Stress resistance analysis of yeast strains with mitotic exit-related gene deletions

Abstract

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.