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Age- and Temperature-Dependent Somatic Mutation Accumulation in Drosophila melanogaster

Garcia, Ana Maria, Calder, R. Brent, Dollé, Martijn E. T., Lundell, Martha, Kapahi, Pankaj, Vijg, Jan·2010·PLoS Genetics

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Figure 1 Comparative analysis of somatic mutations in mice and flies. (A) Schematic depiction of the LacZ-plasmid model in Mus musculus and D. melanogaster. Note that the mouse line 60 contains about 10 head-to-tail organized plasmid copies per integration site, but only two plasmid copies are depicted. For the transgenic flies, each line harbors only 1 copy of the pUR288 plasmid. Individual plasmids can be rescued by excision of genomic DNA with Hind III (H) or Pst I (P). After purification from the mouse or fly genomic DNA, self-ligation and transformation into Escherichia coli C (ΔlacZ, galE -) host cells, individual plasmids are recovered in the form of ampicillin-resistant colonies. A small amount of transformants is plated on medium containing X-gal, to determine the total number of plasmids rescued (titer plate). The remainder is plated on media supplemented with the lactose-analogue p-gal, to select only the cells harboring a mutant lacZ (selective plate). The mutant frequency is the ratio of the colonies on the selective plate versus colonies on the titer plate (times the dilution factor). Location and direction of the integration site of the pP[CaSper]vector is shown for line 11. For the mouse, the location and direction of the integrated pUR288 concatamers is shown for line 60. LacZ = lacZ reporter gene; P5′ = 5′ end of (pP[CaSper]); P3′ = 3′ end of (pP[CaSper]); white = the white selection marker. (B) Direct comparison of spontaneous somatic mutation frequency in mice (3-month old; liver) and flies (1–2-days old; whole body). White bars represent the frequency of point mutations and black bars the frequency of DNA rearrangements. The frequency of all mutations in the fly is greater than that of mouse liver (one-tailed Welch Two Sample t-test, p = 9.04e−05). Error bars are Standard Deviations.
Figure 1 Comparative analysis of somatic mutations in mice and flies. (A) Schematic depiction of the LacZ-plasmid model in Mus musculus and D. melanogaster. Note that the mouse line 60 contains about 10 head-to-tail organized plasmid copies per integration site, but only two plasmid copies are depicted. For the transgenic flies, each line harbors only 1 copy of the pUR288 plasmid. Individual plasmids can be rescued by excision of genomic DNA with Hind III (H) or Pst I (P). After purification from the mouse or fly genomic DNA, self-ligation and transformation into Escherichia coli C (ΔlacZ, galE -) host cells, individual plasmids are recovered in the form of ampicillin-resistant colonies. A small amount of transformants is plated on medium containing X-gal, to determine the total number of plasmids rescued (titer plate). The remainder is plated on media supplemented with the lactose-analogue p-gal, to select only the cells harboring a mutant lacZ (selective plate). The mutant frequency is the ratio of the colonies on the selective plate versus colonies on the titer plate (times the dilution factor). Location and direction of the integration site of the pP[CaSper]vector is shown for line 11. For the mouse, the location and direction of the integrated pUR288 concatamers is shown for line 60. LacZ = lacZ reporter gene; P5′ = 5′ end of (pP[CaSper]); P3′ = 3′ end of (pP[CaSper]); white = the white selection marker. (B) Direct comparison of spontaneous somatic mutation frequency in mice (3-month old; liver) and flies (1–2-days old; whole body). White bars represent the frequency of point mutations and black bars the frequency of DNA rearrangements. The frequency of all mutations in the fly is greater than that of mouse liver (one-tailed Welch Two Sample t-test, p = 9.04e−05). Error bars are Standard Deviations.CC BY · PubMed Central
Figure 2 Somatic mutations as a function of life span at different temperatures. (A) Survivalship of female flies grown at 18, 25 and 29°C (p<0.0001 for all comparisons by log-rank test). (B) The same for male flies (p<0.0001 for all comparisons by log-rank test). (C) Mutation frequency in flies maintained at different temperatures with age. Through linear regression analysis, the slopes of all mutation frequencies with chronological time are significant at the p<.01 level except for female flies at 18°C (see Figure S2). The slopes of the different temperatures are significant for females (p = 3.33e−05) and males (p = 4.89e−05). (D) Mutation accumulation at 18 and 29°C in male flies (data are independent of Figure 2C). (E) Mutation frequency of flies as a function of remaining survival time at a given temperature (data are from Figure 2A–2C). Black = 18°C, green = 25°C, red = 29°C, circle = female, triangle = male. Error bars are standard deviations.
Figure 2 Somatic mutations as a function of life span at different temperatures. (A) Survivalship of female flies grown at 18, 25 and 29°C (p<0.0001 for all comparisons by log-rank test). (B) The same for male flies (p<0.0001 for all comparisons by log-rank test). (C) Mutation frequency in flies maintained at different temperatures with age. Through linear regression analysis, the slopes of all mutation frequencies with chronological time are significant at the p<.01 level except for female flies at 18°C (see Figure S2). The slopes of the different temperatures are significant for females (p = 3.33e−05) and males (p = 4.89e−05). (D) Mutation accumulation at 18 and 29°C in male flies (data are independent of Figure 2C). (E) Mutation frequency of flies as a function of remaining survival time at a given temperature (data are from Figure 2A–2C). Black = 18°C, green = 25°C, red = 29°C, circle = female, triangle = male. Error bars are standard deviations.CC BY · PubMed Central
Figure 3 LacZ mutation spectra in D. melanogaster at different temperatures. (A) Frequencies of point mutations (white) and DNA rearrangements (black). Each determination point is based on 60 mutants, taken from the plates used to generate the data in Figure 2C. The accumulation of genome rearrangements was statistically significant (p = 0.0043 in males and p = 0.0260 in females). No significant accumulation of point mutations was detected. (B) Physical maps of the second breakpoint for the genome rearrangements in the fly at 18 and 29°C.
Figure 3 LacZ mutation spectra in D. melanogaster at different temperatures. (A) Frequencies of point mutations (white) and DNA rearrangements (black). Each determination point is based on 60 mutants, taken from the plates used to generate the data in Figure 2C. The accumulation of genome rearrangements was statistically significant (p = 0.0043 in males and p = 0.0260 in females). No significant accumulation of point mutations was detected. (B) Physical maps of the second breakpoint for the genome rearrangements in the fly at 18 and 29°C.CC BY · PubMed Central
Figure 4 Mutation frequency in DNA from the head, thorax, and abdomen. Of young and old, female (A) and male (B) flies maintained at 29°C. *p<0.05; **p<0.01, ***p<1e−5 (t-test of old vs. young). Mutation frequency of old male thorax was significantly different from old male abdomen (p = 9.13e−05) and old male head (p = 0.01092). Mutation frequency of old female thorax was different only from old head p = 0.03318. Error bars are Standard Deviations.
Figure 4 Mutation frequency in DNA from the head, thorax, and abdomen. Of young and old, female (A) and male (B) flies maintained at 29°C. *p<0.05; **p<0.01, ***p<1e−5 (t-test of old vs. young). Mutation frequency of old male thorax was significantly different from old male abdomen (p = 9.13e−05) and old male head (p = 0.01092). Mutation frequency of old female thorax was different only from old head p = 0.03318. Error bars are Standard Deviations.CC BY · PubMed Central

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Metadata

Authors
Garcia, A. M., Calder, R. B., Dollé, M. E. T., Lundell, M., Kapahi, P., & Vijg, J.
Venue
PLoS Genetics
Published
2010-05-13
Volume
6
Issue
5
Pages
e1000950
ISSN
1553-7404
Cited by
62
References
2
Language
en

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Garcia, A. M., Calder, R. B., Dollé, M. E. T., Lundell, M., Kapahi, P., & Vijg, J. (2010). Age- and Temperature-Dependent Somatic Mutation Accumulation in Drosophila melanogaster. PLoS Genetics. https://doi.org/10.1371/journal.pgen.1000950