Mutation Research 760 (2014) 24–32
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
p53-Dependent suppression of genome instability in germ cells Shinji Otozai a,1 , Tomoko Ishikawa-Fujiwara b,1 , Shoji Oda c , Yasuhiro Kamei b,2 , Haruko Ryo d , Ayuko Sato e , Taisei Nomura d , Hiroshi Mitani c , Tohru Tsujimura e , Hidenori Inohara a , Takeshi Todo b,∗ a
Department of Otorhinolaryngology and Head and Neck Surgery, Osaka University School of Medicine, Osaka 565-0871, Japan Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan d Nomura Project, National Institute of Biomedical Innovation, Osaka 565-0085, Japan e Department of Pathology, Hyogo College of Medicine, Hyogo 663-8501, Japan b c
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 7 January 2014 Keywords: Medaka fish Microsatellite instability Radiation Spermatogonial stem cell msh2 p53
a b s t r a c t Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in ␥-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2−/− males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2−/− and wild-type fish. By contrast, irradiated p53−/− fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2−/− fish, but negligible levels in p53−/− fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The maintenance of genome integrity is crucial for the survival of all organisms. However, genome stability is continuously challenged by a diverse array of mutagenic forces, including errors during DNA replication, environmental factors, and endogenous mutagens. To maintain genome integrity against these sources of mutation, several defense pathways have evolved, including DNA repair, the cell cycle checkpoint, and apoptosis [1–3]. However, DNA damage eludes these defense mechanisms in rare cases, resulting in mutation. Induction of mutations exerts a deleterious effect
Abbreviations: CE, fluorescent capillary electrophoresis; DDR, DNA damage response; DSB, double-strand break; ESTR, expanded simple tandem repeat; HRM, high-resolution melting; MMR, mismatch-repair; MSI, microsatellite instability; TILLING, target induced local lesion in genome. ∗ Corresponding author. Tel.: +81 6 6879 3810; fax: +81 6 6978 3819/+81 6 6879 3819. E-mail addresses: [email protected], van.todo.6-4 [email protected] (T. Todo). 1 These authors contributed equally to this work. 2 Present address: NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan. 0027-5107/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2013.12.004
on genome integrity, especially when it occurs in germ cells. Consequently, the mechanism underlying mutagenesis is an important issue in the study of the DNA damage response (DDR). Most of our current knowledge regarding the induction processes of heritable mutations in metazoans comes from studies of phenotypic markers in laboratory model organisms. However, these studies suffered from a serious drawback in that the rates of mutation induction were low. To overcome this drawback, tandem repeat sequences such as minisatellites and microsatellites have been used [4–6]. These tandem repeat sequences are highly unstable, with very high rates of spontaneous mutation and high susceptibility to induction of mutations by environmental stress. In these sequences, changes in the mutation rate can be detected in significantly smaller sample sizes than those required for conventional phenotype-based mutation studies. Minisatellites and microsatellites are conventionally classified based on the number of nucleotides that comprise the repeat unit sequence. Minisatellites consist of variant repeats that range in length from 10 to 100 base pairs (bp), and the number of repeats may exceed 500. They are located at around 1000 sites in the human genome [7]. Microsatellites consist of shorter tandem arrays of simple DNA sequences with core repeat units of 1–6 bp, and the number of repeats may be only 100 or less. Their entire length
S. Otozai et al. / Mutation Research 760 (2014) 24–32
can range up to several hundred bp and they are found at over 100,000 sites in the human genome [8,9]. Mutational changes in the tandem repeat sequence are manifest as alterations in the number of core tandem repeats, and hence allele length, rather than in the primary sequence. This is a sharp contrast to mutations induced at unique DNA sequences. Several studies have measured radiation-induced mutations at minisatellite loci in humans. However, the results of these studies appear to be conflicting. Increased mutation rates were reported in residents of an area contaminated by the Chernobyl accident, of an area exposed to an atmospheric nuclear test at Semipalatinsk, and of an area along the Techa River at which radionucleotides were released [10–13]. By contrast, no significant increase in mutation rates was observed in studies of atomic bomb survivors, Chernobyl clean-up workers, or patients who underwent radiotherapy [14–19]. The genetic effects of radiation were also examined at microsatellite loci in several organisms, including humans, but the results have occasionally been discordant. In humans, there is no clear evidence of increased mutation rates in response to radiation [20–23]. On the other hand, significant increases in mutation rates have been observed in barn swallows [24] and wheat [25] grown in sites highly contaminated by the Chernobyl accident. A similar increase has also been observed in an experimental model animal, the Japanese medaka fish [26,27]. However, dose-dependency was not well established in these studies. Therefore, we attempted to determine whether radiation induces germline mutations at microsatellite loci in a dose-dependent manner and, if so, which DDR pathways are involved in the induction of these mutations. Microsatellite mutations are thought to be generated by ‘slippage’ during DNA replication that results in the gain or loss of repeat units [28,29]. The phenomenon of a high mutation frequency at microsatellite loci is called ‘microsatellite instability’ (MSI). MSI is a prominent phenotype that can result from loss of mismatch-repair (MMR) genes. The MMR pathway corrects mis-paired nucleotides in DNA resulting from replication errors, recombination intermediates, or base mutations caused by DNAdamaging agents [30–32]. Consequently, genomic DNA sequences containing alterations in the number of core tandem repeats are good substrates for MMR enzymes. In fact, mutations within genes in this repair pathway often lead to hereditary nonpolyposis colorectal cancer, which is characterized by a high rate of MSI. The MMR system consists of several key proteins, including Msh2. Another factor playing a crucial role in maintaining genome stability is the tumor suppressor protein p53 [33]. The p53 protein acts as a DNA sequence-specific transcription factor and determines an appropriate cellular response to various stress signals. In response to stress, activated p53 selectively transcribes a set of target genes that initiate various cellular responses, including cell cycle arrest, DNA repair, and apoptosis. These programs either repair the underlying damage or eliminate cells with damaged and mutated genomes before they become nascent tumor cells. Among the vertebrates, small laboratory fish are suitable for the study of gene function in germ cells due to their ease of handling and large numbers of progeny per generation. Reverse genetics, an essential tool in the study of gene function, are available in two teleost species: zebrafish, Danio rerio, and medaka, Oryzias latipes. Several inbred lines are available, especially in medaka. The uniform genome sequences of all animals of a given inbred line provide a good system for the precise analysis of alterations in the genome [34]. Shima and Shimada [35] showed that the mutation response of the medaka male germ cell is comparable to that of the mouse, and therefore medaka could serve as a vertebrate model system of the DDR in germ cells. We made use of these advantages to investigate radiation-induced MSI in medaka fish. msh2−/− and p53−/− mutants were generated, and the efficiency of radiation-induced MSI in their germlines were compared with that in wild-type fish.
25
2. Materials and methods 2.1. Medaka fish The medaka inbred line HdrR was used in this study. Fish were housed at room temperature (26–28 ◦ C) under a 14-h light and 10h dark cycle, and were fed a powdered diet (Tetramin, Tetra Werke Co., Mells, Germany) and brine shrimp (Artemina franciscana) three times per day. This study was conducted according to the Osaka University Animal Experimental Enforcement Rules. 2.2. High-resolution melting (HRM) assay The HRM assay is a recently developed promising technology used to detect variations in DNA [38–40]. The thermal stability of a DNA fragment is determined by its base sequence. When the DNA fragment contains an altered sequence, its duplex stability is changed, leading to a different melting behavior. The HRM assay identifies mutations by detecting differences in melting behavior. To precisely monitor melting behavior during HRM analysis, melting curves are produced using DNA-intercalating dyes that fluoresce in the presence of double-stranded DNA and a specialized instrument that monitors this fluorescence during heating of the DNA. When the temperature increases, the DNA-intercalating dye is released from DNA and fluorescence decreases. Monitoring of this process produces a characteristic melting profile. Changes in the sequence of the DNA fragment, owing to single-nucleotide polymorphisms and/or mutations, alter the melting profile, which is compared to that of wild-type DNA. In the HRM assay, each microsatellite locus was amplified by PCR using primers specific for that locus. HRM assays were performed using a LightScanner (Idaho Technology) or LightCycler (Roche Diagnostics), and the melting curves obtained were analyzed as described previously [37]. Briefly, after exponential background subtraction, melting curves were normalized between 0% and 100%. Normalized and temperature-overlaid curves were viewed on the subtraction plots to magnify differences in the shapes of the melting curves. The subtraction plots were generated by subtracting each curve from the mean wild-type curve, which was defined as that of the most common genotype. The subtraction plot helps to cluster the samples into groups. Clustering of the melting curves for genotype identification was performed manually using the LightScanner software. All mutations detected by the HRM assay were re-confirmed by sequencing. Sequencing reactions were performed using BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA) on the ABI 3730xl sequencing platform. 2.3. Generation of mutant medaka fish lines To generate mutant fish, the medaka TILLING (Target Induced Local Lesion In Genome) library [36] was screened using the HRM method as previously described [37]. The mutant fish obtained in this manner were out-crossed with wild-type fish of the inbred strain HdrR for more than five generations. Genotyping was performed by PCR amplification of DNA fragment containing the identified mutation and subsequent re-sequencing. The sequences of all oligonucleotides used in this study are listed in Supplementary Table S1. 2.4. -Ray irradiation and sample preparation Mutant fish were usually maintained as heterozygotes with one wild-type allele, and these heterozygotes were crossed to obtain homozygous mutants. Eggs were collected following singlepair mating. For non-irradiated control samples, 4-month-old homozygous mutant males were mated with wild-type females
26
S. Otozai et al. / Mutation Research 760 (2014) 24–32
and eggs were collected from each cross. Several sets of singlepair mating were performed, and approximately 400 embryos were collected in total from each type of cross. The exception was non-irradiated msh2−/- p53−/− males, from which only 155 embryos were obtained. For the irradiated samples, 5–10 4-monthold homozygous mutant males were exposed to ␥-rays from a 137 Cs source at a dose rate of 0.93 Gy/min (Gamma Cell 6000 Elan, MDS Nordion, Ottawa, Canada). Thirty days after irradiation, offspring were obtained by mating each irradiated male with a non-irradiated female. Finally, approximately 400 embryos were collected in total from several sets of single-pair mating. The exceptions were 2-Gy-irradiated msh2−/− male fish, 2-Gyirradiated p53−/− male fish, 4-Gy-irradiated p53−/− male fish, and 2-Gy-irradiated msh2−/− p53−/− male fish, from which 288, 275, 192, and 133 embryos were obtained, respectively. For each genotype, the numbers of males from which embryos were obtained are as follows: seven non-irradiated wild-type fish, six 2-Gyirradiated wild-type fish, five 4-Gy-irradiated wild-type fish, six non-irradiated msh2−/− fish, one 2-Gy-irradiated msh2−/− fish, four 4-Gy-irradiated msh2−/− fish, three non-irradiated p53−/− fish, five 2-Gy-irradiated p53−/− fish, nine 4-Gy-irradiated p53−/− fish, two non-irradiated msh2−/- p53−/− fish, and two 2-Gy-irradiated msh2−/− p53−/− fish. The numbers of offspring obtained from each male are summarized in Supplementary Table S2. After culture for 14 days at room temperature, genomic DNA was extracted from these fry as previously described [37]. msh2−/− p53−/− male fish only produced a small number of fertilized eggs after irradiation; therefore, offspring were obtained by in vitro fertilization with female drR fish, the parental inbred strain of HdrR.
MSI, the HRM assay and the CE assay were first compared in terms of their sensitivity and specificity for the detection of MSI. In the CE assay, each microsatellite locus was amplified by PCR. Primers used for PCR amplification were generated by adding M13forward or M13-reverse tag sequences to the 5 ends of the primer sequences used in the HRM assay. This allowed a second PCR primer set to be used that was fluorescently labeled for detection on a DNA analyzer/sequencer. The reaction mixture was diluted 10-fold with KOD-Plus buffer (Toyobo, Japan), and 1 l was used for the second PCR. The second PCR was performed using fluorescently labeled M13 primers. The M13-forward and M13-reverse primers were labeled with 6-FAM and VICTM (Applied Biosystems), respectively. The fluorescently labeled PCR products were analyzed using a capillary sequencer as previously described [23]. 2.7. Statistical analysis Statistical analyses were conducted using Fisher’s exact test. To evaluate the possibility that radiation exposure increases the germline mutation rate, P values were obtained from two-sided likelihood-ratio tests, with values
Contents lists available at ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
p53-Dependent suppression of genome instability in germ cells Shinji Otozai a,1 , Tomoko Ishikawa-Fujiwara b,1 , Shoji Oda c , Yasuhiro Kamei b,2 , Haruko Ryo d , Ayuko Sato e , Taisei Nomura d , Hiroshi Mitani c , Tohru Tsujimura e , Hidenori Inohara a , Takeshi Todo b,∗ a
Department of Otorhinolaryngology and Head and Neck Surgery, Osaka University School of Medicine, Osaka 565-0871, Japan Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan d Nomura Project, National Institute of Biomedical Innovation, Osaka 565-0085, Japan e Department of Pathology, Hyogo College of Medicine, Hyogo 663-8501, Japan b c
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 2 December 2013 Accepted 27 December 2013 Available online 7 January 2014 Keywords: Medaka fish Microsatellite instability Radiation Spermatogonial stem cell msh2 p53
a b s t r a c t Radiation increases mutation frequencies at tandem repeat loci. Germline mutations in ␥-ray-irradiated medaka fish (Oryzias latipes) were studied, focusing on the microsatellite loci. Mismatch-repair genes suppress microsatellite mutation by directly removing altered sequences at the nucleotide level, whereas the p53 gene suppresses genetic alterations by eliminating damaged cells. The contribution of these two defense mechanisms to radiation-induced microsatellite instability was addressed. The spontaneous mutation frequency was significantly higher in msh2−/− males than in wild-type fish, whereas there was no difference in the frequency of radiation-induced mutations between msh2−/− and wild-type fish. By contrast, irradiated p53−/− fish exhibited markedly increased mutation frequencies, whereas their spontaneous mutation frequency was the same as that of wild-type fish. In the spermatogonia of the testis, radiation induced a high level of apoptosis both in wild-type and msh2−/− fish, but negligible levels in p53−/− fish. The results demonstrate that the msh2 and p53 genes protect genome integrity against spontaneous and radiation-induced mutation by two different pathways: direct removal of mismatches and elimination of damaged cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The maintenance of genome integrity is crucial for the survival of all organisms. However, genome stability is continuously challenged by a diverse array of mutagenic forces, including errors during DNA replication, environmental factors, and endogenous mutagens. To maintain genome integrity against these sources of mutation, several defense pathways have evolved, including DNA repair, the cell cycle checkpoint, and apoptosis [1–3]. However, DNA damage eludes these defense mechanisms in rare cases, resulting in mutation. Induction of mutations exerts a deleterious effect
Abbreviations: CE, fluorescent capillary electrophoresis; DDR, DNA damage response; DSB, double-strand break; ESTR, expanded simple tandem repeat; HRM, high-resolution melting; MMR, mismatch-repair; MSI, microsatellite instability; TILLING, target induced local lesion in genome. ∗ Corresponding author. Tel.: +81 6 6879 3810; fax: +81 6 6978 3819/+81 6 6879 3819. E-mail addresses: [email protected], van.todo.6-4 [email protected] (T. Todo). 1 These authors contributed equally to this work. 2 Present address: NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan. 0027-5107/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2013.12.004
on genome integrity, especially when it occurs in germ cells. Consequently, the mechanism underlying mutagenesis is an important issue in the study of the DNA damage response (DDR). Most of our current knowledge regarding the induction processes of heritable mutations in metazoans comes from studies of phenotypic markers in laboratory model organisms. However, these studies suffered from a serious drawback in that the rates of mutation induction were low. To overcome this drawback, tandem repeat sequences such as minisatellites and microsatellites have been used [4–6]. These tandem repeat sequences are highly unstable, with very high rates of spontaneous mutation and high susceptibility to induction of mutations by environmental stress. In these sequences, changes in the mutation rate can be detected in significantly smaller sample sizes than those required for conventional phenotype-based mutation studies. Minisatellites and microsatellites are conventionally classified based on the number of nucleotides that comprise the repeat unit sequence. Minisatellites consist of variant repeats that range in length from 10 to 100 base pairs (bp), and the number of repeats may exceed 500. They are located at around 1000 sites in the human genome [7]. Microsatellites consist of shorter tandem arrays of simple DNA sequences with core repeat units of 1–6 bp, and the number of repeats may be only 100 or less. Their entire length
S. Otozai et al. / Mutation Research 760 (2014) 24–32
can range up to several hundred bp and they are found at over 100,000 sites in the human genome [8,9]. Mutational changes in the tandem repeat sequence are manifest as alterations in the number of core tandem repeats, and hence allele length, rather than in the primary sequence. This is a sharp contrast to mutations induced at unique DNA sequences. Several studies have measured radiation-induced mutations at minisatellite loci in humans. However, the results of these studies appear to be conflicting. Increased mutation rates were reported in residents of an area contaminated by the Chernobyl accident, of an area exposed to an atmospheric nuclear test at Semipalatinsk, and of an area along the Techa River at which radionucleotides were released [10–13]. By contrast, no significant increase in mutation rates was observed in studies of atomic bomb survivors, Chernobyl clean-up workers, or patients who underwent radiotherapy [14–19]. The genetic effects of radiation were also examined at microsatellite loci in several organisms, including humans, but the results have occasionally been discordant. In humans, there is no clear evidence of increased mutation rates in response to radiation [20–23]. On the other hand, significant increases in mutation rates have been observed in barn swallows [24] and wheat [25] grown in sites highly contaminated by the Chernobyl accident. A similar increase has also been observed in an experimental model animal, the Japanese medaka fish [26,27]. However, dose-dependency was not well established in these studies. Therefore, we attempted to determine whether radiation induces germline mutations at microsatellite loci in a dose-dependent manner and, if so, which DDR pathways are involved in the induction of these mutations. Microsatellite mutations are thought to be generated by ‘slippage’ during DNA replication that results in the gain or loss of repeat units [28,29]. The phenomenon of a high mutation frequency at microsatellite loci is called ‘microsatellite instability’ (MSI). MSI is a prominent phenotype that can result from loss of mismatch-repair (MMR) genes. The MMR pathway corrects mis-paired nucleotides in DNA resulting from replication errors, recombination intermediates, or base mutations caused by DNAdamaging agents [30–32]. Consequently, genomic DNA sequences containing alterations in the number of core tandem repeats are good substrates for MMR enzymes. In fact, mutations within genes in this repair pathway often lead to hereditary nonpolyposis colorectal cancer, which is characterized by a high rate of MSI. The MMR system consists of several key proteins, including Msh2. Another factor playing a crucial role in maintaining genome stability is the tumor suppressor protein p53 [33]. The p53 protein acts as a DNA sequence-specific transcription factor and determines an appropriate cellular response to various stress signals. In response to stress, activated p53 selectively transcribes a set of target genes that initiate various cellular responses, including cell cycle arrest, DNA repair, and apoptosis. These programs either repair the underlying damage or eliminate cells with damaged and mutated genomes before they become nascent tumor cells. Among the vertebrates, small laboratory fish are suitable for the study of gene function in germ cells due to their ease of handling and large numbers of progeny per generation. Reverse genetics, an essential tool in the study of gene function, are available in two teleost species: zebrafish, Danio rerio, and medaka, Oryzias latipes. Several inbred lines are available, especially in medaka. The uniform genome sequences of all animals of a given inbred line provide a good system for the precise analysis of alterations in the genome [34]. Shima and Shimada [35] showed that the mutation response of the medaka male germ cell is comparable to that of the mouse, and therefore medaka could serve as a vertebrate model system of the DDR in germ cells. We made use of these advantages to investigate radiation-induced MSI in medaka fish. msh2−/− and p53−/− mutants were generated, and the efficiency of radiation-induced MSI in their germlines were compared with that in wild-type fish.
25
2. Materials and methods 2.1. Medaka fish The medaka inbred line HdrR was used in this study. Fish were housed at room temperature (26–28 ◦ C) under a 14-h light and 10h dark cycle, and were fed a powdered diet (Tetramin, Tetra Werke Co., Mells, Germany) and brine shrimp (Artemina franciscana) three times per day. This study was conducted according to the Osaka University Animal Experimental Enforcement Rules. 2.2. High-resolution melting (HRM) assay The HRM assay is a recently developed promising technology used to detect variations in DNA [38–40]. The thermal stability of a DNA fragment is determined by its base sequence. When the DNA fragment contains an altered sequence, its duplex stability is changed, leading to a different melting behavior. The HRM assay identifies mutations by detecting differences in melting behavior. To precisely monitor melting behavior during HRM analysis, melting curves are produced using DNA-intercalating dyes that fluoresce in the presence of double-stranded DNA and a specialized instrument that monitors this fluorescence during heating of the DNA. When the temperature increases, the DNA-intercalating dye is released from DNA and fluorescence decreases. Monitoring of this process produces a characteristic melting profile. Changes in the sequence of the DNA fragment, owing to single-nucleotide polymorphisms and/or mutations, alter the melting profile, which is compared to that of wild-type DNA. In the HRM assay, each microsatellite locus was amplified by PCR using primers specific for that locus. HRM assays were performed using a LightScanner (Idaho Technology) or LightCycler (Roche Diagnostics), and the melting curves obtained were analyzed as described previously [37]. Briefly, after exponential background subtraction, melting curves were normalized between 0% and 100%. Normalized and temperature-overlaid curves were viewed on the subtraction plots to magnify differences in the shapes of the melting curves. The subtraction plots were generated by subtracting each curve from the mean wild-type curve, which was defined as that of the most common genotype. The subtraction plot helps to cluster the samples into groups. Clustering of the melting curves for genotype identification was performed manually using the LightScanner software. All mutations detected by the HRM assay were re-confirmed by sequencing. Sequencing reactions were performed using BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA) on the ABI 3730xl sequencing platform. 2.3. Generation of mutant medaka fish lines To generate mutant fish, the medaka TILLING (Target Induced Local Lesion In Genome) library [36] was screened using the HRM method as previously described [37]. The mutant fish obtained in this manner were out-crossed with wild-type fish of the inbred strain HdrR for more than five generations. Genotyping was performed by PCR amplification of DNA fragment containing the identified mutation and subsequent re-sequencing. The sequences of all oligonucleotides used in this study are listed in Supplementary Table S1. 2.4. -Ray irradiation and sample preparation Mutant fish were usually maintained as heterozygotes with one wild-type allele, and these heterozygotes were crossed to obtain homozygous mutants. Eggs were collected following singlepair mating. For non-irradiated control samples, 4-month-old homozygous mutant males were mated with wild-type females
26
S. Otozai et al. / Mutation Research 760 (2014) 24–32
and eggs were collected from each cross. Several sets of singlepair mating were performed, and approximately 400 embryos were collected in total from each type of cross. The exception was non-irradiated msh2−/- p53−/− males, from which only 155 embryos were obtained. For the irradiated samples, 5–10 4-monthold homozygous mutant males were exposed to ␥-rays from a 137 Cs source at a dose rate of 0.93 Gy/min (Gamma Cell 6000 Elan, MDS Nordion, Ottawa, Canada). Thirty days after irradiation, offspring were obtained by mating each irradiated male with a non-irradiated female. Finally, approximately 400 embryos were collected in total from several sets of single-pair mating. The exceptions were 2-Gy-irradiated msh2−/− male fish, 2-Gyirradiated p53−/− male fish, 4-Gy-irradiated p53−/− male fish, and 2-Gy-irradiated msh2−/− p53−/− male fish, from which 288, 275, 192, and 133 embryos were obtained, respectively. For each genotype, the numbers of males from which embryos were obtained are as follows: seven non-irradiated wild-type fish, six 2-Gyirradiated wild-type fish, five 4-Gy-irradiated wild-type fish, six non-irradiated msh2−/− fish, one 2-Gy-irradiated msh2−/− fish, four 4-Gy-irradiated msh2−/− fish, three non-irradiated p53−/− fish, five 2-Gy-irradiated p53−/− fish, nine 4-Gy-irradiated p53−/− fish, two non-irradiated msh2−/- p53−/− fish, and two 2-Gy-irradiated msh2−/− p53−/− fish. The numbers of offspring obtained from each male are summarized in Supplementary Table S2. After culture for 14 days at room temperature, genomic DNA was extracted from these fry as previously described [37]. msh2−/− p53−/− male fish only produced a small number of fertilized eggs after irradiation; therefore, offspring were obtained by in vitro fertilization with female drR fish, the parental inbred strain of HdrR.
MSI, the HRM assay and the CE assay were first compared in terms of their sensitivity and specificity for the detection of MSI. In the CE assay, each microsatellite locus was amplified by PCR. Primers used for PCR amplification were generated by adding M13forward or M13-reverse tag sequences to the 5 ends of the primer sequences used in the HRM assay. This allowed a second PCR primer set to be used that was fluorescently labeled for detection on a DNA analyzer/sequencer. The reaction mixture was diluted 10-fold with KOD-Plus buffer (Toyobo, Japan), and 1 l was used for the second PCR. The second PCR was performed using fluorescently labeled M13 primers. The M13-forward and M13-reverse primers were labeled with 6-FAM and VICTM (Applied Biosystems), respectively. The fluorescently labeled PCR products were analyzed using a capillary sequencer as previously described [23]. 2.7. Statistical analysis Statistical analyses were conducted using Fisher’s exact test. To evaluate the possibility that radiation exposure increases the germline mutation rate, P values were obtained from two-sided likelihood-ratio tests, with values
