Transferring the p27-responsive domain. (A) Myb protein structures. The diagrams summarize the structures of A-Myb (□) and c-Myb (▦) and illustrate the conserved DNA binding, transcriptional activation, and negative regulatory domains. The highly conserved DNA-binding domain is labeled and shaded black. The diagrammed recombinant proteins were constructed by swapping cDNA fragments at the conserved EcoRI (E) and HincII (H) sites, as indicated. Regions of c-Myb involved in interactions with cyclin D1 and CDK6 or that confer p27 responsiveness are indicated at the bottom. (B) Activation of A-Myb/c-Myb recombinants. QT6 cells were transfected with a Myb-responsive reporter plasmid as described in Figure 4 along with plasmids expressing c-Myb, A-Myb, or the recombinant Myb proteins CEA, CHA, AEC,or AHC, either alone or along with a plasmid expressing p27. The data are plotted as fold activation by p27, relative to the activity observed by the various Myb proteins alone. (C) Target gene activation. QT6 cells were transfected with combinations of plasmids expressing NF-M and various Myb proteins or swap constructs either with or without p27, as indicated at the top. After 2 days, the samples were harvested and analyzed by Northern blotting for Mim1 then stripped and reprobed for lysozyme and beta-actin. Not shown: Western blotting showed that p27 expression had little or no effect on the expression or stability of the Myb proteins.