Venom gland transcriptomes of two elapid snakes (Bungarus multicinctus and Naja atra) and evolution of toxin genes

<p>Abstract</p> <p>Background</p> <p>Kraits (genus <it>Bungarus</it>) and cobras (genus <it>Naja</it>) are two representative toxic genera of elapids in the old world. Although they are closely related genera and both of their venoms are very toxic, the compositions of their venoms are very different. To unveil their detailed venoms and their evolutionary patterns, we constructed venom gland cDNA libraries and genomic bacterial artificial chromosome (BAC) libraries for <it>Bungarus multicinctus </it>and <it>Naja atra</it>, respectively. We sequenced about 1500 cDNA clones for each of the venom cDNA libraries and screened BAC libraries of the two snakes by blot analysis using four kinds of toxin probes; <it>i.e</it>., three-finger toxin (3FTx), phospholipase A2 (PLA2), kunitz-type protease inhibitor (Kunitz), and natriuretic peptide (NP).</p> <p>Results</p> <p>In total, 1092 valid expressed sequences tags (ESTs) for <it>B. multicinctus </it>and 1166 ESTs for <it>N. atra </it>were generated. About 70% of these ESTs can be annotated as snake toxin transcripts. 3FTx (64.5%) and <it>β </it>bungarotoxin (25.1%) comprise the main toxin classes in <it>B. multicinctus</it>, while 3FTx (95.8%) is the dominant toxin in <it>N. atra</it>. We also observed several less abundant venom families in <it>B. multicinctus </it>and <it>N. atra</it>, such as PLA2, C-type lectins, and Kunitz. Peculiarly a cluster of NP precursors with tandem NPs was detected in <it>B. multicinctus</it>. A total of 71 positive toxin BAC clones in <it>B. multicinctus </it>and <it>N. atra </it>were identified using four kinds of toxin probes (3FTx, PLA2, Kunitz, and NP), among which 39 3FTx-postive BACs were sequenced to reveal gene structures of 3FTx toxin genes.</p> <p>Conclusions</p> <p>Based on the toxin ESTs and 3FTx gene sequences, the major components of <it>B. multicinctus </it>venom transcriptome are neurotoxins, including long chain alpha neurotoxins (<it>α</it>-ntx) and the recently originated <it>β </it>bungarotoxin, whereas the <it>N. atra </it>venom transcriptome mainly contains 3FTxs with cytotoxicity and neurotoxicity (short chain <it>α</it>-ntx). The data also revealed that tandem duplications contributed the most to the expansion of toxin multigene families. Analysis of nonsynonymous to synonymous nucleotide substitution rate ratios (<it>dN</it>/<it>dS</it>) indicates that not only multigene toxin families but also other less abundant toxins might have been under rapid diversifying evolution.</p

Some toxic and enzymatic activities of Bothrops ammodytoides (yarará ñata) venom

Fil: de Roodt, Adolfo R. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Dolab, Jorge A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Hajos, S E. Cátedra de Inmunologı́a de la Facultad de Farmacia y Bioquı́mica de la Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Gould, Eduardo G. Fundación de Estudios Biológicos, Pte. Perón, Buenos Aires; Argentina.Fil: Dinápoli, H. Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, CONICET, Buenos Aires; Argentina.Fil: Troiano, J C. Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, CONICET, Buenos Aires; Argentina.Fil: Gould, J. Cátedra de Inmunologı́a de la Facultad de Farmacia y Bioquı́mica de la Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Dokmetjian, Christian ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina. Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, CONICET, Buenos Aires; Argentina.Fil: Carfagnini, Julio C. Cátedra de Patologı́a General, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires; Argentina.Fil: Fernández, Teresa. Fundación de Estudios Biológicos, Pte. Perón, Buenos Aires; Argentina.Fil: Amoroso, Marcela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Segré, Liliana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Vidal, Juan C. Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, CONICET, Buenos Aires; Argentina.Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom