Mutational analysis of Ac ORFa protein in vivo

--Ralf Lütticke, Ulrike Courage and Reinhard Kunze

The development of a transient in vivo transposition assay (Houba-Herin et al., MGG 224:17-23, 1990) enables us to test in vitro mutated Ac ORFa protein for a loss or reduction of transposase function. We have constructed a set of ORFa protein mutants by introducing 6bp linkers into the MaeI restriction sites of the Ac cDNA. These mutations were introduced into a plasmid which contains a 5'-terminally deleted derivative of the Ac cDNA behind the 2'-promoter. From this plasmid an ORFa protein derivative starting at amino acid 103 of the 807 codon ORFa and preceded by 7 out-of-frame ATGs is expressed (see also report by Becker, Lütticke and Starlinger, this issue).

Twelve mutants were created, each carrying a single 6bp linker-insertion. None of these affect the DNA binding domain of the ORFa protein. Four mutants induced Ds excisions from the cotransfected reporter plasmid with the same efficiency as the wildtype ORFa protein. Of these, the most N-terminal insertion behind amino acid 51 of the wildtype ORFa protein is located in the leader region, and therefore cannot alter the properties of the truncated ORFa protein. However, three insertions within the truncated ORFa, behind amino acids 623, 754 and 771 do not disturb transposase function either. These three insertions are located in protein regions with no obvious homology to the open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila.

An insertion of two amino acids behind ORFa residue 270 destroys transposase function. This position is situated within the downstream helix of the putative helix-loop-helix motif. Although this finding is no evidence for the formation of a helix-loop-helix structure, it demonstrates that this segment is essential for the transposase (see also report of Feldmar and Kunze, this issue).

Insertions behind ORFa residues 369, 390, 445, 462, 577, 585 and 709 abolish the capacity to mobilize a Ds element, too. They all are located within protein regions with strong sequence homologies to the open reading frames of Tam3 from Antirrhinum majus and Hobo from Drosophila (Feldmar and Kunze, EMBO J. 10:4003-4010, 1991). These results indicate that essential transposase functions different from the DNA binding reaction are located in the C-terminal two thirds of the Ac ORFa protein. We are presently testing if less well conserved regions of the ORFa protein are more tolerant against two amino acid insertions.


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