**9. Fungal metallothioneins**

Fungal MTs, study are more sporadic and are described usually as copper binding proteins. Still, few are characterized functionally in the positions of metal organization in chemical properties or complementation tests in mutants of metal sensitive yeasts. Reputed MTs are mostly designated within collections of ESTs (Expressed Sequence Tag) based on sequence resemblance. These are occasion for the MT like arrangements that are present in the mycorrhizea, specifically in *Pisolithus tinctorius* (ectomycorrhizal fungi), the ericoid fungus (*Oidiodendron maius)* and the arbuscular mycorrhizal fungi (*Gigaspora rosea*, *Gigaspora margarita* and *Glomus intraradices)* [31, 32].

The *P. involutus* gene (*Pimt1*) codes for short MT (34 amino acids) and contains domain bearing classical C–X–C motifs, compared with longer and canonical MTs that usually have 2 Cys-rich domains. These are found in the other fungal MTs too; it appears to be that length is not critical required for binding of metal, as even some shorter protein of MT has proved to efficiently chelate the metal ions [33].

Heterologous expression systems for demonstrating unambiguously that *Pimt1* gene product can sequester metal ions, therefore conferring *in vivo* protection against the metals, in particular with cadmium and copper [34]. The functional complementation assays was done using 3 distinct mutants of metal-hypersensitive yeast. Similar approach was done for the characterization of 2 MT- encoding genes from the endomycorrhizal fungi [31]. It explains in what way Genome Omission Project for *Saccharomyces* offered materials towards the efficient genomics researches in area of the metal forbearance. Availability of abundant metal sensitive strains lets *in vivo* segmentation of the metal protection mechanism and ultimately lead for identifying specific molecular roles that are played by the DNA sequence of interest.

There are more evidence for MTs role or MT-like proteins in the mutualistic and the pathogenic interactions in between fungi and plants. The genes of fungal MT are mostly regulated transcriptionally in the life stages mainly, during the colonization of plant. The first report was in 1995, when the 2 genes similar to MTs were expressed uniquely during appressorium formation by of conidia in *Colletotrichum gloeosporioides* which are induced by the host surface wax. The 2 putative MT genes from biotrophic pathogen (*Uromyces fabae)* are upregulated strongly in the parasitic mycelium that are colonizing tissues of leaves [35]. So far as the mycorrhizal fungi are concerned, the *Pimt1* was found in macroarray experiments as upregulated in ectomycorrhizal tissues compared with the saprotroph growth condition. In contrast, MTs from arbuscular mycorrhizal fungi appear to be downregulated when the fungus colonizes the root tissues. It is worth noting that plant MTs are also upregulated in ectomycorrhizal associations of *Betula pendula* with *Eucalyptus globulus* and *P. involutus* with *P. tinctorius*, respectively. To support further MTs role in the plant–fungus interaction, Tucker *and* his co-workers described one unusual MT-like protein with 22 amino acids long with 6 cysteines in *Magnaporthae grisea*

(fungal pathogen), that showed high affinity to zinc. Gene has no effect in the metal tolerance and it is shown to confer the pathogenicity, through playing role in the biochemical differentiation of appressorium cell wall [33].

### **10. Cyanobacterial metallothionein**

Numerous mechanisms of metal resistance exist in cyanobacteria. The first mechanism involves extracellular binding. Cells might synthesize and release some organic materials that could chelate the metals and reduce their bioavailability or metal ions might bound to outer cell surface [36]. These complex forms are not readily transported into cell due to its complexity and structure. Secondly, cells could increase excretion rate of some metal ions using the energy-driven efflux pumps [37]. The sequestration of internal metal, a 3rd resistance mechanism, is a key mechanisms through which the bacteria combats the exposure of the heavy metal and their subsequent accumulation. In cyanobacteria, metal ion sequestration inside the cell are performed by the Class II MTs.

The Class II MTs are a cysteine-rich, thiol-containing and are metal-binding proteins that can sequester the metals, thereby stopping the buildup of potentially some deadly free metals in cell. Metal binding happens by interactions of the metals with thiol clusters of the cysteine remainders. MTs gene was organized as operon termed as smt locus, that contains together the metallothionein protein (smtA) and the regulatory protein, repressor (smtB) genes. The SmtBs, are the managing repressor of the countenance from smtA promoter- operator province. The MTs countenance, from gene to the efficient protein, was encouraged through metals and regulation of the transcript to mRNA is based on the interface among the metals and repressor protein that regulates transcript, over by the interface by a thiol clusters that are contemporary on the repressor type of protein [38, 39].

#### **11. Mammalian metallothionein**

The MT genes were readily induced by several toxicologic and physiologic stimuli. Since the MT cysteines are conserved across the species, it is suspected that the cysteines are essential for the function and the MTs are required for the life. In attempts for determining the MT function(s), researches were carried out with 4 different experimental paradigms: (i) the animals were injected with chemicals that induce MT; (ii) the cells adapted to grow and survive in higher MT concentrations that induces toxicants; (iii) the cells are transfected with the MT gene; and (iv) the MT-null mice and the MT transgenic. Very often, the reports from the researches with the first 3 approaches had indicated several purposes of MT in the cell biology is as follows: the MT (a) is "warehouse" for the "Zn" (b) the free radical hunter (c) protect from cadmium harmfulness. However, researches with MT transgenic with the null rats did not toughly reinforced initial 2 proposed purposes then they sturdily sustenance its purpose in the protection in contradiction of the cadmium harmfulness. Recurrent cadmium management to the MT null rats resulted in the nephron harmfulness at the 1/10 of the quantity that products the toxicity in the control rats. Researches in human being showed as 7% of overall populations had some renal malfunction by cadmium exposures. Consequently, if the human being did not consume the MT, "standard" exposure of cadmium will be toxic to human beings [17]. Thus, it appears that during evolution, the MT ability to protect against the Cd toxicity would had taken a chief role for the life processes maintenance, as compared with its other proposed functions (*i.e.* storehouse for zinc and free radical scavenger) [40, 41].
