The overall goal of this procedure is to study age-dependent genomic instability in yeast by combining the chronological lifespan with a set of simple DNA damage and mutation assays. This is accomplished by first sampling chronological aging cultures every two days and checking for viability by colony forming unit or CFU formation. The next step of the procedure is to examine the DNA mutations by sampling aging cultures and plating cells on mutation specific selection medium.
Ultimately, results can be obtained that show changes in DNA mutations during yeast chronological aging through normalizing the mutation assay results to the viability. This method can help answer key question in the aging field, such as the mechanisms that link aging and genomic instability in aria To assay for yeast chronological lifespan or CLS first inoculate a single yeast colony into one milliliter of synthetic complete glucose, or SDC medium and incubate overnight with shaking at 30 degrees Celsius on the following day. Dilute the overnight culture into 10 milliliters of fresh SDC medium to an OD 600 of 0.05 to 0.1 incubate with shaking at 30 degrees Celsius for three days.
On day three, when cells are in stationary phase, dilute each Eloqua 10, 000 times in autoclave water, then remove two 10 microliter aliquots from the flask and plate 10 microliters of each diluted culture on A-Y-E-P-D plate. Incubate the plate at 30 degrees Celsius until colonies arise. The number of colonies arising or colony forming units from the day three culture is considered 100%survival.
Continue removing aliquots from the aging culture in subsequent days for plating. Adjusting the dilution factors accordingly to ensure about 20 to 100 colonies in the CFU assay. Sampling is stopped when the viability of the culture has decreased below 1%typically between days 11 and 13, cell viability can also be studied in cells aged on plates.
To begin this assay inoculate a single colony into one milliliter of synthetic complete glucose medium, and incubate overnight with shaking at 30 degrees Celsius. When the culture has grown to about 10 to the eighth cells per milliliter, dilute the culture with autoclave water to 100 to 200 cells per 10 microliters and 1000 cells per 10 microliters. For each strain, prepare a set of eight to 20 trytophan dropout SDC plates labeled according to plating density.
To ensure 10 to 100 colonies can be counted in anticipation of decreased viability during aging plate two aliquots of 10 to 30 microliters of diluted culture onto each plate. Incubate the plate set at 30 degrees Celsius for the duration of the lifespan analysis on the day of plating and every two days subsequently, remove one plate and dropwise add 0.5 milliliters of trytophan to allow viable cells to grow. Incubate the plate at 30 degrees Celsius for an additional 48 to 72 hours.
After 48 to 72 hours. Count the CFU for the viability for the day of tryptophan edition to assay four DNA damage and mutation frequency during chronological aging. A number of strange with special features are grown in parallel with the wild type strain for the yeast CLS assay.
Spontaneous mutation frequency can be evaluated by measuring the frequency of canna vanning resistance in a chronologically aging culture. Mutations in the can one gene which encodes the plasma arginine permease render cells resistant to the arginine analog L Canavan Canavan resistant cells will grow on arginine dropout Synthetic complete medium plates supplemented with L Canavan sulfate measurement of trip positive reversion frequency in a strain containing an amber mutation in the trip. One coating sequence allows the estimation of the rate of base substitution during yeast chronological aging trip.
Positive reversion cells will grow on tryptophan dropout plates. The lyse negative strain EH one 50 harbors a plus four shift in the open reading frame, which results in ox atrophy for lysine, and this can be reversed by small insertion or deletion mutations. The revertants will be selected on lysine dropout plates to detect gross chromosomal rearrangements or gcr.
A mutant strain is used in which HX T 13 located 7.5 kilobases telomeric to can one on chromosome five is disrupted by a U three cassette mutations in both can one and U three genes render cells resistant to L Canavan and five fluoro orotic acid respectively. Since the frequency of point mutations occurring in both genes is low analysis of the frequency of resistance to both El Canavan and five fluoro orotic acid provides an estimation of gcr to monitor the level of homologous and homologous recombination. During chronological aging mutants are generated that carry either 100%homologous inverted repeats or 91%homologous.
IRS at the his three locus recombination between the IRS allows the expression of functional his three protein. Therefore, cells in which recombination has occurred will grow on histidine dropout plates supplemented with galactose. On day three, when cells are in stationary phase and appropriate amount of cells will be removed from each aging culture.
Since the same procedure is used for all the assays, only one sample will be processed. For the purpose of this video, pellet the cells by spinning in a benchtop centrifuge. Remove the supernatant and resuspend the cells in one milliliter of autoclave water pellet.
Again, remove the supernatant and resuspend the cell pellet in 100 microliters of water plate cells on the appropriate plates to select for the desired reverts. Incubate plates at 30 degrees Celsius after three to four days count CFUs, the mutation frequency is normalized to the number of viable cells. This graph shows representative results from a typical chronological lifespan assay when the chronological survival of wildtype cells is compared to that of S CH nine delta TOR one delta and RAs two delta mutants.
In liquid culture mutants lacking TOR one s, CH nine, or RAs two show an extended chronological lifespan. Furthermore, deficiencies in s CH nine and RAs two show an additive effect on longevity promotion chronological survival in the presence of various carbon sources. Using the NC two viability assay is shown in this next graph.
Day one, SDC wild type cultures were diluted and plated onto SC trip dropout plates with no carbon source. SC Tripp dropout plates supplemented with glucose ethanol or glycerol orates plates were incubated at 30 degrees Celsius every two days. One plate was retrieved from each set and appropriate nutrients were added to allow growth and colony formation.
Age dependent mutation frequency varies greatly depending on the strain, background, genetic manipulation, culture, conditions, and age. This table shows typical results obtained in a wild type strain. Representative results of trans lesion synthesis is shown in this next image.
The age dependent mutation frequency increase may involve changes in erroneous DNA repair using nuclear extracts and various DNA templates. We can evaluate the translation synthesis or TLS in the chronologically aged cells. Nuclear extracts from three day old stationary phase, wild type, and SCH nine delta mutant cells were analyzed.
TLS products with undamaged template are indicated by solid lines and TLS products with damaged template are indicated by dotted lines. There was no trans lesion synthesis observed in nuclear extracts from the long-lived s CH nine delta mutant free primers are indicated by the open arrow. After watching this video, you should have a good understanding of how to perform chronological lifespan and DNA mutation analysis in yeast.
