BACKGROUND
australian elapids are among the most venomous land snakes of the world.
various bioactive peptide have been purified and characterized from these venoms and a comprehensive survey of the bioactive proteins present in a number of these snake venoms have been reported recently  <cit> .
snakes in austrelaps genus are widely distributed in australia, but are moderately toxic compared to other elapids  <cit> .
presently three species of austrelaps  are known – lowland copperhead , highland copperhead  and pygmy copperhead .
so far, only a small number of proteins have been isolated and characterized from a. superbus venom.
they are mostly phospholipase a <dig>  enzymes  <cit>  and cobra venom factor-like protein  <cit> .
however, no significant data on the venoms of other two species are available.
pigmy copperhead  is smaller in size as compared to a. superbus and a. ramsayi  <cit>  and it has distinguishable white bars to its upper lips, circular eyes and yellowish-brown iris  <cit> .
they mainly feed on small lizards and frogs  <cit>  and the ld <dig> of their venom is  <dig>  mg/kg  <cit> .
in the present study, we have attempted to profile venom components of a. labialis to define its composition and to look for novel proteins.
profiling venom toxins can be achieved via transcriptomics or proteomics approach.
in former approach, the transcripts are directly sequenced from a cdna library constructed from the venom gland, whereas in the latter approach venom proteins are separated using various techniques like lcms, 2d gel electrophoresis and hplc.
we have obtained toxin profile of pygmy copperhead venom by constructing of cdna.
the data revealed that neurotoxins and pla <dig> are the most abundant proteins in this venom.
interestingly, most of the toxin families in this cdna library have truncated transcripts.
we propose that the lower venom toxicity and subsequent decreased size of these snakes could be due to unusually high degree of deletions or insertions  in their toxin genes resulting in truncated, most likely inactive products.
methods
construction of library and dna sequencing
venom glands were dissected and extracted from an euthanized a. labialis snake captured in kangaroo island, south australia .
total rna was extracted using rneasy® mini kit from qiagen .
the purity and the concentration were spectrophotometrically determined.
first strand cdnas were synthesized from  <dig> ng of total rna according to protocol of creator™ smart™ cdna library construction kit obtained from clontech laboratories .
amplification of full-length double-stranded cdna was carried out using pcr-based protocol.
double-stranded cdna pcr products  were purified and subjected to ta cloning.
ligation products were transformed into the competent top <dig> e. coli strain obtained from invitrogen  and plated on lb/amp/iptg/x-gal for blue/white screening.
a bidirectional pgem®-t easy vector system obtained from promega  plasmid-based cdna library was titered with  <dig>  ×  <dig> cfu/ml.
individual colonies were picked randomly and the presence of insert was confirmed by ecori digestion.
only clones containing inserts larger than  <dig> bp in length were selected for further dna sequencing.
dna sequencing reactions were carried out using the abi prism® bigdye® terminator cycle sequencing ready reaction kit  according to manufacturer's instructions .
dna sequencing was carried out using abi prism®  <dig> automated dna sequencer.
sequencing was repeated at least twice in all singletons to make sure there was no error created by sequencing step.
cloning snake venom protein with two kunitz domains
pcr amplification of protein containing two kunitz-type domain from rna pool was performed using a forward primer designed from the signal peptide-encoding region  and the reverse primer from library construction kit 30-3').
long pcr enzyme mix  was used in the amplification reaction.
pcr was performed as follows:  <dig> cycles of one step each at 95°c for  <dig> s, 58°c for  <dig> s, 68°c for  <dig> min followed by a final extension step at 68°c for  <dig> min.
pcr products were subjected to 1% agarose gel and visualized using ethidium bromide staining.
amplified pcr products were gel purified and cloned to pgem®-t easy vector system and sequenced subsequently.
analysis of sequence data by bioinformatics tools
the dna sequences were analyzed after trimming the adaptor sequences  and the putative functions of the gene products were predicted by batch blasting sequence results in grid blast system.
the signal peptide was predicted using online signalp  <dig>  server.
clustering of sequences was performed using fastgroupii  <cit>  and sequences were aligned using clustalw  <cit> .
jalview  <cit>  was employed to make some necessary editing on sequence alignments.
phylogenetic trees were generated using phyml  <cit> , which uses maximum likelihood method to build phylogenetic tree.
RESULTS
compositions of cdna library
we randomly selected and isolated  <dig> clones.
of these only  <dig> clones, which had larger than  <dig> bp inserts, were sequenced and analyzed.
these ests were categorized into different clusters on the basis of sequence similarities.
members of each clusters has been sequenced completely and the full length sequences were submitted to ncbi with accession numbers from eu <dig> to eu <dig>  eu <dig> and eu <dig>  blast search against nucleotide, protein and est database, revealed that 60% of the est belong to putative toxins, while 32% belong to cellular transcripts and 8% of the est were unknown transcripts .
these relative ratios may change, if more est clones are sequenced and analyzed.
toxin genes
putative toxin genes found in the cdna library were further grouped based on their sequence similarities to known toxin superfamilies .
in total, we found six superfamilies of the toxin genes in the cdna library of a. labialis.
these families, in order of abundance, are three-finger toxin family , phospholipase a <dig> family , kunitz-type proteinase inhibitor family , cysteine-rich protein family , metalloproteinase family  and c-type lectin family  .
three-finger toxin family
three-finger toxin family is a well characterized non-enzymatic polypeptide family named after their canonical protein folds.
members of this family contain about 60– <dig> amino acid residues  <cit>  and exhibit various pharmacological activities such as neurotoxic, cytotoxic, cardiotoxic, anticoagulant and antiplatelet effects  <cit> .
three-finger family forms the major group of toxins  in this cdna library, and majority of mature proteins are 66– <dig> amino acid residues in length.
we identified a total of  <dig> sequences  with 60%–80% identities to known long-chain neurotoxins  and  <dig> sequences  with 60%–85% identical to short-chain neurotoxins .
long-chain neurotoxins
clone 8a  is the most dominant long-chain neurotoxin and other clones  are its isoforms with few amino acid substitutions .
most of these substitutions are due to single nucleotide changes.
since these isoforms are represented by many clones, these sequence differences are not artifacts of sequencing.
however, clone 5b has  <dig> substitutions and hence may have distinct specificity compared to other long-chain neurotoxins.
further, a dinucleotide  deletion in front of the typical stop codon position followed by a dinucleotide  insertion  in the gene resulted in a frame shift and additional five amino acid residues in clone 5b .
however, two clones  are distinctively shorter due to deletion of nucleotides a <dig> and g <dig> respectively , resulting in the frame shift and truncated peptides.
there were eight clones identical to clone  <dig> suggesting that this observation is not due to sequencing error.
the segments after the deletion show significant identity with other complete transcripts.
similar phenomenon was observed in sea snake aipysurus eydouxii in which a dinucleotide deletion leads to the truncation of the only three-finger neurotoxin found in the venom gland library and the loss of toxicity  <cit> .
in contrast, in clone  <dig> deletion of a <dig> leads to a potentially much longer protein product.
in this case, three of the conserved cysteine residues at the c-terminal end are lost due to the frame shift.
three distinct neurotoxin sequences  compared to clone 8a and its closer sibling toxins were also found in the cdna library.
clones  <dig> and  <dig> encode for slightly longer proteins with  <dig> and  <dig> amino acid residues respectively as compared to other clones.
in these cases there is a single nucleotide substitution at consensus stop codon  present in the majority of these putative long-chain neurotoxins.
in clone  <dig>  a dinucleotide  deletion in the stop codon followed by a dinucleotide  insertion and a dinucleotide deletion  has been found.
most of the putative full length long-chain neurotoxins possess the functionally conserved residues such as lys <dig>  trp <dig>  asp <dig>  phe <dig>  arg <dig> and lys <dig> that are shown to participate in the binding to torpedo nicotinic acetylcholine receptor  in the case of α-cobratoxin from naja naja venom  <cit> .
however, lys <dig>  is replaced by the negatively charged glu <dig> .
this charge reversal may significantly reduce its interaction with torpedo nachr as mutation of k <dig> to e <dig> in α-cobratoxin cause  <dig> fold decrease in binding affinity  <cit> .
further, neutrally charged ala <dig>  which is specific for binding to α7-nachr in α-cobratoxin, is replaced by the positively charged arg <dig> with the exception of clone 5b .
this substitution did not affect the binding affinity to the receptor in the case of α-cobratoxin  <cit> .
the majority of a. labialis long-chain neurotoxins form a distinctive group almost completely separated from the other long-chain neurotoxins .
phylogenetically, this separation seems to start quite early in the evolution time frame as indicated by early branching pattern originating from the root.
interestingly, three distinct clones  form a separate cluster as compared to other long-chain neurotoxins and appear to have a completely different phylogenetic origin.
short-chain neurotoxins
two representatives of short-chain neurotoxins, clone 6a and 12b were found in the library .
their amino acid sequences are identical except for the substitution of thr <dig> by ala. both short-chain neurotoxins have one additional cys residue at the fourth position .
appearance of cys at 4th position is observed for short-chain neurotoxins from sea snake venom  <cit> .
they have functionally invariant residues such as lys <dig>  trp <dig>  asp <dig>  arg <dig> and lys <dig> which are important in the binding of erabutoxin a  to torpedo nachr  <cit> .
interestingly, one of the binding residues, phe <dig> found in erabutoxin-a, is replaced by his in a. labialis short-chain neurotoxins which might effect its binding to the receptor.
phospholipase a <dig> family
phospholipase a <dig>  enzymes are esterolytic enzymes which hydrolyze glycerophospholipids at the sn- <dig> position of the glycerol backbone releasing lysophospholipids and free fatty acids.
snake venom pla <dig> enzymes are one of the well studied superfamily of snake venom enzymes  <cit>  which play an important role in immobilization and capture of prey.
in addition to their role in digestion of preys, they exhibit variety of pharmacological effects such as neurotoxic, myotoxic, cardiotoxic, anticoagulant, antiplatelet and edema-inducing effects  <cit> .
a total of  <dig> pla <dig> cdna clones were obtained which makes up the second abundant family.
the deduced amino acid sequences are 65% to 97% identical to other pla <dig> sequences and were grouped into three clusters and four singletons.
all pla <dig> cdna sequences obtained from this snake belong to group ia.
clones  <dig>   <dig>   <dig> and  <dig> are the most abundant clones in this library and are closely related to the pla <dig> sequences of a. superbus  <cit>  of the same genus.
however, they are different from the other pla <dig> enzymes due to the presence of three tryptophan residues in the β-wing.
at present, role of these trp residues are not known.
these pla <dig> enzymes together with five pla <dig> isoforms of a. superbus   <cit>  form a distinguishable cluster shown in phylogram generated by phyml method .
this cluster is well separated from the other pla <dig> enzymes having one tryptophan residue in the β-wing.
clones  <dig> and  <dig> have significant difference in amino acid sequences of other a. labialis pla <dig> enzymes.
clone  <dig> encodes for the full length protein sequence, whereas clone  <dig> encodes for a truncated product due to a single nucleotide  addition at position  <dig>   leading to frame shift and premature truncation .
these clones form a distinct cluster along with aad <dig>  .
kunitz-type serine protease inhibitor family
kunitz-type of protease inhibitors are  <dig> residues long which possess the conserved sequence motif of  <dig> cysteines   <cit>  and functionally belong to bovine pancreatic trypsin inhibitor  family  <cit> .
this family of snake venom protein is known to involve in coagulation, fibrinolysis and inflammation through interaction with various proteases  <cit> .
<dig> cdna clones in this library belong to kunitz-type serine protease inhibitor family.
the sequences of this group are 77%–90% identical to mulgin isoforms  and textilinin isoforms  .
textilinins are shown to be distinct plasmin inhibitors and have a 60% bleeding reduction in murine model  <cit> .
it will be interesting to study the role of these kunitz-type serine proteases inhibitors in blood clotting cascade.
however one of the clones, clone  <dig> was found to be truncated prematurely due to transversion of one nucleotide  at position  <dig> .
this clone was resequenced to confirm the transversion is not due to sequencing error.
a unique clone  encoding protein with two tandem kunitz-type protease inhibitor domains was found in this cdna library.
the entire cdna sequence is  <dig>  bp long  and the deduced peptide has  <dig> amino acid residues with potential glycosylation site.
its presence was further confirmed by pcr using gene specific and reverse primer from cdna construction kit.
we sequenced  <dig> clones and seven isoforms with minor changes in the amino acid sequence were found .
they are 40% identical to bikunin, a serine proteinase inhibitor with two kunitz domains  <cit> .
bikunin from human placenta exhibits a potent inhibitory effect to some serine proteases crucial in the blood coagulation and fibrinolysis, such as factor xia and plasmin  <cit> .
this is the first report of this novel member of kunitz-type serine protease inhibitor family from snake venom.
cysteine-rich protein family
cysteine-rich secretory proteins  are abundantly found in mammalian reproductive tracts and play important role in sperm maturation and immune system  <cit> .
they have also been found in venoms of reptiles.
the conserved sequence in crisps span through out the protein with  <dig> cysteine residues and  <dig> of them resides in the c-terminal end  <cit> .
crisps have been purified and characterized from various snake venoms  <cit> .
functions of majority of these proteins are unknown.
some of them are known to block cyclic nucleotide-gated ion channels  <cit>  while several others block potassium-stimulated smooth muscle contraction  <cit> .
<dig> cdna clones encoding crisps making up 8% of the library has been observed.
this family has one cluster and four singletons .
clone  <dig> has a dinucleotide deletion  in position  <dig> and series of single nucleotide deletion of a in positions  <dig>   <dig>   <dig> and  <dig>  single nucleotide deletion of t, c and g at position  <dig>   <dig> and  <dig> respectively.
clone  <dig> has two single nucleotide deletions of a <dig> and a <dig>  clone  <dig> has also two single nucleotide deletions of a <dig> and g <dig>  clones  <dig> and  <dig> have an insertion of one a at position  <dig> while clone  <dig> has another insertion of t in position  <dig>  in case of clone  <dig> there is an insertion of t and a at position  <dig> and  <dig>  deletions of t <dig>  a <dig>  a <dig>  a <dig> and a <dig> .
due to these insertions in clone  <dig> and  <dig> c-terminal end has encoded different amino acid residues as compared to other crisps.
clones  <dig>   <dig>   <dig> and  <dig> have several deletions in the orf leading to frame shift and premature truncation.
thus, all of them are prematurely terminated losing the c-terminal domain where most of the conserved cysteine residues are found .
thus the truncated transcript might not be functional.
however, the n-terminal region have high identity to snake venom crisps , but low identity to human, murine, frog crisps .
metalloproteinase family
snake venom metalloproteinases are more abundantly found in viper venoms, but they are also reported from elapid family  <cit> .
they are synthesized as zymogens in the venom gland and contain a propeptide which is cleaved off during maturation.
they have a common zinc binding site with a consensus sequence of hexxhxxgxxh  <cit> .
they are classified into different types  on the basis of the other domains that are present in these complexes  <cit> .
this family of enzymes are responsible for haemorrhagic  <cit> , local myonecrotic  <cit> , antiplatelet  <cit> , edema-inducing and other inflammatory effects  <cit> .
we have found  <dig> clones which makes 3% of the entire cdna library, however they code for only the signal peptide and the n-terminal region similar to trigramin precursor and mocarhagin .
this is due to the loss of two nucleotides at position  <dig> downstream of atg leading to premature termination of the protein.
thus, if proteins of this family are present in the venom, they would have only partial propeptide without any other functional domains of metalloproteinase or disintegrin.
c-type lectin family
c-type lectins are non-enzymatic proteins that bind to mono- and oligosaccharides in presence of ca2+  <cit> .
generally they contain the highly conserved domain called the carbohydrate recognition domain   <cit> .
c-type lectins and related proteins have been frequently reported from viperidae snake venoms.
venom c-type lectin related proteins are known to disrupt the normal functioning of haemostatic mechanism by interfering in the normal platelet receptor-ligand interaction, binding to coagulation factors and other important proteins in coagulation cascade  <cit> .
only few of them have been reported from elapids  <cit>  including australian elapids  <cit> .
this family has seven clones which form one cluster.
the primary structure shows 71% and 74% identical to c-type lectins found in bungarus fasciatus and b. multicinctus  <cit> , 56% identical to galactose-binding lectin found in bitis arietans  <cit> , but less than 37% identical to c-type lectin related proteins.
the deduced amino acid sequence reveals the conserved carbohydrate binding domain as well as the ca2+ interaction amino acid residues .
the odd cysteine present is likely to involved in interchain disulphide bond  <cit> .
the presence of only one cluster of  <dig> clones indicates that it may exist as homodimer similar to other c-type lectins.
further, phylogenetic analysis  revealed that this putative c-type lectin is more closely related to galactose-binding proteins and forms a cluster distinct from that of c-type lectin like proteins that bind to blood coagulation factors or platelet receptors.
thus, this clone most likely encodes a homodimeric c-type lectin.
cellular transcripts
<dig> clones were identified as cellular trancripts which were grouped into  <dig> clusters and  <dig> singleton which constituted 32% of the entire library .
this suggests that harvesting the venom gland after 3– <dig> days after milking increases the chances of obtaining mostly high abundant toxin genes.
we identified ribosomal proteins, creatine kinase, dehydrogenase, muscle troponin, cytochrome b, parvalbumin, heparin binding protein and protein disulphide isomerase  in this library.
in addition, polyadenylated 12s and 16s rrnas were also found.
although polyadenylation is the distinctive features of mrna, polyadenylation of rrna has been observed in yeast  <cit> , leishmania  <cit>  as well as in other higher organisms including human  <cit> .
these polyadenylated rrnas are degraded subsequently through polyadenyled-stimulated rna degradation pathway  <cit> .
unknown sequences
8% of the cdna sequences  of the library did not show any significant match to toxins or other metabolic genes and hence were categorized as unknown sequences.
bioinformatics analysis of these sequences showed only partial or poor homology to protein sequences of other organism.
their functional role is not known.
accelerated rate of deletions and insertions in toxin genes
evolution of large multigene families in snake venom occurs through "birth and death" process  <cit> .
toxin genes appear to undergo duplication followed by accelerated evolution to give rise to new genes  <cit> .
some of the genes either get deleted from the genome due to unequal cross-over and other phenomena or become non-functional and degenerate into pseudogenes  <cit> .
such birth and death phenomenon allows the snake to adapt to target various preys.
in a. labialis, we observed unusually accelerated rate of deletions and insertions in toxin cdna clones resulting in the death of functional toxin genes.
out of the  <dig> cdnas encoding toxins , only  <dig> encode full length proteins.
a total of  <dig> cdnas encode for truncated products due to deletions  or insertions .
thus  <dig> % of the toxin cdnas encode non-functional products.
even the main toxin groups are severely affected.
for example, six neurotoxin genes and three pla <dig> genes were truncated.
interestingly, entire families of metalloproteinases and crisps appear to be lost due to deletions and insertions.
although some of the long-chain neurotoxin genes encode full length functional proteins, there are some deletions at their c-terminal ends.
these observations were made not only in singletons, but also in a number of clusters of toxin genes.
in contrast none of the cellular transcripts  showed deletions or insertions in nucleotide level.
thus, the only toxin cdnas show this unusually high rate of deletions and insertions in a. labialis.
the phenomenon of such high rate of accelerated insertions and deletions in only the toxin genes  as with the accelerated evolution of exons of the toxin genes  <cit>  will be of great interest.
recently, we showed that in marbled sea snake  the only neurotoxin gene has a dinucleotide deletion resulting in the loss of viable neurotoxin  <cit> .
this explains the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus.
we proposed that this loss could be a secondary result of the adaptation of aipysurus eydouxii to a new dietary habit – feeding exclusively on fish eggs and, thus, the snake no longer requires its venom for prey capture  <cit> .
further, this snake is physically smaller in size compared to aipysurus laevis.
similarly, venom of austrelaps labialis is relatively less toxic compared to austrelaps superbus and austrelaps ramsayi.
the ld <dig> of a. labialis   <cit>  is higher as compared to its close relative a. superbus whose ld <dig> is  <dig>  mg/kg when injected subcutaneouly  <cit> .
further, a. labialis are smaller in size compared to others.
thus, rapid rate of deletions/insertions, as with accelerated evolution of toxin genes, may have significant influence in the evolution and survival of a. labialis.
in a previous study, chijiwa et al.
<cit>  showed that the protein composition of venoms from protobothrops flavoviridis  of okinawa island was different compared to that of snakes from the main island.
in okinawa snakes, some of the main components, such as myotoxic pla <dig> enzymes bpi and bpii, were absent.
the genes encoding these proteins lost segments in their exon and intron and have become pseudogenes.
this loss of bpi and bpii genes may not explain the overall decrease in the venom toxicity  <cit> .
interestingly, haemorrhagic metalloprotease, hr1b from the venom of okinawa snakes is  <dig> fold less active.
however, the reasons for this decrease in activity are not clearly understood.
a detailed study of the entire transcriptome of their venom glands may help in understanding whether the observed regional variation in okinawa snakes is similar to our findings with a. labialis snake.
it would also be interesting to examine the transcriptome of a. labialis inhabiting the mainland to determine whether the observed accelerated deletions/insertions in the genes of the kangaroo island inhabitant is also a regional variation.
CONCLUSIONS
in the present study we have presented the toxin profile of a. labialis by constructing cdna library using its venom gland tissue.
neurotoxin and pla <dig> are the most abundant family of toxin in this snake venom.
however it is interesting to note that some of the toxin transcripts have been found to be terminated prematurely either due to insertion or deletion of nucleotides.
due to these insertion and deletion some of the genes might not code for functional proteins.
such a higher rate of insertion and deletion might be responsible for its lower toxicity and play crucial role evolution of toxin genes.
authors' contributions
rd and nnbt have and carried out the experiments, analyzed data and wrote the manuscript.
ar has analyzed and interpreted the data.
rmk is the principal investigator who designed the experiment, analyzed the data and critically reviewed the manuscript.
all the authors have approved the final form of the manuscript.
supplementary material
additional file 1
nucleotide sequences of long-chain neurotoxins showing insertions and deletions of nucleotides.
nucleotide sequences were aligned using clustalw.
gaps are indicated with dots, insertion with arrow, deletion with asterisk and stop codon with square box.
click here for file
additional file 2
nucleotide sequences of pla <dig> showing insertions and deletions of nucleotides.
nucleotide sequences were aligned using clustalw.
gaps are indicated with dots, insertion with arrow and stop codon with square box.
click here for file
additional file 3
nucleotide sequences of crisps showing insertions and deletions of nucleotides.
nucleotide sequences were aligned using clustalw.
gaps are indicated with dots, insertion with arrow, deletion with asterisk and stop codon with square box.
click here for file
acknowledgements
this work was supported from the grant   from biomedical research council, agency for science, technology and research, singapore.

 australian elapids are among the most venomous land snakes of the world. various bioactive peptide have been purified and characterized from these venoms and a comprehensive survey of the bioactive proteins present in a number of these snake venoms have been reported recently. snakes in austrelaps genus are widely distributed in australia, but are moderately toxic compared to other elapids. presently three species of austrelaps are known – lowland copperhead, highland copperhead and pygmy copperhead. so far, only a small number of proteins have been isolated and characterized from a. superbus venom. they are mostly phospholipase a enzymes and cobra venom factor-like protein. however, no significant data on the venoms of other two species are available. pigmy copperhead is smaller in size as compared to a. superbus and a. ramsayi and it has distinguishable white bars to its upper lips, circular eyes and yellowish-brown iris. they mainly feed on small lizards and frogs and the ld of their venom is mg/kg. in the present study, we have attempted to profile venom components of a. labialis to define its composition and to look for novel proteins. profiling venom toxins can be achieved via transcriptomics or proteomics approach. in former approach, the transcripts are directly sequenced from a cdna library constructed from the venom gland, whereas in the latter approach venom proteins are separated using various techniques like lcms, 2d gel electrophoresis and hplc. we have obtained toxin profile of pygmy copperhead venom by constructing of cdna. the data revealed that neurotoxins and pla are the most abundant proteins in this venom. interestingly, most of the toxin families in this cdna library have truncated transcripts. we propose that the lower venom toxicity and subsequent decreased size of these snakes could be due to unusually high degree of deletions or insertions in their toxin genes resulting in truncated, most likely inactive products. methods construction of library and dna sequencing venom glands were dissected and extracted from an euthanized a. labialis snake captured in kangaroo island, south australia. total rna was extracted using rneasy® mini kit from qiagen. the purity and the concentration were spectrophotometrically determined. first strand cdnas were synthesized from ng of total rna according to protocol of creator™ smart™ cdna library construction kit obtained from clontech laboratories. amplification of full-length double-stranded cdna was carried out using pcr-based protocol. double-stranded cdna pcr products were purified and subjected to ta cloning. ligation products were transformed into the competent top e. coli strain obtained from invitrogen and plated on lb/amp/iptg/x-gal for blue/white screening. a bidirectional pgem®-t easy vector system obtained from promega plasmid-based cdna library was titered with × cfu/ml. individual colonies were picked randomly and the presence of insert was confirmed by ecori digestion. only clones containing inserts larger than bp in length were selected for further dna sequencing. dna sequencing reactions were carried out using the abi prism® bigdye® terminator cycle sequencing ready reaction kit according to manufacturer's instructions. dna sequencing was carried out using abi prism® automated dna sequencer. sequencing was repeated at least twice in all singletons to make sure there was no error created by sequencing step. cloning snake venom protein with two kunitz domains pcr amplification of protein containing two kunitz-type domain from rna pool was performed using a forward primer designed from the signal peptide-encoding region and the reverse primer from library construction kit 30-3'). long pcr enzyme mix was used in the amplification reaction. pcr was performed as follows: cycles of one step each at 95°c for s, 58°c for s, 68°c for min followed by a final extension step at 68°c for min. pcr products were subjected to 1% agarose gel and visualized using ethidium bromide staining. amplified pcr products were gel purified and cloned to pgem®-t easy vector system and sequenced subsequently. analysis of sequence data by bioinformatics tools the dna sequences were analyzed after trimming the adaptor sequences and the putative functions of the gene products were predicted by batch blasting sequence results in grid blast system. the signal peptide was predicted using online signalp server. clustering of sequences was performed using fastgroupii and sequences were aligned using clustalw. jalview was employed to make some necessary editing on sequence alignments. phylogenetic trees were generated using phyml, which uses maximum likelihood method to build phylogenetic tree.  compositions of cdna library we randomly selected and isolated clones. of these only clones, which had larger than bp inserts, were sequenced and analyzed. these ests were categorized into different clusters on the basis of sequence similarities. members of each clusters has been sequenced completely and the full length sequences were submitted to ncbi with accession numbers from eu to eu eu and eu blast search against nucleotide, protein and est database, revealed that 60% of the est belong to putative toxins, while 32% belong to cellular transcripts and 8% of the est were unknown transcripts. these relative ratios may change, if more est clones are sequenced and analyzed. toxin genes putative toxin genes found in the cdna library were further grouped based on their sequence similarities to known toxin superfamilies. in total, we found six superfamilies of the toxin genes in the cdna library of a. labialis. these families, in order of abundance, are three-finger toxin family, phospholipase a family, kunitz-type proteinase inhibitor family, cysteine-rich protein family, metalloproteinase family and c-type lectin family. three-finger toxin family three-finger toxin family is a well characterized non-enzymatic polypeptide family named after their canonical protein folds. members of this family contain about 60– amino acid residues and exhibit various pharmacological activities such as neurotoxic, cytotoxic, cardiotoxic, anticoagulant and antiplatelet effects. three-finger family forms the major group of toxins in this cdna library, and majority of mature proteins are 66– amino acid residues in length. we identified a total of sequences with 60%–80% identities to known long-chain neurotoxins and sequences with 60%–85% identical to short-chain neurotoxins. long-chain neurotoxins clone 8a is the most dominant long-chain neurotoxin and other clones are its isoforms with few amino acid substitutions. most of these substitutions are due to single nucleotide changes. since these isoforms are represented by many clones, these sequence differences are not artifacts of sequencing. however, clone 5b has substitutions and hence may have distinct specificity compared to other long-chain neurotoxins. further, a dinucleotide deletion in front of the typical stop codon position followed by a dinucleotide insertion in the gene resulted in a frame shift and additional five amino acid residues in clone 5b. however, two clones are distinctively shorter due to deletion of nucleotides a and g respectively, resulting in the frame shift and truncated peptides. there were eight clones identical to clone suggesting that this observation is not due to sequencing error. the segments after the deletion show significant identity with other complete transcripts. similar phenomenon was observed in sea snake aipysurus eydouxii in which a dinucleotide deletion leads to the truncation of the only three-finger neurotoxin found in the venom gland library and the loss of toxicity. in contrast, in clone deletion of a leads to a potentially much longer protein product. in this case, three of the conserved cysteine residues at the c-terminal end are lost due to the frame shift. three distinct neurotoxin sequences compared to clone 8a and its closer sibling toxins were also found in the cdna library. clones and encode for slightly longer proteins with and amino acid residues respectively as compared to other clones. in these cases there is a single nucleotide substitution at consensus stop codon present in the majority of these putative long-chain neurotoxins. in clone a dinucleotide deletion in the stop codon followed by a dinucleotide insertion and a dinucleotide deletion has been found. most of the putative full length long-chain neurotoxins possess the functionally conserved residues such as lys trp asp phe arg and lys that are shown to participate in the binding to torpedo nicotinic acetylcholine receptor in the case of α-cobratoxin from naja naja venom. however, lys is replaced by the negatively charged glu. this charge reversal may significantly reduce its interaction with torpedo nachr as mutation of k to e in α-cobratoxin cause fold decrease in binding affinity. further, neutrally charged ala which is specific for binding to α7-nachr in α-cobratoxin, is replaced by the positively charged arg with the exception of clone 5b. this substitution did not affect the binding affinity to the receptor in the case of α-cobratoxin. the majority of a. labialis long-chain neurotoxins form a distinctive group almost completely separated from the other long-chain neurotoxins. phylogenetically, this separation seems to start quite early in the evolution time frame as indicated by early branching pattern originating from the root. interestingly, three distinct clones form a separate cluster as compared to other long-chain neurotoxins and appear to have a completely different phylogenetic origin. short-chain neurotoxins two representatives of short-chain neurotoxins, clone 6a and 12b were found in the library. their amino acid sequences are identical except for the substitution of thr by ala. both short-chain neurotoxins have one additional cys residue at the fourth position. appearance of cys at 4th position is observed for short-chain neurotoxins from sea snake venom. they have functionally invariant residues such as lys trp asp arg and lys which are important in the binding of erabutoxin a to torpedo nachr. interestingly, one of the binding residues, phe found in erabutoxin-a, is replaced by his in a. labialis short-chain neurotoxins which might effect its binding to the receptor. phospholipase a family phospholipase a enzymes are esterolytic enzymes which hydrolyze glycerophospholipids at the sn- position of the glycerol backbone releasing lysophospholipids and free fatty acids. snake venom pla enzymes are one of the well studied superfamily of snake venom enzymes which play an important role in immobilization and capture of prey. in addition to their role in digestion of preys, they exhibit variety of pharmacological effects such as neurotoxic, myotoxic, cardiotoxic, anticoagulant, antiplatelet and edema-inducing effects. a total of pla cdna clones were obtained which makes up the second abundant family. the deduced amino acid sequences are 65% to 97% identical to other pla sequences and were grouped into three clusters and four singletons. all pla cdna sequences obtained from this snake belong to group ia. clones and are the most abundant clones in this library and are closely related to the pla sequences of a. superbus of the same genus. however, they are different from the other pla enzymes due to the presence of three tryptophan residues in the β-wing. at present, role of these trp residues are not known. these pla enzymes together with five pla isoforms of a. superbus form a distinguishable cluster shown in phylogram generated by phyml method. this cluster is well separated from the other pla enzymes having one tryptophan residue in the β-wing. clones and have significant difference in amino acid sequences of other a. labialis pla enzymes. clone encodes for the full length protein sequence, whereas clone encodes for a truncated product due to a single nucleotide addition at position leading to frame shift and premature truncation. these clones form a distinct cluster along with aad. kunitz-type serine protease inhibitor family kunitz-type of protease inhibitors are residues long which possess the conserved sequence motif of cysteines and functionally belong to bovine pancreatic trypsin inhibitor family. this family of snake venom protein is known to involve in coagulation, fibrinolysis and inflammation through interaction with various proteases. cdna clones in this library belong to kunitz-type serine protease inhibitor family. the sequences of this group are 77%–90% identical to mulgin isoforms and textilinin isoforms. textilinins are shown to be distinct plasmin inhibitors and have a 60% bleeding reduction in murine model. it will be interesting to study the role of these kunitz-type serine proteases inhibitors in blood clotting cascade. however one of the clones, clone was found to be truncated prematurely due to transversion of one nucleotide at position. this clone was resequenced to confirm the transversion is not due to sequencing error. a unique clone encoding protein with two tandem kunitz-type protease inhibitor domains was found in this cdna library. the entire cdna sequence is bp long and the deduced peptide has amino acid residues with potential glycosylation site. its presence was further confirmed by pcr using gene specific and reverse primer from cdna construction kit. we sequenced clones and seven isoforms with minor changes in the amino acid sequence were found. they are 40% identical to bikunin, a serine proteinase inhibitor with two kunitz domains. bikunin from human placenta exhibits a potent inhibitory effect to some serine proteases crucial in the blood coagulation and fibrinolysis, such as factor xia and plasmin. this is the first report of this novel member of kunitz-type serine protease inhibitor family from snake venom. cysteine-rich protein family cysteine-rich secretory proteins are abundantly found in mammalian reproductive tracts and play important role in sperm maturation and immune system. they have also been found in venoms of reptiles. the conserved sequence in crisps span through out the protein with cysteine residues and of them resides in the c-terminal end. crisps have been purified and characterized from various snake venoms. functions of majority of these proteins are unknown. some of them are known to block cyclic nucleotide-gated ion channels while several others block potassium-stimulated smooth muscle contraction. cdna clones encoding crisps making up 8% of the library has been observed. this family has one cluster and four singletons. clone has a dinucleotide deletion in position and series of single nucleotide deletion of a in positions and single nucleotide deletion of t, c and g at position and respectively. clone has two single nucleotide deletions of a and a clone has also two single nucleotide deletions of a and g clones and have an insertion of one a at position while clone has another insertion of t in position in case of clone there is an insertion of t and a at position and deletions of t a a a and a. due to these insertions in clone and c-terminal end has encoded different amino acid residues as compared to other crisps. clones and have several deletions in the orf leading to frame shift and premature truncation. thus, all of them are prematurely terminated losing the c-terminal domain where most of the conserved cysteine residues are found. thus the truncated transcript might not be functional. however, the n-terminal region have high identity to snake venom crisps, but low identity to human, murine, frog crisps. metalloproteinase family snake venom metalloproteinases are more abundantly found in viper venoms, but they are also reported from elapid family. they are synthesized as zymogens in the venom gland and contain a propeptide which is cleaved off during maturation. they have a common zinc binding site with a consensus sequence of hexxhxxgxxh. they are classified into different types on the basis of the other domains that are present in these complexes. this family of enzymes are responsible for haemorrhagic, local myonecrotic, antiplatelet, edema-inducing and other inflammatory effects. we have found clones which makes 3% of the entire cdna library, however they code for only the signal peptide and the n-terminal region similar to trigramin precursor and mocarhagin. this is due to the loss of two nucleotides at position downstream of atg leading to premature termination of the protein. thus, if proteins of this family are present in the venom, they would have only partial propeptide without any other functional domains of metalloproteinase or disintegrin. c-type lectin family c-type lectins are non-enzymatic proteins that bind to mono- and oligosaccharides in presence of ca2+. generally they contain the highly conserved domain called the carbohydrate recognition domain. c-type lectins and related proteins have been frequently reported from viperidae snake venoms. venom c-type lectin related proteins are known to disrupt the normal functioning of haemostatic mechanism by interfering in the normal platelet receptor-ligand interaction, binding to coagulation factors and other important proteins in coagulation cascade. only few of them have been reported from elapids including australian elapids. this family has seven clones which form one cluster. the primary structure shows 71% and 74% identical to c-type lectins found in bungarus fasciatus and b. multicinctus, 56% identical to galactose-binding lectin found in bitis arietans, but less than 37% identical to c-type lectin related proteins. the deduced amino acid sequence reveals the conserved carbohydrate binding domain as well as the ca2+ interaction amino acid residues. the odd cysteine present is likely to involved in interchain disulphide bond. the presence of only one cluster of clones indicates that it may exist as homodimer similar to other c-type lectins. further, phylogenetic analysis revealed that this putative c-type lectin is more closely related to galactose-binding proteins and forms a cluster distinct from that of c-type lectin like proteins that bind to blood coagulation factors or platelet receptors. thus, this clone most likely encodes a homodimeric c-type lectin. cellular transcripts clones were identified as cellular trancripts which were grouped into clusters and singleton which constituted 32% of the entire library. this suggests that harvesting the venom gland after 3– days after milking increases the chances of obtaining mostly high abundant toxin genes. we identified ribosomal proteins, creatine kinase, dehydrogenase, muscle troponin, cytochrome b, parvalbumin, heparin binding protein and protein disulphide isomerase in this library. in addition, polyadenylated 12s and 16s rrnas were also found. although polyadenylation is the distinctive features of mrna, polyadenylation of rrna has been observed in yeast, leishmania as well as in other higher organisms including human. these polyadenylated rrnas are degraded subsequently through polyadenyled-stimulated rna degradation pathway. unknown sequences 8% of the cdna sequences of the library did not show any significant match to toxins or other metabolic genes and hence were categorized as unknown sequences. bioinformatics analysis of these sequences showed only partial or poor homology to protein sequences of other organism. their functional role is not known. accelerated rate of deletions and insertions in toxin genes evolution of large multigene families in snake venom occurs through "birth and death" process. toxin genes appear to undergo duplication followed by accelerated evolution to give rise to new genes. some of the genes either get deleted from the genome due to unequal cross-over and other phenomena or become non-functional and degenerate into pseudogenes. such birth and death phenomenon allows the snake to adapt to target various preys. in a. labialis, we observed unusually accelerated rate of deletions and insertions in toxin cdna clones resulting in the death of functional toxin genes. out of the cdnas encoding toxins, only encode full length proteins. a total of cdnas encode for truncated products due to deletions or insertions. thus % of the toxin cdnas encode non-functional products. even the main toxin groups are severely affected. for example, six neurotoxin genes and three pla genes were truncated. interestingly, entire families of metalloproteinases and crisps appear to be lost due to deletions and insertions. although some of the long-chain neurotoxin genes encode full length functional proteins, there are some deletions at their c-terminal ends. these observations were made not only in singletons, but also in a number of clusters of toxin genes. in contrast none of the cellular transcripts showed deletions or insertions in nucleotide level. thus, the only toxin cdnas show this unusually high rate of deletions and insertions in a. labialis. the phenomenon of such high rate of accelerated insertions and deletions in only the toxin genes as with the accelerated evolution of exons of the toxin genes will be of great interest. recently, we showed that in marbled sea snake the only neurotoxin gene has a dinucleotide deletion resulting in the loss of viable neurotoxin. this explains the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. we proposed that this loss could be a secondary result of the adaptation of aipysurus eydouxii to a new dietary habit – feeding exclusively on fish eggs and, thus, the snake no longer requires its venom for prey capture. further, this snake is physically smaller in size compared to aipysurus laevis. similarly, venom of austrelaps labialis is relatively less toxic compared to austrelaps superbus and austrelaps ramsayi. the ld of a. labialis is higher as compared to its close relative a. superbus whose ld is mg/kg when injected subcutaneouly. further, a. labialis are smaller in size compared to others. thus, rapid rate of deletions/insertions, as with accelerated evolution of toxin genes, may have significant influence in the evolution and survival of a. labialis. in a previous study, chijiwa et al. showed that the protein composition of venoms from protobothrops flavoviridis of okinawa island was different compared to that of snakes from the main island. in okinawa snakes, some of the main components, such as myotoxic pla enzymes bpi and bpii, were absent. the genes encoding these proteins lost segments in their exon and intron and have become pseudogenes. this loss of bpi and bpii genes may not explain the overall decrease in the venom toxicity. interestingly, haemorrhagic metalloprotease, hr1b from the venom of okinawa snakes is fold less active. however, the reasons for this decrease in activity are not clearly understood. a detailed study of the entire transcriptome of their venom glands may help in understanding whether the observed regional variation in okinawa snakes is similar to our findings with a. labialis snake. it would also be interesting to examine the transcriptome of a. labialis inhabiting the mainland to determine whether the observed accelerated deletions/insertions in the genes of the kangaroo island inhabitant is also a regional variation.  in the present study we have presented the toxin profile of a. labialis by constructing cdna library using its venom gland tissue. neurotoxin and pla are the most abundant family of toxin in this snake venom. however it is interesting to note that some of the toxin transcripts have been found to be terminated prematurely either due to insertion or deletion of nucleotides. due to these insertion and deletion some of the genes might not code for functional proteins. such a higher rate of insertion and deletion might be responsible for its lower toxicity and play crucial role evolution of toxin genes. authors' contributions rd and nnbt have and carried out the experiments, analyzed data and wrote the manuscript. ar has analyzed and interpreted the data. rmk is the principal investigator who designed the experiment, analyzed the data and critically reviewed the manuscript. all the authors have approved the final form of the manuscript. supplementary material additional file 1 nucleotide sequences of long-chain neurotoxins showing insertions and deletions of nucleotides. nucleotide sequences were aligned using clustalw. gaps are indicated with dots, insertion with arrow, deletion with asterisk and stop codon with square box. click here for file additional file 2 nucleotide sequences of pla showing insertions and deletions of nucleotides. nucleotide sequences were aligned using clustalw. gaps are indicated with dots, insertion with arrow and stop codon with square box. click here for file additional file 3 nucleotide sequences of crisps showing insertions and deletions of nucleotides. nucleotide sequences were aligned using clustalw. gaps are indicated with dots, insertion with arrow, deletion with asterisk and stop codon with square box. click here for file acknowledgements this work was supported from the grant from biomedical research council, agency for science, technology and research, singapore.
in this present study, an attempt has been made to describe the toxin profile of a. labialis from kangaroo island using the cdna library of its venom glands. however, cellular trancripts from the same venom glands are not affected. interestingly, the cdna library reveals that most of the toxin families have truncated transcripts due to insertion or deletion of nucleotides. they include putative long-chain and short-chain neurotoxins, phospholipase a kunitz-type protease inhibitor, crisps, c-type lectins and metalloproteases. we sequenced clones which represent the common families of toxin genes present in snake venom. this unusual higher rate of deletion and insertion of nucleotide in toxin genes may be responsible for the lower toxicity of a. labialis venom of kangroo island and have significant effect on evolution of toxin genes. the pygmy copperhead is one of the less studied species.
 toxin profiling helps in cataloguing the toxin present in the venom as well as in searching for novel toxins. the former helps in understanding potential pharmacological profile of the venom and evolution of toxins, while the latter contributes to understanding of novel mechanisms of toxicity and provide new research tools or prototypes of therapeutic agents.  the pygmy copperhead is one of the less studied species. in this present study, an attempt has been made to describe the toxin profile of a. labialis from kangaroo island using the cdna library of its venom glands. we sequenced clones which represent the common families of toxin genes present in snake venom. they include putative long-chain and short-chain neurotoxins, phospholipase a kunitz-type protease inhibitor, crisps, c-type lectins and metalloproteases. in addition, we have also identified a novel protein with two kunitz-type domains in tandem similar to bikunin.  interestingly, the cdna library reveals that most of the toxin families have truncated transcripts due to insertion or deletion of nucleotides. these truncated products might not be functionally active proteins. however, cellular trancripts from the same venom glands are not affected. this unusual higher rate of deletion and insertion of nucleotide in toxin genes may be responsible for the lower toxicity of a. labialis venom of kangroo island and have significant effect on evolution of toxin genes. 