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: 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: 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: 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: 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: 7Citation - Scopus: 8Checkpoint Deficient Rad53-11 Yeast Cannot Accumulate Dntps in Response To Dna Damage(Elsevier Ltd., 2007) Koç, Ahmet; Merrill, Gary F.Deoxyribonucleotide pools are maintained at levels that support efficient and yet accurate DNA replication and repair. Rad53 is part of a protein kinase regulatory cascade that, conceptually, promotes dNTP accumulation in four ways: (1) it activates the transcription of ribonucleotide reductase subunits by inhibiting the Crt1 repressor; (2) it plays a role in relocalization of ribonucleotide reductase subunits RNR2 and RNR4 from nucleus to cytoplasm; (3) it antagonizes the action of Sml1, a protein that binds and inhibits ribonucleotide reductase; and (4) it blocks cell-cycle progression in response to DNA damage, thus preventing dNTP consumption through replication forks. Although several lines of evidence support the above modes of Rad53 action, an effect of a rad53 mutation on dNTP levels has not been directly demonstrated. In fact, in a previous study, a rad53-11 mutation did not result in lower dNTP levels in asynchronous cells or in synchronized cells that entered the S-phase in the presence of the RNR inhibitor hydroxyurea. These anomalies prompted us to investigate whether the rad53-11 mutation affected dNTP levels in cells exposed to a DNA-damaging dose of ethylmethyl sulfonate (EMS). Although dNTP levels increased by 2- to 3-fold in EMS treated wild-type cells, rad53-11 cells showed no such change. Thus, the results indicate that Rad53 checkpoint function is not required for dNTP pool maintenance in normally growing cells, but is required for dNTP pool expansion in cells exposed to DNA-damaging agents.Article Evidence for the Presence of a Second Electron Donor for the Cytoplasmic Thioredoxins in the Yeast Saccharomyces Cerevisiae(TUBITAK, 2006) Koç, Ahmet; Karakaya, Hüseyin Çağlar; Ünlü, Ercan SelçukIn yeast, the cytoplasmic thioredoxin system is composed of NADPH, thioredoxin reductase-1 (TRR1) and 2 thioredoxin genes (TRX1, TRX2). In this study, using yeast knockout mutants for TRR1, TRX1 and TRX2 genes, the role of the thioredoxin system in methionine sulfoxide reduction was investigated. Cells lacking both TRX1 and TRX2 genes simultaneously were not able to reduce methionine sulfoxides to methionine; however, mutants missing the TRR1 gene were able to reduce methionine sulfoxides to methionine, which showed that electrons could be transferred from NADPH to thioredoxins in the absence of TRR1. Similar results were observed for 3-phosphoadenosine 5-phosphosulfate reduction in the inorganic sulfate assimilation pathway. Results from both assays suggested that yeast cells have additional cytoplasmic thioredoxin reductase activity that could compensate for methionine sulfoxide reduction and sulfate assimilation in the absence of TRR1. This report also constitutes the first evidence that thioredoxins are the in vivo electron donors for methionine sulfoxide reductases in yeast.