Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio

Key findings

Kawakami et al. identified and cloned the full-length Tol2 transposase transcript from zebrafish embryos injected with Tol2-tyr plasmid using 3' and 5' RACE. The complete transcript of 2156 nucleotides contained four exons and three introns, encoding a predicted protein of 649 amino acids. Amino acid sequence analysis revealed strong similarity to transposases of the hAT family (hobo, Ac, Tam3), particularly showing conserved NH2- and COOH-terminal domains.

A novel transient embryonic excision assay was developed by co-injecting in vitro transcribed Tol2 mRNA with a nonautonomous (Tol2-tyr)ΔRV plasmid containing a transposase deletion into zebrafish fertilized eggs. DNA analysis 8 hours post-injection detected excision products in 100% of co-injected embryos (56 of 56 embryos) versus 0% in embryos receiving plasmid alone (0 of >50 embryos). Sequencing of six independent excision products revealed three with precise wild-type tyrosinase sequences and three with near-precise sequences containing few nucleotide additions.

Deletion analysis demonstrated that Tol2 sequences lacking the first intron could not be excised in the co-injection assay, indicating this region contains essential cis-regulatory elements required for transposition. The excision products showed characteristic hAT family transposon footprints, confirming authentic transposase-mediated excision rather than illegitimate recombination.