Creation of an Aconitase Overexpression Strain Of

Creation of an Aconitase Overexpression Strain of

Saccharomyces cerevisiae for Lifespan Analysis

Steve Nunes

A Thesis Submitted to the Faculty of

The Wilkes Honors College

In Partial Fulfillment of the Requirements for the Degree of

Bachelor of Arts in Liberal Arts and Sciences

With a Concentration in Biology

Wilkes Honors College of

Florida Atlantic University

Jupiter, Florida

May 2012

Creation of an Aconitase Overexpression Strain of Saccharomyces cerevisiae for

Lifespan Analysis

Steve Nunes

This thesis was prepared under the direction of the candidate’s thesis advisor, Dr.

Paul A. Kirchman, and has been approved by the members of her/his supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences.

SUPERVISORY COMMITTEE:

______

Dr. Paul A. Kirchman Date

______

Dr. James K. Wetterer Date

______

Dr. Jeffrey Buller Date Dean, Wilkes Honors College ii

ABSTRACT Author: Steve Nunes

Title: Creation of an Aconitase Overexpression Strain of Saccharomyces cerevisiae for

Lifespan Analysis

Institution: Wilkes Honors College of Florida Atlantic University

Thesis Advisor: Dr. Paul A. Kirchman

Degree: Bachelor of Arts in Liberal Arts and Sciences

Concentration: Biology

Year: 2012

In my thesis work, I attempted to construct a plasmid that would allow stable integration of genes into the Saccharomyces cerevisiae yeast genome under the control of the repressible TetO promoter. The yeast ACO1 gene was cloned under the control of the

TetO operator and the tTA transactivator. This construct was inserted into yeast cells in order to observe the effects of aconitase overexpression on aging. Unfortunately, the transformed cells appeared incapable of aconitase expression as determined by glutamic acid auxptrophy, a phenotype of aconitase mutants. We have sequenced the pIT1ACO1 plasmid and have found many abnormalities in the promoter region. If the plasmid can be made to function as intended, the resulting yeast strain can be used in the future to determine if aconitase plays an important role in cellular aging.

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TABLE OF CONTENTS

List of Figures ...... v

Literature Review ...... 1

Methods ...... 9

Results ...... 11

Discussion ...... 14

References ...... 18

LIST OF FIGURES

Figure 1A: Electrophoresis of pIT1 digest with HindIII ...... 11

Figure 1B: SfiI digest of pIT1 plasmid ...... 11

Figure 2: Yeast colonies containing pITACO1 plasmid ...... 14

Figure 3: Expected pIT1ACO1 structure ...... 15

Literature Review

Immortality has always been an elusive dream that humans have fantasized about, no doubt leading to research on aging. The aging process is a widely studied phenomenon that occurs naturally to the cells of most living things, although some organisms seem to age more slowly and have a very long lifespan, suggesting different systems of defense against aging. Even bacteria, which were thought to simply divide infinitely and be immortal, have shown signs of aging. Some bacteria have been found to divide in asymmetrical ways; preexisting components of the mother bacteria are forced onto one of the daughter cells while the other daughter cell acquires completely new components (Ksiazek, 2010). While the final objective of lifespan research is to hopefully improve the human lifespan, research in this area must be done on model organisms such as yeast, flies, and rats. Yeast (Saccharomyces cerevisiae) are most often used for lifespan analysis because they are a very simple unicellular organism: The DNA of yeast can be manipulated fairly easily though recombination, the age of yeast cells can be easily determined through the number of budding cycles the yeast go though, and yeast have a shorter lifespan than other model organisms, allowing for multiple experiments to be done in short periods of time.

The Free Radical Theory of Aging was originally proposed by Denham Harman in

1957 to help explain the cellular aging process (Biesalski, 2002). This highly popularized theory suggests that the major cause of cell death due to age is in fact oxidative damage to cellular proteins and DNA caused by byproducts from the metabolic

processes of the cell (Biesalski, 2002). The theory also supports another theory known as the Rate of Living Hypothesis, which claims that there is an inverse relationship between the metabolic rate and the life span of an organism (Biesalski, 2002). In fact, mitochondria produce almost 90% of the reactive oxygen species found in organisms through the electron transport chain, thus organisms with higher metabolic rates would produce more reactive oxygen species (Bratic and Trifunovic, 2010). There has also been evidence showing that the decline of membrane potential of the mitochondria also leads to the increase of oxidant production and cell death (Shigenaga et al.,1994). It has been suggested that these oxygen radicals would interact with cellular components and damage proteins involved in glycolysis and in the tricarboxylic acid cycle as well as mitochondrial DNA, leading to the eventual death of the cell. Reactive oxygen species can also be produced by peroxisomal fatty acid metabolism, cytochrome P450 reactions, and phagocytic cells (Biesalski, 2002).

Although the cell is constantly producing these harmful molecules, enzymes called antioxidants are used in cells to help prevent protein and DNA damage caused by free radicals. Pamplona and Constantini (2011) defined antioxidants to be “any mechanism, structure and/or substance that prevents, delays, removes or protects against oxidative nonenzymatic chemical modification (damage) to a target molecule.”

It is hypothesized that animals, especially those that live longer, have more resistant structures to prevent oxidative damage. Specifically, long-lived animals are thought to generate reactive species at a slower rate and have macromolecules that are less sensitive to oxidative damage (Pamplona and Constantini, 2011).

The enzyme superoxide dismutase (SOD) is one example of an antioxidant free radical elimination system that has been heavily studied. Superoxide dismutase exists in three different forms requiring different coffactors: Cu/Zn SOD, Fe SOD, and Mg SOD.

The main goal of SOD is to catalyze reactive oxygen species, specifically superoxide, into oxygen and hydrogen peroxide, which is then converted into water and oxygen by the enzyme catalase. In an effort to decrease the damage done by these reactive oxygen species, and perhaps increase the lifespan of cells, scientists have to tried to overexpress and/or mutate antioxidants such as SOD in cells in an attempt to increase the defenses of the cell against oxidative damage.

Anirban Paul experimented with mitochondrial manganese SOD (MnSOD, coded for by the SOD2 gene and localized to mitochondria) to see if reduced activity would decrease cellular lifespan. Paul was able to generate Drosophila mutants that progressively reduce SOD2 expression and MnSOD function. Lifespan was progressively shortened with reduced SOD2 expression and mitochondrial aconitase activity was also found to be significantly reduced in the mutants. Although this data suggests a link between MnSOD activity and lifespan it was found that the lifespan and age dependent mortality varied exponentially with MnSOD activity suggesting that there are other factors contributing to senescence (Paul et al., 2007). Attempted overexpression of Cu/Zn SOD produced results that do not necessarily strengthen SOD's link to lifespan.

Ryan Doonan created an overexpression Cu/Zn SOD line of Caenorhabditis elegans and observed a slight increase in lifespan. However, Doonan found that the increase in lifespan is not due to SOD reducing the number of reactive oxygen species. Instead

Doonan showed that Cu/Zn SOD enhances dauer larva formation which in turn increases lifespan of the C. elegans (Doonan et al., 2008).

While genetic manipulations of antioxidants have produced some uncertain conclusions there has been recent evidence suggesting the existence of additional defenses against cellular oxidative damage. Pamplona and Constantini (2011) review three lines of defense: Inherent resistance of macromolecules to oxidative damage, internal oxidative balance regulatory components, and external oxidative balance regulatory components.

Resistance of macromolecules to oxidative damage is thought to be an evolutionary adaptation that is supported by recent evidence. In fact, it is thought that glucose, an energy rich molecule that has poor susceptibility to oxidation, is a main source of energy for many organisms because it is a stable reacting carbohydrate, and so oxidation of the molecule is easier to control than other possible sources of energy

(Pamplona and Constantini, 2011). Also, any of the highly reactive glycolytic intermediates are found in lower cellular concentrations. This may be a possible evolutionary adaptation for preventing oxidative damage, particularly in birds, which have higher blood glucose levels than mammals. Birds have lower glucose cellular permeability and a decreased sensitivity to insulin to signaling, preventing glucose from entering cells and acquiring higher intracellular concentrations of the glycolytic intermediates (Pamplona and Constantini, 2011).

Other macromolecules that exhibit higher rates of oxidation are found in lower quantities throughout the body when compared to other similar macromolecules.

Unsaturated fatty acids are macromolecules that are found in cells and are the most susceptible to oxidative damage due to the number of double bounds found in the molecule. Species that are known to have a longer lifespan on average have the least amount of unsaturated fatty acids in their cellular membranes when compared to the amounts of other fatty acids (Hulbert et al., 2007). Methionine is an amino acid that is more susceptible to oxidation by free radicals when compared to the other amino acids, and when oxidized can prevent methionine from acting as a methyl donor, causing loss of protein functionality. This may be one of the reasons why methionine content in proteins is found to be a smaller percentage (Pamplona and Constantini, 2011).

Internal oxidative balance regulatory components are physiological mechanisms meant to regulate the rate of mitochondrial free radical generation. The internal mechanisms are thought to be linked with the complex I and III of the electron transport chain since these are the major sites of reactive oxygen species generation (Pamplona and

Constantini, 2011). This mechanism is thought to work in four ways: modulation of the concentration of complexes I and III, reduction state of complex I, modulation of free radical production by uncoupling proteins, and posttranslational modifications. Lower concentrations of complexes I and III produces less radicals however, if more energy is needed then there must be a higher concentration. The second mechanism controls the degree of reduction of complexes I and III, the higher the degree of reduction the higher the rate of reactive oxygen species production. The third internal mechanism focuses around regulation of uncoupling proteins. Uncoupling proteins respond to overproduction of superoxide by catalyzing uncoupling, this lowers proton motive force

and decreases superoxide production. The last internal mechanism involves postranslational modifications of complexes I and III. Glutathionylation of complex I increases superoxide production by the complex (Pamplona and Constantini, 2011).

The external oxidative balance regulatory components involve environmental conditions surrounding the mitochondria that can influence free radical generation.

These environmental conditions include local oxygen concentrations, locations of proteins, and membrane composition (Pamplona and Constantini, 2011). The first mechanism involves hypoxia-inducible transcription factors (HIFs). HIFs promote expression of genes that help cells respond to a hypoxic or anoxic status. This can be done through a variety of mechanisms, seals for instance will store oxygen in blood and skeletal muscles (Pamplona and Constantini, 2011). A second mechanism involves cardiolipin resistance to oxidative damage. Cardiolipin is a phospholipid that contains many unsaturated fatty acids with low degrees of unsaturation. It is located within the inner mitochondrial membrane and influences the activity of complexes I and III as well as other proteins. It is thought that the cardiolipin was evolutionarily selected for regulation of complexes I and III because of its ability to resist oxidation due to its low unsaturation degree (Pamplona, 2008).

In addition to the antioxidants found in cells there are also proteins that are used to repair the damage done by the free radicals. There are many proteins involved in DNA repair, such as the excision repair mechanism, which replaces incorrect nucleotides in

DNA which could be caused by oxidative damage (David et al., 2007). Other groups of enzymes degrade oxidatively damaged proteans, repair oxidized amino acids, remove

phspholiped fatty acyl chains, and detoxify systems against carbonyl compounds

(Pamplona and Constantini, 2011).

Which are the most important cellular components to protect to insure longevity remains an open question. Liang-Jun Yan isolated mitochondrial proteins from houseflies between five and fifteen days of age and compared oxidative damage. The oxidative damage to the proteins in the mitochondria was shown by the carbonyl content, which is created when free radicals react with proteins. The damage was measured by the intensity of immuno staining and compared to the carbonyl content between proteins in the mitochondria. Yan uncovered that aconitase is the only mitochondrial protein that showed any evidence of oxidation over time. Also, the oxidation of aconitase was shown to decrease the activity the enzyme progressively as more damage is exhibited (Yan et al.,1997) which agrees with the research done by Anirban Paul when he worked with

SOD.

Recently there has been evidence to show that yeast cells control distribution of aconitase during budding to help preserve the life of the colony. Kingler found that mother cells of yeast will preferentially donate active aconitase enzymes to the daughter cell instead of inactive aconitase that has been damaged (Kingler et al., 2010). However, as the mother cell ages, the amount of aconitase that has not been damaged by free radical oxidation decreases. Eventually the mother cell will start to give damaged aconitase to the daughter cell. These daughter cells have been found to have a shorter lifespan than the daughter cells that were produced earlier that did not inherit the damaged aconitase (Kingler et al., 2010). This research suggests that aconitase is a factor

when it comes to cellular aging.

The recognition that aconitase is specifically targeted by oxygen radicals has made it a protein of interest for researchers studying the cellular aging process.

Aconitase is also known as citrate hydro-lyase and is an enzyme of the Krebs cycle

(tricarboxylic acid cycle) located in the mitochondrial matrix. In yeast cells aconitase also takes part in the glyocylate cycle which allows cells to use simple carbon compounds as a carbon source when glucose isn't available. Yeast cells also require functional aconitase in order to synthesize glutamate. A yeast strain lacking the aconitase gene

(ACO1) will be a glutamate auxotroph, these yeast cells will be unable to grow on acetate, lactate, ethanol, or glycerol (Gangloff et al., 1990).

The purpose of this experiment is to further the research of the aconitase protein and its link to cellular aging. A yeast overexpression strain will be created using a promoter region encoding and responsive to the tetracycline activator complex. This promoter will be used in order to control the expression of the aconitase gene. With the creation of this strain, a lifespan analysis of yeast can be done. If the overexpression does increase the lifespan of yeast cells this will support the hypothesis that damage to aconitase is a factor limiting cellular longevity.

METHODS

Construction of pIT1 and pIT ACO1

The pIT1 plasmid was constructed in two steps. A 1.8 kb HindIII-XbaI fragment from pCM185 was cloned into pIS373, which had been digested with the same enzymes.

The resulting plasmid was then cut EcoRI-XbaI and a 1.1 kb fragment from pCM185 generated by digestion with the same enzymes was cloned.

Construction of pIT ACO1

The pIT1 was digested using SfiI in a large scale digest to create linear strand of

DNA. This strand was then dephosphorylated using the procedure recommended by the supplier (Promega). The ACO1 gene was excised from pGEM ACO1 using SfiI and was ligated together with the dephosphorylated pIT, yielding the pIT ACO1 plasmid.

Correct orientation of the fragment was confirmed by digesting with XbaI.

Yeast transformation

Transformation of yeast cells was done by first growing cell to 2 x 107 cells/mL, then harvesting the cells by centrifugation at 3000 g for 5 min. Cells were washed in

25mL of sterile water and resuspended in 1mL of sterile water. A 1mL sample of salmon sperm was heated at 99° C for 5 min then chilled in an ice water bath. Cell suspension was then transfered to a 1.5mL microcentrifuge tube and centrifuged for 30 sec. The supernatant was then discarded. Sterile water was added until the final volume was 1mL and the cells were vortexed to suspend in the water. About 100uL of the cells were

transferred into a 1.5mL microfuge tube. Transformation Mix was then made using the following: 240uL of PEG 3500 50% w/v, 36uL of LiAc 1.0 M, 50uL heated salmon sperm, 34uL pIT ACO1 plasmid that had been digested with AscI. This tranformation mix was then put into the tube containing the yeast cells and mixed. The yeast cells were then incubated at 42° C over a two day period. The yeast cells were then selected for proper integration using the method described by Ivan Sadowski (Sadowski et al., 2007), requiring both uracil prototrophy and resistance to 5-FC. To allow “disintegration” of the pIS373 vector portion of the integrated DNA, transformants were grown without section in YPD medium overnight. After selecting for loss of URA3 on plates containing 5-FOA, the activity of aconitase was screened by growth on synthetic deficient plates lacking glutamic acid. The pIT ACO1 plasmid was then extracted from E. coli that had previously been transformed and sent to be sequenced.

RESULTS

Correct ligation of the pIT plasmid was shown by digestion with the HindIII enzyme producing 2.5 and 5 kb fragments (Figure 1A), as well as by expression of the ampicillin resistant gene on pIS373.

Figure 1. A: Gel electrophoresis showing digest of pIT1 with HindIII. The bands shown by the top arrow is the 5kb fragment and the band below it is the 2.5kb fragment. B: . Gel electrophoresis showing large scale digest of pIT plasmid using SfiI. The DNA bands on the far left are from the 1kb DNA ladder. The bright band shown is the digested pIT DNA at 7.6 kb. Digestion of the pIT ACO1 plasmid with SfiI showed a 7.6 kb fragment of DNA as well as a part of undigested DNA (Figure 1B).

Proof of insertion of ACOI was also shown through XbaI enzyme digestion and

gel elctrophoresis giving the expected fragment lengths of 2.9 and 7 kb fragments (not shown). Proper integration of the pIT ACO1 plasmid into the yeast chromosome was shown by selection on media lacking uracil. Colonies from this selection process were then grown in YPD media. These colonies were then selected for loss of the URA3 gene on 5-FOA and 5-FC containing media (Figure 2). Sequencing of pIT ACO1 showed that the promoter region contained abnormalities that were not expected and may have resulted from defective pCM185 plasmid.

Figure 2. Synthetic media plate containing leucine, lycine, uracil, 5-FOA, and 5-FC. Plate shows yeast cells that have lost the URA3 gene and that have disrupted fyc1 gene.

Figure 3. Anticipated pIT ACO1 plasmid structure.

DISCUSSION

The selection process showed that proper integration of the pIT1 component occurred however, these components were shown to have incorrect nucleotides when sequenced. Lacking the correct promoter, the pIT ACO1 plasmid did contain the correct

ACOI gene as well as the vector from the pIS373 plasmid. This being said, there are some processes that did not work as expected.

The incorporation of the tetO and tTA genes from the pCM185 plasmid into the pIS373 plasmid proved to be one of the most difficult parts of the experiment. A variety of different enzymes were tried and discarded during this process due to the difficulty of isolating the correct size fragment and incorporation into the pIS373 plasmid. It was originally planned to use the PvuII and EcoRI enzymes to excise the promoter region from the pCM185 fragment however a proper digest was unable to be completed. If this digest worked correctly the fragment would have been inserted into the pIS373 plasmid which would be linearized using EcoRI and EcoCRI enzymes. It is currently thought that the inability to correctly digest the pCM185 plasmid may have been a result of the acquired pCM185 plasmid being incorrect.

The aconitase gene that was used, ACO1, is found in Saccharomyces cerevisiae, and was a PCR product cloned within pGEM. In order to excise the ACO1 gene from the plasmid the enzyme SfiI needed to be used. The SfiI enzyme digests by forming a tetramer (four SfiI subunits bound together) around two SfiI restriction sites (Wentzell et al., 1995). When digesting DNA with only one SfiI restriction site the digest efficiency is reduced and higher concentrations of the DNA is needed in order to cleave the DNA

(even then it still may not be possible). This is because circular DNA can form supercoils and prevent SfiI from binding to two DNA molecules at once (Wentzell et al., 1995). On the first attempt of pGEM digest the using SfiI very little of the DNA was digested, and not enough to perform a ligation. CJ Kwan was able to clone ACO1 gene into the SfiI site by manipulating DNA and enzyme concentrations. A large scale digest of the pIT plasmid was done using SfiI in order to acquire plenty of linearized pIT.

The dephosphorylation of pIT was required in order to prevent the religation of pIT to itself. After dephosphorylation, the DNA was run on electrophoresis gel in order to purify the DNA from the CIP to allow for ligation to ACO1. After ligating the ACO1 gene to the pIT plasmid and transforming E. coli cells, proof of proper ligation was shown through enzymatic digest with XbaI which resulted in fragments of 2.9 and 7 kb.

Proper ligation was also shown through ampicillin resistance which is provided by the pIT plasmid.

The integration process of the pIT1 plasmid into yeast cells was done using disintegration vectors described by Sadowski et al. (2007). Confirmation of the integration of the pIT1 plasmid into the yeast chromosome was first done through selection on plates without uracil. Yeast cells that did not obtain the URA3 gene from the pIT ACO1 plasmid would not survive on this media allowing for only yeast cells that have accepted pIT ACO1 into their chromosome. Growth on non selective media allowed for the yeast cells to lose the URA3 gene at a high frequency during recombination, and either regeneration of the marker gene (fcy1) and loss of the inserted

DNA or disruption of the marker gene and retention of the inserted ACO1 construct,

which allowed for resistance to 5-FC.

The fcy1 gene encodes for cytosine deaminase which can convert 5-fluorocytosine

(5-FC) into 5-fluorouracil, a toxic compound. Since the yeast cells with the disrupted fcy1 gene are desired, these cells were placed in media containing 5-FC to select against cells containing the intact fcy1 gene. Loss of the URA3 gene was also selected for by placing 5-Fluoroorotic acid in the media also. This compound will be converted into 5- fluorouracil by 5-phosphate decarboxylase, which is encoded by URA3. Through these compounds it was possible to select for loss of URA3 and retention of the tet controllable promoter-ACO1 construct in the chromosome.

When ACO1 isn't expressed the cells develop auxotrophy towards glutamate. In order to test that the yeast strain that was created was correctly expressing ACO1, yeast cells carrying a deletion of the normal chromosomal ACO1 gene were transformed with pIT ACO1 then plated on media lacking glutamic acid. The yeast were be able to grow on the glutamic acid containing media regardless of whether the ACO1 gene was being expressed. However, there was no growth on the plates lacking glutamic acid indicating the ACO1 gene was not being expressed.

Sequencing of the pIT ACO1 showed that the promoter region was defective and contained many abnormalities that were not expected. It is uncertain yet if the pIT1 plasmid can be salvaged to form a correct aconitase overexpression strain. The incorrect promoter region would need to be excised from the pIT1 plasmid. This might be done using SfoI and PmeI, which are both blunt end restriction enzymes. The excised promoter may then be replaced with either the correct tTA transactivator and tetO genes,

or with a different promoter capable of overexpressing the ACO1 gene. Afterwards, the plasmid can be integrated into yeast cells and a lifespan analysis may be done to observe the effects of aconitase overexpression on yeast lifespan.

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