Date

2013

Author

Yılmaz, Tülay

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In this study differential expression of the two proteasome associated VAT genes (TVNO382 and TVN0947) of Thermoplasma volcanium GSS1 (Tpv) cells challenged by extracellular stresses, i.e., pH, heat-shock and hydrogen peroxide were investigated using quantitative RT-PCR and Western blotting/hybridization techniques. We also performed a comprehensive transcriptome analysis of the Tpv as response to environmental stresses by genome-wide expression arrays. Quantitative RT-PCR analyses revealed that VAT genes elicited specific response under heat-shock (at 65ºC and 70ºC), pH stress (at pH 4.0) and oxidative stress (at 0.02 mM H2O2) over 120 min. Western hybridization corroborated the qRT-PCR results at protein level. These results suggested a critical role for VAT proteins possibly in association with the 20S proteasome in Tpv cells for adaptation to external stress, especially heat-shock. Microarray analyses revealed that Tpv cells’ gene expression over 60 min stress exposure was essentially down-regulated. Within this group of genes those functionally linked to energy production and conversion, transcription and translation were significantly and extensively inhibited. Response to heat stress (at 65ºC) could be linked to elevated expression of genes encoding heat-shock related genes (e.g. GrpE) whereas the stress response to pH and oxidative stresses was characterized by up-regulation of membrane and transport related genes. Among the stress responsive genes sugar permease (TVN1145) and GrpE (TVN0489) genes were selected for verification of the microarray data by qRT-PCR technique. The results demonstrated a consistent trend in the expression patterns of the selected genes by two techniques. Collectively, our results suggest that the transcription and translation are retarded to prevent error prone synthesis of proteins and to avoid energy requiring synthetic processes. The energy balance may be maintained by activation of carbohydrate transport and metabolism under all stress conditions. Tpv also modulates stress specific genes (e.g., GrpE-Heat-shock, redoxins-Oxidative stress, transporters-pH stress) as part of its stress defense mechanism(s).

Subject Keywords

Archaebacteria., Nucleotides., Polymerase chain reaction., Gene expression., Thermoplasma volcanium.

URI

http://etd.lib.metu.edu.tr/upload/12615917/index.pdf
https://hdl.handle.net/11511/22656

Collections

Graduate School of Natural and Applied Sciences, Thesis