The Journal of Heredity 2001:92(4)
© 2001 The American Genetic Association 92:352-355
Brief Communication |
Unique Mutations in Mitochondrial DNA of Senescence-Accelerated Mouse (SAM) Strains
From the Department of Aging Angiology, Research Center on Aging and Adaptation, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan (Mizutani, Chiba, Higuchi, and Mori), and Department of Gene Therapy, Gifu International Institute of Biotechnology, Yagi Memorial Park, Nataki, Gifu, Japan (Tanaka).
Address correspondence to Masayuki Mori, Ph.D. at the address above or e-mail: masamori{at}sch.md.shinshu-u.ac.jp
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Abstract |
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Mitochondrial DNA (mtDNA) is exclusively inherited maternally and hence could offer a good method for tracing the lineage of mouse strains. We examined the mtDNA sequence of senescence-accelerated mouse (SAM) strains as well as other laboratory strains of inbred mice to deduce the ancestral strain of SAM. Four unique mutations were identified at bases 2256, 10,847, 11,181, and 13,053 in SAM strains. The mutations were not found in other mouse strains including AKR/J, one of the parental strains of SAM. Comparison of the mtDNA sequences also led to the consensus mtDNA sequence of laboratory strains of inbred mice. The seven laboratory strains of common inbred mice showed polymorphisms at base 9348, thymine repeat from base 9818, and adenine repeat from base 9821, and could be classified into five types by combination of the differences. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, the polymorphisms would provide a promising clue to ascertain the ancestral strain(s) of SAM. The polymorphism in mtDNA could be used to ascertain the genealogy of other mouse strains as well.
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Introduction |
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More than 450 inbred strains of mice have been described. The origin and relationships of many of these strains have also been described (Beck et al. 2000). However, some of the other strains have uncertain lineage. A series of senescence-accelerated mouse (SAM) strains is a case in point. The senescence-accelerated mouse prone (SAMP) strains have been established by selective inbreeding for the accelerated senescence from a progeny of inadvertent crossing between AKR/J and unspecified strain(s) (B strain) (Takeda et al. 1997). Several lines were separated from the progeny and established as inbred senescence-accelerated mouse resistant (SAMR) strains with a normal senescence profile. Currently nine SAMP and three SAMR strains exist. SAMP strains are bone and develop normally. After sexual maturation, however, age-specific death rate of SAMP mice increases steeply as they age (accelerated senescence). Consequently SAMP strains show markedly shorter average life spans (9.7 months) compared to SAMR strains (13.3 months). Another important feature of SAM is that they exhibit various age-associated diseases, such as senile amyloidosis, senile osteoporosis, and deficit in learning and memory in a strain-specific manner. SAM mice have been used worldwide in gerontologic research. Although the mechanism of accelerated senescence has not yet been elucidated, the fact that it could be fixed by breeding implies that the accelerated senescence is under genetic control. The genes responsible for the accelerated senescence might have descended from the B strain, as AKR/J mice do not exhibit the accelerated senescent phenotype. If this were the case, then specification of the mouse strain crossed to AKR/J would be of value to reveal the genes responsible for the accelerated senescence in the SAMP strains. Xia et al. (1999) determined the genetic profile of SAMP and SAMR strains with microsatellite marker loci on chromosomes. They found that each chromosome is a complex mosaic of AKR/J-derived and unspecified B strain-derived segments, and that all SAM strains are genetically diverged from each other. They also observed three alleles at several loci, suggesting that the unspecified strain was plural or genetically heterogeneous. However, the study did not lead to identification of the parental strain(s) of SAM.
Mitochondrial DNA (mtDNA) is exclusively inherited maternally, does not recombine, and remains unchanged through generations if it does not undergo germline mutations. We postulated that the B strain had distinct mtDNA from the AKR/J strain and the mtDNA has been maintained intact in the current SAM strains. In the present study we investigated the mtDNA sequences of SAM strains and compared them with those of AKR/J, as well as other mouse strains.
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Materials and Methods |
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Mouse Strains
DNA samples of all SAM mice used in this study were obtained from a mouse colony at the Institute for Frontier Medical Sciences, Kyoto University. AKR/J, C3H/He, A/J, BALB/c, DBA/2, C57BL/6J mice were purchased from Japan SLC. An SJL/J mouse was obtained from the National Institute of Genetics, Japan. One mouse was examined for each strain. Ten Slc:ddY mice, all born from a different mother, were purchased from Japan SLC. DNA was isolated from the liver of mice using the G NOME DNA kit (BIO 101). DNA samples of six CD-1/Crj and six ICR/Jcl mice were a gift from Dr. M. Ebukuro (Central Institute for Experimental Animals, Japan).
PCR Amplification and DNA Sequencing
Oligonucleotide primers for the analysis of mouse mtDNA were designed based on the reported mouse mtDNA (GenBank accession no. V00711). The entire mtDNA was obtained as a contig of six overlapping fragments (A–F) by PCR amplification from total liver DNA using the following primers:
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PCR amplification was performed using an AccuTaq LA DNA polymerase (Sigma) following the manufacturer's instructions. The cycling parameters for PCR were initial denaturation for 1 min at 94°C; followed by 35 cycles of 30 sec at 94°C, 30 sec at 60°C (for A and E) or 55°C (for B, C, D, and F), and 2.5 min at 68°C; final elongation for 1 min at 68°C. The PCR products were purified using the UltraClean PCR Clean Up Kit (MO BIO Laboratories) and directly sequenced using 46 primers and a BigDye Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) following the manufacturer's instructions. The 46 primers were arranged to have the same orientation, which anneal to the L strand at intervals of about 250–500 bp. The sequencing products were run on an ABI310 DNA Sequencer (Applied Biosystems). The entire mtDNA sequences were determined for SAMP8, SAMR1, SAMP1, AKR/J, and C3H/He mice. Only polymorphic sites were determined for the other strains.
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Results |
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Polymorphisms in mtDNA of SAM
We initially determined the entire mtDNA sequences of SAMP8, SAMR1, and AKR/J. The size of the mtDNA sequence of SAMP8 and SAMR1 was 16,299 bp, while that of AKR/J was 16,300 bp. Comparison of the sequences revealed five variations among the three strains at base positions 2256, 9821, 10,847, 11,181, and 13,053 (Figure 1 and Table 1). The mtDNA sequence of the AKR/J mouse was different from that of any of the SAM strains. The size difference between SAMP8 and AKR/J was due to the different number of adenine repeats starting from position 9821 (Table 1). Then we examined the five polymorphic sites of eight other SAMP and four other SAMR strains. The strains could be classified into three types (I, II, and III) (Table 1). Subsequently we determined the entire mtDNA sequence of SAMP1 (type II mtDNA), but no other polymorphism was found. All SAM strains had the thymine to cytosine transition at position 13,053. The strain distribution pattern at base 2256 (thymine or cytosine) coincided completely with that at base 11,181 (adenine or guanine). The transitional change from guanine to adenine at position 10,847 distinguished the type III mtDNA from the type II mtDNA.
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Comparison of the Polymorphic Bases with Other Mouse Strains
We performed blast search with the mouse mtDNA sequences in the GenBank, but could not find mouse mtDNA sequences with the four transitions. We then examined the five polymorphic bases in mtDNA of five inbred strains of different origin: SJL/J, A/J, BALB/c, DBA/2, and C57BL/6J. Transition mutations at bases 2256, 10,847, 11,181, and 13,053 were not found in these strains (Table 1). We extended our investigation to closed colony strains. Ten Slc:ddY, six CD-1/Crj, and six ICR/Jcl mice were examined for base 13,053, but all had T, different from C in SAM.
mtDNA Sequence of C3H/He Strain
When we compared the mtDNA sequence of AKR/J to the reported mouse mtDNA from the L cell line (V00711), we found 19 differences including base changes and insertions/deletions. As the L cell line is derived from the C3H strain (Bibb et al. 1981), we attempted to verify the entire mtDNA sequence of a C3H/He mouse. Eighteen of the 19 differences were identical between C3H/He and AKR/J (Table 2). Base 9348 was confirmed to be adenine as originally reported for V00711, instead of guanine in AKR/J. The number of adenine repeats starting from base 9821 was nine, different from eight in V00711. In addition, the number of thymine repeats starting from base 9818 was four, different from three in V00711.
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Discussion |
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Unique Mutations in mtDNA of SAM
We found five variant sites in mtDNA sequences among SAM and AKR/J strains. One of the variations was a difference in the number of adenine repeats starting from position 9821. The polymorphism was actually found in mtDNA sequences from other inbred mouse strains and could be found in the GenBank data (accession no. V00711, L07095, and L07096), implying that it is a common polymorphism in the mouse population. The number of adenine repeats ranged from 8 to 10 times depending on the strain (Table 1). The transitions at positions 2256, 10,847, and 13,053 in SAM were also in the repeats of the same bases. DNA sequences consisting of the same base repeats are known to be highly unstable and prone to replication errors (Richards and Sutherland 1994).
The four transitional changes at positions 2256, 10,847, 11,181, and 13,053 were not found in mouse strains other than SAM, suggesting that they are very rare polymorphisms. The distribution pattern of the transitions suggested that the transitions occurred in the order 13,053, 2256, and 11,181, and 10,847. Of interest, the mtDNA sequence of the AKR/J mouse was different from that of any of the SAM strains. This result implies that a female mouse was crossed to the AKR/J. It is hardly conceivable that the five mutations occurred during the 30 years spent establishing SAM strains after the inadvertent crossing to AKR/J. Rather it would be reasonable to postulate that three types of mitochondrial DNA existed at the inadvertent crossing. However, it is uncertain whether or not the three female mice each had one type of mtDNA. It might also be possible that one mouse had the three types of mtDNA in the state of heteroplasmy, and each type was fixed at random in the SAM strains. The polymorphisms may provide a promising clue to ascertain the ancestral strain(s) of SAM. Although we could not identify mouse strains with the same type of mtDNA as SAM in this study, screening of other strains should eventually reveal the parental strain(s) of SAM. There is a successful example of tracking down the culprit for genetic contamination of NZB mice by mtDNA typing (Yonekawa et al. 1986).
Consensus mtDNA Sequence of Laboratory Strains of Inbred Mice
MtDNA sequence has been used for the study of polymorphic histocompatibility antigen encoded on mtDNA of mice (Loveland et al. 1990) or phylogenetic analysis of members of the house mouse (Boissinot and Boursot 1997; Prager et al. 1996, 1998). Optimal interpretation of experimental observations by these kinds of studies requires precise mtDNA sequence data. We determined the entire mtDNA sequence of five inbred mouse strains in this study. To our knowledge there have been only three entire mouse mtDNA sequences (V00711, L07095, and L07096) deposited to the DNA data bank. L07096 is from the MilP strain (Mus domesticus), a descendant of a wild female mouse caught near Pavia, Italy (Loveland et al. 1990). V00711 is the first to be reported for the entire mouse mtDNA sequence (Bibb et al. 1981) and has long been regarded as a reference for mouse mtDNA. V00711 is actually for the LA9 cell line derived from the C3H/An strain. The L cell, a parental cell line of LA9 was established in 1943 (Earle 1943). LA9 was then selected as a clone resistant to 8-azaguanine by treating L cells with the reagent (Littlefield 1963). Hence it would not be surprising if mutations had accumulated in mtDNA by 1981, when the cells were used for mtDNA isolation. Actually we found 20 differences between V00711 and C3H/He, and 18 of the different bases in C3H/He and AKR/J were identical. L07095 is for the inbred mouse, NZB/BINJ. However, both restriction analysis (Ferris et al. 1983; Yonekawa et al. 1982) and sequencing of mtDNA (Loveland et al. 1990) revealed that NZB has a distinct mtDNA from the other inbred mouse strains. Most "old" inbred mouse strains, including C3H/He, AKR/J, BALB/c, A/J, SJL/J, DBA/2, and C57BL/6J, but not NZB, yield the same restriction enzyme cleavage patterns of mtDNA (Ferris et al. 1982; Yonekawa et al. 1982). We found two differences between C3H/He and AKR/J; guanine or adenine differences at base 9348 and the number of thymine repeats starting from base 9818. Subsequently we investigated the two polymorphic sites of BALB/c, A/J, SJL/J, DBA/2, and C57BL/6J. BALB/c and A/J had adenine at base 9348, while SJL/J, DBA/2, and C57BL/6J had guanine (Table 1), indicating that it is a common polymorphism in the inbred mouse strains. The number of thymine repeats from base 9818 was three in all strains examined.
From these results it might be rational to propose the mtDNA sequence of AKR/J strain revealed in this study as the consensus mtDNA sequence of old laboratory strains of inbred mice. The seven inbred mouse strains could be classified into five types by a combination of differences at base 9348, thymine repeats from base 9818, and adenine repeats from base 9821. We cannot infer precisely when these mutations arose. The mutations could have occurred before or after the initiation of the establishment of laboratory mice. We should be able to elucidate it by the examination of mtDNA of other mouse strains and referring it to their genealogy.
Effects of Mutations on Mitochondrial Function
Another question is whether or not the polymorphisms identified in this study have effects on the function of mitochondria. An intriguing feature in SAM is the age-associated mitochondrial dysfunction in the brain of SAMP8 mice (Fujibayashi et al. 1998; Nakahara et al. 1998; Nishikawa et al. 1998). Reduced activity of respiratory complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase) is a characteristic of the SAMP8 brain (Fujibayashi et al. 1998), potentially correlated with the age-associated deterioration in learning and memory of the strain (Miyamoto et al. 1992). The reduced activity of respiratory complex III, however, is not likely to be the result of a defect of the mitochondrial cytochrome b gene, because no difference was observed between the DNA sequences of the gene from SAMP8 compared to that from SAMR1 or AKR/J mice. The transitions at bases 10,847 and 13,053 are silent mutations. The polymorphism at position 2256 is in the coding region for the 16S ribosomal RNA (rRNA). The stretch of adenine residues including base 9821 constitutes the D arm of Arg-tRNA. We have no clues to deduce the effects of these four polymorphisms.
The transition at base 11,181 leads to amino acid substitution from serine to glycine at the 339th amino acid (S339G) in ND4 protein (Figure 1). Amino acids 338–340 of ND4 (His, Ser, and Arg) are conserved among mouse, rat, human, bovine, seal, and whale. In humans, substitution of amino acid 340 of ND4 from arginine to histidine leads to Lebers hereditary optic neuropathy (Wallace et al. 1988). The optic neuropathy is not observed in strains with S339G, including SAMP8. Although the serine to glycine substitution is conservative, it may be associated with the reduced activity of ND4 and complex I in SAMP8 mice. Further study will be necessary to resolve the issue.
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Acknowledgments |
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We thank Dr. M. Ebukuro (Department of Genetics, Central Institute for Experimental Animals, Japan) for providing DNA samples of CD-1/Crj and ICR/Jcl mice. We thank Dr. T. Shiroishi (Mammalian Genetics Laboratory, National Institute of Genetics, Japan) for providing us the SJL/J mouse. We also thank Drs. H. Yonekawa (Tokyo Metropolitan Institute of Medical Science), H. Shisa (Saitama Cancer Center Research Institute), and H. Katoh (Institute for Experimental Animals, Hamamatsu University School of Medicine) for helpful discussion. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan (no. 11680819 to M.M. and K.H.). The mtDNA sequence data revealed in this research have been submitted to DDBJ and have been assigned the accession numbers AB042432 (for AKR/J), AB042523 (SAMR1), AB042524 (SAMP1), AB042809 (SAMP8), and AB049357 (C3H/He).
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Footnotes |
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Corresponding Editor: Roger H. Reeves
Received October 14, 2000
Accepted March 31, 2001
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