Molecular Cloning Of CDNA Encoding For MT-Ⅰ/-Ⅱ And Sequence Analysis Of Nucleotide/Protein In Yak

Metallothioneins (MTs) are low-molecular-weight (approximately 7000 daltons),cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrateand vertebrate animals. Since their first description as cadmium- and znic-bindingproteins in horse kidney, the genes and proteins have been characterized from diffirentorganisms in many animal, such as human, horse, bovine, sheep, pig, rabbit and mouse,etc. They have an unusual amino acid composition in that they have no aromatic aminoacids and a single polypeptide chain of 61 amino acids that one-third of their residues arecysteines (20 residues in mammal MTs at invariant positions). Metallothioneins normallybind with d10 metal ions, such as Zn2+, Cd2+, Hg2+, and Cu+. MTs have been considered toplay a central role in detoxification of heavy metals and free radicals and immuneresponses. In both prokaryotes and eukaryotes, it acts as a free radical scavengerprotecting against oxidative damage, and protects against toxicity of alkylating anticancerdrugs. The MT multigene family is composed of at least four isoforms: MT-I, -II, -III and-IV. In the present study, the domestic yak (B.Grunniens) metallothionein-I and -II cDNAcoding sequence and MT-I cDNA 3'-end full length sequence was amplified and fishedby RT-PCR and 3'-RACE using the primer YMTSP1, YMTSP2, YMTSP3 and M13 PrimersM4. The fragments of target DNA were selected by electrophoresis. The plasmid librarywas constructed with T4 polymerase by ligating the DNA fragments into the Hind III andXho I site of the cloning plasmid pZeroT Vector. Then, by performed following steps:isolation of library DNA, transfection of competent E.Coli. DH5a, extraction of DNAfrom transfected cells, restriction identification of recombinant plasmids with Hind IIIand Xho I and sequencing, we obtained the yak MT-I and MT-II coding sequence(Accession No: AY513744 and AY513745, respectively) and MT-I cDNA 3'-end fulllength sequence. The cDNA sequences of MT-I and MT-II were subjected to Blastn searching in CBIand the results showed that the nucleotide sequences of yak MT-I and MT-II betweendifferent species mammal are comparatively conservative. The yak cDNA sequences coding for yak MT-I/-II protein composed of 61 aminoacids, respectively. There are lots of conserved tripetide, such as C-X-C, C-C-X-C-C,C-X-X-C, etc. and they are comparatively conservative in their evolution. By homology comparatvie modeling, we predicted the molecular spatial structures ofyak MT-I/-II, Metallothionein is composed of a and ?domains that are linked by aconserved tripeptide KKS.