title: Molecular cloning and characterization of cDNAs encoding biosynthetic precursors for the antimicrobial peptides japonicin-1Ja, japonicin-2Ja, and temporin-1Ja in the Japanese brown frog, Rana japonica.
abstract: Using a combination of reverse-transcription polymerase chain reaction and the 5'- and/or 3'-rapid amplification of cDNA ends, we cloned, from a Japanese brown frog (Rana japonica) skin total RNA preparation, cDNAs encoding biosynthetic precursors for the antimicrobial peptides (AMPs) japonicin-1Ja (FFPIGVFCKIFKTC), japonicin-2Ja (FGLPMLSILPKALCILLKRKC), and temporin-1Ja (ILPLVGNLLNDLL.NH2). These peptides were previously isolated from an extract of R. japonica skin. The present study is the first report to describe the molecular cloning of the cDNA encoding a japonicin-2 family peptide. The nucleotide and deduced amino acid sequence analyses revealed that the hypothetical precursor protein of japonicin-2Ja, as well as japonicin-1Ja and temporin-1Ja, is organized similarly to those of typical amphibian AMP precursors, with a highly conserved signal peptide, a relatively well conserved intervening sequence, and a hypervariable AMP mature region. Antimicrobial assays for synthetic replicates of cyclic and linear japonicin-2Ja revealed that the intramolecular disulfide bond is necessary for activity. A semi-quantitative analysis by real-time RTPCR using TaqMan probes revealed that the relative values of preprojaponicin-2Ja mRNA expression levels in the skin, skeletal muscle of hind leg, kidney, testis, small intestine, and stomach total RNA sample specimens in adult R. japonica were 6.5×10(5), 9.6, 2.0, 1.6, 1.6, and 1.0, respectively. The presence of preprojaponicin-2Ja mRNAs in the cytoplasm of glandular cells in R. japonica dorsal skin glands was demonstrated by means of in situ hybridization using digoxigenin-labeled cRNA probes for the precursor.
authors.affiliation: Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Koyama
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; D000818:Animals; D000890:Anti-Infective Agents; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011898:Ranidae / Q000378:metabolism*
chemical_list: D000890:Anti-Infective Agents; D018076:DNA, Complementary; D012333:RNA, Messenger