--Reinhard Kunze and Peter Starlinger
Crude nuclear protein extracts from insect cells infected with baculovirus containing the Ac coding sequence or from cells infected with wildtype baculovirus (Fusswinkel et al., MNL 62:47, 1988) were tested for DNA binding activity with various DNA fragments by a mobility shift assay. Fragments containing the terminal 181bp of the Ac 5'-end (the end from which transcription starts) and 147bp from the 3'-end, respectively, were incubated with the protein extracts. Both fragments were retarded during electrophoresis after incubation with the recombinant, but not wildtype extracts. Three other DNA fragments not derived from the Ac ends were not retarded by either extract. The Ac fragment-protein complexes did not enter into 3.5 % polyacrylamide gels, but only in agarose gels, and formed rather a smear than a uniform band.
In order to determine the DNA sequence which is recognized by the Ac protein the Ac 5'-end-fragment was cleaved with PvuI, yielding two fragments containing the outermost 75bp (5'o) and the 106 inner nucleotides (5'i) of the Ac 5'-end fragment: In the mobility shift assay the 5'i-fragment was more efficiently retarded than the 5'o-fragment, which contains the 11bp inverted terminal repeat of Ac.
Since DNase I footprinting experiments have been unsuccessful so far, we examined the ends of Ac for peculiar sequence motifs. The hexamer motif AAACGG is repeated 6 times in the 5'-end and 2 times in the 3' end of Ac. Synthetic oligodeoxynucleotides with this sequence were ligated and cloned into pUC19. Fragments containing this motif behaved undistinguishably from the Ac 5'- or 3'-end fragments in the gel retardation experiment. However, DNA-fragments containing an AAAGGG motif were not retarded at all.
We conclude that the Ac protein expressed in the baculovirus system is capable of binding specifically to the ends of Ac. In particular, it can bind to an AAACGG motif which is repeated several times in the terminal sequences of Ac, but it does not recognize an AAAGGG motif. However, the AAACGG motif cannot be the only recognition sequence, because the 5'o fragment of Ac contains no complete copy of this motif but is retarded in the mobility shift assay. Also, the Ds1 element does not contain this motif.
The Ac element can be inactivated by methylation. In this context
it is conspicuous that the AAACGG motif contains a potential methylation
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