Worm Breeder's Gazette 13(2): 104 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Tc1 and Tc3 are members of a family of transposable elements found in many different species (for example: Henikoff (1992), New Biologist 4:382-388). The Tc1 -likefamily shares no homology with other groups of transposable elements, except for a motif within the coding region of the transposase gene which it has in common with integrases of retroviruses and transposases of bacterial IS elements (Doak et al. (1994), Proc. natl. Acad. Sci. USA in press). This motif includes 2 conserved aspartic acid residues and a glutamic acid residue: the latter two are separated by 35 amino acids. This motif was first identified in a comparison of retroviral/retrotransposon integrase proteins and IS3 transposases (Fayet et al. (1990), Mol. Microbiol. 4:1771-1777; Khan et al. ( 1990), Nucl. Acids Res. 19:851-860). A mutation of each of these amino acids abolishes the catalytic functions of these proteins as shown in vitro (Drelich et al. (1992), Virology 188:459-468; Engelman and Craigie (1992), J. Virol. 66:6361-6369; Van Gent et al. (1992), Proc. Natl. Acad. Sci. USA 89:9598-9602; Kulkosky et al. (1992), Mol. Cell. Biol. 12:2331-2338).
We have assayed the biological relevance of the D-D35E motif for Tc3 transposition in vivo by mutagenising each of the three conserved amino acids. The aspartic acid residues at position 144 and 231 and the glutamic acid residue at position 266 were changed to valine residues. As controls we made two additional mutations outside the D-D35E motif: a glutamic acid residue at position 213 and an aspartic acid residue at position 254 were both changed to a valine residue. The first residue (E213) is not conserved between Tc1 and Tc3 or any other member of the Tc1 -likefamily, the second residue (D254) is conserved in several transposable elements ( Tc1 ,C. briggsae Tc2 and Minos). The mutant genes and the wild type Tc3A gene were fused to the hsp-16 promoter and assayed in transgenic nematodes as described before (Van Luenen et al., (1993), EMBO J. 12:2513-2520).
The activity of the transposase protein was determined in two different assays. Transposition of endogenous Tc3 elements was detected using PCR, and excision of Tc3 elements was detected by Southern blot analysis (Van Luenen et al. (1993), EMB0 J. 12:2513-2520). The three mutants within the D-D35E motif were no longer capable of transposition or excision. The non-conserved mutant (E213V) was normally active. The conserved, non-D-D35E mutant (D254V) was also active, but somewhat reduced in activity compared to the wild type transposase.
The presence of a motif that is similar between bacterial IS transposases, retroviral integrases and transposases of the Tc1 -likefamily suggests a possible common ancestry. Our mutagenesis data show that these residues are as important for Tc1 jumping as they are for the other transposases and integrases. This may suggest not only a conservation in sequence but also a conservation in function.