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