Akademik Veri Yönetim Sistemi
Tezin Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye
Tezin Onay Tarihi: 2008
Öğrenci: FÜSUN DOLDUR
Danışman: SEMRA KOCABIYIK
Özet:Chaperonins (Hsp60 chaperones) comprise a class of oligomeric, high-molecular-weight chaperones that have the unique ability to fold some proteins that cannot be folded by simpler chaperone systems. The term “thermosome” is used for molecular chaperonins from Archaeal organisms since they accumulate to high levels upon heat-shock. In this study first time, we have cloned and sequenced two Hsp60 subunit genes (α and β) from a thermoacidophilic archaeon Thermoplasma volcanium. For cloning we have followed a PCR based strategy. Amplification of Hsp60 α gene from chromosomal DNA of T. volcanium yielded a product of 1939 bp amplicon and that of Hsp60 β gene yielded a product of 1921 bp amplicon. After ligation of the PCR fragments to pDrive vector, recombinant plasmids were transferred into E. coli TG-1 competent cells and recombinant colonies were selected by blue/white screening. The cloning of two subunit genes were confirmed by restriction mapping and by sequencing. Both subunit genes were then subcloned to pUC18 vector consequtively to construct a co-expression vector. Both subunit genes were expressed under control of their own promoters leading to production of active Hsp60 chaperonin (thermosome). Chaperone activity of the recombinant thermosome was shown by using pig citrate synthase enzyme as substrate. Thermosome induced refolding was observed when renaturation was carried out at 50°C for 2,5 h. Under this condition, citrate synthase activities associated with control and test were mA412/min:19.0 and mA412/min:24.0 respectively. Clustal W Version 1.82 was used for multiple sequence alignments of Hsp60 and Hsp60 proteins of T. volcanium and other Hsp60 proteins from various eukaryotes, bacteria and archaea. The highest sequence similarity was found between α subunit proteins of T. volcanium and T. acidophilum (94%) and β subunit proteins of T. volcanium and T. acidophilum (93%). Clusters of orthologous groups and conserved domain database searches revealed the phylogenetic relationships between Hsp60 and Hsp60 subunits of T. volcanium thermosome and other Hsp60 proteins from various eukaryotes, bacteria and archaea. Induction of both subunit genes under heat shock (65°C, 70°C and 75°C for 2h) and under oxidative stress (imposed by 0,008 mM, 0,01 mM, 0,02 mM, 0,03 mM and 0,05 mM H2O2) conditions was studied by Real-Time PCR technique and amplified cDNA band density analysis.