Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Citation - WoS: 9Citation - Scopus: 10Characterization of Long Living Yeast Deletion Mutants That Lack Mitochondrial Metabolism Genes Dss1, Ppa2 and Afg3(Elsevier, 2019) Muid, Khandaker Ashfaqul; Kimyon, Önder; Reza, Shahadat Hasan; Karakaya, Hüseyin Çağlar; Koç, AhmetMolecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2 Delta, dss1 Delta, and afg3 Delta) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondria] DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.Article Citation - WoS: 2Citation - Scopus: 2Characterization of the Beta1 Gene, Which Might Play a Role in Beta Vulgaris Subsp. Maritima Salt Tolerance(Türkiye Klinikleri Journal of Medical Sciences, 2017) Uysal, Özge; Çakıroğlu, Çiğdem; Koç, Ahmet; Karakaya, Hüseyin ÇağlarSalinity stress has a negative impact on plant growth, which affects homeostasis and productivity. The uptake of nonessential salt ions changes the osmotic balance of the cell and causes dehydration. Higher plants develop salt tolerance mechanisms to avoid dehydration. Sea beet (Beta vulgaris subsp. maritima) is a halophytic ancestor of cultivated sugar beet that displays salt stress tolerance. In this study, we screened a B. vulgaris subsp. maritima cDNA library in Saccharomyces cerevisiae strain Ab11c (ena1Δ, nha1/4Δ, nhx1Δ), which is deficient in sodium transport, to find sodium-detoxifying genes. We identified a cDNA construct, named BETA1, providing salt tolerance to yeast cells. This gene had no previously described function. Intracellular sodium measurements demonstrated no significant differences between yeast cells expressing BETA1 or a sham vector, suggesting that sodium was not effluxed in BETA1-expressing cells. Transcriptionally, BETA1 mRNA levels were induced immediately in leaves and later in the root system in response to the salt stress. Our results suggest that the BETA1 gene is part of the salt tolerance network in B. vulgaris subsp. maritima.Article Citation - WoS: 19Citation - Scopus: 22Absence of Superoxide Dismutase Activity Causes Nuclear Dna Fragmentation During the Aging Process(Academic Press Inc., 2014) Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koç, AhmetSuperoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.Article Citation - WoS: 5Citation - Scopus: 4Characterization of a Cdna From Beta Maritima That Confers Nickel Tolerance in Yeast(Elsevier Ltd., 2014) Bozdağ, Gönensin Ozan; Kaya, Alaattin; Koç, Ahmet; Noll, Gundula A.; Prüfer, Dirk; Karakaya, Hüseyin ÇağlarNickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.Article Citation - WoS: 11Citation - Scopus: 14Proteomic Changes During Boron Tolerance in Barley (hordeum Vulgare) and the Role of Vacuolar Proton-Translocating Atpase Subunit E(Türkiye Klinikleri Journal of Medical Sciences, 2011) Atik, Ahmet Emin; Bozdağ, Gönensin Ozan; Akıncı, Ersin; Kaya, Alaattin; Koç, Ahmet; Yalçın, Talat; Karakaya, Hüseyin ÇağlarBoron is an essential micronutrient for plants and animals; however, it can be toxic when present at high concentrations. The purpose of this study was to understand the mechanisms of boron tolerance in the Turkish barley (Hordeum vulgare) Anadolu cultivar. For this purpose, 2-dimensional electrophoresis (2-DE) was used to screen differentially expressed proteins for both control and boron-stressed Anadolu barley genotypes. Seven proteins were revealed by 2-DE: 1) ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo large chain), 2) TLP5, a thaumatin-like protein, 3) PR5, a basic pathogenesis-related protein, 4) a RNase S-like protein, 5) a PSI type III chlorophyll a/b-binding protein, 6) a light-harvesting complex I LHC I, and 7) the vacuolar proton-translocating ATPase subunit E protein. These were found to be upregulated in response to boron treatment. Even though the protein encoded by the V-ATPase subunit E gene was overexpressed, its transcript level was downregulated by boron treatment. Heterologous expression of the barley V-ATPase subunit E gene in yeast provided boron resistance to yeast cells. These results indicated that the V-ATPase subunit E gene was functional and conferred tolerance to toxic boron levels in yeast and might play a role in the overall boron tolerance of barley. © TÜBITAK.Article Citation - WoS: 12Citation - Scopus: 14Roles of Atr1 Paralogs Ymr279c and Yor378w in Boron Stress Tolerance(Elsevier Ltd., 2011) Bozdağ, Gönensin Ozan; Uluışık, İrem; Gülcüler, Gülce Sıla; Karakaya, Hüseyin Çağlar; Koç, AhmetBoron is a necessary nutrient for plants and animals, however excess of it causes toxicity. Previously, Atr1 and Arabidopsis Bor1 homolog were identified as the boron efflux pump in yeast, which lower the cytosolic boron concentration and help cells to survive in the presence of toxic amount of boron. In this study, we analyzed ATR1 paralogs, YMR279c and YOR378w, to understand whether they participate in boron stress tolerance in yeast. Even though these genes share homology with ATR1, neither their deletion rendered cells boron sensitive nor their expression was significantly upregulated by boron treatment. However, expression of YMR279, but not YOR378w, from the constitutive GAPDH promoter on a high copy plasmid provided remarkable boron resistance by decreasing intracellular boron levels. Thus our results suggest the presence of a third boron exporter, YMR279c, which functions similar to ATR1 and provides boron resistance in yeast.Article Citation - WoS: 18Citation - Scopus: 19Assessment of Chronological Lifespan Dependent Molecular Damages in Yeast Lacking Mitochondrial Antioxidant Genes(Elsevier Ltd., 2010) Demir, Ayşe Banu; Koç, AhmetThe free radical theory of aging states that oxidative damage to biomolecules causes aging and that antioxidants neutralize free radicals and thus decelerate aging. Mitochondria produce most of the reactive oxygen species, but at the same time have many antioxidant enzymes providing protection from these oxidants. Expecting that cells without mitochondrial antioxidant genes would accumulate higher levels of oxidative damage and, therefore, will have a shorter lifespan, we analyzed oxidative damages to biomolecules in young and chronologically aged mutants lacking the mitochondrial antioxidant genes: G. RX2, CCP1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, and PRX1. Among these mutants, ccp1Δ, trx3Δ, grx5Δ, prx1Δ, mutants were sensitive to diamide, and ccs1Δ and sod2Δ were sensitive to both diamide and menadione. Most of the mutants were less viable in stationary phase. Chronologically aged cells produced higher amount of superoxide radical and accumulated higher levels of oxidative damages. Even though our results support the findings that old cells harbor higher amount of molecular damages, no significant difference was observed between wild type and mutant cells in terms of their damage content. © 2010 Elsevier Inc.Article Citation - WoS: 30Citation - Scopus: 32Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast(American Chemical Society, 2010) Kaya, Alaattin; Koç, Ahmet; Lee, Byung Cheon; Fomenko, Dmitri E.; Rederstorff, Mathieu; Krol, Alain; Lescure, Alain; Gladyshev, Vadim N.Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments. © 2010 American Chemical Society.Article Citation - WoS: 55Citation - Scopus: 64Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast(American Society for Microbiology, 2009) Kaya, Alaattin; Karakaya, Hüseyin Çağlar; Fomenko, Dmitri E.; Gladyshev, Vadim N.; Koç, AhmetBoron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1△ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1△ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1△ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.Article Citation - WoS: 17Citation - Scopus: 19Effect of Thioredoxin Deletion and P53 Cysteine Replacement on Human P53 Activity in Wild-Type and Thioredoxin Reductase Null Yeast(American Chemical Society, 2009) Stoner, Christopher S.; Pearson, George D.; Koç, Ahmet; Merwin, Jason R.; Lopez, Nathan I.; Merrill, Gary FredericReporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (K d = 0.9 μM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Δtrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.