Ty1 Retrotransposition and DNA Mutation Frequency Under Caloric Stress and Oxidative Stress in  Saccharomyces cerevisiae B...
How are cellular stresses related to aging? <ul><li>Reducing caloric intake  Burhans et al. DOI: 10.4161/cc.8.14.8852 </li...
Dietary Restriction Increases CLS <ul><li>Caloric restriction increases CLS  </li></ul><ul><li>Kaeberlein et al. Nature 46...
Ty1 Retrotransposition in  Saccharomyces cerevisiae <ul><li>Reinsertion of mobile genetic elements via reverse transcripti...
Projects <ul><li>Does varying Glucose concentration at 10%, 2%, and 0.2% influence Ty1 and mutational frequency? </li></ul...
Identifying Ty1 Mobility www.wadsworth.org org/resnres/bios/curcio.htm SC-His (x7)  YPD (x14) Cultured 4 Days @ 20  C Pla...
<ul><li>Ty1 mobility hindered under low Glucose conditions? </li></ul>1  2  3  1  2
DNA Mutation Assay <ul><li>Cultured 4 Days @ 20  C </li></ul><ul><li>Plated @ 30  C </li></ul><ul><li>Counted after 4 Da...
<ul><li>DNA Mutations are lower at 0.2% Glucose Media Conditions </li></ul>1  2  3  1  2
Single Deletion of SOD1 and ATF1 on Ty1 Mobility and DNA Mutational Frequencies <ul><li>Increases in ROS leads to greater ...
Transformations via KanMx cassette <ul><li>KanMX cassette replaces ORF in mutant deletion strains </li></ul><ul><li>Transf...
2000 1500 1000 500 Ladder  WT  ATF1  M7ATF1  SOD1  M7SOD1  WT  ATF1  SOD1  WT  SOD1    “Check”  Trans  Trans  ATF1 PCR Pri...
Ty1 Mobility and DNA Mutations in Mutants <ul><li>Higher mutations occurring within ATF1 mutant </li></ul><ul><li>Ty1 mobi...
Conclusions <ul><li>Caloric restriction on yeast strains appear to reduce Ty1 mobility in cells grown with 0.2%  </li></ul...
Further Work <ul><li>Adapt short-term caloric restriction experiments to longer lifespan experiments </li></ul><ul><li>Alt...
Acknowledgements <ul><li>Professor Patrick Maxwell </li></ul><ul><li>Bianca Pier </li></ul><ul><li>Biology Graduate Progra...
References <ul><li>Burhans, W. C. and M. Weinberger (2009). &quot;Acetic acid effects on aging in budding yeast.&quot;  Ce...
PCR Results DNA Ladder M7 ATF1 M7 SOD1 ATF1 SOD1 DNA Ladder M7 ATF1 ATF1 500  1Kbp  1.5  2  2.5 500  1Kbp  1.5  2  2.5
500  1000  1500 2000 Ladder PCR “Check” of Transformants  Finding Transformant  Colonies
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  • Genetic stresses and changes within yeast genome Retro. Could affect genome stability Influences for aging…short-term effects…age related stress----genetic changes? Burhans et al. DOI: 10.4161/cc.8.14.8852 Acetyltransferase----Ethanol---&gt;acetic acid Superoxidative Dismutase removes super oxide anions (in humans as well)
  • In accordance with lifespan If less calories results in longer CLS….would expect Ty1 mobility to decrease as well Higher Glucose should result in greater Ty1 mobility then… 10% should decrease lifespan
  • Occurs in S. cerevisiae. Do retrotansposition events be a result of increased genomic stress? How do we assess Ty1 mobility in experiments/lab?
  • Affecting ROS with deletions…
  • Genetic strains have genetic retrotranscript indicator genes. Within Ty1 element This is how we select for retro. Events Selected on SC-His minus plates
  • Not seeing a greater frequency of Ty1 mobility as Glucose increases beyond 2% Still….might be consistent with lifespan
  • DNA mutations as a frequency of canavanin resistance.
  • Undetectable in .2%…..probably not zero, but so low that it is hard to get even one colony
  • Select for Kanamycin resistance on YPD +G418 Sufate plates
  • Arrow pointing to said mutants with KanMx gene product SOD1=464bp ATF1=1577bp
  • SOD1 Can- value is less than this value
  • Sizes. How mutants were made
  • KanMx reverse primer, with upstream check primer for particular gene Has KanMx gene in it…colonies #4 (ATF1) and #5 (SOD1).
  • Maxwell Rotation Presentation

    1. 1. Ty1 Retrotransposition and DNA Mutation Frequency Under Caloric Stress and Oxidative Stress in Saccharomyces cerevisiae By: David VanHoute Maxwell Lab 4/15/11
    2. 2. How are cellular stresses related to aging? <ul><li>Reducing caloric intake Burhans et al. DOI: 10.4161/cc.8.14.8852 </li></ul><ul><li>Oxidative stresses </li></ul><ul><li>SOD1 </li></ul><ul><li> ROS,  Ty1 Retrotransposition, Stoycheva et al. DOI: 10.1002/yea.1749 </li></ul><ul><li>Is aging influenced by genomic stress? </li></ul>www.impactaging.com
    3. 3. Dietary Restriction Increases CLS <ul><li>Caloric restriction increases CLS </li></ul><ul><li>Kaeberlein et al. Nature 464, 513-519| doi:10.1038/nature08981 </li></ul><ul><li>At 10%, 2%, and 0.2% Glucose concentrations? </li></ul><ul><li>Can varying stresses regulate aging and genomic stability? </li></ul>Kaeberlein et al. Nature 464, 513-519| doi:10.1038/nature08981 ATF1
    4. 4. Ty1 Retrotransposition in Saccharomyces cerevisiae <ul><li>Reinsertion of mobile genetic elements via reverse transcription. </li></ul><ul><li>Mobile element is copied out, then reinserted somewhere else in the genome </li></ul><ul><li>Ty1 might cause genetic change associated with aging </li></ul>www.wadsworth.org
    5. 5. Projects <ul><li>Does varying Glucose concentration at 10%, 2%, and 0.2% influence Ty1 and mutational frequency? </li></ul><ul><li>How do SOD1 and ATF1 deletions impact Ty1 and mutational frequency? </li></ul>
    6. 6. Identifying Ty1 Mobility www.wadsworth.org org/resnres/bios/curcio.htm SC-His (x7) YPD (x14) Cultured 4 Days @ 20  C Plated @ 30  C Counted after 4 Days
    7. 7. <ul><li>Ty1 mobility hindered under low Glucose conditions? </li></ul>1 2 3 1 2
    8. 8. DNA Mutation Assay <ul><li>Cultured 4 Days @ 20  C </li></ul><ul><li>Plated @ 30  C </li></ul><ul><li>Counted after 4 Days </li></ul>SC-Arg + Can. (x7) YPD (x14)
    9. 9. <ul><li>DNA Mutations are lower at 0.2% Glucose Media Conditions </li></ul>1 2 3 1 2
    10. 10. Single Deletion of SOD1 and ATF1 on Ty1 Mobility and DNA Mutational Frequencies <ul><li>Increases in ROS leads to greater cellular damage and Mitochondrial dysfunction. </li></ul><ul><li>How is Ty1 mobility and DNA mutations affected by SOD1 deletions and ATF1 deletions? </li></ul>SC-His (x7) YPD (x14) Cultured 23 hours @ 20  C Plated @ 30  C Counted after 4 Days
    11. 11. Transformations via KanMx cassette <ul><li>KanMX cassette replaces ORF in mutant deletion strains </li></ul><ul><li>Transformants constructed from KanMx PCR product </li></ul><ul><li>Grow on selective plates for Kanamycin resistance </li></ul><ul><li>PCR “Check” with Kanrev. and upstream gene primer </li></ul>KANMX
    12. 12. 2000 1500 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
    13. 13. Ty1 Mobility and DNA Mutations in Mutants <ul><li>Higher mutations occurring within ATF1 mutant </li></ul><ul><li>Ty1 mobility values are congruent </li></ul><ul><li>SOD1 deletion results due to poor viability? </li></ul>
    14. 14. Conclusions <ul><li>Caloric restriction on yeast strains appear to reduce Ty1 mobility in cells grown with 0.2% </li></ul><ul><li>Not at 10%? </li></ul><ul><li>ATF1 deletion shows similar Ty1 frequencies as WT </li></ul><ul><li>SOD1 deletion reduces Ty1 mobility? </li></ul>www.impactaging.com
    15. 15. Further Work <ul><li>Adapt short-term caloric restriction experiments to longer lifespan experiments </li></ul><ul><li>Alter procedure of mutant experiments to account for reduced growth in SOD1 mutants </li></ul><ul><li>Analyze other genomic stresses that may give insight into DNA damage and aging. </li></ul>
    16. 16. Acknowledgements <ul><li>Professor Patrick Maxwell </li></ul><ul><li>Bianca Pier </li></ul><ul><li>Biology Graduate Program </li></ul>
    17. 17. References <ul><li>Burhans, W. C. and M. Weinberger (2009). &quot;Acetic acid effects on aging in budding yeast.&quot; Cell Cycle 8 (14): 2300-2302. </li></ul><ul><li>Garfinkel, D. J. (2005). &quot;Genome evolution mediated by Ty elements in Saccharomyces.&quot; Cytogenetic and Genome Research 110 (1-4): 63-69 </li></ul><ul><li>Kaeberlein, M. (2010). &quot;Lessons on longevity from budding yeast.&quot; Nature 464 (7288): 513-519. </li></ul><ul><li>Servant, G., C. Pennetier, et al. (2008). &quot;Remodeling yeast gene transcription by activating the Ty1 long terminal repeat retrotransposon under severe adenine deficiency.&quot; Molecular and Cellular Biology 28 (17): 5543-5554. </li></ul><ul><li>Stamenova, R., M. Dimitrov, et al. (2008). &quot;Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation.&quot; Cryobiology 56 (3): 241-247. </li></ul><ul><li>Stoycheva, T., M. Pesheva, et al. (2010). &quot;The role of reactive oxygen species in the induction of Tyl retrotransposition in Saccharomyces cerevisiae.&quot; Yeast 27 (5): 259-267. </li></ul>
    18. 18. PCR Results DNA Ladder M7 ATF1 M7 SOD1 ATF1 SOD1 DNA Ladder M7 ATF1 ATF1 500 1Kbp 1.5 2 2.5 500 1Kbp 1.5 2 2.5
    19. 19. 500 1000 1500 2000 Ladder PCR “Check” of Transformants Finding Transformant Colonies

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