**5. Comparison of trocarin D and TrFX genes**

In the previous sections, we have described how the venom prothrombin activators have been modified to gain certain characteristics, such as resistance to inactivation, which enables them to function better as toxins. However, differential and tissue-specific expression of venom prothrombin activators and their plasma coagulation factors is also important for their respective physiological roles. The expression of toxins should be venom gland-specific and inducible to higher levels. This is so the snake can protect itself against its own venom toxins and replenish its venom supply quickly. Conversely, plasma coagulant factors are mainly expressed in the liver at constituently low levels so that they can be activated to induce blood coagulation during vascular injuries.

To understand how the venom prothrombin activators are regulated for tissue specificity and level of expression, we determined the gene structure of trocarin D and TrFX. Based on the cDNA sequences of trocarin D and TrFX, and that of mammalian FX gene, primers were designed, and the gene sequences were determined using genomic DNA PCR and genome walking strategies (Reza et al. 2006). The gene organizations of trocarin D and TrFX are identical (eight exons and seven introns). The intron sequences were highly similar to each other with only differences in the promoter and intron 1 regions (Figure 8). Such similarities strongly support our findings that there are two closely related, parallel prothrombin activator systems, and that the venom prothrombin activators are "recruited" from plasma coagulation factors through gene duplication. The duplicated FX gene was subsequently modified and "recruited" for expression in the venom gland as a venom prothrombin activator.

Fig. 8. Gene organization comparison of trocarin D and TrFX. Exons are shown as numbered boxes. The differences in the promoter and intron 1 regions are indicated in yellow (insertions in trocarin D gene) and green (deletions in TrFX gene) (Reza et al., 2007).
