BACKGROUND
snake venom is a mixture of proteins and polypeptides which can be divided into enzymatic and non-enzymatic families  <cit> .
three-finger toxins are non-enzymatic polypeptides which belong to a well characterized superfamily of snake venom toxins.
structurally they have similar folds, with three β-sheeted loops  that are stabilized by 4– <dig> disulfide bridges present in the central core  <cit> .
despite their structural similarity, they differ widely in their molecular targets.
for example, members of this family target various receptor/ion channel proteins such as α1-nachrs, l-type calcium channels and integrin αiibβ <dig> .
such a wide diversity in their molecular targets is due to changes in their primary sequences, while keeping the basic molecular scaffold intact.
analysis of amino acid sequences and gene structures will help elucidate the molecular evolution of these functionally important toxins.
*nachr, nicotinic acetylcholine receptor
as with other snake venom proteins  <cit> , three-finger toxins are also encoded by a multigene family  <cit>  and contain functionally diversified isoforms.
these venom protein families have evolved through a process of gene duplication followed by accelerated point mutations in the protein coding region  <cit> .
for example, in phospholipase a <dig> genes, the dn/ds ratio in the coding regions is higher than in the non-coding regions  <cit> .
such adaptive darwinian evolution plays an important role in the evolution of novel gene functions, leading to functional diversity in each superfamily of toxins.
the accelerated rate of mutations in venom proteins likely provides a competitive edge in predator-prey interactions  <cit> .
until recently, three-finger toxins were thought to be present only in venoms of the snake families elapidae  <cit> .
however, we and others have demonstrated their presence in the venoms of colubrid and viperid snakes  <cit> .
recently we constructed a cdna library from the venom glands of sistrurus catenatus edwardsii  and identified a three-finger toxin family  in the venom gland transcriptome  <cit> .
as three-finger toxins are uncommon in viperid venoms, we performed rt-pcr using a separate pool of rna as template and found five transcripts that encode three-finger toxins  <cit> .
they have a  <dig> residue signal peptide followed by a mature protein consisting of 64– <dig> residues, and the ten conserved cysteine residues, which form five disulfide bridges characteristic of most three-finger toxins, are also present.
these viperid toxins belong to the non-conventional three-finger toxin subfamily that has the fifth disulfide bridge in the first loop  <cit> , and they were named 3ftx  <dig> through 3ftx  <dig>  3ftx  <dig> and  <dig> differ only in one residue in the mature protein, whereas all others are distinct isoforms.
all of them have nearly identical signal peptide sequences, but the sequence identity in the mature protein region is often low  with the exception of 3ftx  <dig> and  <dig>  .
a systematic comparison of amino acid sequences of the mature proteins indicate that some segments are highly conserved  between two or three isoforms, whereas other regions are not  .
such similarities and dissimilarities in various other segments were observed among these three-finger toxins and they appear to be evolving through "switching" of various segments.
to understand the molecular evolution of these toxins, we obtained the gene sequences of these three-finger toxins using genomic dna  pcr and genomewalking approaches.
this is the first report of gene structure of three-finger toxins from snakes of the family viperidae.
the analyses of their gene sequences show that segment "switching" occurs only in the exons, not in the introns.
this phenomenon of exchange is likely an important contributor to the gene evolution of this family of toxins which exhibit numerous distinct pharmacological effects.
RESULTS
gene structure
to understand the evolution of the three-finger toxins in s. catenatus edwardsii venom, we determined their gene structures .
we obtained the full length gene of 3ftx  <dig> by gdna pcr.
however, we obtained only partial genes  of the other four toxins by gdna pcr even after several attempts, perhaps due to suboptimal annealing of the primers or the thermal cycling profile.
we performed genomewalking to obtain the remaining gene segments from exon i to exon ii .
at least  <dig> clones were sequenced from each pcr product to obtain the gene sequences of all five three-finger toxins .
the cdna sequences were used to determine the exon-intron boundaries , which follow the gt-ag rule of splice-donor and -acceptor sites  <cit> .
genes of all five three-finger toxins have similar architecture, with two introns and three exons, similar to those of elapid three-finger toxin genes.
sequence data was deposited in genbank under accession numbers eu  <dig>  eu  <dig>  eu  <dig>  eu  <dig> and eu  <dig>  respectively.
the gene sequences were used to determine the phylogenetic relationship with three-finger toxin genes from the snake families elapidae and colubridae.
the phylogenetic tree was constructed using dnaman, and viperid three-finger toxin genes form a separate cluster in the tree .
intron i
intron i plays an important role in the expression of various genes  <cit>  and is the most variable region in elapid three-finger toxin genes  <cit> .
sequence alignment of intron i of the viperid genes revealed that it can be divided into segments similar to elapid three-finger toxin genes  <cit> .
segments i, ii, v, vii and x are conserved in all the genes except erabutoxin c gene .
in 3ftx  <dig>   <dig>   <dig> and  <dig>  segments iii and iv are missing.
new additional segments iiia, va and vc are present in 3ftx  <dig>   <dig>   <dig> and  <dig>  3ftx  <dig> has an insertion , whereas 3ftx  <dig> has one additional segment identified as vd .
interestingly, the additional segments in viperid three-finger gene are short or long nucleotide repeats.
segment va in 3ftx  <dig>   <dig>   <dig> and  <dig> has 19– <dig> continuous "taa" repeats, while the vb in 3ftx  <dig> has  <dig> continuous "gat" repeats.
these shorter repeats may represent microsatellite sequences.
the segment vd in 3ftx  <dig> has three different repeats: two repeats of tatttcattccattccatattttcgattctattcctgttctg , three repeats of tctattctattccactcc sequences  and  <dig> and  <dig> continuous repeats of ctatt  .
addition/deletion of segments in intron i is also observed in elapid and hydrophiid genes and was linked to the evolutionary diversification of these snake toxin genes  <cit> .
intron i of all viperid three-finger genes are nearly identical; among 3ftx  <dig>   <dig>   <dig> and  <dig> there is only a short insertion  in 3ftx  <dig>  3ftx  <dig> is distinct from other 3ftxs.
it has two additional segments  and segment iiia is missing .
the additional segments are either deleted  or added  in the gene of three-finger toxins during their evolution, but the role of these insertions/deletions in the expression of the three-finger toxins is currently unknown.
interestingly, the region of exon-intron boundary is similar to elapid and hydrophiid genes and seems to be conserved among all of them.
intron ii
intron ii of the elapid and hydrophiid three-finger toxin genes is conserved and was thought to be not segmented  <cit> .
however, comparison of the intron ii sequences of viperid genes reveals that it also can be divided into segments, similar to intron i .
segments i, iii and v are conserved in all three-finger genes.
however, 3ftx  <dig>   <dig>   <dig> and  <dig> genes have an additional common segment , and 3ftx  <dig> has one additional segments .
thus, segmentation appears to be a common structural feature of both introns, and the insertion/deletion of segments may contribute to their regulation of expression.
further, analysis of segments may facilitate understanding the evolutionary history of this unusual gene structural feature.
segment switching in exons
exons also appeared to have segments .
as expected because of overall size, the segments in exons are much smaller as compared to those in introns .
analysis of the gene sequences reveals that the gain/loss of segments occurs in both introns and exons .
the segments in introns, when present, show high similarities .
in contrast, while some exon segments show high sequence identity , other segments show low identity .
such similarity/dissimilarity of segments is more prominent in exon ii and exon iii.
in exon ii, there are four different kinds of segment ii; in 3ftx  <dig> and  <dig> they are identical, whereas in the other genes they are totally different .
in the same way, segment v is similar in 3ftx  <dig>   <dig> and  <dig>  whereas in 3ftx  <dig> and  <dig> it is different.
segment vi is absent in 3ftx  <dig> but present in the other genes; this segment is similar in 3ftx  <dig> and  <dig> but different in 3ftx  <dig> and  <dig>  thus exon ii appears to have evolved through "switching" of segments, although the origins of the "new" segments are not known.
similarly, exon iii also appears to have evolved through "switching" of segments.
however, this phenomenon is not observed in introns of these genes, although there are additions/deletions of a few short segments in introns .
the switching of segments in exons has far reaching effects on the biological effects of the toxins.
the changed segments will affect the overall surface properties of the expressed protein  and hence the function.
in contrast, the small additions/deletions in introns will not affect the nature of the protein product, but may affect its expression.
it is important to note that in spite of this segment switching, positions of cysteine residues are conserved, thereby maintaining similar disulfide pairing, folding architecture and the overall three-finger fold of the mature protein.
there are several possibilities that could explain the observed switching of segments.
<dig>  splicing variations: the difference in isoforms of some proteins due to change of segments can be easily explained based on splicing variation  <cit> .
however, unlike these proteins, the segment switching in viperid 3ftx occurs within exon ii and exon iii.
among three-finger toxins, long-chain neurotoxins arose from short-chain neurotoxins through an error in the splicing site  <cit> .
such an error leads to insertion of a short segment with the fifth disulfide in the second loop.
in viperid toxin genes, however, the insertions of segments do not occur at the intron-exon boundaries, so the mechanism of insertions/deletions or switching of segments does not occur due to errors in splicing.
<dig>  recombination: distinct genes encoding isoforms of proteins are also generated through recombination of two related/unrelated genes  <cit> .
in general, the segments involved in such recombination events are fairly large , and the segments that are exchanged in exons of 3ftxs are probably much too small.
therefore, canonical recombination may not be involved in these exchange events.
<dig>  accumulation of point mutations: accelerated point mutations in three-finger toxins are common and they lead to the evolution of several isoforms  <cit> .
although these point mutations occur in exon segments, they may not explain such a distinct change in the sequences of segments.
this possibility requires the repeated occurrence and accumulation of multiple point mutations within each segment.
further, all the intermediates have to be selected through evolution.
the absence of intermediate isoforms, however, contradicts this possibility; and
<dig>  independent recruitment events: venom protein genes are thought to be recruited to the venom gland genome by gene duplication of a normal physiologically important gene and recruitment of the duplicated gene for expression in the venom gland  <cit> .
it is possible, but not probable, that each of these isoforms has an independent origin and their ancestral three-finger toxin genes were recruited at different times.
high similarity across numerous 3ftx genes in exon i, and introns i and ii, supports instead a single recruitment and lineage of these genes and not multiple recruitment events.
in the unlikely events of independent recruitments, introns will have to undergo convergent evolution to explain the high similarity while the exons will be undergoing divergent evolution.
therefore, segment switching results in divergence of functional regions of exons  while maintaining the basic fold, rather than convergence upon a single scaffold motif during independent recruitment.
although the mechanism of the exchange of segments in exons is unknown, it is apparent that these events play important roles in the evolution of these toxins, in addition to the role that accelerated point mutations in the exons plays in toxin evolution  <cit> .
these point mutations appear to alter the interaction surfaces of toxins  <cit> .
however, they affect one residue at a time and a smaller molecular surface, and they may help in fine-tuning of functional sites of the molecule for interaction with specific molecular targets.
they may  enhance the affinity to a specific receptor or ion channel;  change the specificity to another closely related receptor or ion channel; and  change the species specificity of the toxin to a particular receptor.
in contrast, accelerated exchange of larger segments drastically changes parts of the interacting loops or toxin surface.
therefore, these exchanges of segments may help in switching the molecular targets of toxins and hence affecting their pharmacological properties.
we propose that accelerated segment switch in exons to alter targeting  is a phenomenon which plays an important role in "remodeling" a toxin toward a different and novel receptor target .
asset and evolution of molecular surfaces
to understand the impact of switching of segments on the molecular surfaces of three-finger toxins in s. catenatus edwardsii, we modeled all four distinct three-finger toxins.
as shown in figure  <dig>  the three β-sheeted loops in these toxins are distinctly different from one another, as most of them are replaced by segment switching .
further, the electrostatic potentials of these toxins indicate that the charge distributions on their molecular surfaces are also different.
3ftx  <dig> and  <dig> have more acidic residues on the surface as compared to 3ftx  <dig> and  <dig> .
this drastic difference in the charge residues on the surface is due to the exchange of segments , but retention of the similar molecular fold.
such a change in the charged residues might play an important role in switching the molecular targets.
since most of the functional sites are located on these β-sheeted loops , it is logical to propose that all of these novel viperid toxins have distinct pharmacological properties.
therefore, asset phenomena affect the molecular surfaces of three-finger toxins significantly and alter their molecular targets, playing a crucial role in the evolution of the three-finger toxins.
CONCLUSIONS
systematic analyses of gene sequences of sistrurus catenatus edwardsii  three-finger toxins indicate that short segments in exons ii and iii are changed more rapidly compared to intron segments.
we propose that such a phenomenon  of accelerated segment switching in exons has the effect of rapidly altering the molecular surface properties.
this mechanism of rapid change can provide a selective advantage to venomous snakes in predator/prey coevolutionary arms races, resulting in a diversity of structurally similar toxins in a single venom and allowing the venom toxins repertoire to stay a step ahead of prey defensive responses  <cit> .
thus asset plays an important role in changing the molecular target and hence the pharmacology of these toxins.

 snake venom is a mixture of proteins and polypeptides which can be divided into enzymatic and non-enzymatic families. three-finger toxins are non-enzymatic polypeptides which belong to a well characterized superfamily of snake venom toxins. structurally they have similar folds, with three β-sheeted loops that are stabilized by 4– disulfide bridges present in the central core. despite their structural similarity, they differ widely in their molecular targets. for example, members of this family target various receptor/ion channel proteins such as α1-nachrs, l-type calcium channels and integrin αiibβ. such a wide diversity in their molecular targets is due to changes in their primary sequences, while keeping the basic molecular scaffold intact. analysis of amino acid sequences and gene structures will help elucidate the molecular evolution of these functionally important toxins. *nachr, nicotinic acetylcholine receptor as with other snake venom proteins, three-finger toxins are also encoded by a multigene family and contain functionally diversified isoforms. these venom protein families have evolved through a process of gene duplication followed by accelerated point mutations in the protein coding region. for example, in phospholipase a genes, the dn/ds ratio in the coding regions is higher than in the non-coding regions. such adaptive darwinian evolution plays an important role in the evolution of novel gene functions, leading to functional diversity in each superfamily of toxins. the accelerated rate of mutations in venom proteins likely provides a competitive edge in predator-prey interactions. until recently, three-finger toxins were thought to be present only in venoms of the snake families elapidae. however, we and others have demonstrated their presence in the venoms of colubrid and viperid snakes. recently we constructed a cdna library from the venom glands of sistrurus catenatus edwardsii and identified a three-finger toxin family in the venom gland transcriptome. as three-finger toxins are uncommon in viperid venoms, we performed rt-pcr using a separate pool of rna as template and found five transcripts that encode three-finger toxins. they have a residue signal peptide followed by a mature protein consisting of 64– residues, and the ten conserved cysteine residues, which form five disulfide bridges characteristic of most three-finger toxins, are also present. these viperid toxins belong to the non-conventional three-finger toxin subfamily that has the fifth disulfide bridge in the first loop, and they were named 3ftx through 3ftx 3ftx and differ only in one residue in the mature protein, whereas all others are distinct isoforms. all of them have nearly identical signal peptide sequences, but the sequence identity in the mature protein region is often low with the exception of 3ftx and. a systematic comparison of amino acid sequences of the mature proteins indicate that some segments are highly conserved between two or three isoforms, whereas other regions are not. such similarities and dissimilarities in various other segments were observed among these three-finger toxins and they appear to be evolving through "switching" of various segments. to understand the molecular evolution of these toxins, we obtained the gene sequences of these three-finger toxins using genomic dna pcr and genomewalking approaches. this is the first report of gene structure of three-finger toxins from snakes of the family viperidae. the analyses of their gene sequences show that segment "switching" occurs only in the exons, not in the introns. this phenomenon of exchange is likely an important contributor to the gene evolution of this family of toxins which exhibit numerous distinct pharmacological effects.  gene structure to understand the evolution of the three-finger toxins in s. catenatus edwardsii venom, we determined their gene structures. we obtained the full length gene of 3ftx by gdna pcr. however, we obtained only partial genes of the other four toxins by gdna pcr even after several attempts, perhaps due to suboptimal annealing of the primers or the thermal cycling profile. we performed genomewalking to obtain the remaining gene segments from exon i to exon ii. at least clones were sequenced from each pcr product to obtain the gene sequences of all five three-finger toxins. the cdna sequences were used to determine the exon-intron boundaries, which follow the gt-ag rule of splice-donor and -acceptor sites. genes of all five three-finger toxins have similar architecture, with two introns and three exons, similar to those of elapid three-finger toxin genes. sequence data was deposited in genbank under accession numbers eu eu eu eu and eu respectively. the gene sequences were used to determine the phylogenetic relationship with three-finger toxin genes from the snake families elapidae and colubridae. the phylogenetic tree was constructed using dnaman, and viperid three-finger toxin genes form a separate cluster in the tree. intron i intron i plays an important role in the expression of various genes and is the most variable region in elapid three-finger toxin genes. sequence alignment of intron i of the viperid genes revealed that it can be divided into segments similar to elapid three-finger toxin genes. segments i, ii, v, vii and x are conserved in all the genes except erabutoxin c gene. in 3ftx and segments iii and iv are missing. new additional segments iiia, va and vc are present in 3ftx and 3ftx has an insertion, whereas 3ftx has one additional segment identified as vd. interestingly, the additional segments in viperid three-finger gene are short or long nucleotide repeats. segment va in 3ftx and has 19– continuous "taa" repeats, while the vb in 3ftx has continuous "gat" repeats. these shorter repeats may represent microsatellite sequences. the segment vd in 3ftx has three different repeats: two repeats of tatttcattccattccatattttcgattctattcctgttctg, three repeats of tctattctattccactcc sequences and and continuous repeats of ctatt. addition/deletion of segments in intron i is also observed in elapid and hydrophiid genes and was linked to the evolutionary diversification of these snake toxin genes. intron i of all viperid three-finger genes are nearly identical; among 3ftx and there is only a short insertion in 3ftx 3ftx is distinct from other 3ftxs. it has two additional segments and segment iiia is missing. the additional segments are either deleted or added in the gene of three-finger toxins during their evolution, but the role of these insertions/deletions in the expression of the three-finger toxins is currently unknown. interestingly, the region of exon-intron boundary is similar to elapid and hydrophiid genes and seems to be conserved among all of them. intron ii intron ii of the elapid and hydrophiid three-finger toxin genes is conserved and was thought to be not segmented. however, comparison of the intron ii sequences of viperid genes reveals that it also can be divided into segments, similar to intron i. segments i, iii and v are conserved in all three-finger genes. however, 3ftx and genes have an additional common segment, and 3ftx has one additional segments. thus, segmentation appears to be a common structural feature of both introns, and the insertion/deletion of segments may contribute to their regulation of expression. further, analysis of segments may facilitate understanding the evolutionary history of this unusual gene structural feature. segment switching in exons exons also appeared to have segments. as expected because of overall size, the segments in exons are much smaller as compared to those in introns. analysis of the gene sequences reveals that the gain/loss of segments occurs in both introns and exons. the segments in introns, when present, show high similarities. in contrast, while some exon segments show high sequence identity, other segments show low identity. such similarity/dissimilarity of segments is more prominent in exon ii and exon iii. in exon ii, there are four different kinds of segment ii; in 3ftx and they are identical, whereas in the other genes they are totally different. in the same way, segment v is similar in 3ftx and whereas in 3ftx and it is different. segment vi is absent in 3ftx but present in the other genes; this segment is similar in 3ftx and but different in 3ftx and thus exon ii appears to have evolved through "switching" of segments, although the origins of the "new" segments are not known. similarly, exon iii also appears to have evolved through "switching" of segments. however, this phenomenon is not observed in introns of these genes, although there are additions/deletions of a few short segments in introns. the switching of segments in exons has far reaching effects on the biological effects of the toxins. the changed segments will affect the overall surface properties of the expressed protein and hence the function. in contrast, the small additions/deletions in introns will not affect the nature of the protein product, but may affect its expression. it is important to note that in spite of this segment switching, positions of cysteine residues are conserved, thereby maintaining similar disulfide pairing, folding architecture and the overall three-finger fold of the mature protein. there are several possibilities that could explain the observed switching of segments. splicing variations: the difference in isoforms of some proteins due to change of segments can be easily explained based on splicing variation. however, unlike these proteins, the segment switching in viperid 3ftx occurs within exon ii and exon iii. among three-finger toxins, long-chain neurotoxins arose from short-chain neurotoxins through an error in the splicing site. such an error leads to insertion of a short segment with the fifth disulfide in the second loop. in viperid toxin genes, however, the insertions of segments do not occur at the intron-exon boundaries, so the mechanism of insertions/deletions or switching of segments does not occur due to errors in splicing. recombination: distinct genes encoding isoforms of proteins are also generated through recombination of two related/unrelated genes. in general, the segments involved in such recombination events are fairly large, and the segments that are exchanged in exons of 3ftxs are probably much too small. therefore, canonical recombination may not be involved in these exchange events. accumulation of point mutations: accelerated point mutations in three-finger toxins are common and they lead to the evolution of several isoforms. although these point mutations occur in exon segments, they may not explain such a distinct change in the sequences of segments. this possibility requires the repeated occurrence and accumulation of multiple point mutations within each segment. further, all the intermediates have to be selected through evolution. the absence of intermediate isoforms, however, contradicts this possibility; and independent recruitment events: venom protein genes are thought to be recruited to the venom gland genome by gene duplication of a normal physiologically important gene and recruitment of the duplicated gene for expression in the venom gland. it is possible, but not probable, that each of these isoforms has an independent origin and their ancestral three-finger toxin genes were recruited at different times. high similarity across numerous 3ftx genes in exon i, and introns i and ii, supports instead a single recruitment and lineage of these genes and not multiple recruitment events. in the unlikely events of independent recruitments, introns will have to undergo convergent evolution to explain the high similarity while the exons will be undergoing divergent evolution. therefore, segment switching results in divergence of functional regions of exons while maintaining the basic fold, rather than convergence upon a single scaffold motif during independent recruitment. although the mechanism of the exchange of segments in exons is unknown, it is apparent that these events play important roles in the evolution of these toxins, in addition to the role that accelerated point mutations in the exons plays in toxin evolution. these point mutations appear to alter the interaction surfaces of toxins. however, they affect one residue at a time and a smaller molecular surface, and they may help in fine-tuning of functional sites of the molecule for interaction with specific molecular targets. they may enhance the affinity to a specific receptor or ion channel; change the specificity to another closely related receptor or ion channel; and change the species specificity of the toxin to a particular receptor. in contrast, accelerated exchange of larger segments drastically changes parts of the interacting loops or toxin surface. therefore, these exchanges of segments may help in switching the molecular targets of toxins and hence affecting their pharmacological properties. we propose that accelerated segment switch in exons to alter targeting is a phenomenon which plays an important role in "remodeling" a toxin toward a different and novel receptor target. asset and evolution of molecular surfaces to understand the impact of switching of segments on the molecular surfaces of three-finger toxins in s. catenatus edwardsii, we modeled all four distinct three-finger toxins. as shown in figure the three β-sheeted loops in these toxins are distinctly different from one another, as most of them are replaced by segment switching. further, the electrostatic potentials of these toxins indicate that the charge distributions on their molecular surfaces are also different. 3ftx and have more acidic residues on the surface as compared to 3ftx and. this drastic difference in the charge residues on the surface is due to the exchange of segments, but retention of the similar molecular fold. such a change in the charged residues might play an important role in switching the molecular targets. since most of the functional sites are located on these β-sheeted loops, it is logical to propose that all of these novel viperid toxins have distinct pharmacological properties. therefore, asset phenomena affect the molecular surfaces of three-finger toxins significantly and alter their molecular targets, playing a crucial role in the evolution of the three-finger toxins.  systematic analyses of gene sequences of sistrurus catenatus edwardsii three-finger toxins indicate that short segments in exons ii and iii are changed more rapidly compared to intron segments. we propose that such a phenomenon of accelerated segment switching in exons has the effect of rapidly altering the molecular surface properties. this mechanism of rapid change can provide a selective advantage to venomous snakes in predator/prey coevolutionary arms races, resulting in a diversity of structurally similar toxins in a single venom and allowing the venom toxins repertoire to stay a step ahead of prey defensive responses. thus asset plays an important role in changing the molecular target and hence the pharmacology of these toxins. 
thus the phenomenon of accelerated segment switch in exons to alter targeting may play an important role in the evolution of three-finger toxins, resulting in a family of toxins with a highly conserved structural fold but widely varying biological activities. such a "switching" of segments in exons may result in drastically altering the molecular surface topology and charge, and hence the molecular targets of these three-finger toxins. here we report five gene structures of three-finger toxins from a viperid snake, sistrurus catenatus edwardsii. venom proteins are encoded by multigene families that have evolved through a process of gene duplication followed by accelerated evolution in the protein coding region. these toxin genes are structured similarly to elapid and hydrophiid three-finger toxin genes, with two introns and three exons. both introns and exons show distinct patterns of segmentation, and the insertion/deletion of segments may define their evolutionary history.
 snake venoms consist primarily of proteins and peptides showing a myriad of potent biological activities which have been shaped by both adaptive and neutral selective forces. venom proteins are encoded by multigene families that have evolved through a process of gene duplication followed by accelerated evolution in the protein coding region.  here we report five gene structures of three-finger toxins from a viperid snake, sistrurus catenatus edwardsii. these toxin genes are structured similarly to elapid and hydrophiid three-finger toxin genes, with two introns and three exons. both introns and exons show distinct patterns of segmentation, and the insertion/deletion of segments may define their evolutionary history. the segments in introns, when present, are highly similar to their corresponding segments in other members of the gene family. in contrast, some segments in the exons show high similarity, while others are often distinctly different among corresponding regions of the isoforms.  ordered, conserved exon structure strongly suggests that segments in corresponding regions in exons have been exchanged with distinctly different ones during the evolution of these genes. such a "switching" of segments in exons may result in drastically altering the molecular surface topology and charge, and hence the molecular targets of these three-finger toxins. thus the phenomenon of accelerated segment switch in exons to alter targeting may play an important role in the evolution of three-finger toxins, resulting in a family of toxins with a highly conserved structural fold but widely varying biological activities. 