Pakistan Research Repository

Isolation and Characterization of Cadmium Metallothionein Gene from Local Ciliates

Nighat, Rukhsana (2010) Isolation and Characterization of Cadmium Metallothionein Gene from Local Ciliates. PhD thesis, University of the Punjab, Lahore.

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Abstract

The ciliates, Tetrahymena tropicalis lahorensis (T. t. lahorensis) and Paramecium sp., were isolated from industrial effluents.After the optimization of growth conditions for both the organisms the cultures were maintained in the laboratory conditions and their resistance to cadmium was checked. The non toxic dose of cadmium was found to be 16μg/ml and 18μg/ml for Paramecium sp. and T.t. lahorensis respectively.Paramecium sp. was capable to remove 84% of cadmium from the culture medium after 96 hours and 96% of cadmium was reduced after 96 hours when T.t. lahorensis was exposed to cadmium.This ability of these locally isolated ciliates can be exploited for bioremediation of cadmium contaminated industrial waste water. First cadmium metallothionein like gene (PMCd1) from a ciliate, Paramecium sp. and a novel cadmium inducible metallothionein (TMCd1) gene has been identified and sequenced from the locally isolated ciliate; Tetrahymena tropicalis lahorensis isolated from industrial wastewater. Both the genes (PMCd1 and TMCd1) are intronless, encoding 612 and 471 nucleotides respectively, with TGA as the stop codon and TAA coding for glutamine. The coding region of PMCd1 comprises 203 amino acids, including 37 cysteine residues with a conserved structural pattern in the form of recurring structural motifs, arranged in 17 cys-x-ycys,1 cys-cys and cys contexts. Both, the deduced amino acids and nucleotide sequence differ, not only from other animal metallothioneins (MTs), but also from the previously characterized Tetrahymena Cu and Cd-MTs.The TMCd1 is quite different from the previously reported MT genes in Tetrahymena pyriformis and Tetrahymena pigmentosa. However, it shows 78% homology with four different Cd-MT genes reported from Tetrahymena thermophila. The amino acids sequence of TMCd1 has a special feature of three CCCX6CCX6CCCX6CC and two CCX6CXCX2CXCC intragenic tandem repeats with a conserved structural pattern of cysteine.The translated protein of TMCd1 contains 30.12% cysteine residues, which is characteristic of typical Tetrahymena Cd inducible MT genes.A TATA box is located in the 5` flanking region at nucleotide 34-38 upstream region of ATG.The truncated TMCd1 fusion protein with GST tag was also expressed and purified by affinity column chromatography.

Item Type:Thesis (PhD)
Uncontrolled Keywords:Conditions, Chromatography, Isolation, Cadmium, Metallothionein, Characteristic, Local, Upstream, Ciliates, Protein, Organisms, Gene, Structural
Subjects:Biological & Medical Sciences (c) > Biological Sciences(c1)
ID Code:6037
Deposited By:Mr. Javed Memon
Deposited On:25 Apr 2011 09:41
Last Modified:19 Aug 2011 10:42

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