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.
3.
4.
5.
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.
8.
9.
10.
11.
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
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--->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).
![How are cellular stresses related to aging? ,[object Object],[object Object],[object Object],[object Object],[object Object],www.impactaging.com](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)