Maxwell Rotation Presentation
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Ty1 Retrotransposition and DNA Mutation Frequency Under Caloric Stress and
Oxidative Stress in Saccharomyces cerevisiae
By: David VanHoute
Maxwell Lab
4/15/11
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How are cellular stresses related to aging?
• Reducing caloric intake Burhans et al. DOI: 10.4161/cc.8.14.8852
• Oxidative stresses• SOD1
ROS, Ty1 Retrotransposition, Stoycheva et al. DOI: 10.1002/yea.1749
• Is aging influenced by genomic stress?
www.impactaging.com
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Dietary Restriction Increases CLS
• Caloric restriction increases CLS Kaeberlein et al. Nature 464, 513-519| doi:10.1038/nature08981
• At 10%, 2%, and 0.2% Glucose concentrations?
• Can varying stresses regulate aging and genomic stability?
Kaeberlein et al. Nature 464, 513-519| doi:10.1038/nature08981
ATF1
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Ty1 Retrotransposition in Saccharomyces cerevisiae
• Reinsertion of mobile genetic elements via reverse transcription.
• Mobile element is copied out, then reinserted somewhere else in the genome
•Ty1 might cause genetic change associated with aging
www.wadsworth.org
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Projects
1. Does varying Glucose concentration at 10%, 2%, and 0.2% influence Ty1 and mutational frequency?
2. How do SOD1 and ATF1 deletions impact Ty1 and mutational frequency?
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Identifying Ty1 Mobility
www.wadsworth.org
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SC-His (x7) YPD (x14)
Cultured 4 Days @ 20C
Plated @ 30C
Counted after 4 Days
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Ty1 mobility hindered under low Glucose conditions?
1 2 3 1 2
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DNA Mutation Assay
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Plated @ 30C
Counted after 4 Days
SC-Arg + Can. (x7) YPD (x14)
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DNA Mutations are lower at 0.2% Glucose Media Conditions
1 2 3 1 2
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Single Deletion of SOD1 and ATF1 on Ty1 Mobility and DNA Mutational Frequencies
• Increases in ROS leads to greater cellular damage and Mitochondrial dysfunction.
• How is Ty1 mobility and DNA mutations affected by SOD1 deletions and ATF1 deletions?
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SC-His (x7) YPD (x14)
Cultured 23 hours @ 20C
Plated @ 30C
Counted after 4 Days
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Transformations via KanMx cassette
• KanMX cassette replaces ORF in mutant deletion strains
• Transformants constructed from KanMx PCR product
• Grow on selective plates for Kanamycin resistance
• PCR “Check” with Kanrev. and upstream gene primer
KANMX
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20001500
1000
500
Ladder WT ATF1 M7ATF1 SOD1 M7SOD1 WT ATF1 SOD1 WT SOD1 “Check” Trans Trans ATF1
PCR Primer Products and Resulting Transformants
Mutant “check” products with KanMx
insertion
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Ty1 Mobility and DNA Mutations in Mutants
• Higher mutations occurring within ATF1 mutant• Ty1 mobility values are congruent• SOD1 deletion results due to poor viability?
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Conclusions
• Caloric restriction on yeast strains appear to reduce Ty1 mobility in cells grown with 0.2%
• Not at 10%?• ATF1 deletion shows
similar Ty1 frequencies as WT
• SOD1 deletion reduces Ty1 mobility?
www.impactaging.com
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Further Work
• Adapt short-term caloric restriction experiments to longer lifespan experiments
• Alter procedure of mutant experiments to account for reduced growth in SOD1 mutants
• Analyze other genomic stresses that may give insight into DNA damage and aging.
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Acknowledgements
• Professor Patrick Maxwell
• Bianca Pier
• Biology Graduate Program
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References1. Burhans, W. C. and M. Weinberger (2009). "Acetic acid effects on aging in
budding yeast." Cell Cycle 8(14): 2300-2302.
2. Garfinkel, D. J. (2005). "Genome evolution mediated by Ty elements in Saccharomyces." Cytogenetic and Genome Research 110(1-4): 63-69
3. Kaeberlein, M. (2010). "Lessons on longevity from budding yeast." Nature 464(7288): 513-519.
4. Servant, G., C. Pennetier, et al. (2008). "Remodeling yeast gene transcription by activating the Ty1 long terminal repeat retrotransposon under severe adenine deficiency." Molecular and Cellular Biology 28(17): 5543-5554.
5. Stamenova, R., M. Dimitrov, et al. (2008). "Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation." Cryobiology 56(3): 241-247.
6. Stoycheva, T., M. Pesheva, et al. (2010). "The role of reactive oxygen species in the induction of Tyl retrotransposition in Saccharomyces cerevisiae." Yeast 27(5): 259-267.
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PCR Results
DNA Ladder
M7 ATF1
M7 SOD1ATF1
SOD1
DNA Ladder
M7 ATF1
ATF1
500 1Kbp 1.5 2 2.5
500 1Kbp 1.5 2 2.5
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500 1000 1500 2000Ladder
PCR “Check” of Transformants
Finding Transformant Colonies