**4. Phylogenetic relationship between snake venom and plasma prothrombin activators**

A phylogenetic tree of the snake venom and plasma prothrombin activators with other known FX sequences was constructed to understand their evolutionary relationships using zebrafish FX as the out group (Reza et al. 2006; St Pierre et al. 2005). All the reptilian sequences form a monophyletic group (Reza et al. 2006) (Figure 7). Within the reptilian clade, group C and D prothrombin activators appear as two separate clades on the tree. This indicates that, despite their similarities, group C and D prothrombin activators have originated independently. Interestingly, the PFX2 sequence is found nested within the group C prothrombin activators. This supports the hypothesis that PFX2 is an evolutionary intermediate of PCCS from PFX1. Based on the topology of the phylogenetic tree, it is suggested that these snake venom prothrombin activators have been "recruited" through independent evolutionary events (Reza et al. 2006) (Figure 7).

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 269

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

Fig. 8. Gene organization comparison of trocarin D and TrFX. Exons are shown as numbered

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

The overlapping promoter regions of trocarin D and TrFX were characterized by comparing them against previously characterized human (Hung et al. 2001; Hung and High 1996) and murine (Wilberding and Castellino 2000) FX promoter regions (Reza et al. 2007) (Figure 9). Based on these comparisons, four conserved *cis*-regulatory elements in the trocarin D and TrFX promoter regions were identified (Figure 9): (i) a CCAAT box (Hung et al. 2001; Hung and High 1996; Wilberding and Castellino 2000), (ii) a gut-specific transcription factor GATA-4 binding site (Hung et al. 2001), (iii) a liver-specific transcription factor HNF-4 (Hung and High 1996), and (iv) multiple Sp1/Sp3 binding

Comparison of the trocarin D and TrFX promoter regions reveals that trocarin D has a 264 bp insertion (Figure 8 and 9). This 264 bp is located from -33 to -297 bp upstream of the trocarin D start codon (ATG) (Figure 9). This insertion is postulated to play a major role in the recruitment of the duplicated TrFX gene by causing it to be exclusively expressed in the venom gland as the procoagulant toxin, trocarin D. Hence, it was termed *V*enom *R*ecruitment/*S*witch *E*lement (*VERSE*). This segment was characterized for its *cis*-elements and gene-regulatory role using luciferase assays in primary venom gland cells and mammalian cell lines (Kwong et al. 2009). The *VERSE* promoter was found to be responsible for the elevation of expression levels, but not tissue-specific expression, of trocarin D. In terms of *cis*-element characterization, besides confirming the presence of two TATA-boxes, one GATA box and one Y-box, three novel *cis*-elements were also identified (Figure 9). Functionally, it is found that both TATA boxes (TLB2 and TLB3) are functional. However, TLB2 is the primary TATA box which initiates and directs transcription start site (Kwong et

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).

**5.1** *Cis***-elements in trocarin D promoter region** 

**Promoter Intron 1**

sites (Hung et al. 2001).

al. 2009) (Figure 9).

activator.

**TrFX**

**Trocarin D**

Fig. 7. Phylogenetic relationships of snake venom and plasma prothrombin activators with other known FX sequences (Reza et al. 2006). "vPA" is an abbreviation for venom prothrombin activators. Arrows indicate the three independent "recruitment" events of snake venom prothrombin activators.
