A method for efficient extraction of bovine papilloma virus-based minichromosomes that preserves native chromatin structure

Aiming to create an adequate model for investigation of the molecular mecha nisms involved in transcriptional regulation by steroid hormones, a number of cell lines carrying bovine papillomavirus (BPV) based constructs contain ing the mouse mammary tumor virus long terminal repeat (LTR) were establish ed (Ostrowski et al., Mol. Cell. Biol, 3, 2945-2957, 1983). However, all ou r attempts to extract from the cells such minichromosomes as nucleoprotein complexes using a method previously described (Ostrowski, Nucleic Acids Res . 15, 6957-6971, 1987) failed. Here, we show that this failure was attribut able to DNA rearrangements in most of the cell lines, resulting in the inte gration of the BPV-based constructs into the host cell genome. We have iden tified two cell lines where the constructs are episomal. Micrococcal nuclea se digestion of the nuclei demonstrated the presence of nucleosomes positio ned over the episomal MMTV LTR. We managed to optimize conditions for prepa ration of nuclei and minichromosomes, which allowed extraction of approxima tely 40% of the minichromosomes, most of them being in circular superhelica l form. Our data show clearly that the main factor preventing the release o f minichromosomes from the nuclei is the presence of polyamines in the cell lysis buffer. The organization of MMTV promoter chromatin was unaffected b y the extraction procedure, suggesting that these minichromosomes could be valuable templates for in vitro transcription studies and to identify prote ins involved in chromatin remodeling during transcription.