Elucidation of DNA replication mechanism of Protobothrops flavoviridis (Habu snake)

• Project/Area Number: 16580079
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Sojo University
• Principal Investigator: ODA-UEDA Naoko Sojo University, Faculty of Biotechnology and Life Science, Associate professor, 生物生命学部, 助教授 (70211828)
• Co-Investigator(Kenkyū-buntansha): OHNO Motonori Sojo University, Faculty of Biotechnology and Life Science, Professor, 生物生命学部, 教授 (30038434)
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
• Keywords: error-prone DNA polymerase / snake (Habu) venom / Protobothrops (Trimeresurus) flavoviridis / error-prone DNAポリメラーゼ / snake venom / error-prone DNA polymerase / Trimeresurus flavoviridis

Research Abstract

We have found that snake venom isozymes with a variety of physiological activities from Protobothrops (formally known as Trimesurus) flavoviridis had evolved in an accelerated manner, and believed that mostlikely the diversity in such venomous isozyme genes had produced by accumulation of point mutations. To understand the molecular mechanism underlying mutation in P.flavoviridis, we have cloned P.flavoviridis homolog of the Escherichia coli dinB gene which encode DNA polymerase IV and known as error-prone DNA polymerase. Total RNA from P.flavoviridis oocyte was used as template for first strand cDNA synthesis by used the SMART cDNA Library construction kit. Degenerate primers were designed based on conserved sequence in the Xenopus laevis (frog) and Gallus gallus (chicken). Multiple rounds of 5' and 3' rapid amplification of cDNA ends were used to extend the putative P.flavoviridis genes using BD SMART RACE cDNA ampification kit. Fibroblast cells from the embryo of P.flavoviridis were prepared for chromosome mapping and fluoresence in situ hybridaization. Multiple alignments were constructed with the CLUSTAL X program.
The P.flavoviridis DinB sequence of cDNA 2882 bp contains an ORF of 2.5 kiobases (kb) that can encode a protein of 833 amino acids. The amino acid sequence of P.flavoviridis DinB protein contain N-terminal nucleotidyl transferase domain, two tandem HhH domain implicated of DNA binding, and nuclear locarization signal like domain and proliferating cell nuclear antigen domain as being conserved in DinB superfamily, and share significant identity (〜84%) with G.gallus (chiken). In a phylogenetic tree of DinB superfamily, P.flavoviridis DinB make a branch with G.gallus out of other eukaruote such as H.sapiens or mouse. Fluoresence in situ hybridization of chromosome represents that P.flavoviridis dinB gene localized to chromosome 6 (macrochromosome) unlike G.gallus's which localized to microchromosome.

Research Products

• [Journal Article] Amino acid sequence of a basic aspartate-49-phospholipase A_2 from Trimeresurus flavorirdis and phylogenic analysis of Crotalinae venom phosholipase A_2 (2005)
  • Author(s): Chijiwa, T., Abe, L., Ogawa, T., Nikandrov, M.N., Hattori, S., Oda-Ueda, N., Ohno,M.
  • Journal Title: Toxicon 46 Pages: 185-195
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Diversity and accelerated evolution of C-type lectin-like proteins from snake venom (2005)
  • Author(s): Ogawa, T., Chijiwa, T., Oda-Ueda, N., Ohno, M., Molecular Toxicon, 45, 1-14(2005)
  • Journal Title: Toxicon 45 Pages: 1-14
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Amino acid sequence of a basic aspartate-49-phospholipase A_2 from Trimeresurus flavoviridis and phylogenic analysis of Crotalinae venom phospholipase A_2 (2005)
  • Author(s): Chijiwa, T., Abe, L., Ogawa, T., Nikandrov, N.N., Hattori, S., Oda-Ueda, N., Ohno, M.
  • Journal Title: Toxicon 46 Pages: 185-195
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Molecular Diversity and accelerated evolution of C-type lectin-like proteins from snake venom (2005)
  • Author(s): Ogawa, T., Chijiwa, T., Oda-Ueda, N., Ohno, M.
  • Journal Title: Toxicon 45 Pages: 1-14
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Amino Acid Sequence of a basic Aspartate-49-phospholipase A2 from Trimeresurus flavoviridis Venom and Phylogenetic Analysis of Crotalinae Venom phospholipase A2 (2005)
  • Author(s): Chijiwa, T.Abe, L., Ogawa, T., Nikandrov, N.N., Hattori, S., Oda-Ueda, N., Ohno, M
  • Journal Title: Toxicon 46 Pages: 185-195
• [Journal Article] Molecular diversity and acclerated evolution C-type lectin like proteins from snake venom (2005)
  • Author(s): Ogawa, T., Chijiwa, T., Oda-Ueda, N., Ohno, M
  • Journal Title: Toxicon 45 Pages: 1-14
• [Journal Article] Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom (2005)
  • Author(s): Ogawa, T., Chijiwa, T., Oda-Ueda, N, Ohno, M.
  • Journal Title: Toxicon Vol 45,No 1 Pages: 1-14
• [Journal Article] ハブ毒を科学する (2004)
  • Author(s): 上田直子, 千々岩宗仁, 大野素徳
  • Journal Title: 化学と生物 42 Pages: 687-693
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Science in Protobothrops flavoviridis venom.(review article) (2004)
  • Author(s): Oda-Ueda, N., Chijiwa, T., Ohno, M.
  • Journal Title: Kagaku to Seibutsu. [Chemistry and biology] 42 Pages: 687-693
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] ハブ毒を科学する (2004)
  • Author(s): 上田直子, 千々岩崇仁, 大野素徳
  • Journal Title: 化学と生物 Vol 42,No 10 Pages: 687-693
• [Book] ライフサイエンスの機器分析 6章 4項 (2004)
  • Author(s): 上田 直子
  • Publisher: 三共出版