Download - Kennedy Lab Research
My Internship in the Kennedy LabStudying calorie restriction and cell respiration in yeast cells—and how these factors affect the aging process
Jason FreebergKennedy LabThe Buck Institute for Research on AgingMarch 12th to June 10th, 2013
Overview of Calorie Restriction
It has been well documented that calorie restriction (CR) slows the aging process in many species of animals Results include resistance to oxidative stress, enhanced DNA
repair, delayed onset of age-related diseases, even increased lifespan
However, a mechanic understanding of this process is still unknown We know the results of CR, but we don’t know why this
happens
My section of the Kennedy Lab set out to test if and how cellular respiration is a factor in the calorie restriction process
Overview of Cellular Respiration
General WorkflowCreate the rho0 yeast cells
Check for presence of mDNA
Selective plating DAPI staining
PCR test for mDNA Begin lifespan tests and calorie
restriction
Test for Sir2 expression in the cells
Quantify the buildup of ERC’s
Generating Cells without Mitochondrial DNA
Yeast cells lacking mitochondrial DNA (mDNA) are incapable of respiration, so this is how our lab was able to test the effects of respiration on calorie restricted cells Rho0 cells lack mDNA
I replicate plated yeast cells on media treated with Ethidium Bromide (EtBr) to create rho0 cells The EtBr “tangles” with the mDNA, and the daughter cells
will then lack the mDNA And bam! rho0 cells!
Testing for Presence of mDNA After the EtBr treatment, I created glucose and glycerol
plates I split these down the middle, and Z-streaked the left sides
with wild type yeast, and the right side with the rho0 yeast
The wildtype yeast (i) easily grows on both mediums, while the rho0 yeast (ii) is unable to grow on glycerol The rho0 cells can’t respire, so growth on the glycerol is
impossible
Testing for Presence of mDNA Alternatively, mDNA can be visualized through
fluorescence staining and imaging microscopy DAPI is a fluorescent stain for the DNA in a cell, we can
then image it under a microscope after we excite the DAPI molecule
DAPI will show as a blue color DNA is present anywhere is a blue stain on the cells Specifically, the DAPI bonds to the DNA regions with
high A-T concentration
DAPI Stains for mDNAWildtype
+ for mDNARho0 strain- for mDNA
Testing for Presence of mDNA
L 1 2 3 4 5 6 7 x 8L: base-pair ladder
1-4: 2820 rho0
5-7: 2823 rho0
x: skipped, broken well
8: wildtype (+)
PCR for mitochondrial DNA Should be no bands for rho0 because there is no
mDNA to replicate
Testing for Presence of mDNA PCR for mitochondrial DNA
Should be no bands for rho0 because there is no mDNA to replicate
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
1-4: 2820 rho0
5-7: 2823 rho0
8: wildtype (+)
Testing for Chromosomal DNA
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
As a second control, I did PCR for a section of the yeast chromosomal DNA There should be bands for all samples, even the rho0
1-4: 2820 rho0
5-7: 2823 rho0
8: wildtype (+)
Conclusions from my Time at the Buck I created Rho0 cells by
treatment with EtBr I checked the treatment
using… Glucose and glycerol plating DAPI staining for DNA PCRs for mDNA
The results show that the rho0 cells I made did lose their mDNA
Acknowledgements
Dr. Scott Tsuchiyama, my mentor Dr. Julie Mangada, head of Education Outreach Dr. Brian Kennedy, Buck Inst. CEO and PI of the
Kennedy Lab Camille Madfes, our School to Career Liaison Dr. Lafevre-Bernt, our Biotech II instructor