The maize Myb transcription factor C1 depends on the basic helix-loop-helix (bHLH) proteins R or B for regulatory function, but the closely related Myb protein P does not. We have used the similarity between the Myb domains of C1 and P to identify residues that specify the interaction between the Myb domain of C1 and the N-terminal region of R. Substitution of four predicted solvent-exposed residues in the first helix of the second Myb repeat of P with corresponding residues from C1 is sufficient to confer on P the ability to physically interact with R. However, two additional Myb domain amino acid changes are needed to make the P regulatory activity partially dependent on R in maize cells. Interestingly, when P is altered so that it interacts with R, it can activate the Bz1 promoter, normally regulated by C1 ؉ R but not by P. Together, these findings demonstrate that the change of a few amino acids within highly similar Myb domains can mediate differential interactions with a transcriptional coregulator that plays a central role in the regulatory specificity of C1, and that Myb domains play important roles in combinatorial transcriptional regulation. Combinatorial interactions between transcription factors are of central importance to regulation of gene expression in eukaryotes. These interactions can either modulate transcription factor activity or contribute to the biological specificity of factors with very similar DNA-interaction motifs. Elucidation of the mechanisms by which proteins with very similar DNA-binding domains achieve regulatory specificity remains a fundamental question in biology today.Proteins containing the Myb-homologous DNA-binding domain are widespread in eukaryotes (reviewed in refs. 1 and 2). The vertebrate c-myb gene plays an essential regulatory role in the proliferation and differentiation of hematopoietic cells. Besides c-myb, at least two other myb-related genes (A-myb and B-myb) are present in vertebrates (3). The products of these genes have Myb domains, each consisting of three head-to-tail Myb motifs (R1, R2, and R3). Oncogenic versions of c-myb, such as v-myb, contain only R2 and R3, as do hundreds of plant Myb-domain proteins (4). Myb domains formed by the R2 and R3 Myb motifs bind DNA. Each Myb motif contains three ␣-helices, and the third helix of each Myb motif makes sequencespecific DNA contacts. The second and third helices of each Myb motif form a helix-turn-helix structure when bound to DNA, similar to motifs found in the repressor and in homeo domains (5). In addition to their well-established roles in DNA binding, Myb domains are also emerging as important protein-protein interaction motifs. These Myb domain-mediated proteinprotein interactions play key roles in the biological specificity of the corresponding factors (6-13). However, the mechanisms by which protein-protein interactions contribute to the regulatory specificity of Myb domain proteins are poorly understood.In f lowering plants, several hundred genes containing the conserved Myb DNA-binding domain have b...

Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA͞DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green f luorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green f luorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10 ؊4 , which is 1-3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.