Amplification and sequencing of mu-conotoxin homologs from selected neogastropod species

• Main Authors: Bulan, Joy Charisse O., Heralde, Francisco M., III, Duque, Richelle C., Ignacio, Cherry Mae G., Javellana, Noel Francis B., Rocamora, Frances Maureen C., Diaz, Ma. Aiko Angela A., Cantalejo, Ma. Angela Clarissa D.R., Monje, Virginia D.
• Format: text
• Published: Animo Repository 2003
• Subjects: Neogastropoda; Conus; Marine toxins; Biology
• Online Access: https://animorepository.dlsu.edu.ph/faculty_research/6862
• Institution: De La Salle University

Mu-conotoxins are members of the M-superfamily of disulfide rich conopeptides that share a similar cysteine scaffold (CC-C-C-CC). They are excellent ligands for sodium channels with high binding specificity and are excellent research tools for neuroscience and neuropharmacology. Mu-conotoxins have been isolated in several cone snails and the precursors have been cloned form venom duct tissues. In this paper, mu-conotoxin homologs are amplified from seven different neogastropod snais namely Conus consors, C. floridulus, C. mustelinus, Turridrupa certhina, Turris crispa, terebra babylonia, and Terebra cumingi. Total RNA was extracted from the venom dcut using Trizol® (Invitrogen), reverse-transcribes to cDNA and amplified through the BD Smart TM cDNA library construction kit (Clontech). Mu-conotoxins were amplified using the primers Mu-C (5’-CAAGA(A/G)GGATCGATA-3’) and Mu-3’ (5’-ACTGACATC(A/G)TTTTACTTATTC-3’). Results from BLAST (basic local alignment search tool) show that the C. consors data has a high homology with a reported cone snail mu-conotoxin precursor sequence while all the sequences from the other species hit for 28S rRNA or mouse chromosome 9 genes. The cloned mu-conotoxin sequence of C. consors does not have the complete signal sequence but exhibited the correct cysteine scaffold. These findings show that although mu-conotoxin genes are amplified form the University of Utah donated primers (Mu-C and Mu-3’), the same primers also cross react and amplify unrelated gene sequences and therefore necessitates a redesign for improved specificity.