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The formation of nucleoprotein complexes between the Escherichia coli initiator protein DnaA and the replication origin oriC was analysed in vitro by bandshift assays and electron microscopy. DnaA protein binds equally well to linear and supercoiled oriC substrates as revealed by analysis of the binding preference to individual DnaA boxes (9-mer repeats) in oriC, and by a competition band-shift assay. DnaA box R4 (oriC positions 260-268) binds DnaA preferentially and in the oriC context with higher affinity than expected from its binding constant. This effect depends on oriC positions 249 to 274, is enhanced by the wild-type sequence in the DnaA box R3 region, but is not dependent on Dam methylation or the curved DNA segment to the right of oriC. DnaA binds randomly to the DnaA boxes R1, M, R2 and R3 in oriC with no apparent cooperativity: the binding preference of DnaA to these sites was not altered for templates with mutated DnaA box R4. In the oriC context, DnaA box R1 binds DnaA with lower affinity than expected from its binding constant, i.e. the affinity is reduced to approximately that of DnaA box R2. Higher protein concentrations were required to observe binding to DnaA box M, making this low-affinity site a novel candidate for a regulatory DnaA box.

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Molecularly cloned proviral DNA of avian replication‐defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5′ terminus, and a 1.3‐kb segment of the v‐myc transforming gene near the 3′ terminus. These v‐myc sequences are closely related to the v‐myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c‐myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v‐mil, which are unrelated to v‐myc, or to other oncogenes or structural genes of the avian leukemia‐sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v‐mil, termed c‐mil. Analyses of chicken c‐mil clones isolated from a recombinant DNA library of the chicken genome reveal that c‐mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v‐mil is expressed via genome‐sized mRNA as a gag‐related hybrid protein, p100gag‐mil, while v‐myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell‐derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.