Figure 1.
Architecture of spider-toxin genes.
Intron-exon organization for genes encoding (A) μ-diguetoxin-Dc1a from the American desert spider Diguetia canities [15]; (B) 24 different disulfide-rich venom peptides from the Chinese tarantulas Haplopelma hainanum and Haplopelma huwenum [16]–[18]. In panel (A), the colors denote exons encoding the signal peptide, propeptide, and mature toxin. In panel (B), the entire toxin prepropeptide precursor is encoded by an intronless ORF.
Figure 2.
Primary and tertiary structure of δ-hexatoxins.
(A) Alignment of the amino acid sequences of the lethal toxins δ-hexatoxin-Hv1a (and its paralog δ-hexatoxin-Hv1b), δ-hexatoxin-Ar1a, δ-hexatoxin-Hi1a, and δ-hexatoxin-Iw1a from the Australian funnel-web spiders Hadronyche versuta, Atrax robustus, Hadronyche infensa, and Illawarra wisharti, respectively. Identical amino acids are boxed in black and conservative substitutions are shaded grey. (B) Ribbon representation of the three-dimensional structure of δ-hexatoxin-Hv1a (PDB code 1VTX) [26]. β-Strands and 310-helix are shown in orange and blue, respectively. The N- and C-termini are labeled. The three disulfide bonds shown in green form an inhibitor cystine knot motif while the disulfide bridge that connects the C-terminal Cys residue to the core ICK region is colored red.
Figure 3.
The gene encoding δ-hexatoxin-Hi1a is intronless.
(A) Schematic of the putative δ-hexatoxin gene showing the region that each primer set (L1–L5) is designed to amplify. (B) Gels showing the PCR products obtained using each of the designed primer sets L1–L5: (i) cDNA template; (ii) gDNA template; (iii) Southern Blot. For each gel, ML denotes 1 kb molecular-weight ladder, while L1–L5 denote the primer sets A–E shown in Table 1.
Table 1.
Primer sets used to amplify specific regions of the transcript and gene encoding δ-hexatoxin-Hi1a from cDNA and gDNA templates, respectively (letters in brackets denote lanes in gels in Fig. 3B).
Figure 4.
Architecture of gene encoding the lethal toxin from Australian funnel-web spiders.
Alignment of the cDNA and gDNA sequences obtained for δ-hexatoxin-Hi1a. The nucleotide sequences are identical except at a single position (T versus C, highlighted in grey) that does not alter the encoded protein sequence. The stop codon is denoted by an asterisk. A schematic of the toxin precursor showing the signal peptide, propeptide, mature toxin, and 3’ untranslated region in yellow, blue, purple and white, respectively, is shown above the sequences. The protein sequence (i.e., a translation of the cDNA/gDNA) is shown sandwiched between the cDNA and gDNA sequences.
Figure 5.
Architecture of genes encoding ICK toxins from Australian funnel-web spiders.
Alignment of cDNA and gDNA sequences of (A) ω-hexatoxin-Hi2a and (B) U3-hexatoxin-Hi1a. Stop codons are indicated by an asterisk. A schematic of the toxin precursors showing the signal peptide, propeptide, and mature toxin in yellow, blue, and purple, respectively, is shown above the sequences. The protein sequences (i.e., a translation of the cDNA/gDNA) are shown sandwiched between the cDNA and gDNA sequences. There are several codons where a difference between the cDNA and gDNA sequences leads to a difference in the sequence of the encoded protein; in these cases the amino acid encoded by the cDNA and gDNA are shown in black and red, respectively. The “GR” sequence underlined in green at the C-terminus of the mature-toxin region of ω-hexatoxin-Hi2a in panel (A) is a C-terminal amidation signal [34].