**7. Conclusion**

Gene duplication has played a major role in the development of snake venom toxins. Our findings on venom prothrombin activators and blood coagulation factors have captured the first molecular evidence of gene duplication. The characterization of the differences in their genes, i.e. *VERSE* segment and intron 1 of trocarin D, has increased our understanding of gene regulation of snake venom toxins. It is shown that the *VERSE* segment is responsible for the elevation of gene expression and that the intron 1 is probably responsible venom gland-specific expression (unpublished observations). We identified three novel *cis*-elements in the *VERSE* segment, and these play important roles in gene regulation. It would be interesting to further characterize them and their *trans*factor partners to determine how various *trans*-factors interact with each other to regulate gene expression. The answers to some of these questions will increase our overall understanding of gene regulation.

## **8. References**


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

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

Gene duplication has played a major role in the development of snake venom toxins. Our findings on venom prothrombin activators and blood coagulation factors have captured the first molecular evidence of gene duplication. The characterization of the differences in their genes, i.e. *VERSE* segment and intron 1 of trocarin D, has increased our understanding of gene regulation of snake venom toxins. It is shown that the *VERSE* segment is responsible for the elevation of gene expression and that the intron 1 is probably responsible venom gland-specific expression (unpublished observations). We identified three novel *cis*-elements in the *VERSE* segment, and these play important roles in gene regulation. It would be interesting to further characterize them and their *trans*factor partners to determine how various *trans*-factors interact with each other to regulate gene expression. The answers to some of these questions will increase our overall

Bos, M.H., M.Boltz, L.St Pierre, P.P.Masci, J.de Jersey, M.F.Lavin, and R.M.Camire. 2009.

Boulikas, T. 1993. "Nature of DNA sequences at the attachment regions of genes to the

Braud, S., C.Bon, and A.Wisner. 2000. "Snake venom proteins acting on hemostasis."

Chester, A. and G.P.Crawford. 1982. "In vitro coagulant properties of venoms from

Chow, G. and R.M.Kini. 2001. "Exogenous factors from animal sources that induce platelet

Cockerill, P.N. and W.T.Garrard. 1986. "Chromosomal loop anchorage of the kappa

immunoglobulin gene occurs next to the enhancer in a region containing

through procoagulant adaptations." *Blood*. 114:686-692.

nuclear matrix." *J.Cell Biochem.* 52:14-22.

Australian snakes." *Toxicon*. 20:501-504.

aggregation." *Thromb.Haemost.* 85:177-178.

topoisomerase II sites." *Cell*. 44:273-282.

"Venom factor V from the common brown snake escapes hemostatic regulation

(Ma et al. 2001; Ma et al. 2002).

resistance to inactivation.

understanding of gene regulation.

*Biochimie*. 82:851-859.

**7. Conclusion** 

**8. References** 


Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 275

Mann, K.G., M.F.Hockin, K.J.Begin, and M.Kalafatis. 1997. "Activated protein C cleavage of factor Va leads to dissociation of the A2 domain." *J.Biol.Chem.* 272:20678-20683.

Masci, P.P., A.N.Whitaker, and J.de Jersey. 1988. "Purification and characterization of a

McMullen, B.A., K.Fujikawa, W.Kisiel, T.Sasagawa, W.N.Howald, E.Y.Kwa, and

Minh, L.T., M.A.Reza, S.Swarup, and R.M.Kini. 2005. "Gene duplication of coagulation

Morita, T. 2004. "C-type lectin-related proteins from snake venoms." *Curr.Drug* 

Morita, T. and S.Iwanaga. 1978. "Purification and properties of prothrombin activator from

Moura-da-Silva, A.M., R.D.Theakston, and J.M.Crampton. 1996. "Evolution of disintegrin

Nesheim, M.E., W.M.Canfield, W.Kisiel, and K.G.Mann. 1982. "Studies of the capacity of

Nesheim, M.E., J.B.Taswell, and K.G.Mann. 1979. "The contribution of bovine Factor V and Factor Va to the activity of prothrombinase." *J.Biol.Chem.* 254:10952-10962. Nishida, S., T.Fujita, N.Kohno, H.Atoda, T.Morita, H.Takeya, I.Kido, M.J.Paine, S.Kawabata,

Owen, W.G. and C.M.Jackson. 1973. "Activation of prothrombin with *Oxyranus scutellatus*

Pittman, D.D., K.N.Tomkinson, D.Michnick, U.Selighsohn, and R.J.Kaufman. 1994.

Rao, V.S., J.S.Joseph, and R.M.Kini. 2003a. "Group D prothrombin activators from snake

scutellatus (Taipain snake) venom." *Thromb.Res.* 3:705-714.

the venom of Echis carinatus." *J.Biochem.(Tokyo)*. 83:559-570.

prothrombin-activating enzyme from the venom of the Australian rough-scaled

prothrombin activator from the venom of the Australian brown snake, Pseudonaja

B.Weinstein. 1983. "Complete amino acid sequence of the light chain of human blood coagulation factor X: evidence for identification of residue 63 as beta-

factor V and origin of venom prothrombin activator in Pseudonaja textilis snake."

cysteine-rich and mammalian matrix-degrading metalloproteinases: gene duplication and divergence of a common ancestor rather than convergent

factor Xa to protect factor Va from inactivation by activated protein C." *J.Biol.Chem.*

and S.Iwanaga. 1995. "cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom." *Biochemistry*.

"Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity." *Biochemistry*.

venom are structural homologues of mammalian blood coagulation factor Xa."

Markland, F.S. 1998. "Snake venoms and the hemostatic system." *Toxicon*. 36:1749-1800. Marsh, N.A., T.L.Fyffe, and E.A.Bennett. 1997. "Isolation and partial characterization of a

Markland, F.S. 1997. "Snake venoms." *Drugs*. 54 Suppl 3:1-10.

textilis textilis." *Biochem.Int.* 17:825-835.

*Thromb.Haemost.* 93:420-429.

evolution." *J.Mol.Evol.* 43:263-269.

257:1443-1447.

34:1771-1778.

33:6952-6959.

*Biochem.J.* 369:635-642.

snake (Tropidechis carinatus)." *Toxicon*. 35:563-571.

hydroxyaspartic acid." *Biochemistry*. 22:2875-2884.

*Targets.Cardiovasc.Haematol.Disord.* 4:357-373.


Inoue, K. and T.Morita. 1993. "Identification of O-linked oligosaccharide chains in the

Joseph, J.S., M.C.Chung, K.Jeyaseelan, and R.M.Kini. 1999. "Amino acid sequence of

Joseph, J.S. and R.M.Kini. 2001. "Snake venom prothrombin activators homologous to blood

Kalafatis, M., M.D.Rand, and K.G.Mann. 1994. "The mechanism of inactivation of human

Kini, R.M. 2002. "Molecular moulds with multiple missions: functional sites in three-finger

Kini, R.M. 2004. "Platelet aggregation and exogenous factors from animal sources."

Kini, R.M. and G.Chow. 2001. "Exogenous inhibitors of platelet aggregation from animal

Kini, R.M. and R.Doley. 2010. "Structure, function and evolution of three-finger toxins: mini

Kini, R.M. and H.J.Evans. 1990. "Effects of snake venom proteins on blood platelets."

Kini, R.M., T.Morita, and J.Rosing. 2001. "Classification and nomenclature of prothrombin activators isolated from snake venoms." *Thromb.Haemost.* 86:710-711. Kornalik, F. and B.Blomback. 1975. "Prothrombin activation induced by Ecarin - a

Kwong, S., A.E.Woods, P.J.Mirtschin, R.Ge, and R.M.Kini. 2009. "The recruitment of blood

Landan, G., A.Bdolah, Z.Wollberg, E.Kochva, and D.Graur. 1991a. "Evolution of the sarafotoxin/endothelin superfamily of proteins." *Toxicon*. 29:237-244. Landan, G., A.Bdolah, Z.Wollberg, E.Kochva, and D.Graur. 1991b. "Evolution of the sarafotoxin/endothelin superfamily of proteins." *Toxicon*. 29:237-244. Liebich, I., J.Bode, I.Reuter, and E.Wingender. 2002. "Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs)." *Nucleic Acids Res.* 30:3433-3442. Lynch, V.J. 2007. "Inventing an arsenal: adaptive evolution and neofunctionalization of

Ma, D., A.Armugam, and K.Jeyaseelan. 2001. "Expression of cardiotoxin-2 gene. Cloning,

Ma, D., A.Armugam, and K.Jeyaseelan. 2002. "Alpha-neurotoxin gene expression in Naja

characterization and deletion analysis of the promoter." *Eur.J.Biochem.* 268:1844-

sputatrix: identification of a silencer element in the promoter region."

prothrombin converting enzyme from Echis carinatus venom." *Thromb.Res.* 6:57-

coagulation factor X into snake venom gland as a toxin: the role of promoter Cis-

moieties in the activation of factor X." *Eur.J.Biochem.* 218:153-163.

similarity to coagulation factor Xa." *Blood*. 94:621-631.

coagulation factor Xa." *Haemostasis*. 31:234-240.

toxins." *Clin.Exp.Pharmacol.Physiol*. 29:815-822.

sources." *Thromb.Haemost.* 85:179-181.

*Toxicon*. 28:1387-1422.

*Curr.Drug Targets.Cardiovasc.Haematol.Disord.* 4:301-325.

proteins with multiple targets." *Toxicon*. 56:855-867.

elements in its expression." *Thromb.Haemost.* 102:469-478.

snake venom phospholipase A2 genes." *BMC.Evol.Biol.* 7:2.

31880.

63.

1850.

*Arch.Biochem.Biophys.* 404:98-105.

activation peptides of blood coagulation factor X. The role of the carbohydrate

trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural

factor V and human factor Va by activated protein C." *J.Biol.Chem.* 269:31869-


Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 277

Speijer, H., J.W.Govers-Riemslag, R.F.Zwaal, and J.Rosing. 1986. "Prothrombin activation by

St Pierre, L., S.T.Earl, I.Filippovich, N.Sorokina, P.P.Masci, J.De Jersey, and M.F.Lavin. 2008.

St Pierre, L., P.P.Masci, I.Filippovich, N.Sorokina, N.Marsh, D.J.Miller, and M.F.Lavin. 2005.

Stenflo, J., A.Lundwall, and B.Dahlback. 1987. "beta-Hydroxyasparagine in domains

Stocker, K., H.Hauer, C.Muller, and D.A.Triplett. 1994. "Isolation and characterization of

Suzuki, K., B.Dahlback, and J.Stenflo. 1982. "Thrombin-catalyzed activation of human

Tans, G., J.W.Govers-Riemslag, J.L.van Rijn, and J.Rosing. 1985. "Purification and properties

Thorelli, E., R.J.Kaufman, and B.Dahlback. 1998. "Cleavage requirements of factor V in tissue-factor induced thrombin generation." *Thromb.Haemost.* 80:92-98. van der Neut, K.M., R.J.Dirven, H.L.Vos, G.Tans, J.Rosing, and R.M.Bertina. 2004b. "Factor

van der Neut, K.M., R.J.Dirven, H.L.Vos, G.Tans, J.Rosing, and R.M.Bertina. 2004a. "Factor

van Drunen, C.M., R.W.Oosterling, G.M.Keultjes, P.J.Weisbeek, R.van Driel, and

Walker, F.J., W.G.Owen, and C.T.Esmon. 1980. "Characterization of the prothrombin

Wang, C., M.Eufemi, C.Turano, and A.Giartosio. 1996. "Influence of the carbohydrate moiety on the stability of glycoproteins." *Biochemistry*. 35:7299-7307. Welton, R.E. and J.N.Burnell. 2005. "Full length nucleotide sequence of a factor V-like

Wilberding, J.A. and F.J.Castellino. 2000. "Characterization of the murine coagulation factor

venomous snakes." *Cell Mol.Life Sci.* 65:4039-4054.

protein S." *Proc.Natl.Acad.Sci.U.S.A*. 84:368-372.

coagulation factor V." *J Biol Chem*. 257:6556-6564.

Arg-506, and Arg-679." *J.Biol.Chem.* 279:6567-6575.

Arg-506, and Arg-679." *J.Biol.Chem.* 279:6567-6575.

thaliana." *Nucleic Acids Res.* 25:3904-3911.

X promoter." *Thromb.Haemost.* 84:1031-1038.

elapids." *Mol.Biol.Evol.* 22:1853-1864.

venom." *Toxicon*. 32:1227-1236.

*J.Biol.Chem.* 260:9366-9372.

*Biochemistry*. 19:1020-1023.

*Toxicon*. 46:328-336.

261:13258-13267.

an activator from the venom of Oxyuranus scutellatus (Taipan snake)." *J.Biol.Chem.*

"Common evolution of waprin and kunitz-like toxin families in Australian

"Comparative analysis of prothrombin activators from the venom of Australian

homologous to the epidermal growth factor precursor in vitamin K-dependent

Textarin, a prothrombin activator from eastern brown snake (Pseudonaja textilis)

of a prothrombin activator from the venom of Notechis scutatus scutatus."

Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306,

Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306,

S.C.Smeekens. 1997. "Analysis of the chromatin domain organisation around the plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis

activator from the venom of Oxyuranus scutellatus scutellatus (taipan venom)."

subunit of oscutarin from Oxyuranus scutellatus scutellatus (coastal Taipan)."


Rao, V.S. and R.M.Kini. 2002. "Pseutarin C, a prothrombin activator from Pseudonaja textilis

Rao, V.S., S.Swarup, and R.M.Kini. 2003b. "The nonenzymatic subunit of pseutarin C, a

Rao, V.S., S.Swarup, and R.M.Kini. 2004. "The catalytic subunit of pseutarin C, a group C

to mammalian blood coagulation factor Xa." *Thromb.Haemost.* 92:509-521. Reza, A., S.Swarup, and R.M.Kini. 2005a. "Two parallel prothrombin activator systems in

Reza, M.A., L.T.Minh, S.Swarup, and R.M.Kini. 2006. "Molecular evolution caught in action:

Reza, M.A., S.Swarup, and R.M.Kini. 2005b. "Gene structures of trocarin D and coagulation

Reza, M.A., S.Swarup, and R.M.Kini. 2007. "Structure of two genes encoding parallel

Rosing, J. and G.Tans. 1991. "Inventory of exogenous prothrombin activators. For the

Rosing, J. and G.Tans. 1992. "Structural and functional properties of snake venom

Schieck, A., E.Habermann, and F.Kornalik. 1972. "The prothrombin-activating principle

Silva, M.B., M.Schattner, C.R.Ramos, I.L.Junqueira-de-Azevedo, M.C.Guarnieri,

Snow, C.M., A.Senior, and L.Gerace. 1987. "Monoclonal antibodies identify a group of

nuclear pore complex glycoproteins." *J.Cell Biol.* 104:1143-1156.

Pseudonaja textilis (brown snake)." *J.Thromb.Haemost.* 4:1346-1353.

scaled snake." *Pathophysiol.Haemost.Thromb.* 34:205-208.

Haemostasis." *Thromb.Haemost.* 65:627-630.

*Schmiedebergs Arch.Pharmacol.* 274:7-17.

cloning." *Biochem.J.* 369:129-139.

prothrombin activators." *Toxicon*. 30:1515-1527.

Xa-Va complex." *Thromb.Haemost.* 88:611-619.

1354.

93:40-47.

CRC Press. 173-205.

venom: its structural and functional similarity to mammalian coagulation factor

prothrombin activator from eastern brown snake (Pseudonaja textilis) venom, shows structural similarity to mammalian coagulation factor V." *Blood*. 102:1347-

prothrombin activator from the venom of Pseudonaja textilis, is structurally similar

Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of venom prothrombin activator with blood coagulation factor X." *Thromb.Haemost.*

gene duplication and evolution of molecular isoforms of prothrombin activators in

factor X, two functionally diverse prothrombin activators from Australian rough

prothrombin activators in Tropidechis carinatus snake: gene duplication and recruitment of factor X gene to the venom gland." *J.Thromb.Haemost.* 5:117-126. Robin Doley, Xingding Zhou, and R.M.Kini. 2009. "Snake Venom Phospholipase A2

Enzymes." In Stephen P.Mackessy, editor, *Handbook of Venoms and Toxins of Reptiles*.

Subcommittee on Nomenclature of Exogenous Hemostatic Factors of the Scientific and Standardization Committee of the International Society on Thrombosis and

from Echis carinatus venom. II. Coagulation studies in vitro and in vivo." *Naunyn* 

M.A.Lazzari, C.A.Sampaio, R.G.Pozner, J.S.Ventura, P.L.Ho, and A.M.Chudzinski-Tavassi. 2003. "A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular


**15** 

*1,2China 3USA* 

**A Puroindoline Mutigene Family** 

Feng Chen1,2, Fuyan Zhang1,

Craig F. Morris3 and Dangqun Cui1,2

*of Food Crops in Henan Province, Zhengzhou* 

**Associated with Yield-Related Traits** 

*1Department of Agronomy, Henan Agricultural University, Zhengzhou 2Key Laboratory of Physiological Ecology and Genetic Improvement* 

*Nutrition Facility East, Washington State University, Pullman,* 

**Exhibits Sequence Diversity in Wheat and is** 

*3USDA-ARS, Western Wheat Quality Laboratory, E-202 Food Science and Human* 

Kernel texture (grain hardness) is a leading quality characteristic of bread wheat (*Triticum aestivum* L.) as it dramatically influences the milling and processing properties, and consequently determines the classification and marketing of grain (Bhave and Morris 2008a, b). The word puroindoline is derived from the Greek word "*puros"* meaning wheat and "*indoline"* describing the indole ring of tryptophan (Gautier et al. 1994). Puroindolines, composed of puroindoline a and b, are amphipathic proteins of ca. 13,000 Da, and share homology with grain softness protein (GSP), purothionins, lipid transfer proteins, and other members of the prolamin super-family of proteins (Shewry and Halford 2002). Puroindoline proteins possess a characteristic tryptophan-rich domain and cysteine backbone; isoforms occur in the starchy endosperm of the Triticeae. Their secondary structure, determined by infrared and Raman spectroscopies, is comprised of approximately 30% *α*-helices, 30% *β*sheets, and 40% unordered structure at pH 7.4 in solution (Bihan et al. 1996). Puroindoline genes are present throughout the Triticeae tribe of the Poaceae (Gramineae), including wheat (*Triticum* sp.), rye (*Secale* sp.), barley (*Hordeum* sp.), and the wild relatives of wheat (*Aegilops* sp. and *Triticum* sp.). In *Triticum aestivum*, puroindolines exist as two expressed genes, Puroindoline a and Puroindoline b, and are located on the distal end of the short arm of chromosome 5 (5DS). An exception to this general situation lies with the tetraploid (AABB) wheats (*T. turgidum*), which include cultivated durum (ssp. *durum*). Apparently during the allotetraploidization formation of T. *diccocoides*, the wild ancestor of cultivated durum, both the A- and B-genome Puroindoline loci were eliminated due to transposable element insertion and two large deletions in the *Hardness* loci caused by illegitimate DNA recombination (Chantret et al. 2005). Consequently, hexaploid wheat, *T. aestivum*, possesses

**1. Introduction** 

