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CFUs measurement

CFUs measurement

Time (days)

Figure 19.1. Chronological life-span measurement and its use for the isolation of long-lived mutants. Populations of mutagenized yeast or a pool of deletion mutants obtained from the Yeast Knock-out (YKO) collection are grown in minimal medium containing glucose (SDC). After 2-3 days, cell growth stops and ethanol accumulates in the medium. At day 3 yeast are either kept in this ethanol-rich medium or switched to water. Cell viability is measured every two days by diluting the yeast cultures and plating an appropriate number of cells onto rich medium (YPD) plates to monitor the colony forming units (CFUs). When yeast are switched to water, the cultures are washed every two days to avoid the cell division that might be caused by the accumulation of nutrients released from the dead yeast. Mutants that are still alive when the majority of the population is dead are isolated and then retested individually to monitor their mean and maximum life span.

Time (days)

Figure 19.1. Chronological life-span measurement and its use for the isolation of long-lived mutants. Populations of mutagenized yeast or a pool of deletion mutants obtained from the Yeast Knock-out (YKO) collection are grown in minimal medium containing glucose (SDC). After 2-3 days, cell growth stops and ethanol accumulates in the medium. At day 3 yeast are either kept in this ethanol-rich medium or switched to water. Cell viability is measured every two days by diluting the yeast cultures and plating an appropriate number of cells onto rich medium (YPD) plates to monitor the colony forming units (CFUs). When yeast are switched to water, the cultures are washed every two days to avoid the cell division that might be caused by the accumulation of nutrients released from the dead yeast. Mutants that are still alive when the majority of the population is dead are isolated and then retested individually to monitor their mean and maximum life span.

it is of key importance to reproduce conditions similar to those under which these pathways have evolved. Although the chronological life-span paradigm may appear to be a starvation phase that does not resemble the life span of higher eukaryotes, nondividing yeast are not starving but are slowly utilizing the nutrients stored intracellularly at the end of the growth phase. An environment that lacks nutrients may not be common for certain mammals, but it is very common for microorganisms. In some circumstances even mammals have learned how to respond to long periods of starvation. For example, black bears and turkish hamsters alternate between a high and a low metabolism hibernation phase, in which stored nutrients are utilized to survive. The longer the period turkish hamsters spend under hibernation, the longer the life span (Lyman, 1981). Similarly, yeast forced to enter the low metabolism stationary phase by incubation in water survive longer than yeast grown and incubated in SDC medium, which maintain high metabolic rates (Fabrizio et al., 2004a).

In addition to the high metabolism postdiauxic life span (SDC), and the low metabolism stationary phase, under particularly severe starvation conditions, diploid S. cerevisiae can form haploid spores that may survive for years in a dormant state. However, most yeast diploid organisms enter and remain in stationary phase, and only a minority of diploid organisms form spores (Codon et al., 1995). All of our life-span studies are performed using haploid strains that behave similarly to diploid cell under most conditions but do not sporulate. Yeast spores may be the equivalent of the worm dauer larva, which also live much longer than adult worms (Guarente, 2001; Riddle, 1988). The food supply determines whether worms grow and become metabolically active adults or exit development at the L2 larva stage to enter the low-respiration dauer larva stage. In the following sections we will describe how the ability to survive of long-lived S. cerevisiae and C. elegans mutants appears to be linked to the entry into phases with similarities to starvation-response phases such as the spore or dauer.

Survival in SDC: Postdiauxic phase Most of our chronological life-span studies are performed by monitoring survival in the high metabolism postdiauxic phase (SDC medium). The SDC studies are started by diluting overnight cultures to an initial density of 1-2 106 cells/ml (OD600 of 0.1-0.2) in 10-50 ml of synthetic complete medium containing 2% glucose (SDC) as well as a 4-fold excess of the supplements Trp, Leu, Ura, and His (for DBY746-derived strains). The SDC medium contains glucose, yeast nitrogen base, agar, ammonium sulfate (nitrogen source), sodium phosphate, vitamins, metals, and salts. Yeast cultures are incubated at 30°C in flasks with a volume/medium ratio of 5:1, shaking at 220 rpm. After approximately 10 hours of growth, the glucose concentration in the medium reaches very low levels, and yeast switch from a fermentation- to a respiration-based metabolism. During fermentative growth, ethanol is accumulated and released from the cells. In wild-type DBY746 and SP1 cultures the level of ethanol undergoes an age-dependent decline, suggesting that it is used as a carbon source

(VL, unpublished results). When yeast organisms are incubated in SDC, the diauxic shift is followed by a postdiauxic phase, in which growth continues slowly until approximately 48 hours, and then stops (Figure 19.1). In the postdiauxic phase metabolic rates remain high until days 4-6 (day 0 = dilution day). The final density reached at day 3 varies from strain to strain and is usually between 7 and 15 (OD600). The mean survival of wild-type strains depends on their genetic background and ranges from 6 to 7 days (DBY746 or SP1) to 15-20 days (S288C or BY4700). A diauxic-shift-like switch from fermentation to respiration may also occur after reducing the glucose concentration in the medium from 2 to 0.5%. This form of calorie restriction increases respiration rates and causes an extension of the yeast replicative life span (Lin et al., 2002).

In a standard postdiauxic experiment we monitor survival by measuring the ability of an individual yeast cell/organism to form a colony (colony forming units or CFUs) within three days of plating onto YPD plates. CFUs are normally monitored until at least 99.9% of the population dies. The number of CFUs at day 3 is considered to be the initial survival (100% survival) and is used to determine the age-dependent mortality. Day 3 was selected considering that in our wild-type strains DBY746 and SP1 the population density does not normally increase after day 3, suggesting that the great majority of the cells have stopped dividing. To confirm that the loss of CFUs correlates with death, we have used a live/dead fluorescence assay to monitor the percentage of live cells over time. After staining a sample of cells with the FUN-1 dye, which confers red fluorescence to live cells and green/yellow fluorescence to dead cells, we have estimated the live/dead ratio and found a very significant correlation with the CFUs-based viability data (Fabrizio et al., 2003).

The ''postdiauxic phase'' differs for organisms incubated in SDC and those incubated in rich YPD medium (Werner-Washburne et al., 1996). In fact, incubation in YPD promotes a 6-7 day postdiauxic phase characterized by slow growth and low respiration followed by entry into a nondividing hypometabolic stationary phase in which organisms are highly resistant to multiple stresses and survive for up to 3 months. By contrast, incubation in SDC triggers the entry into an alternative postdiauxic phase in which only minimal growth occurs after 48 hours and metabolic rates remain high until the population begins to die.

Survival in water/YPD: Stationary phase Yeast incubated in YPD or water survive much longer than yeast grown and maintained in SDC/ethanol medium (Figure 19.1). In fact, the mean life span of strains DBY746 and SP1 in water is approximately 3 times longer than in SDC (15-20 days). Our experiments are usually repeated in water to simulate an alternate environment that may be commonly encountered by yeast in the wild and also to confirm that the extended life span of a particular mutant is not an artifact caused by regrowth. In fact, we do not monitor survival in YPD, since this rich medium may promote growth after the culture reaches the maximum density. After more than 99% of wild-type DBY746 and SP1 yeast incubated in SDC dies, in about 50% of the studies a better-adapted subpopulation is able to grow back by utilizing the nutrients released by dead cells (Fabrizio et al., 2004a). A similar phenomenon called "gasping" is observed for populations of bacteria (Zambrano and Kolter, 1996). By contrast, incubation of yeast in YPD appears to promote major increases in viability before the majority of the population has died, suggesting that some growth may occur when viability is high. Growth would create a mixed population containing both young and old organisms and possibly only young organisms, which would invalidate the survival studies.

For life-span studies in water, yeast are grown in and incubated for 3 days in SDC and are then washed with sterile distilled water and resuspended in sterile water (Figure 19.1). Viability is monitored by measuring CFUs every 2 days. The cells are washed 3 times with water every 2 days to remove all the nutrients released by dead yeast. Incubation in water and the removal of nutrients released by dead organisms minimize the chance of growth during long-term survival in stationary phase. Incubation in water decreases metabolic rates and may increase life span simply by promoting a slower rate of senescence.

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