Peptidomics pp 159-176 | Cite as

An Efficient Protocol for DNA Amplification of Multiple Amphibian Skin Antimicrobial Peptide cDNAs

  • Shawichi Iwamuro
  • Tetsuya Kobayashi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 615)

Abstract

Antimicrobial peptides (AMPs) play an important role in the host’s innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes. While the nucleotide sequences of the AMP coding region in precursors are hypervariable, those of other regions, including the 5- and 3-untranslated regions (UTRs), are highly or relatively conserved in different precursors. Such nucleotide sequence conservation suggests an efficient strategy for molecular cloning of the antimicrobial peptide genes by 3-rapid amplification of cDNA ends (3-RACE) and reverse transcriptase polymerase chain reaction (RT-PCR) methods using specific primers. With this strategy in mind we have established an efficient protocol suitable for amplification of multiple cDNAs encoding amphibian AMP precursor proteins.

Key words

Amphibian skin antimicrobial peptides molecular cloning 3-RACE RT-PCR 

Notes

Acknowledgments

The authors wish to thank the members in the laboratory of Regulatory Biology in Toho University for their useful suggestions for the preparation of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research (19570063) from the Japan Society for the Promotion of Science to S.I. and T.K.

References

  1. 1.
    Boman, H. G. (1995) Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 61–92.PubMedCrossRefGoogle Scholar
  2. 2.
    Ganz, T. and Lehrer, R. I. (1999) Antibiotic peptides from higher eukaryotes: biology and applications. Mol. Med. Today 5, 292–297.PubMedCrossRefGoogle Scholar
  3. 3.
    Hancock, R. E. and Diamond, G. (2000) The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8, 402–410.PubMedCrossRefGoogle Scholar
  4. 4.
    Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415, 389–395.PubMedCrossRefGoogle Scholar
  5. 5.
    Radek, K. and Gallo, R. (2007) Antimicrobial peptides: natural effectors of the innate immune system. Semin. Immunopathol. 29, 27–43.PubMedCrossRefGoogle Scholar
  6. 6.
    Nicolas, P., Vanhoye, D. and Amiche, M. (2003) Molecular strategies in biological evolution of antimicrobial peptides. Peptides 24, 1669–1680.PubMedCrossRefGoogle Scholar
  7. 7.
    Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanated-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.PubMedCrossRefGoogle Scholar
  8. 8.
    Duda, T. F., Jr., Vanhoye, D. and Nicoras, P. (2002) Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides. Mol. Biol. Evol. 19, 858–864.PubMedGoogle Scholar
  9. 9.
    Chen, T., Li, L., Zhou, M., Rao, P., Walker, B. and Shaw, C. (2006) Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs. Peptides 27, 42–48.PubMedCrossRefGoogle Scholar
  10. 10.
    Chen, T., Zhou, M., Rao, P., Walker, B. and Shaw, C. (2006) The Chinese bamboo leaf odorous frog (Rana (Odorana) versabilis) and North American Rana frogs share the same families of skin antimicrobial peptides. Peptides 27, 1738–1744.PubMedCrossRefGoogle Scholar
  11. 11.
    Suzuki, H., Iwamuro, S., Ohnuma, A., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Taylor, C. K., Abel, P. W. and Conlon, J. M. (2007) Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii. Peptides 28, 505–514.PubMedCrossRefGoogle Scholar
  12. 12.
    Ohnuma, A., Conlon, J. M., Kawasaki, H. and Iwamuro, S. (2006) Developmental and triiodothyronine-induced expression of genes encoding preprotemporins in the skin of Tago’s brown frog Rana tagoi. Gen. Comp. Endocrinol. 146, 242–250.PubMedCrossRefGoogle Scholar
  13. 13.
    Ohnuma, A., Conlon, J. M., Yamaguchi, K., Kawasaki, H., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H. and Iwamuro, S. (2007) Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs. Peptides 28, 524–532.PubMedCrossRefGoogle Scholar
  14. 14.
    Suzuki, H., Conlon, J. M. and Iwamuro, S. (2007) Evidence that the genes encoding the melittin-related peptides in the skins of the Japanese frogs Rana sakuraii and Rana tagoi are not orthologous to bee venom melittin genes: developmental- and tissue-dependent gene expression. Peptides 28, 2061–2068.PubMedCrossRefGoogle Scholar
  15. 15.
    Koyama, T. and Iwamuro, S. (2008) Molecular cloning of a cDNA encoding atypical antimicrobial and cytotoxic brevinin-1Ja from the skin of the Japanese brown frog, Rana japonica. Zool. Sci. 25, 487–491.PubMedCrossRefGoogle Scholar
  16. 16.
    Reilly, D. S., Tomassini, N. and Zasloff, M. (1994) Expression of magainin antimicrobial peptide genes in the developing granular glands of Xenopus skin and induction by thyroid hormone. Dev. Biol. 162, 123–133.PubMedCrossRefGoogle Scholar
  17. 17.
    Kim, J. B., Iwamuro, S., Knoop, F. C. and Conlon, J. M. (2001) Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. J. Peptide Res. 58, 349–356.CrossRefGoogle Scholar
  18. 18.
    Barchiesi, F., Colombo, A. L., McGough, D. A. and Rinaldi, M. G. (1994) Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards’ proposed standards. J. Clin. Microbiol. 32, 2494–2500.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Shawichi Iwamuro
    • 1
  • Tetsuya Kobayashi
    • 2
  1. 1.Department of Biology, Faculty of ScienceToho University, FunabashiChibaJapan
  2. 2.Department of Regulation Biology, Faculty of ScienceSaitama UniversitySaitamaJapan

Personalised recommendations