Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in genome condensation, and influence transcription and replication. Properties of the DNA sequence, such as curvature and flexibility, direct the location of nucleosomes. DNA sequences that position nucleosomes have been identified and rules that govern their properties have been formulated. However, DNA sequences that are refractory to nucleosome formation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequences by selecting those that refrain from nucleosome formation from a large pool of synthetic DNA fragments with a central region of 146 random base-pairs fitted with adapters for PCR amplification. These were used for in vitro salt-induced reconstitution of nucleosomes under thermodynamic equilibrium conditions. Fragments that did not form nucleosomes were purified, amplified by PCR, and the reconstitution was repeated. After 17 rounds of negative selection, the material was highly enriched in sequences reluctant to form nucleosomes. Cloning and sequencing revealed that 35% of the molecules had long repeats of TGGA, and their affinity for histone octamers was about half that of average DNA.
Copyright 1998 Academic Press