**4. Classes of metallothionein**

Based upon their similarities in structure, the MTs are divided into 3 classes: Class I: This type present in mammalians, oysters, some fishes, crabs and mussels as Cys **-** Cys proteins.

Class II: Occurs in fungi, plants, yeasts, drosophila, cyanobacteria and nematodes in the form of Cys – x **-** Cys.

Class III: Presence noticed in phytochelatins that present in all plants, some fungi and also algae as Cys – x – x - Cys.

The MTs belongs to the class I & II are been produced through direct mRNA translation. Whereas, the class III MTs belongs to the metal-binding polypeptides and have peptide bonds which are biologically synthesized as some of the enzymatic products. All vertebrates that are examined contain 2 or more distinct isoforms of MT, that are grouped into 2 classes, designated as MT-I and MT-II, depending upon elution position from the DEAE cellulose. In many cases each of the class actually consists of various different proteins that were designated as MT-IA, MT-IB, MT-IC, etc.

General characteristics of Metallothioneins are,


MT expression in the crops serve as the key index in selecting for the heavy metal tolerance. In attempting for shedding light on their function, the researchers relied on the RNA blot hybridization for studying genes expression of MT during the development and also in response to several other environmental factors. Detailed localization of the MT mRNAs or gene promoter activity is obtained through reporter gene expression studies and *in situ* hybridization. The RT–PCR are evaluated as an alternative for Northern blot analysis for quantifying mRNA levels of MT. But the sensitivities of the two techniques were identical for detecting the induced mRNA levels in MT. Thus the above results suggests that the RT–PCR would be a quantitative and a sensitive method for evaluating gene

expression of MT [20]. Most of the MT genes are been expressed at a higher level in the plant tissues, in the terms of the transcript abundance. The direct evidence are from the rice gene profiling experiments using SAGE (serial analysis of gene expression) protocol. The transcripts from four MT genes comprised about 3% of the transcripts in 2 weeks old seedlings. Metallothionin gene belonging to Type II contributes about 1.26% of all transcripts that was found to be most expressed form in that tissue. The MT, 2 forms of transcripts as Type 3 genes counted as extra 1.25% of mRNAs. Numerous MT genes that are identified through differential screening of the cDNA libraries indicates that the RNAs encoding to MTs are higher in most of the other plant species also.

In general, affinity of the metal ions for binding sites follows the typical order of thiolate model complexes, *i.e.,* Zn (II) < Pb (II) < Cd (II) < Cu (I), Ag (I), Hg (II), Bi (III) [15]. The MT are usually detected by its virtue of high content of the metals (detected by labelling with radionuclides or by atomic absorption spectrophotometer) or by cysteine. Some of the procedures include, reversed phase High Pressure Liquid Chromatography (HPLC) gel filtration be elevated as a variety of mammalian metallothionins and utilized for immune-electrophoresis, radioimmunoassay and immunodiffusion. These protocols are mainly helpful for the detection of minimum quantity in MT. Basic structure of MT are examined by several biochemical and bio-physical techniques such as NMR spectroscopy, UV, ESR,CD partial proteolysis and X-ray crystallography.
