Fine structure of the origin-proximal DNAase I-hypersensitive region in wild-type and EC mutant polyoma.

The chromatin of wild-type polyoma virus displays a unique DNAase I highly sensitive region in situ in the infected nuclei, extending for about 260 nucleotides from the origin of replication to the beginning of the late region. We show that this highly sensitive region is not homogeneous. It displays a well defined pattern of differential sensitivity along its 260 nucleotides, including one protected subregion and two hypersensitive sites, which concern a unique residue or very few nucleotides. All these features were mapped to a precision of +/- 5 bp relative to the DNA nucleotide sequence. In parallel, we studied a PyEC mutant, whose sequence is grossly rear-ranged in this very region. This allows the PyEC to overcome the block in the expression of the early genes in the mouse embryonal carcinoma PCC4 cell line. We show that this mutant, however, displays an identical highly sensitive region with the same fine structure. The mapping of this structure on the mutant nucleotides sequence coincides with that of the wild-type relative to any arbitrary point on the late side of the rearrangement; however, 60% of the mutant molecules display this unique local chromatin structure, instead of 20% for the wild-type ones. Finally, the sequence determinism of this singularity is discussed, as well as its possible role in the control of the early transcription and the establishment of the PyEC phenotype..