The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding The v-myb oncogene of the avian myeloblastosis virus (AMV) causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells but not fibroblasts in vitro (for a review, see reference 29). The related E26 avian leukemia virus causes acute erythroid leukemia in chickens and transforms avian erythroid and myeloid cells in vitro (36,43). The c-myb proto-oncogene from which v-myb arose is expressed at high levels predominantly in immature hemopoietic cells including those of the erythroid, lymphoid, and myeloid lineages (8, 16). The expression of c-myb declines as these cells mature (11,48). c-myb itself appears to control this differentiation process because the expression of exogenous c-myb or v-myb can block or reverse the differentiated phenotype of various hemopoietic cells (3,10,12,33,37,47). myb-related genes have also been identified in mammals, Drosophila melanogaster, Zea mays, Saccharomyces cerevisiae, and Dictyostelium discoideum (23,39,41,44,46; R. Firtel, and J. Lipsick, unpublished data).The protein products of v-myb and c-myb are present in the cell nucleus, bind DNA in a sequence-specific fashion, and trans-activate gene expression in transient DNA transfection assays (4-6, 22, 24a, 25, 27, 38, 49). Consistent with these findings, the myb-related BASI gene of S. cerevisiae encodes a genetically defined transcription factor which binds directly to DNA (46). In addition, the myb-related product of the Cl locus ofZ. mays controls several unlinked, developmentally regulated genes involved in the synthesis of anthocyanin pigments (41).A number of oncogenes which encode nuclear proteins (GGGCCC) to the end-filled HindlIlresistant neo fragment of the pUC-NEO plasmid (31). This DNA was digested with SmaI to trim excess linkers, purified by agarose gel electrophoresis, and then cloned into the SmaI site of pUC18 to create the plasmid pANA (Fig. 1). This plasmid was then digested with SmaI to release a 1.4-kilobase neo fragment flanked by single Apal linkers. ApaI was chosen because it does not cut within v-myb; however, the method can easily be adapted for other restriction sites.The plasmid pUC-MYB-dE was used as a target for mutagenesis (21). Plasmid DNA was digested with the desired restriction enzyme, and linear DNA was purified by agarose gel electrophoresis and then ligated to the 1.4-kilobase neo fragment described above. Digestion of the target plasmid with restriction enzymes with multiple recognition sites was limited by titration of ethidium bromide to maximize the yield of linear DNA (40). In rare cases, certain individual sites (e.g., Avall at 960) were never mutagenized by this method, suggesting a strong restriction enzyme site preference during partial digestion in the presence of ethidium bromide. Target DNAs produced by enzymes which generated three-nucleot...