LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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Chronological lifespan analysis of stress-resistant yeasts

(Graduate School, 2024-06-28) Akaydın, Aslı Nur ; Çakar, Zeynep Petek ; 521211101 ; Molecular Biology - Genetics and Biotechnology

Saccharomyces cerevisiae has an important place in human life with its wide usage in various processes like fermentation, brewing, bread and wine production from the oldest times in history. Since its genome sequencing in 1996, it has become one of the most well-known and studied model organisms in many different areas of biology such as cell biology, biotechnology, cancer and aging research. Compared to other model organisms, its ease in genetic manipulation and cultivation conditions made it a convenient host for the production of heterologous proteins and economically valuable products. Yeast shares 30% of homology with many human genes, thus it is a convenient platform to study eukaryotic cell metabolism along with disease models. Aging is a common process all living organisms share and involves numerous metabolic and physiological changes that usually result from accumulated damage and deterioration. Despite the developed technology and improved living conditions through every passage of human life, it is estimated that the aging population will cover one-fifth of the whole population of the world by end of the century and age related diseases will cause a socioeconomic burden to governments. Studying aging in humans is complicated because of the long lifespan and economic and ethical concerns. Although a wide range of organisms share similar aging patterns, using yeast provides a convenient and accurate eukaryotic model with a shorter lifespan and easier growth capability. There are two main approaches to studying aging in yeast: chronological lifespan (CLS) and replicative lifespan (RLS). CLS seeks to analyze the lifespan of undivided yeast cells after they enter the stationary phase, mostly caused by decreased nutrients or toxic metabolite accumulation. RLS defines the number of cell divisions a cell undergoes before its death. CLS analysis is particularly useful for analyzing the response and survival of the cell against certain stress factors and modeling G0 cells that arrest their cell cycle. Metabolic engineering is an effective biotechnological approach for improving metabolic processes and product formation of the organism by altering the existing mechanisms or introducing new ones through the usage of recombinant DNA technology. In classical metabolic engineering, information on metabolic, genetic and physiological data of the strain is gathered, then the manipulations on relevant factors are employed to obtain the desired phenotype. Yet in the inverse metabolic engineering approach, for example when evolutionary engineering is employed, the desired phenotype is achieved using laboratory-based evolutionary settings. In this strategy, yeast strains can become resistant to certain stress factors or produce desired molecules throughout the increased stress treatment during culture. After obtaining the desired phenotype, yeast strains are examined by genomic or transcriptomic analyses to further determine the molecular changes in the genome or transcriptome. With more advanced xix technologies such as CRISPR-Cas9, altered genetic traits can be transferred to wild type strains to evoke the same resistant phenotype. In this study, previously obtained stress-resistant S. cerevisiae strains were analyzed for their CLS and viability performances. Stress factors selected for this purpose were antimycin, boron and freeze-thaw stresses which can affect the production efficiency or viability of yeast strains. In parallel with the general evolutionary engineering strategy, strains were obtained by increasing the stress levels gradually, which is the concentration of the compound in the case of antimycin and boron, and repeat numbers in the case of freeze-thaw stress, in selection cultures until the resistant population is achieved. It was shown previously that the evolved strains could become cross resistant to other stress types or their longevity can be affected by the process. The aim of this study was to determine which type of stress resistances can extend or shorten the CLS of the yeast thus affecting the lifespan of the industrial and laboratory yeast strains. For this aim, both quantitative and semi-quantitative CLS analyses were carried out. In the semi-quantitative CLS experiment, OD600 values of the yeast strains were set to 6 before they were spotted onto agar plates every 2nd day with serial dilutions. The longevity of the resistant strains was compared with their control strains visually, based on the growth on the plates. According to the results, P8 which is the freeze-thaw stress-resistant, industrial polyploid strain had a longer CLS than its industrial reference strain, whereas the antimycin and boron-resistant strains did not have a longer CLS than their reference strain. In the second part of the study, quantitative CLS analysis was done by spreading the long-lived industrial P8 strain along with its industrial reference strain R625 and the laboratory reference strain 905 to agar plates. The longevity was measured by counting colony-forming units (CFUs). The experiment was repeated until the viability of the cultures was reduced to 0.0001% from day 0 of the experiment where the viability was accepted as 100%. In the second part of the study, further validating the results from the semi-quantitative analysis, P8 exhibited longer CLS compared to its industrial reference strain and could live until the 10th day of the experiment. Among the various stress-resistant strains tested in this study, only the freeze-thaw stress-resistant, industrial P8 strain was found to have a longer CLS. However, the antimycin and boron-resistant yeast strains did not show a longer CLS compared to their laboratory reference strain. Since the freeze-thaw response was previously associated with oxidative stress response and nutrient metabolism alterations, the longer CLS of the freeze-thaw stress-resistant industrial strain can be related to changes in respective pathways that originated from the evolutionary engineering process. In the scope of the research done for this study, despite being studied in other organisms, the effect of boron resistance on longevity was studied in yeast for the first time. Similarly, antimycin resistance was examined for its effect on longevity in yeast for the first time, as well. Further studies to analyze genomic and transcriptomic changes that occurred by the acquired resistance can be performed and these changes can be transferred to wild-type or reference strains to assess the viability and CLS profiles.
Comparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain

(Graduate School, 2022) Şimşek, Burcu Tuğba ; Çakar, Zeynep Petek ; 737560 ; Molecular Biology - Genetics and Biotechnology Programme

Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.
Evolutionary engineering of freeze-thaw stress-resistant yeasts without using chemical mutagenesis

(Graduate School, 2023-06-20) Balaban, İrem ; Çakar, Zeynep Petek ; 521211107 ; Molecular Biology - Genetics and Biotechnology

Saccharomyces cerevisiae, also known as budding yeast or baker's yeast is a unicellular microorganism from the fungi kingdom. It has been consistently used in winemaking, brewing and baking bread throughout human history. After 1930s, laboratory studies were conducted to obtain strains with increased product quality. Today, S. cerevisiae is the most popular yeast strain due to its good fermentative abilities. S. cerevisiae with high fermentation performance and tolerance to environmental stresses is preferred for industrial applications. During bread production, yeast cells are exposed to a variety of environmental stresses including freeze–thaw, high sugar concentrations, air-drying and oxidative stress. Stress conditions cause a decline in cell growth rate, product yield and quality. Cells give responses to stress conditions, as environmental stress response (ESR) and stress-specific response. ESR mechanism is not specific to the stress factor and it can be used to explain the cross-resistance of the yeast cells against various stress types. One of the reasons for cross-resistance is the use of the same transcription factors as a response to a variety of different stresses. S. cerevisiae is exposed to freeze-thaw stress during the cryopreservation and frozen dough process. Freeze-thaw stress causes physiological injuries to cells. At high freezing rates, formation of intracellular ice crystals causes cellular damages; while at low freezing rates formation of extracellular ice crystals causes cellular dehydration. The thawing process causes oxidative stress which leads to oxidative damage on proteins, nucleic acids and other biomolecules inside the cell. Studies conducted in S. cerevisiaes' stress-specific response against freeze-thaw stress revealed cells focus on regulating the contents of the cell membrane, protecting cell wall integrity, increasing degradation of damaged proteins from stress and increasing overall protein synthesis under stress conditions. Cryoprotective agents can be added to decrease ice crystal formation under freezing conditions. Alternatively, yeast levels in the product can be increased to increase product yield. However, these methods can decrease product quality and increase cost. Thus, stress-resistant S. cerevisiae strains are preferred for industrial applications. Stress-resistant strains can be obtained by metabolic engineering. Evolutionary engineering is an inverse metabolic engineering method that mimics the natural evolution process. In this approach, the desired phenotype is selected first and the genes responsible for the phenotype are determined later by reverse engineering methods. In this study, freeze-thaw resistant yeast strains were obtained with the evolutionary engineering method. A reference yeast strain was exposed to freeze-thaw stress in the form of pulse stress selection. The evolved strains obtained under stress conditions generally show mutations mainly in their stress-induced genes. This allows ease in reverse engineering studies to determine genes related to the applied stress. Freeze-thaw stress was applied in the form of pulse stress selection to maintain the survival rate of cells with increasing stress levels and to induce selective pressure. In this study, a S. cerevisiae CEN.PK113-7D reference strain was exposed to gradually increasing freeze-thaw stress until the final population was obtained. The final population was obtained after 10 cycles of freeze-thaw stress application. Ten mutant individuals were randomly selected from the final population and their resistance to freeze-thaw stress was tested with the spot assay method. Four evolved strains labeled as FT-1, FT-5, FT-6 and FT-9 that showed the highest freeze-thaw resistance were selected for detailed analysis. Further physiological characterizations of the evolved strains were made by cross resistance analysis. FT-1, FT-6 and FT-9 showed cross-resistance to potassium chloride (KCl) and iron stress. KCl, at high concentrations, causes hyperosmotic stress to the cell. This cross-resistance could be the result of a similar response mechanism activated by the cell to protect itself from dehydration caused by freezing stress. Metals such as iron increase generation of ROS in cell and cause oxidative stress. The cross resistance to iron stress could be the result of activation of similar pathways used by the cell as a response to oxidative stress caused by thawing process. All evolved strains tested showed resistance to boric acid. Boric acid disrupts cell wall synthesis in S. cerevisiae. The freezing process also causes cell wall damage in S. cerevisiae. Inducing cell wall synthesis due to freezing stress may also result with increased resistance to boric acid. The aim of this study was to obtain freeze-thaw stress-resistant S. cerevisiae strains from a reference laboratory strain, without using chemical mutagenesis, by evolutionary engineering. Physiological characterization of the evolved strains was also performed by determining their cross-resistance to selected stress factors. Further genomic, transcriptomic and proteomic analyses could be performed on the selected FT-9 strain to identify the genes, pathways and molecular mechanisms responsible for resistance against freeze-thaw stress and the pathways that cause cross-resistance to selected stress factors.
Evolutionary engineering of rapamycin-resistant yeast

(Graduate School, 2022-12-02) Esen, Ömer ; Çakar, Zeynep Petek ; 521191120 ; Molecular Biology-Genetics and Biotechnology

The budding yeast Saccharomyces cerevisiae, a unicellular eukaryotic microorganism, is widely used in many industrial processes such as baking, alcohol fermentation, biofuel and recombinant protein production as well as in basic research to understand the complex biological processes of advanced eukaryotic organisms, including humans due to its well-characterized genome and proteome, ease of growth and manipulation, as well as the similarity of its genes and pathways to higher organisms. Rapamycin is a macrolide compound that is produced by the bacterium Streptomyces hygroscopicus. In the medical field, rapamycin and its analogs are used to prevent organ transplant rejection, coat coronary stents and treat tumor cells. Many key metabolic pathways including cell growth and lifespan, protein synthesis and ribosome biogenesis, regulation of cell cycle and size, environmental stress response, nutrient uptake, starvation control, and autophagy in S. cerevisiae and also other higher eukaryotes are affected by rapamycin due to its inhibition of the target of rapamycin (TOR). Fkbp12 forms a complex with rapamycin that interacts with TOR resulting in its inhibition where the exact mechanism is still unknown. Fkbp12, immunophilin, is conserved in its structure and function among eukaryotes from yeasts to mammalians. Fpr1 found in S. cerevisiae is an orthologue of Fkbp12 found in humans. In this study, an inverse metabolic engineering strategy, evolutionary engineering was used to obtain rapamycin stress-resistant S. cerevisiae. Thus, serial batch cultivation of the S. cerevisiae CEN.PK113-7D reference strain under gradually increasing rapamycin stress was carried out. Before selection, a screening experiment was performed and 3 ng/ml rapamycin stress was determined as the initial stress level. During the selection process, the concentration of rapamycin in the medium was gradually increased from 3 ng/ml to 200 ng/ml over 61 daily passages or populations. From the final population, fifteen individual colonies were randomly selected which were named R1 to R15. Every individual (R1 to R15) was highly resistant to rapamycin stress in comparison to the reference strain (905). Afterward, R1, R3, R7, R12 and R14 were selected to continue for genetic stability test in which they were found to be genetically stable and their resistance to rapamycin was shown to be permanent. Genetically stable strains were tested by spot assay for their cross-resistance or sensitivity against various stress types, including 0.5 mM NiCl2, 2.5 mM CrCl3, 3 mM CoCl2, 17.5 mM MnCl2, 50 mM NH4Fe(SO4)2, 10 mM AlCl3, 20 mM CuCl2, 15 mM LiCl, 50 mM H3BO4, 100 µM AgNO3, 0.5 M NaCl, 15 mM caffeine, 4 mM vanillin, 200 ng/ml propolis, 1mM coniferyl aldehyde and 200 ng/ml cycloheximide. R12 was found to be the mutant with the highest number of cross-resistance and sensitivities: R12 strain was cross-resistant to CuCl2, NH4Fe(SO4)2, NaCl, coniferyl aldehyde, vanillin, cycloheximide, propolis; and sensitive to AlCl3, CoCl2, H3BO4 and AgNO3 in comparison to the reference strain (905). In order to determine the cell wall integrity of the rapamycin-resistant mutant (R12), lyticase susceptibility assay was performed. The result of this experiment showed that R12 resisted lyticase more than 905, under nonstress condition. On the other hand, under rapamycin stress, 905 resisted lyticase more than R12. Furthermore, the presence of rapamycin stress did not change the lyticase resistance of the evolved strain R12, in comparison to the nonstress condition. The chronological lifespan of rapamycin resistant strain (R12) was determined by using semi-quantitative and also quantitative chronological lifespan analysis. The result of the experiments correlated with each other in which R12 was found to have a shorter CLS, in comparison to 905. Comparative whole genome sequencing analysis of the rapamycin-resistant mutant (R12) revealed four single nucleotide variations (SNVs). These SNVs were located in four different genes. The precise functions of these genes in rapamycin response and resistance in yeast should be examined in greater detail in future studies. In conclusion, a rapamycin-stress-resistant and genetically stable S. cerevisiae strain (R12) was successfully obtained by using evolutionary engineering in this thesis study, and characterized at genomic and physiological levels. These results indicate that TOR pathway-related changes occurred in rapamycin-resistant mutant in order to overcome the high levels of rapamycin stress. However, in order to fully comprehend the molecular basis of rapamycin-resistance of R12, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Genomic analysis of freeze-thaw stress-resistant Saccharomyces cerevisiae

(Graduate School, 2024-07-12) Güney, Çağla ; Çakar, Zeynep Petek ; 521211104 ; Molecular Biology-Genetics and Biotechnology

The budding yeast Saccharomyces cerevisiae is a well-known unicellular eukaryotic organism widely used in industry for making bread and for ethanol production. In addition, it is highly used as a eukaryotic model organism for research purposes in molecular biology and genetics. Since its genome sequence is well-known for several years and it has homologous genes with other eukaryotes including humans, S. cerevisiae is used as model organism for studying higher eukaryotic organisms. Additional advantages of S. cerevisiae are its easy manipulation and well-known growth conditions. Freeze-thaw stress is a physical stress type, and freeze-thaw stress resistance is highly desired in S. cerevisiae. Freeze-thaw stress causes physiological damage to cell. Freezing step causes ice crystal formation and cellular dehydration and damages the cell. Thawing step creates damage as a result of ROS formation. S. cerevisiae is faced with this stress type while used in bakery industry. First, the dough prepared with yeast is frozen and stored. Then, the dough is thawed for the baking process. This would cause the drop of gassing power of the yeast. The drop in rising power is due to cyclic Adenosine Monophosphate (cAMP)-Protein Kinase A (PKA) pathway. There is a glucose-induced increase of cAMP which results in the activation of PKA. Then, trehalase breaks down the trehalose, which is an important component in yeast stress resistance, including resistant to freeze- thaw stress. Thus, freeze-thaw stress resistance is important for improving the efficiency of bread making process and other yeast bioprocesses. In this study, genomic and physiological analysis of previously obtained freeze-thaw stress-resistant S. cerevisiae strains were performed. The freeze-thaw stress-resistant mutant strains were obtained by an inverse metabolic engineering strategy, evolutionary engineering, without using any chemical mutagenesis. After batch selection, genetic stability of the mutants was determined to understand if the resistance to freeze-thaw stress was an adaptation, or it was caused by a permanent genomic change. The selected FT1, FT5, FT6 and FT9 mutant strains were found to be genetically stable. Determination of the cell wall integrity was done using lyticase susceptibility assay. The assay was performed with the genetically stable, freeze-thaw stress-resistant mutant FT9. Results of the lyticase susceptibility assay demonstrated that the freeze-thaw stress-resistant mutant FT9 was resistant to lyticase degradation more than the reference strain (905), under both stress and nonstress conditions. Comparative whole genome sequencing analysis of the freeze-thaw stress-resistant mutant (FT9) revealed only one single nucleotide variation (SNV). The SNV was located on CDC25 gene, and it was a missense SNV. As a result of the missense SNV on this gene encoding a cell division cycle (Cdc) protein, threonine was replaced by lysine (T1415K). In conclusion, a previously obtained freeze-thaw stress-resistant S. cerevisiae evolved strain was characterized in this study at genomic and physiological levels. The results revealed that cAMP/PKA pathway-related changes occurred in the freeze-thaw stress-resistant mutant strain in order to protect it against damage. However, in order to fully comprehend the molecular basis of freeze-thaw stress resistance of this strain, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Inverse metabolic engineering of KCl-resistant Saccharomyces cerevisiae

(Graduate School, 2022-06-20) Morkoç, Ogün ; Çakar, Zeynep Petek ; 521181116 ; Molecular Biology - Genetics and Biotechnology

Bu çalışmada, KCl tuzu kaynaklı hiperosmotik strese dayanıklı S. cerevisiae suşları elde etmek için, tersine metabolik mühendislik yaklaşımı olan evrimsel mühendislik kullanılmıştır. Evrimsel mühendislik ile elde edilen suşların fizyolojik ve metabolik analizleri yapılmıştır. Evrimsel mühendislik için seçilim deneyi, kademeli olarak artan KCl stresi altında gerçekleştirilmiştir.
Stress resistance analysis of yeast strains with mitotic exit-related gene deletions

(Graduate School, 2023-07-06) Gargı, Rüveyda ; Çakar, Zeynep Petek ; Çaydaşı Koca, Ayşe ; 521201122 ; Molecular Biology – Genetics and Biotechnology

Saccharomyces cerevisiae (budding yeast) is a unicellular eukaryotic organism that is utilized as a model organism in scientific studies. It provides a simpler system to understand the cellular processes of a eukaryotic cell and requires low cost compared to other higher eukaryotes. The genome of S. cerevisiae has been sequenced and genes within the genome have been identified. Depending on the study's goals, homologous recombination can be employed to remove, tag, or introduce new genes. S. cerevisiae has two stages in its life cycle: haploid and diploid. During haploid stage, cells go through mitosis to form two daughter cells, which then split from the mother cell. Haploid cells can also mate to form diploid cells. S. cerevisiae has four stages in its cell cycle: G1, S, G2 and M. DNA replication occurs during S phase, while chromosome segragation and mitosis occurs during M phase. After the nucleus division, cytokinesis occurs which is the step where daughter cells separate from each other. The gap phases between M phase and S phase are required to ensure that new daughter cells have time to grow to maintain cell size in each generation. Cyclins and cyclin dependent kinases (Cdks) regulate cell cycle events by phosphorylating their target molecules. Cdk inhibitors, synthesis and degradation of cyclins, and phosphorylation of Cdk at regulatory sites are three ways the cell uses to adjust the activity of cyclin/Cdk complexes. Cdk inhibitors are used to inhibit the activity of cyclin/Cdk complexes. The synthesis and degradation of cyclins are used to control the activity of these complexes. There are several different cyclins dedicated to different stages and transitions in cell cycle of budding yeast. Mitotic Exit Network (MEN) is a signaling pathway that operates exit from mitosis, control spindle orientation and initiate cytokinesis. It is regulated by changes in the activity of its components and their ability to interact with each other. Cdc14 activation and Cdk1 inactivation are required to exit from mitosis, which is achieved by degradation of mitotic cyclins and the expression of Cdk1 inhibitor when the substrates of Cdc14 are dephosphorylated. Mitotic exit network starts with the GTPase protein Tem1 which is controlled by Bfa1-Bub2 complex and Lte1. Activated Tem1 passes the signal to protein kinase Cdc15 which activates the Dbf2-Mob1 complex, promoting Cdc14 release. At the beginning of anaphase, polo kinase Cdc5 phosphorylates Bfa1 to inhibit the GAP activity of the complex, allowing Bfa1 to not inhibit the exit from mitosis. Once Cdc14 is released by FEAR network, it dephosphorylates Cdc15 to enable its active form. The DNA Damage Checkpoint (DDC) is a checkpoint that arrests cell cycle when DNA is damaged. If detected during G1 phase, cell cycle stops at G1/S transition and DNA replication slows down during S phase. Additionally, the presence of chromosome damage during G2 activates G2/M DDC. Spindle Assembly Checkpoint (SAC) is a checkpoint that ensures the chromosome attachment to mitotic spindle. The protein phosphatase-1 participates in the proper microtubule attachment to kinetochores. Once bipolar attachment of sister chromatids to mitotic spindle is compromised, kinetochores pass the signal to prevent cohesin cleavage and chromosome segregation. Cell cycle is arrested at metaphase. Spindle Position Checkpoint (SPOC) is a checkpoint that ensures the proper positioning of mitotic spindle. If there is a misalignment, the checkpoint delays mitotic exit. The activity of Bfa1-Bub2 is regulated by Kin4 in SPOC. When the mitotic spindle aligns properly, Kin4 localizes at mother cell cortex and the SPB stays in the mother cell. However, when there is a misalignment, Kin4 locates on both of the SPBs and phosphorylates Bfa1. Glc7 also participates in the inhibition of mitotic exit by dephosphorylating Bfa1 and inhibiting the Cdc5 phosphorylation. The aim of this study is to investigate the effects of potential stressors on S. cerevisiae strains with specific gene alterations including deletions of BFA1, SPO12, KIN4 genes and the overexpression of protein phosphatase 1 (Glc7). In order to test the impact of stressors, chemicals that potentially affect mitotic exit pathway was used to expose these yeast strains with different alterations. The changes in growth of strains was tested by spot assay. By comparing the responses of the wild-type strains and the strains with gene mutations, this study aimed to inform about the specific roles of these genes in mediating the cellular response to various stress conditions. At first, the stressors that were tested on yeast cells were rapamycin, aluminum, chromium, nickel, maganese and hydrogen peroxide. It was then decided to test rapamycin-like chemicals such as caffeine, coniferyl aldehyde and propolis. According to the coniferyl aldehyde results, vanillin and spermine which are similar to coniferyl aldehyde were also tested on yeast strains. The sensitivity difference between different strains of yeast was found to be affected by selection markers used while obtaining deletions. Strains used in the first part of the study were transformed with cassettes containing different selection marker genes like klTRP1, hphNT1 and his3MX6. Spot assay results of yeast strains under rapamycin treatment showed an interesting result. BKY091-1 strain (ESM356-1 BFA1-3HA-hphNT1) was sensitive compared to ESM356-1 even though the only difference was the 3HA-hphNT1 tag. Therefore, PCR-based tagging with hphNT1 gene (coding for hygromycin B phosphotransferase) was considered to result in the difference between these two control groups. According to previous studies, hygromycin B treatment can affect cells during the selection process. The glucose consumption, lactic acid production and expression of glucose-related genes are increased in Tc7 (human colon adenocarcinoma) cells after the selection with hygromycin. The morphology of these cells also changed after selection. Therefore, hygromycin B treatment can interfere with the cellular processes of yeast cells. This thesis study also showed the importance of using the same selectable marker, according to spot assay results of yeast cells under rapamycin stress. Under the stress conditions caused by caffeine, propolis and vanillin, there was a difference between trp positive control groups and the trp-negative control. The trp-negative control ESM356-1 was more sensitive compared to control groups that have trp gene. Conversely, under the rapamycin treatment, the trp-negative control ESM356-1 displayed slightly higher resistance. Thus, it was decided to compare results of other strains with the trp-positive controls. The coniferyl aldehyde results were interesting, because different alterations in genes that were expected to have opposite results showed similar growth in yeast cells under the coniferyl aldehyde treatment. bfa1∆ (AKY314-1), spo12∆ (RGY001-1), kin4∆ (RGY002-2) and Gal1-Glc7-GFP (DKY163) strains were resistant to coniferyl aldehyde treatment when compared with their control groups. It is known that Bfa1 is important for the checkpoint activations. Kin4 phosphorylates Bfa1 to inhibit the phosphorylation by Cdc5. Glc7 dephosphorylates Bfa1 replaced from SPBs, inhibiting the Cdc5 phosphorylation and inhibition of Bfa1 phosphorylation results in the inhibition of mitotic exit. However, under the coniferyl aldehyde treatment, both deletion of BFA1 and KIN4 and GLC7 expression led to resistance. bfa1∆ and kin4∆ strains showed that inhibiting checkpoint activation and progressing with the mitotic exit might result into advantages while dealing with the stress created by coniferyl aldehyde. The Gal1-Glc7-GFP strain displayed resistance to coniferyl aldehyde treatment. Glc7 has other functions other than contributing to the inhibition of mitotic exit, such as dephosphorylation by Glc7 that reverses the activation of two checkpoints: the Spindle Assembly Checkpoint and the DNA damage checkpoint. When there is an overexpression of Glc7, it causes chromosome missegregation due to the checkpoint bypass. When DNA is damaged, chromatin is marked by the phosphorylation of Histone 2A (Hta2) in order to activate repairing factors. Cell cycle continues by the dephosphorylation of Hta2 and Rad53, once the damage is fixed. Considering these activities of Glc7, the resistance to coniferyl aldehyde observed in the Gal1-Glc7-GFP strain might also depend on the checkpoint bypass. Like in the bfa1∆ and kin4∆ cells, not inhibiting cell cycle at mitotic exit or at other phases might have given an advantage to the Gal1-Glc7-GFP strain under coniferyl aldehyde treatment. The Gal1-Glc7-GFP strain showed resistance to propolis, compared to its control group. It is known that propolis can lead to reduced growth rate, oxidative stress, DNA damage and mitochondrial dysfunction in yeast cells. Propolis can also generate reactive oxygen species (ROS) in cells which can cause oxidative damage to nucleic acids and proteins. Response against the oxidative stress is achieved by the coordination of many defence systems in yeast cells. In S. cerevisiae, the HOG pathway is essential for the oxidative stress response, consisting of MAP kinase (MAPK) Hog1 and MAP kinase kinase (MAPKK) Pbs2. Glc7 also interacts with the stress response pathways and cell cycle checkpoints. It is considered to have a role in adjusting the activity of Hog1 pathway through Pbs2 and it regulates the activity of Slt2 contributing to the oxidative stress response. Therefore, the resistance to propolis in the Gal1-Glc7-GFP strain can be related to the oxidative stress response.

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