Lu, D.Y., 1986. Evolutionary implications of the non-transcribed spacer region of ribosomal DNA repeating units in various subspecies of Mus musculus. Mol. Biol. Evol. 3, 126±137. Tanooka, H., Ootsuyama, A., Shiroishi, T., Moriwaki, K., 1995. Distribution of the p53 pseudogene among mouse species and subspecies. Mammal. Genome 6, 360±362.
H. Tanooka et al. / Gene 270 (2001) 153±159 Yonekawa, H., Gotoh, O., Tagashira, Y., Matsushima, Y., Shi, L.I., Cho, W.S., Miyashita, N., Moriwaki, K., 1981. Evolutionary relationships among ®ve subspecies of Mus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA. Genetics 98, 801± 816. Yonekawa, H., Moriwaki, K., Gotoh, O., Miyashita, N., Migita, S.,
159
Bonhomme, F., Hiorth, J.P., Petras, M.L., Tagashira, Y., 1982. Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA. Differentiation 22, 222±226. Zakut-Houri, R., Oren, M., Bienz, B., Lavie, V., Hazum, S., Givol, D., 1983. A single gene and a pseudogene for the cellular tumour antigen p53. Nature 306, 594±597.