title: Cloning and expression of genes enocoding antimicrobial peptides and bradykinin from the skin and brain of Oki Tago's brown frog, Rana tagoi okiensis.
abstract: Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK. In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis). RT-PCR of brain RNA resulted in the amplification of cDNAs encoding ranatuerin-2TOc and ranatuerin-2TOd that contained the amino acid substitutions Lys(6)-->Arg and Ala(14)-->Thr, respectively compared with ranatuerin-2TOb. cDNAs encoding preprobrevinin-1TOa and preprotemporin-TOa were amplified from brain RNA as well as a second preprotemporin cDNA that contained a 10-nucleotide insertion that introduced a frame shift resulting in a premature stop codon. A cDNA encoding a novel peptide, DK25 (DVNDLKNLCAKTHNLLPMCAMFGKK) was amplified from brain RNA but neither DK25 nor its putative post-translationally modified form, DF22-amide (DVNDLKNLCAKTHNLLPMCAMF.NH(2)) displayed antimicrobial or hemolytic activities.
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Shoro
authors.initials: S
authors.lastname: Tazato
authors.forename: J Michael
authors.initials: JM
authors.lastname: Conlon
authors.forename: Shawichi
authors.initials: S
authors.lastname: Iwamuro
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029845:Amphibian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D001920:Bradykinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D009928:Organ Specificity; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011898:Ranidae / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000378:metabolism*
chemical_list: D029845:Amphibian Proteins; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D020033:Protein Isoforms; D011498:Protein Precursors; D011506:Proteins; D012333:RNA, Messenger; C121230:ranatuerin; C104501:temporin; C078413:brevinin-1 protein, Rana; D001920:Bradykinin