DNA binding activities of c-Myc purified from eukaryotic cells.

c-Myc is a nuclear phosphoprotein which contains both a leucine zipper and a helix-loop-helix dimerization motif. These are adjacent to a basic region believed to make specific contacts with DNA upon dimerization. We report the purification of full-length c-Myc to near homogeneity from two independent eukaryotic systems: the baculovirus overexpression system using an insect cell host, and Chinese hamster ovary cells containing heat-inducible c-myc genes. The DNA binding capabilities of these preparations were characterized. Both preparations contain two distinct activities that bind specifically to sequences with a core of CACGTG. The Myc protein is solely responsible for one of these binding activities. Specific sequences that bound to c-Myc were selected from a large pool of random DNA sequence. Sequencing of individual binding sites selected by this procedure yielded a 12-base consensus, PuACCACGTGCTC, for c-Myc binding. Both protein preparations additionally demonstrated a distinct complex, containing both c-Myc and a copurifying 26-29-kDa protein, that bound to DNA with higher affinity than Myc alone. Selection of specific DNA sequences by this complex revealed a consensus binding site similar to the 12-base consensus described above. These data demonstrate that c-Myc isolated from eukaryotic cells is capable of sequence-specific DNA binding and further refine the optimal sequence for c-Myc binding. These protein preparations should prove useful in further characterizing the biochemical properties of c-Myc.     

Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori

Summary The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene. Offprint requests to: S. Ichimura