Table 1.
List of primers used for amplification of transcripts within specific venom protein families.
Table 2.
RNA and mRNA isolation protocols used to obtain extracellular RNA within venom, and the resulting yields and cDNA amplification success.
Fig 1.
Agarose gel electrophoresis showing cDNA transcripts amplified from mRNA in venomous snake venoms.
Various amounts of fresh venom (A) and lyophilized venom (B) from Crotalus simus tzabcan were used to determine optimal amplification conditions for phospholipase A2 transcripts. Regions of phospholipase A2 Mojave toxin subunit A (sA) and subunit B (sB) cDNAs were amplified to demonstrate transcript detectability in Crotalus scutulatus scutulatus venom (C). Three-finger toxin cDNA sequences were also amplified from mRNA derived from venoms of rear-fanged snakes (D).
Fig 2.
Aligned Middle American Rattlesnake (Crotalus simus tzabcan) C-type lectins (A) and serine proteases (B). A) Four unique venom-based C-type lectin transcripts (asterisks) were identified for C. s. tzabcan and aligned to other crotaline species. Identical nucleotide sequences are shaded and corresponding GenBank accession numbers are as follows: Crotalus_adamanteus (AEJ31974.1), Deinagkistrodon_acutus (AAM22790.1), Crotalus_d_terrificus (Q719L8.1), and Crotalus_o_helleri (AEU60004.1). B) Venom-based serine proteases cDNA sequences (asterisks) were also obtained from C. s. tzabcan and were aligned with toxins from several other species; identical nucleotide sequences are shaded, and the catalytic triad composed of Ser195, Asp102, and His57 associated with thrombin-like activity in snake venom serine proteases are identified (arrowheads). Isoform 3 from C. s. tzabcan is a partial sequence. GenBank accession numbers are as follows: Agkistrodon_p_leucostoma (HQ270466.1), Bothrops_asper (DQ247724.1), Crotalus_d_terrificus7 (EU360954.1), Crotalus_d_terrificus4 (EU360952.1), Crotalus_d_terrificus3 (EU360951.1), Crotalus_d_durissus (DQ164401.1), Sistrurus_c_edwardsi (DQ464239.1), Trimeresurus_mucrosquamatus (X83225.1), Crotalus_adamanteus (HQ414118.1), Calloselasma_rhodostoma (L07308.1), Deinagkistrodon_acutus (AY861382.1), Trimeresurus_stejnegeri (AF545575.1), and Crotalus_atrox (AF227153.1).
Table 3.
Venom protein transcripts amplified from the venom of the Middle American Rattlesnake (Crotalus simus tzabcan) for the four dominant protein families present.
Table 4.
Relationship between the number of colony picks and the number of observed unique phospholipase A2 isoforms.
Fig 3.
Aligned Group IIA phospholipase A2 acidic (A) and basic (B) subunit isoforms. The neurotoxic, heterodimeric PLA2 complex of crotoxin/Mojave toxin homologs consists of both an acidic A subunit and basic B subunit. A) Sequence alignments of the acidic (A) subunit of crotoxin or Mojave toxin homologs with identical residues shaded; the conserved signal peptide region is indicated by the red bar, and the Ca2+ binding loop is indicated by the blue bar. Regions of the A subunit which are post-translationally cleaved in the mature protein are indicated by red brackets below sequences. Sequences similar to crotoxin/Mojave toxin acidic subunit A derived from from venom (asterisks) were discovered in Crotalus oreganus concolor and C. simus tzabcan venoms. GenBank accession numbers of known toxins are as follows: Crotalus_s_scutulatus_MojaveTX (U01026.1), Crotalus_d_terrificus_CrotoxinA (X12606.1), Sistrurus_c_tergeminus_SistruxinA (Q6EAN6.1), and Gloydius_intermedius_GintexinA (AID56658.1). B) Sequence alignments of the basic (B) subunit of crotoxin/Mojave toxin homologs with identical residues shaded; the conserved signal peptide region is indicated by the red bar, and the Ca2+ binding loop is indicated by the blue bar. The asparagine-6 (N6) associated with neurotoxic PLA2 functionality is indicated by arrowheads. Sequences similar to the crotoxin or Mojave toxin basic B subunits derived from venom (asterisks) were discovered in Crotalus oreganus concolor, C. simus tzabcan, and C. basiliscus venoms, with N6 sequences also found in C. m. nigrescens and C. o. cerberus venoms. GenBank accession numbers of known toxins are as follows: Crotalus_s_scutulatus_MojaveTX (U01027.1), Crotalus_d_terrificus_CrotoxinB (X12603.1), Crotalus_v_viridis_N6 (AF403138.1), Sistrurus_c_tergeminus_N6 (AY355169.1), Sistrurus_c_tergeminus_SistruxinB (Q6EER2.1), Bothriechis_schlegelii_N6 (AY355168.1), Protobothrops_mucrosquamatus (AF408409.1), and Deinagkistrodon_acutus (X77649.1).
Fig 4.
Aligned Pseudechis Group IA phospholipase A2 (PLA2) mature protein sequences.
Amplified PLA2 sequences from Pseudechis porphyriacus venom (asterisks) were aligned to known PLA2 sequences from the same species and known PLA2 sequences from two other Pseudechis species. Sequences from P. porphyriacus venom showed 99% and 100% identity to P. porphyriacus mature PLA2 protein sequences that had been obtained from venom gland tissue or via Edman degradation.
Fig 5.
Aligned sequences of non-conventional three-finger toxins (3FTxs) from venoms of rear-fanged and elapid snakes.
Venom-derived 3FTx sequences (asterisks) obtained from Boiga irregularis, B. dendrophila, B. nigriceps, B. cynodon, Oxybelis fulgidus, Ahaetulla prasina, and Trimorphodon biscutatus lambda were aligned with various other rear-fanged and Elapidae species; identical nucleotide sequences are shaded. GenBank accession numbers are as follows: Trimorphodon_biscutatus_Tri3 (EU029678.1), Trimorphodon_biscutatus_Tri2 (EU029677.1), Telescopus_dhara_Tel4 (EU029686.1), Boiga_dendrophila_denmo (DQ366293.1), Boiga_irregularis_irditoxinB (DQ304539.1), Boiga_irregularis_irditoxinA (DQ304538.1), Boiga_irregularis_1f (GBSH01000015.1), Thrasops_jacksoni_Thr3 (EU029685.1), Dispholidus_typus_Dis1 (EU029674.1), Telescopus_dhara_Tel1 (EU029675.1), Thrasops_jacksoni_Thr5 (EU036635.1), Trimorphodon_biscutatus_Tri1 (EU029675.1), Naja_atra (AF031472.1), Bungarus_multicinctus (AF056400.1), Ophiophagus_hannah (FJ952515.1), Psammophis_mossambicus_Psa1 (EU029669.1), Leioheterodon_madagascariensis (EU029676.1), Bungarus_candidus (AY057878.1), and Dendroaspis_angusticeps (AF241871.1).
Fig 6.
Bayesian sequence similarity tree depicting non-conventional three-finger toxin (3FTx) relationships.
3FTx cDNA sequences derived from venom (asterisks) were obtained from Boiga irregularis, B. dendrophila, B. nigriceps, B. cynodon, Oxybelis fulgidus, Ahaetulla prasina and Trimorphodon biscutatus lambda, and sequence relationships with various known rear-fanged and Elapidae 3FTxs are shown. Posterior probabilities are shown for each node, and GenBank accession numbers correspond to sequences in Fig 6.
Fig 7.
Alsophis portoricensis venom PIII metalloproteinase sequence aligned with amino acid sequences from rear-fanged and elapid snake species.
Identical residues are shaded, demonstrating PIII metalloproteinase sequence conservation in these diverse species. Only a partial sequence of the complete transcript from A. portoricensis (asterisks) was used for the alignment. Genbank accession numbers are as follows: Philodryas_chamissonis (AJB84503.1), Philodryas_olfersii (ACS74987.1), Cerberus_rynchops (ADJ51055.1), Pseudechis_porphyriacus (ABQ01133.1), and Demansia_vestigiata (ABK63559.1).
Fig 8.
Bayesian sequence similarity tree depicting viper Group II phospholipase A2 (PLA2) relationships.
PLA2 cDNA sequences were obtained from Crotalus simus tzabcan, C. cerastes, C. molossus nigrescens, C. oreganus concolor, C. oreganus cerberus, C. pricei, C. basiliscus, and Sistrurus miliarius barbouri venoms (asterisks), and their relationships with various published Old and New World viperid venom PLA2s are shown. Newly sequenced crotoxin homologs are indicated by double asterisks (crotoxin A—left side; crotoxin B–right side). GenBank accession numbers are listed within the tree. Published PLA2 sequences are colored corresponding to empirically determined biological activity. Whole clades are highlighted where at least two of the published sequences within the clade have the same experimentally determined activity. Red, neurotoxic; blue, hemolytic; green, edematous; pink, myotoxic.