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

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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.