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제안 방법
- The ligations were electroporated into TB1 Escherichia coli and plated on IPTG, X-Gal, and ampicillin-containing agar plates. Sequencing was performed by using the Dyenamic ET primer and Deza sequencing kit (Amersham Pharmacia, Piscataway, NJ, USA) and an Applied Biosystems 377 DNA sequencer (Perkin-Elmer).
- To classify the newly identified KIFs, molecular evolutionary analysis was performed. Phylogenetic trees were obtained by the MJ method (Fig.
대상 데이터
- Until now, only two kinds of KIF, have been identified in the squid optic lobe. In this study, six new KIFs were identified. And they shared homologous domains which include a putative ATP-binding site and a microtubule-binding site of KHC [7,14].
- Although KIFs have been cloned from several organisms, only two genes have been identified within squid [7]. To improve the understanding of the role KIFs in the giant axon of the squid, based on PCR approach using degenerate oligonucleotide primers, six new members of KIF were identified in the squid. In this report the partial amino acid sequences of these new KIFs are presented with the classification based on molecular evolutionary analysis.
이론/모형
- The motor domain of new cloned KIFs was aligned with CLUSTALW software by the neighbor-joining method. A phylogenetic tree of the motor domain sequences of the KIF was generated using the Maximum Parsimony (MP) method [2], the Neighbor-Joining (NJ) method [11], the Minimum Evolution (ME) method [10], and the Maximum likelihood (ML) method [6] based on the JTT matrix-based model. The confidence limits on the tree were calculated by performing bootstrap sampling 1,000 times.
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