title: Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step "shotgun" cloning.
abstract: The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step "shotgun" cloning approach using a cDNA library constructed from the source material of the peptidomic studies--the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.
authors: Molecular Therapeutics Research, School of Pharmacy, Queen's University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors: Mei
authors: M
authors: Zhou
authors: Lei
authors: L
authors: Wang
authors: Damian E
authors: DE
authors: Owens
authors: Tianbao
authors: T
authors: Chen
authors: Brian
authors: B
authors: Walker
authors: Chris
authors: C
authors: Shaw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D011898:Ranidae / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary