Ty1 Retrotransposition and DNA Mutation Frequency Under Caloric Stress and

Oxidative Stress in Saccharomyces cerevisiae

By: David VanHoute

Maxwell Lab

4/15/11

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

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

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

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?

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

Ty1 mobility hindered under low Glucose conditions?

1 2 3 1 2

DNA Mutation Assay

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Plated @ 30C

Counted after 4 Days

SC-Arg + Can. (x7) YPD (x14)

DNA Mutations are lower at 0.2% Glucose Media Conditions

1 2 3 1 2

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

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

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

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?

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

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.

Acknowledgements

• Professor Patrick Maxwell

• Bianca Pier

• Biology Graduate Program

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.

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

500 1000 1500 2000Ladder

PCR “Check” of Transformants

Finding Transformant Colonies