Heat shock response in thermoplasma volcanium: cloning and differential expression of molecular chaperonin (thermosome) genes

Download

index.pdf

Date

2008

Author

Doldur, Füsun

Metadata

Show full item record

Item Usage Stats

86
views

77
downloads

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.

Subject Keywords

Biology., Genetics.

URI

http://etd.lib.metu.edu.tr/upload/3/12610169/index.pdf
https://hdl.handle.net/11511/18128

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Live-cell imaging of Pol II promoter activity to monitor gene expression with RNA IMAGEtag reporters SHIN, Ilchung; RAY, Judhajeet; Gupta, Vinayak; İlgü, Müslüm; Beasley, Jonathan; BENDICKSON, Lee; MEHANOVIC, Samir; Kraus, George A.; Nilsen-Hamilton, Marit (Oxford University Press (OUP), 2014-01-01) We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from ...
Molecular recognition of poly(A) by small ligands: an alternative method of analysis reveals nanomolar, cooperative and shape-selective binding Persil Çetinkol, Özgül (Oxford University Press (OUP), 2009-02-01) A few drug-like molecules have recently been found to bind poly(A) and induce a stable secondary structure (T(m)approximate to 60 degrees C), even though this RNA homopolymer is single-stranded in the absence of a ligand. Here, we report results from experiments specifically designed to explore the association of small molecules with poly(A). We demonstrate that coralyne, the first small molecule discovered to bind poly(dA), binds with unexpectedly high affinity (K(a) >10(7) M(-1)), and that the crescent sh...
Transverse vibration of pre-stressed beams: an experimental technique for the determination of dynamic viscoelastic material properties of tissue mimicking materials Yazıcıoğlu, Yiğit; Navarro-castillo, K.; Kutluay, U.; Roystonc, T.j. (null; 2008-12-01) An experimental dynamic material property identification technique is presented that is based on the theoretical formulations of a vibrating pre-stressed beam. The technique determines the viscoelastic material properties of tissue mimicking materials that govern their dynamic behavior. Results are presented for a silicone based material (CF11-2188 Nusil Silicone Technology, Carpinteria, CA) that is in the form of a thick string held between fixed supports under tensile pre-stress. The specimens are excited...
Construction of various fusion proteins of recombinant citrate synthase from thermoplasma volcanium Özdoğan, Seda; Kocabıyık, Semra; Department of Biology (2004) In this study, a strategy called gene splicing by overlap extension, 3Gene SOEing4, was used for the construction of the fusion proteins with the purpose of increasing the thermostability of mesophilic enzymes by incorporation of stability domain from a thermostable enzyme. Gene SOEing is a PCR-based approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. In fusion constructs, as th...
Radiation force of ultrasound as shear wave source in Microscopic Magnetic Resonance Elastography Özer, Mehmet Bülent; Xu, Huıhuı; Meral, Can; Royston, Thomas J; Magın, Rıchard L (2005-10-21) Microscopic magnetic resonance elastography (micro‐MRE) is a high‐resolution imaging technique for measuring the viscoelastic properties of small synthetic and biological samples. Taking MRE to the microscopic scale requires stronger static fields, stronger magnetic field gradients, higher performance RF coils, and more compact, higher frequency shear wave actuators. Prior work by our group has been conducted at 11.74 T. A needle attached to a vibrating cantilever beam was placed in contact with the surface...

Citation Formats

F. Doldur, “Heat shock response in thermoplasma volcanium: cloning and differential expression of molecular chaperonin (thermosome) genes,” M.S. - Master of Science, Middle East Technical University, 2008.