Characterization of the Ac sequences required in cis for transposition
--Shivani Chatterjee and Peter Starlinger

This project comprises the investigation of the cis-acting sequences in the termini of the mobile element Ac, which are required for excision. The excision event results from the interaction of the terminal sequences with some trans-acting components, the Ac-encoded protein and perhaps one or several host-encoded factors. Upon the mutation of an essential terminal sequence this interaction should be disturbed and the excision ability of the resulting element should be reduced or even lost.

Mutations were introduced by applying the technique of oligonucleotide-directed mutagenesis and the resulting elements were tested on their excision ability in the "Petunia-filter-assay". In this transient assay system excision events can be visualized as beta-glucuronidase-expressing protoplasts (blue spots). Because deletion experiments had restricted the location of the cis-acting sequences to the terminal 200 bp of each end, the mutations were established in these regions.

A first series of mutations altered the sequence motif GGTAAA, which was protected by nuclear extracts of Ac-free maize (H. A. Becker, HA), implying the involvement of a host-factor. Substitutions of individual copies of this motif did not result in a loss of excision in vivo, indicating either that the host-factor binding is not critical for the excision-reaction or that the loss of a single motif is not severe enough to prevent excision. Another motif that was altered is the hexameric sequence AAACGG, which is reiterated many times in both ends of Ac , and which is bound in vitro by the Ac protein. This motif was substituted block-wise by unrelated sequences. While the substitution of four perfectly repeated motifs resulted in no loss of function, the substitution of a less conserved block of hexameric motifs located in the vicinity led to a nearly tenfold reduction of excision frequency.

The combination mutant with both blocks substituted could not be excised at all. Furthermore, a group of point mutations located in the vicinity of the inverted repeats resulted in a dramatic reduction of excision frequency. Only one of eight of these mutations proximal to the 5' or 3' inverted repeat altered a perfect AAACGG-sequence, whereas the other mutations did not change obvious sequence motifs. The exchange of the inverted repeats of Ac with those of the Tam3 element resulted in a hybrid element that is no longer mobilized by the Ac protein.

From these findings the following conclusions can be tentatively drawn, although the role of the different sequence elements is not yet completely understood:
    1. The inverted repeats are indispensable for transposition.
    2. The blocks of AAACGG hexamers contribute to the excisability of the element. With the exception of one such motif very close to the 3'-terminus, none of these elements seems to play an exclusive role. This does not make it likely that single elements are absolutely necessary for transposition, while the others help in setting up a transposition complex. On the other hand, no such alteration has yet yielded an increase of excisability. This renders the hypothesis unlikely that some of these hexamers bind transposase in a non-productive way and thus help in keeping the transposition rate low.
    3. Sequences between the inverted termini and the AAACGG hexamer block can be point-mutated to yield pronounced decreases in excisability, though they do not bind transposase in vitro. The role of these sequences is yet unclear. 


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