**6. Gene duplication in snake venom toxin diversification**

Besides "recruitment", gene duplication also plays an important role in the diversification of venom toxins. This diversification is essential for the development of novel toxins. This diversification through gene duplication is evident from the many toxin isoforms present in the snake venom. Interestingly, each isoform varies in its function and gene regulation.

Gene duplication has also led to neofunctionalization of venom toxins, which has led to the new families of snake venom toxins (St Pierre et al. 2008) and addition of new members within these families (Fry et al. 2003; Landan et al. 1991b; Lynch 2007; Moura-da-Silva et al. 1996). The three-finger toxin (3FTx) multigene family is a good example of neofunctionalization by gene duplication (Fry et al. 2003). Structurally, all the members of this family have very well-conserved cysteine residues and share a common structure of three beta-stranded loops extending from a central core. However, they exhibit a wide variety of pharmacological effects. For example, acetylcholinesterase inhibition (fasciculin from *Dendroaspis angusticeps* venom), neurotoxicity (α-bungarotoxin from *Bungarus multicinctus* venom), cardiotoxicity (β cardiotoxin from *Ophiophagus hannah* venom), and many others (for details, see (Kini and Doley 2010)). Neofunctionalization occurs when a toxin gene undergoes gene duplication and the duplicated gene is mutated within the functional sites, which often results in new ligand-binding specificities (Kini 2002).

Besides neofunctionalization, changes in gene regulation are also the outcomes of gene duplication. This can be seen in two isoforms present in the venom of *Naja sputatrix*: cardiotoxin and α-neurotoxin (Ma et al. 2001). Besides varying in function, these two isoforms have different expression levels in the venom gland. Cardiotoxin constitutes 60% of the venom while the α-neurotoxin makes up only 3% of the venom. Gene duplication is evident from the gene comparison whereby the structures and amino acid sequence of these

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56.

two toxins are very well-conserved (Ma et al. 2002). The main difference lies in the promoter segment, where it was found that the α-neurotoxin promoter contains a stronger silencer element, which is responsible for significantly reducing its expression level in the venom (Ma et al. 2001; Ma et al. 2002).

In the case of venom prothrombin activators, we have shown that they have been "recruited" from the gene of an ancestral plasma prothrombin activator protein through gene duplication. The duplicated gene underwent modifications in its regulatory and coding regions to gain toxin characteristics. *VERSE* segments were inserted in the promoter regions of trocarin D and PCCS and are responsible for their elevated level of expression. Insertion/deletion segments in their intron 1 regions are postulated to be responsible for venom-gland specific expression. Modifications in the gene-coding regions enable prothrombin activators to function better as toxin by gaining certain characteristics such as resistance to inactivation.
