Natalie Rios

Journal of Undergraduate Research
Volume 6, Issue 6 - March 2005

An Episomal Model for Aging in Saccharomyces cerevisae

ABSTRACT

Saccharomyces cerevisiae, commonly known as a baker’s yeast, has become a key model organism for studying the molecular basis of cellular aging. Aging mechanisms in yeast have been studied for years, and one hypothesis suggests that extrachromosomal rDNA circles (ERCs) accumulate in the mother cell ultimately causing its senescence. To test this hypothesis, autonomously replicating (ARS) plasmids that “mimic” ERCs were constructed. Plasmid transmittance was monitored by measuring mitotic stability. Our results show that ARS-plasmids and ERCs “compete” for retention in the mother cell. Furthermore, we also showed that ARS-plasmids compete with each other when in the presence of one another. Since ERCs retained in the mother cell are rarely passed on to daughter cells due to an asymmetric inheritance process known as mother inheritance bias, we contend that ERCs and ARS-plasmids accumulate due to this mother cell bias inheritance mechanism during the aging process in yeast.

INTRODUCTION

In 1996, Saccharomyces cerevisiae, commonly known as baker’s yeast, became the first eukaryote to have its genome fully sequenced. Like yeast, humans are also eukaryotes, and thus many similarities exist in their genomes. It is estimated that one third of the yeast genes are related to human genes. ¹

All eukaryotes exhibit similarities in their cellular anatomy, and even similarities in cell function. Yeast DNA contains stretches of subunits called bases which encode for proteins that have also been found in human DNA. These protein sequences must play an important role in cell function because they have been conserved by evolution for the billion years that separates humans from yeast. Importantly, certain yeast and human genes seem to be similar in aging functions. Human diseases, such as the Werner Syndrome, which is caused by a mutation to the human WRN protein, have yeast analogs. The Werner Syndrome is a genetic disease that leads to premature aging. Yeast cells with mutations in the SGS1 protein show accelerated aging like those with the Werner Syndrome. Studying the yeast genome and its aging mechanisms will give insight to similar aging mechanisms in humans.²

S. cerevisiae is an excellent model with which to study aging because it is a budding yeast as shown in Fig. 1. Budding is the process by which the mother cell divides and gives rise to a bud which becomes the daughter cell. This division is an asymmetric process which means that the mother cell and daughter cell are not identical and can be tracked individually. The age of the mother cell is determined by the number of daughter cells it produces. A mother cell has a limited capacity to produce daughter cells, and the decline in this capacity with each generation is known as replicative aging.³

Figure 1. Budding Yeast Cell Division. Mother cell(M) gives rise to daughter (D) cells in an asymmetric process.

Aging mechanisms in yeast have been studied for years, and one hypothesis suggests that extrachromosomal rDNA circles (ERCs) accumulate in the mother cell causing its senescence[mss1] . ERCs are the product of homologous recombination at the ribosomal DNA (rDNA) locus, which is transcribed to form ribosomal RNA (rRNA). Since ERCs contain origins of replication, each ERC has the ability to replicate each cell cycle. ERCs are retained by mother cells and rarely passed on to the daughter cells due to an asymmetric inheritance process known as mother cell bias. As the ERCs continue to accumulate, it has been hypothesized that cell functions are hindered, including replication mechanisms, eventually leading to cell senescence.⁴⁵

Figure 2. ERC Model for Regulating Yeast Life Span. ERCs are the product of homologous recombination at the ribosomal DNA (rDNA) locus, which is transcribed to form ribosomal RNA (rRNA). They are retained in the mother cell during mitosis and their accumulation is linked to replicative aging. Obtained from Ref. 4.

However, not all hypotheses agree that the accumulation of ERCs is the cause of aging. Others believe that ERC accumulation is more of an effect of aging[mss1] . Shortened lifespan in yeast may also be due to mutations impairing DNA replication, recombination, and repair. By this view, ERC accumulation has little to do with cell senescence. Previous work by Falcon and Aris tested the ERC hypothesis using a “plasmid-based model.” Their work demonstrated that plasmids accumulate in mother cells and reduce lifespan[mss2]. They showed that plasmid copy number increases with mother cell age and that inheritance of plasmids is affected by ERC levels in the cell. Furthermore, their work also suggests that ERCs are not merely an effect of aging. To continue probing this controversy, we have designed an experiment similar to that of Falcon and Aris with different plasmids. The two types of plasmids used are ARS plasmids and ARS/CEN plasmids, which are shown in Fig.3.

Figure 3. ARS1 and ARS1/CEN4 Plasmids.

Autonomously replicating sequence (ARS) plasmids, which act most like ERCs, are circular DNA molecules with replication origins. In previous experiments they have exhibited a strong mother cell bias, making them an ideal plasmid to mimic ERCs. It is predicted that the ARS plasmids will accumulate in the mother cell and reduce its lifespan.

Autonomously replicating/centromeric sequence (ARS/CEN) plasmids have a centromeric DNA region that attaches the plasmid to the mitotic spindle during replication. This mechanism ensures that the plasmid is effectively passed on to the daughter cell. Like the ARS plasmids, ARS/CEN plasmids also contain a replication origin. However, since ARS/CEN plasmids do not exhibit mother cell bias, they should be distributed throughout the population and not accumulate in any given cell. Thus, it is predicted that ARS/CEN plasmids will not accumulate or reduce lifespan in the mother cell.

Each plasmid will also contain a nutritional marker unique to its type so that cells can be grown under the desired selection. TRP1, URA3, and LEU2 will be used as the markers. If cells are grown in the absence of tryptophan, only the cells that inherited a plasmid with the TRP1 marker will grow. The same holds true for uracil and the URA3 marker, and leucien and the LEU2 marker.

The plasmids were transformed into strains of varying levels of ERCs to monitor the interaction between plasmids and ERCs. To quantify this, the inheritance of the plasmids has to be measured by determining the mitotic stability. Our results show that ARS plasmids are not inherited well by the daughter cells and thus accumulate in the mother cell. In contrast, the ARS/CEN plasmids are inherited by the daughter cells and little accumulation in the mother cell occurs.

These results[mss1] support the findings of Falcon and Aris which state that ARS plasmids and ERCs have similar inheritance mechanisms. Further research shows that when two ARS plasmids are introduced to the same strain, the mitotic stability of each plasmid is affected. This suggests that ARS plasmids compete among themselves much like ERCs compete with the ARS plasmids. It also supports the idea that mother cells have a limited capacity for episomes and thus once that critical threshold is reached, the mother cell begins to pass plasmids and ERCs onto the daughter cells[mss2][mss3].

EXPERIMENTAL

Yeast Strains and Plasmids

Plasmids were transformed into three different yeast strains. The wildtype strain was used to monitor ERC and plasmid interactions in the typical yeast strain. Two mutant strains, SIR2Δ and fob1Δ, were used to determine the effect different levels of ERCs would have on the plasmids. The SIR2Δ strain has a deleted SIR2 gene. Expression of the Sir (silent information regulator) protein complex has been correlated with yeast life span. Yeast strains with a deleted SIR2 show a reduction in life span, while overexpression of SIR2 shows an increase in life span. We chose to work with SIR2Δ because the deletion of the SIR2 gene leads to an increase in ERC levels and a reduction in life span as compared to the wildtype. The fob1Δ, on the other hand, is known to have a reduced level of ERCs and therefore an increased life span as compared to the wildtype.

The wildtype strain, W303AR5 (MAT_ leu2-3,112 his3-11,15 ura3-1 ade2-1 trp1-1 can1-100 RAD5 ADE2::rDNA, [cir+]), was obtained from D.A. Sinclair. The SIR2Δ strain used has a genotype of RMY206-5B SIR2 MAT_ SIR2::HIS3 hmr::TRP1leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100 [RAD5+]. The fob1Δ strain’s genotype is MAT_ fob1::URA3 leu2-3, 112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [RAD5+]. Both strains have the same parent strain HKY580-10D.

Six plasmids were used throughout these experiments. Descriptions of these plasmids can be found in Table 1. Plasmids pJPA113, pJPA133, and pAF31 are ARS plasmids. The ARS/CEN plasmids were pJPA136, pJPA116, and pAF32. The plasmids were transformed in the yeast strains using a standard lithium acetate method described below.

Plasmid	Origin, Insert	Marker	Backbone	Size (kb)
Table 1. Descriptions of Plasmids Used in Experiments
pJPA113	ARS1	URA3	pRS306	4,575
pJPA116	ARS1 , CEN4	URA3	pRS306	5,316
pJPA133	ARS1	LUE2	pRS306	5,698
pJPA136	ARS1 , CEN4	LEU2	pRS306	6,468
pAF31	ARS1	URA3, ADE2	pJPA113	6,828
pAF32	ARS1 , CEN4	URA3, ADE2	pJPA116	7,568

In the lithium acetate method for transforming yeast, the yeast are first grown in YPD medium to an optical density (OD) between 0.2 and 0.4 as measured by a UV/Vis spectrometer at 600 nm. (The YPD medium consists of Bacto Yeast Extract, Bacto Peptone, and Dextrose.) About 2 OD units are needed per plasmid transformation where an OD₆₀₀ of 0.4 in 25 mLs is equivalent to about 10 OD units. Once enough cells have been gathered, they are washed with deionized water and kept on ice. The pellets are then washed with 1 mL of TE/LiOAc. For 2 mL of TE/LiOAc, 200 µL 10X TE, 200 µL 1M LiOAc, and 1.6 sterile ddH₂O were used where 10X TE is made of 100 mM Tris-HCl and 10 mM EDTA at pH of 7.5.

After washing the pellet with TE/LiOAc, the cells are resuspended in 100 µL of TE/LiOAc per 1-2 OD units. In a microfuge tube, 5 µL of 10 mg/mL carrier DNA (HMW, ssDNA, sonicated), 0.5-5 µL transforming DNA, and 100 µL of yeast cells are mixed for each plasmid transformation. A fresh solution of PEG/LiOAc is prepared using 200 µL 10X TE, 200 µL 1M LiOAc, and 1.6 mL of 50% PEG 3400 for 2 mL of solution. To each microfuge tube, 0.6 mL of the PEG/LiOAc solution is added and the tubes are rotated end-over-end for 30 minutes. Afterwards, the tubes are placed in a water bath at 42óC for 15 minutes. Finally, the cells are resuspended in 200 _L of 1X TE of which 100 _L is plated on the appropriate medium.

Mitotic Stabilities

The mitotic stability of each plasmid was determined in order to know about its inheritance. The mitotic stability is defined as the percentage of colony forming units that contain the plasmid in a given population. Each plasmid was transformed into W303AR5, SIR2Δ, and fob1Δ. Five transformants were then grown for each plasmid in each strain for 2 days at 30°C in selective synthetic dextrose (SD) liquid medium. The SD medium is made of Dextrose, Yeast Nitrogen Base, and Ammonium Sulfate. The OD of each cell culture was then measured to determine the volume of cells needed to plate approximately 200-250 colony forming units on nonselective SD medium. This was accomplished using the fact that an OD between 1.1 and 1.5 corresponds to approximately 0.5 – 1 X 10⁷ colony forming units. After 200-250 colony forming units were plated, they were incubated for 2-3 days. Once the cells had grown to a countable size (1-2 mm diameter), they were replica plated onto selective and nonselective plates. Replica plating is a procedure by which cells from colonies on the original (“master”) plate are transferred to new plates. This is done by placing felt sheets on a circular metal block with a slightly smaller diameter than that of the plates. The plates are then placed on the felt so that each colony forming unit transfers cells to the felt. The new plates are then placed on the felt so that cells will be transferred to them as a replica of the original plate. These plates are incubated at 30°C as well and left to grow for 3-4 days. For mitotic stability counts, the plates are replica plated onto selective and nonselective plates. The selective plates contain all the amino acids necessary for the cells to grow except for the amino acid that the plasmid contains. For instance, pJPA133 has a LEU2 selective marker. Therefore, to grow it in selection means to grow it on medium without LEU2 so that only the cells that contain the plasmid can survive.

After the cells are at a countable size, the numbers of cells on the selective and nonselective plates are counted. The ratio of cells on selective to nonselective plates is the mitotic stability. This ratio serves as a measure of how well a plasmid is inherited by a population. A high mitotic stability indicates that the mother cell effectively passes on the plasmid to the daughter cells. A low mitotic stability suggests that the plasmid has asymmetric inheritance and that the mother cell is prone to retaining the plasmid.

RESULTS AND DISCUSSION

The results obtained support the results obtained previously by Falcon and Aris [mss1]. New conclusions as to how plasmids interact with one and other are also presented in these results. They show that plasmids affect each other’s inheritance and suggest that their inheritance mechanisms are similar to ERCs.

Figure 4. Mitotic Stability Results for pJPA133 and pJPA136.

Plasmid and Strain	Mitotic Stability
Table 2. Mitotic Stability Results for pJPA133 and pJPA136
W303 pJPA133	15.55
fob1Δ pJPA133	20.98
sir2Δ pJPA133	24.50
W303 pJPA136	96.35
fob1Δ pJPA136	97.82
sir2Δ pJPA136	95.27

The first plasmids tested were pJPA133 and pJPA113. They were transformed with the wildtype strain, W303, and the mutant strains, fob1_ and sir2_. The results obtained from the mitotic stability experiment are shown below in Table 2 and in Figure 4. The ARS plasmid pJPA133 acted as predicted in all three strains. The mitotic stability of the plasmid was highest in the sir2_ mutant. This is expected because the sir2_ strain has a high level of ERCs. ARS plasmids are known to act like ERCs and thus the rapid accumulation of plasmids and ERCs leads to impairment of the mother cell bias. This result suggests that the mother cell has a limited capacity for episomes. As such, it is reasonable to conclude that the competition of ARS plasmids and ERCs leads to the transmission of the episomes to the daughter cells. Conversely, fob1_ has a reduced level of ERCs as compared to sir2_, and thus the plasmid does not ‘compete’ with ERCs to the same extent as in sir2_. This difference in “level of competition” is reflected in the results since the mitotic stability of pJPA133 in fob1_ is lower than in sir2_. The mother cell has a higher capacity to retain the plasmid in the fob1_ strain since there are less ERCs for the plasmid to compete with. The ARS/CEN plasmid acted as predicted and did not accumulate in any of the strains.

Figure 5. Mitotic Stability Results for pAF31 and pAF32.

Strain and Plasmid	Mitotic Stability
Table 3. Mitotic Stability Results for pAF31 and pAF32
W303 pAF31	15.66
fob1Î pAF31	16.2
sir2Î pAF31	29.15
W303 pAF32	93.91
fob1Î pAF32	95.17
sir2Î pAF32	96.5

The mitotic stability results for pAF31 and pAF32 showed similar trends to those described above. Again, the ARS plasmid had the highest mitotic stability in the sir2_ strain which is known to have increased levels of ERCs. The mitotic stabilities of pAF31 in the wildtype and fob1_ strains were significantly lower than that in the sir2_. This supports the conclusions made earlier with pJPA133. The mitotic stability of pAF32 was in the 90th percentile for all three strains as predicted. CEN plasmids are known not to accumulate in any given cell and thus are inherited efficiently throughout the population.

Figure 6. Mitotic Stability Results for Double Transformants.

Plasmid (MS of plasmid in bold)	Mitotic Stability (MS)
Table 4. Mitotic Stability Results for Single and Double Transformants with W303
113	16.79
133	13.38
113 + 133	24.49
133 + 113	21.36
113 + 136	14.11
133 + 116	14.59
136 + 113	93.19
116 + 133	86.29
136	93.26
116	91.25

Finally, we tested the mitotic stability of plasmids in a double transformant environment. Plasmids pJPA113 and pJPA133 are both ARS plasmids which are predicted to compete with ERCs for retention in the mother cell. Plasmids pJPA116 and pJPA136 are CEN plasmids which do not accumulate in the mother cell. We transformed W303 with all four plasmids alone, and then double transformed W303 with combinations of the plasmids. Our results showed that ARS plasmids in the presence of another ARS plasmid have an increased mitotic stability. The mitotic stability of pJPA113 increased from 17% when alone to 25% when in the presence of another ARS plasmid, pJPA133. Similar results were obtained for pJPA133 in the presence of pJPA113. Normally, the mitotic stability of pJPA133 is 14%, but when transformed with pJPA113, its mitotic stability increased to 21%. The mitotic stability of the ARS plasmids was unaffected when combined with CEN plasmids [mss1].

CONCLUSION

The results show that there is a clear relationship between the accumulation of episomes and the mitotic stability of ARS plasmids. The mitotic stability results of the single transformants with pJPA133 and pAF31 both show that in strains with high ERC levels, the plasmid has a greater chance of getting passed on to the daughter cell. This gives us insight to the aging mechanisms relevant to episomes. It suggests that mother cells have a limited capacity for episomes, and that as this limit is reached, the mother passes on episomes to the daughter cells. Competition among ARS plasmids and ERCs might also be an explanation for the increased mitotic stability of ARS plasmids in high ERC level environments. If the mother cell does have a limited capacity for episomes, then both plasmids and ERCs cannot be retained by the mother cell as it ages. This leads to a ‘competition’ among the episomes to stay in the mother cell.

Even more interesting is the effect that two ARS plasmids have on the mitotic stability of the individual plasmids. Both plasmids compete for retention in the mother cell but neither one seems to be dominant. The mitotic stability of both plasmids increases by similar amounts when in the presence of each other. When the ARS plasmids are in the presence of a CEN plasmid, neither mitotic stability is affected. This suggests that the competition among the ARS plasmids is similar to that among ERCs and ARS plasmids. Thus, it is reasonable to conclude that the inheritance mechanisms of ERCs are the same as those of ARS plasmids.

These conclusions are important to the aging field because they give insight to mechanisms involved with cellular senescence. Understanding these mechanisms in yeast will make studies in higher organisms possible since many analogous genes have been found among eukaryotes. Although ERCs have not been characterized in humans, it is believed that all DNA has the ability to form ERCs. Thus, understanding the role that ERCs play in aging may lead to understanding how DNA is affected by aging and how this leads to senescence in yeasts and higher organisms.

ACKNOWLEDGEMENTS

I would like to thank the following people for their invaluable help and support: Dr. John Aris, Alaric Falcon, Lauren N. Elliot, Gloria Rios, and Joshua Herr.

REFERENCES

“International Team Completes DNA Sequence of Yeast”. National Human Genome Research Institute Press Releases. April 24, 1996. Back
Sinclair, David A., Mills K, and Guarente Leonard. “Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants”. Science, vol. 277 (1997): 1259-60. Back
Sherman, Fred. “Getting Started with Yeast”. Methods of Enzymolgy. ol. 350. New York: Academic Press, 2002. Back
Falcon, Alaric A. and Aris, John P. “Plasmid Accumulation Reduces Life Span in Saccharomyces cerevisiae”. The Journal of Biological Chemistry, vol. 278 (2003): 41607-41617. Back
Sinclair, David A. and Guarente, Leonard. “Extrachromosomal rDNA Circles –A Cause of Aging in Yeast”. Cell, vol. 91 (1997): 1033-1042. Back

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