Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in Joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.

We have sequenced the 480 base pair (bp) repeating unit of the 5S RNA genes of the Dipteran fly Calliphora erythrocephala and compared this sequence to the three known 5S RNA gene sequences from the Dipteran Genus Drosophila (1,2). A striking series of five perfectly conserved homologies identically positioned within the 5' flanks of all four Dipteran 5S RNA coding regions has thus been identified. The spacing (12-13 bp) between all of these homologies is typical of A form rather than B form DNA. Given that the eukaryotic 5S RNA gene specific initiation factor TFIIIA (3) is a DNA unwinding protein (4), a role for these Dipteran 5' flank homologies in initiation site selection on 5S RNA genes transiently unwound for transcription is suggested. One of the Dipteran homology blocks is highly conserved in sequence and position in all but one of the eukaryotic 5S RNA gene sequences known to date (17/18 genes). Its sequence (consensus: TATAAG) and position (average center: -26 bp) are highly reminiscent of the polymerase II gene 'TATA' box (5).