Nuclear factors bind to subterminal sequences of the transposable element Ac --Heinz-Albert Becker, Reinhard Kunze and Peter Starlinger DNA-protein interaction studies revealed that the protein encoded by the transposable element Ac can bind to AAACGG motifs in the subterminal DNA sequences, but not to the 11 bp inverted repeats (Kunze et al., EMBO J. 8:3177, 1989). It is known from some other transposons that host proteins are involved in transposition. Therefore we began to search for cellular factors binding to Ac. Here we report about additional DNA-protein interactions to those observed by the Ac encoded protein.

In electrophoretic mobility shift assays (EMSA) crude nuclear extracts from kernels were investigated for binding activities. 0.2 ng labeled DNA fragments and 2 µg pdIdC as unspecific competitor were incubated with 0.3 µg nuclear protein extract. Three fragments of the Ac ends form strong retarded complexes. Fragment 1 contains the 181 5'-terminal nucleotides of Ac, fragment 2 contains nucleotides 4195-4419, and fragment 3 consists of the 146 3'-terminal bases of Ac.

In homologous competition experiments complex formation of each fragment was strongly reduced by the inclusion of a ca. 10-fold excess of any of these three fragments. Heterologous competition for fragments 1 and 3 was done with two different fragments isolated from pUC19, including the polylinker into which fragments 1 and 3 were subcloned. These two fragments do not interfere with complex formation of fragment 1 and 3. Complex formation with fragment 2 was also not inhibited by the addition of a pUC19 fragment to the binding assay.

When fragment 1 was split at the Ac position 75 and both resulting fragments were tested, the fragment containing positions 76 to 181 was much more strongly retarded. Preliminary results from EMSAs with the isolated 11 bp inverted repeat indicate only a weak complex formation. However, no binding was observed with the isolated AAACGG hexamers which can be bound by the putative transposase.

For further characterisation of the observed protein-DNA interactions indirect DNase I footprints were made. After protein binding the incubation mix for the EMSAs was subjected for one minute to DNase I digestion. The reaction was stopped on ice. Separation was done on a low melting agarose gel. Two bands--free DNA and the retarded DNA, which should contain protected fragments--were excised. The DNA was eluted and separated on a sequence gel.

Fragments 1 and 3 both show two protected sequence regions. In each fragment they have a similar distance. We do not yet know if this similar spacing has a functional meaning. In fragment 1 the observed binding positions lie downstream of position 76. This result is in accordance with the observation of the EMSAs. The binding positions are overlapping with Ac regions containing the AAACGG motifs. So far no protection of the inverted repeats was detectable under indirect DNase I footprint assay conditions applied.

It has to be investigated if there is an interaction of the nuclear proteins with the Ac transposase and if an additional component of the nuclear extracts binds to the 11 bp inverted repeats of the Ac element.


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