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 mechanisms involved in transcriptional regulation by steroid hormones, a number of cell lines carrying bovine papilloma virus (BPV) based constructs containing the mouse mammary tumor virus long terminal repeat (LTR) were established (Ostrowski et al., Mol. Cell. Biol. 3, 2945-2957, 1983). However, all our 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 attributable to DNA rearrangements in most of the cell lines, resulting in the integration of the BPV-based constructs into the host cell genome. We have identified two cell lines where the constructs are episomal. Micrococcal nuclease digestion of the nuclei demonstrated the presence of nucleosomes positioned over the episomal MMTV LTR. We managed to optimize conditions for preparation of nuclei and minichromosomes, which allowed extraction of approximately 40% of the minichromosomes, most of them being in circular superhelical form. Our data show clearly that the main factor preventing the release of minichromosomes from the nuclei is the presence of polyamines in the cell lysis buffer. The organization of MMTV promoter chromatin was unaffected by the extraction procedure, suggesting that these minichromosomes could be valuable templates for in vitro transcription studies and to identify proteins involved in chromatin remodeling during transcription.
Karpova
E
E
Institute of Cytology of the Russian Academy of Sciences, St. Petersburg. kondzac@uni-muenster.de
Tchirkova
I
I
Vorobi'ev
V
V
Richard-Foy
H
H
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000737:chemistry* / Q000235:genetics; D002021:Buffers; D002417:Cattle; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000737:chemistry / Q000302:isolation & purification; D005821:Genetic Techniques; D008836:Micrococcal Nuclease / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification; D011073:Polyamines; D020079:Terminal Repeat Sequences
D002021:Buffers; D002843:Chromatin; D004274:DNA, Recombinant; D009707:Nucleosomes; D011073:Polyamines; D008836:Micrococcal Nuclease