Isolation and characterization of a novel copper‐inducible metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis

Abstract

The two isoforms of copper metallothionein (CuMT) gene of a copper resistant ciliate, Tetrahymena tropicalis lahorensis (Ttl), have been isolated and characterized. The molecular cloning and nucleotide sequencing of cDNAs coding for the two CuMT isoforms revealed that TtlCuMT1 gene has 300, while TtlCuMT2 has 327 nucleotides, both with ATG as the initiation codon and TGA as the translational termination codon. TAG codes for glutamine in TtlCuMT2 gene which is peculiar to Tetrahymena. The deduced or translated TtlCuMT1 and TtlCuMT2 peptide sequences contain 100 and 108 amino acid residues including 28 and 32 cysteine residues, respectively. The amino acid sequences of TtlCuMT1 and TtlCuMT2 have special features of two and three CXCXXCXCXXCXC intragenic tandem repeats with a conserved structural pattern of cysteine, respectively. The predicted tertiary structures of these two isoforms indicate two domains. Domain I and the initial part of domain II showed >98% homology with other Tetrahymena CuMT. On the basis of the differences in the domain II, the metallothionein subfamily 7b can be divided into two groups, one (TtlCuMT1) comprising >100 amino acids and the other (TtlCuMT2) comprising <100 amino acids. This is a novel finding of the present study as no such report on this type of classification exists at the moment. TtlCuMT1 has 95%, while TtlCuMT2 has 97% resemblance with the previously reported CuMT genes of Tetrahymena spp. SDS-PAGE analysis using fluorescent probe as well as coomassie brilliant blue staining also confirmed the presence of metallothionein.

Cd‐Metallothioneins in Three Additional Tetrahymena Species: Intragenic Repeat Patterns and Induction by Metal Ions

“…), T. rostrata (Amaro et al. ), T. tropicalis (Chaudhry and Shakoori ; Shuja and Shakoori ), and T. hegewischi , T. malaccensis , and T. mobilis (this article). CdMTs from T. hegewischi are an important addition; only T. hegewischi and T. pigmentosa are from the australis group, the other major evolutionary group of Tetrahymena species, the remaining species all belong to the borealis group (Chantangsi and Lynn ).…”

“…The subfami-lies show little sequence similarity with one another; they differ in the pattern of clustering of Cys residues and their induction preference under heavy metal exposure (Diaz et al 2007). Previous studies also indicated that Tetrahymena MT polypeptides from the two subfamilies carry out different biological functions in the process of metal homeostasis and detoxification (Amaro et al 2008;Boldrin et al 2002;Chaudhry and Shakoori 2010;Diaz et al 2007;Dondero et al 2004;Fu and Miao 2006;Guo et al 2008;Santovito et al 2001). The highly complex set of Tetrahymena MTs may give insights into functional specificity at the molecular level.…”

Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis

“…Log phase growing cells were starved for 18 h in 50 mM Tris buffer (pH 7.4). The cells were pelleted down and subjected to DNA isolation according to Chaudhry and Shakoori [2010]. Copper metallothionein gene of T. farahensis (TfCuMT) was amplified from genomic DNA using the following set of primers reported by Boldrin et al [2008].…”

“…Most of the MT-1 members are cadmium induced, while MT-2 are copper induced MT. All MT-3 and MT-5 are cadmium MTs, while MT-4 is exclusively copper MTs [Diaz et al, 2007;Chaudhry and Shakoori, 2010].…”

“…Neighborhood of Lys reduces the reactivity of Cys residues and decreases the pKa value [Trinchella et al, 2008]. Up till now Tetrahyemna copper metallothionein (CuMT) has been reported from T. thermophila [Boldrin et al, 2002], T. pigmentosa [Boldrin et al, 2002], T. pyriformis [Fu and Miao, 2006;Santovito et al, 2007], T. rostrata [Amaro et al, 2008], and T. tropicalis [Chaudhry and Shakoori, 2010].…”