Fig. 2. Mutated/modified Brk protein and activity. (A, part i) Domains/motifs in the Brk protein. DBD, DNA binding domain; Q, poly-glutamine; H, histidine rich; A, poly-alanine; 3R, independent repression domain. CiM and GiM, interaction motifs for the co-repressors CtBP and Gro, respectively. (A, parts ii-v) Properties and effects of modified/mutated Brk proteins shown in B-D. (A, part ii) Domains present: present (+), deleted (–) or mutated (M). (A, part iii) Nuclear (N) or cytoplasmic (C) localization (blank spaces, here and in other columns, indicates that they were not tested). (A, part iv) Activity level assessed by effect on the phenotype of adult wings (`–', no repressor activity; `++++', maximal activity; see Materials and methods for details on assigning activity level). (A, part v) The ability of each protein to repress the endogenous sal gene, an omb-lacZ line, and two reporters, vg-QE and UbxB. Y, repressed; N, not repressed; U, we were unable to detect reliable differences in UbxB expression between wild-type and mutant embryos of any genotype. (B) EMS point mutants in the endogenous brk gene. A protein truncated within the DBD, brk^M68, or immediately after it, brk^E427, has no activity, whereas a truncation producing a longer protein, but which still does not include the CiM or GiM, brk^F138, has significant activity and can repress omb, but not the other targets. An amino acid substitution within the DBD, brk^F124, also abolishes activity. (C) Point mutants in a UAS-brk transgene, A438. Four result in amino acid substitutions in the DBD and reduce activity. The fifth mutation, A438-53, results in a truncation immediately after the DBD and has no activity. (D) In vitro mutated/modified UAS-brk transgenes. There are two basic requirements for these transgenes to repress gene expression: the DBD and a repression domain/motif. In addition to the CiM and GiM, there is another independent repressor domain, 3R, located between the DBD and CiM. The DBD plus any one of the three repressor domains/motifs is sufficient to repress some Brk targets, although there is some variability in the ability of individual repressor domains to repress different targets.