Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Characterization of Genes That Play Role in Manganese Tolerance in Different Yeast Species(01. Izmir Institute of Technology, 2021) Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyManganese is an essential element for organisms that can also be toxic. It has been stated that more than 5 mM Mn inhibits the growth of wild-type S. cerevisiae BY4741 strain. In the cases when this amount is exceeded, the stress upon manganese toxicity arises and it leads to a range of responses to normalize the manganese level. However, the genes accountable for that case are unknown. Manganese toxicity is a restrictor factor in the production of agricultural products. Identification and characterization of the genes that play a role in manganese homeostasis are rather essential. In this study, we have used Sanger Centre's Saccharomyces Genome Resequencing Project (SGRP) strains, which are collected from different regions of the world. After screened the whole collection, we have identified four manganese resistant strains; S. cerevisiae BY474, S. paradoxus Y6.5, S. cerevisiae 378604X and S. paradoxus Q74.4. Manganese-related genes were selected via the Saccharomyces genome database (SGD). Expression levels of these genes under manganese stress in most resistant strain Q74.4 analyzed by RT-Q-PCR. As a result, GCR-1 dependent translation factor GDT1 and high-affinity phosphate transporter PHO84 were found to be upregulated in Q74.4 that endure high levels of manganese toxicity. These genes are probably accountable for manganese tolerance in Q74.4 strain. The results arising from that study, will take the lead to the development of biotechnological exercises for manganese bioremediation. Meanwhile, it might help molecular mechanisms to be able to develop resistance to stressful conditions that manganese generates and shed light to further studies.Master Thesis Elucidation of Molecular Mechanisms Conferring Arsenic Tolerance To Yeast Cells(Izmir Institute of Technology, 2016) Işık, Esin; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyArsenic is a highly toxic metalloid available in the environment mainly as arsenite or arsenate. These compounds’ interference with many molecular mechanisms results in several diseases including cancer. Conversely, arsenic is used in therapeutic approaches, however, they are associated with drug resistance. Although some tolerance and toxicity mechanisms of arsenicals in yeast have been enlightened by previous studies, complete understanding, which is important for development of protection and therapy strategies, has not yet been achieved. Comprehensive genome-wide screening is a promising approach for the elucidation of novel genes involved in arsenic-associated mechanisms. The aim in this study was to screen a yeast genome library to characterize novel genes whose overexpression confers resistance to toxic concentrations of arsenate or arsenite in Saccharomyces cerevisiae. The plasmids from the colonies confirmed to be highly-resistant against arsenicals were sequenced to determine the genomic regions and seven genes were selected to clone into expression vectors. The overexpression of Pho86p and Vba3p provided yeast cells with the highest arsenate and arsenite resistance, respectively. Arsenate is a phosphate analogue and taken up by phosphate transporters. Pho86p is an ER-resident protein regulating ER-exit of the phosphate transporter. Therefore, it is reasonable that overexpression of Pho86p provides arsenate resistance. Vacuolar sequestration is a common route for the removal of toxic compounds from the cytosol and Vba3p is a vacuole-located transporter of basic amino acids with a likely role in arsenite resistance. Consequently, the screen in the current study revealed two genes with promising roles for tolerance mechanisms against arsenicals.Master Thesis Characterization of Pollen-E1 Gene Might Play Role in Salt Tolerance in Beta Maritima(Izmir Institute of Technology, 2015) Uysal, Özge; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologySalinity stress has a negative impact on the growth of plants, which affects homeostasis and productivity. The uptake of non-essential salt ions change the osmotic balance of the cell and cause dehydration. Higher plants develop salt tolerance mechanisms to avoid dehydration. In this project, we isolated and characterized salt tolerance genes in Beta maritima plant. For this purpose, functional genomics technique was used by over expressing cDNAs in yeast and colonies can grow toxic salt media isolated and characterized. We found several colonies and we focused on uncharacterized Pollen-E1 gene with an unknown function. Pollen-E1 cDNA confers salt tolerance to yeast cells. Intracellular sodium measurements of Pollen-E1 overexpressed in yeast cells showed decreased salt levels as compared to wild type suggesting that sodium was transported out of the cell. Pollen-E1 protein localized in endomembrane systems in the yeast cells. In mRNA expression analysis, Pollen-E1 mRNA levels induced immediately in leaves and later stages in root systems under salt stress. Our results showed that is the uncharacterized and unknown function Pollen-E1 gene might have some role of regulating salt tolerance in Beta maritima.Master Thesis Construction of Cdna Library From Hordeum Marinum To Indentify Salt Tolerance Genes(Izmir Institute of Technology, 2015) Alkaya, Naki; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologySalt is necessary for plants because of ion homeostasis; however, excess uptake of salt leads to damage in plant cells, which may even result in the death of the plant. Hordeum marinum, also known as sea barley, is a member of Poaceae family that resides in coastal areas, so it is thought that it may have a possible salt tolerance gene or genes. Therefore, this study aims to identify the genes involved in salt tolerance in Hordeum marinum by functional genomics method. After screening, seven transformant yeast colonies found and sequence analyses of these plasmids gave homology to hypothetical protein of Bipolaris oryzae. To confirm salt tolerance of this protein, salt sensitive yeast cell transfected by this candidate gene was checked in high salt concentration containing medium. Based on solid growth assay, these transgenic yeast cells could survive in 1M saline medium. Hence, it is hypothesized that Hordeum marinum and Bipolaris oryzae might have a symbiotic association. It is possible that in this association Bipolaris oryzae may play a role as endophytic fungus that might also confer salt tolerance in Hordeum marinum.Master Thesis Identification and Characterization of Boron Tolerant Genes in Hordeum Vulgare (barley) by Using Mrna Differential Display and Rt-Pcr Techniques(Izmir Institute of Technology, 2007) Akıncı, Ersin; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBoron, is a microelement that plays a role in plant development. In contrast, excess amount of boron is toxic for plants. Turkey is the second country that has the largest boron reserve in the world, thus boron is one of the major problems in agriculture in Turkey. Barley (Hordeum vulgare) is the second widely produced cereal after wheat. Because barley is used in human diet, animal feeding and beer industry it is an economically valuable crop. There are ten different barley varieties in Turkey and these varieties show different genetic variations against boron toxicity. In this study, Hamidiye (boron sensitive) and Anadolu (boron tolerant) varieties were used to identify genes responsible for boron tolerance. RT-PCR and mRNA Differential Display techniques were used from root and leaf samples of Hamidiye and Anadolu varieties grown in laboratory with or without boron conditions. Eight differentially expressed genes identified by using mRNA Differential Display technique. Sequence of these genes gave homology to an eukaryotic translation initiation factor in Arabidopsis thaliana, a chlorophyll a/b binding protein precursor in Triticum aestivum, an elongation factor in Oryza sativa, short-chain dehydrogenase/reductase family protein in Arabidopsis thaliana, a thioredoxin h isoform in Triticum aestivum, a shaggy-like kinase protein in Triticum aestivum, chloroplast genome in Hordeum vulgare, a hypothetical protein in Arabidopsis thaliana. Expression level of six of forty three antiporter genes showed differences between Anadolu and Hamidiye cultivars in Real Time PCR.Master Thesis Molecular Cloning, Overexpression and Biochemical Characterization of Bacterial Amylase for Biotechnological Processes(Izmir Institute of Technology, 2012) Burhanoğlu, Tülin; Şanlı Mohamed, Gülşah; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; Şanlı Mohamed, Gülşah; 04.03. Department of Molecular Biology and Genetics; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologyAmylases are the enzymes that act on glycosidic bond of starch and related polysaccarides. They comprise 25% of enzyme utilised in a variety of industry. It is used to obtain maltose, glucose and maltodextrins in various lenghts during industrial processes. Amylases are widely distributed enzymes in bacteria, fungi, higher plants and animals. Thermophilic enzymes are widely demanded in order to be stable at harsh process conditions. Isolating these enzymes from thermophilic microorganism is increasing trend because of ease of enzyme production. In this study α-amylase gene region from a thermophilic Bacillus sp. isolated from Balçova Geotermal region in İzmir was cloned to compotent E. coli BL 21 cells. Additionally protein expression was reinforced with pKJE7 chaperone plasmid. Cloned gene was sequenced and found as 1542 bp in length. Thermophilic amylase that has a 59.9 kD molecular weight was expressed and purified from this recombinant strain. Mass spectrometric analysis were performed and the enzyme was matched with α-amylase family protein of Geobacillus thermodenitrificans NG80-2 using NCBInr database. The aminoacid sequence of this enzyme was seen to be similar 92% with our obtained enzyme. According to the results of characterization studies, the amylase enzyme was seen to have highest activity at pH 8.0 and 60°C. The enzyme was also showed to have resonable activity between pH5 and 9. 85% of the enzyme activity was retained at 70°C. Furthermore, amylase activities at 65 and 85°C were observed to remain stable for 5 and 2 hours, respectively. It was also showed that the activity was stable and pH7 and 9 for 6 hours. The effects of some metal ions, chemical agents and organic solvents on enzyme activity were examined so, Co+2, Mg+2,Ca+2 was determined to be as inducer for the enzyme activity. Conversely the activity was inhibited by Cu+2. Furthermore methanol, DDT and Triton X-100 was found to have no effect on the enzyme activity.Master Thesis Identification of Salt Stress Responsive Protyeins in Wild Sugar Beet (beta Maritima) Using 2d-Page With Maldi-tof/Tof System(Izmir Institute of Technology, 2012) Çakıroğlu, Çiğdem; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyHigh salinity is one of the abiotic stresses, which affects the homeostasis, growth and productivity of plants. In plants, uptake of the non-essential salt ions negatively affects the anatomy, physiology and metabolism, changes the osmotic balance in cells and causes abundant dehydration. In this case, higher plants develop salt tolerance mechanisms such as induction of related signaling pathways, effluxion of salt ions, accumulation of these toxic ions in their vacuoles, activation of their detoxification mechanisms and production of osmoprotectans. In this study, identification of salt responsive proteins in moderately halophyte wild type sugar beet Beta vulgaris ssp. maritima was aimed. In order to investigate the protein-based natural stress tolerating mechanisms, plants were exposed to 150 mM NaCl and total proteins were extracted. Differentially expressed proteins were identified by proteomic approaches including MALDI-TOF/TOF mass spectrometry combined two dimensional polyacrylamide gel electrophoresis. The results revealed that enzymatic antioxidants and secondary members of antioxidative pathways are responsive in salt stress. In conclusion, these detected proteins demonstrate that antioxidative system may be the major defense mechanism in halophytic plants.Master Thesis Identification and Characaterization of Nickel Tolerance Genes in Beta Maritima(Izmir Institute of Technology, 2009) Bozdağ, Gönensin Ozan; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyNickel is an essential micronutrient for plant growth. However, an excess amount of nickel is toxic for plants and animals. The Beta maritima plant which is known crop as salt and manganese tolerant nature. In this study, we aimed to identify nickel detoxification metabolism genes of Beta maritima by screening plant.s overexpressed cDNA library in yeast cells. After initial screening, three transformants were isolated that were able to grow in the presence of toxic nickel concentration. After the sequence analyses, three genes were named as NIC3, NIC6 and NIC8. We tested the growth rates of the yeast cells those were overexpressing cDNAs in solid mediums and showed similar nickel detoxification patterns. Then intracellular nickel concentrations were measured to see whether mentioned cDNAs pump nickel out of the cell or not. We also analysed the gene expression levels of NIC3 and NIC6 genes after appllying nickel stress to the plants to see the relations between nickel treatment and transcription changes of these two genes. Our data suggest that NIC3, NIC6 and NIC8 genes, besides their other functions, confer yeast cells nickel tolerance and may play roles in heavy metal detoxification for plant cells.Master Thesis Identification and Characterization of Manganese Tolerance Genes in Beta Vulgaris Subsp. Maritima(Izmir Institute of Technology, 2011) Erbaşol, Işıl; Karakaya, Hüseyin Çağlar; Karakaya, Hüseyin Çağlar; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyManganese is an essential element for higher organisms however uptake of excess amount of manganese causes toxicity. Beta vulgaris subsp. maritima, the member of Chenopodiaceae family, is known to tolerate high concentration of sodium. Due to its living conditions, Beta vulgaris subsp. maritima adapted many different stress conditions. Therefore it is an ideal plant for studying plant tolerance mechanisms. In this study, we aimed to identify the genes which are responsible for manganese tolerance in Beta vulgaris subsp. maritima by screening its cDNA library in Saccharomyces cerevisiae cells. After initial screening in the presence of toxic manganese concentration; 2,7mM MnCl2, three resistant yeast colonies were selected. After the sequence and similarity analysis, two genes which might involve in manganese tolerance were identified and named as BmMn1 and BmMn2. The results of solid media tests with different yeast strains which transformed with the genes revealed that BmMn1 provides a remarkable manganese tolerance like BmMn2 and slightly nickel tolerance. They do not show tolerance to other metals such as zinc, cadmium, boron and cobalt. Identified manganese concentrations in pmr1 yeast strainstransformed with BmMn1, BmMn2 or empty vector pointed that BmMn1 and BmMn2 transport excess manganese out of the cell. In addition, GFP localization in the yeast cell proved that the BmMn1 and BmMn2 are located in Golgi apparatus. qRT-PCR analyses of Beta vulgaris subsp. maritima which was exposed to 2mM Mn2+ suggested a dynamic regulation for the expression of these two genes. The results indicate that BmMn1 and BmMn2 have a role in detoxification of excess amount of manganese in Beta vulgaris subsp. maritima.