Isolation and characterization of a novel copper-inducible metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis.
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.
School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan.
Raheela
R
Chaudhry
Abdul Rauf
AR
Shakoori
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017125:Genes, Protozoan; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D020080:Tandem Repeat Sequences; D013768:Tetrahymena / Q000235:genetics* / Q000378:metabolism
D016054:DNA, Protozoan; D020033:Protein Isoforms; D015800:Protozoan Proteins; C007121:copper thionein; D008668:Metallothionein