Isolation and characterization of minichromosome particles that contain a glucocorticoid-modulated promoter.
A procedure for the enrichment of minichromosomes, composed of bovine papilloma virus and the long terminal repeat element of the mouse mammary tumor virus (MTV), from isolated nuclei is described. Up to 60% of the minichromosomes were extracted as nucleoprotein particles. These particles sediment in sucrose gradients as 160S complexes. Hormone-labeled glucocorticoid receptor co-purifies with these complexes in a specific fashion. Between four and six molecules of receptor are bound per minichromosome molecule. Analysis of DNase I hypersensitivity demonstrates that hypersensitive sites are preserved through the purification procedure in a manner that reflects the hormone-dependent in vivo pattern of digestion. These purified minichromosomes will allow features of chromatin structure that may be important for steroid hormone modulation of transcription to be studied in vitro without resorting to destructive nuclease digestion procedures.
Ostrowski
M C
MC
Department of Microbiology and Immunology, Duke University, Durham, NC 27710.
D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics* / Q000648:ultrastructure; D002478:Cells, Cultured; D002875:Chromosomes / Q000187:drug effects / Q000648:ultrastructure*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D003852:Deoxyribonucleoproteins / Q000302:isolation & purification; D003907:Dexamethasone / Q000494:pharmacology*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics* / Q000648:ultrastructure; D008836:Micrococcal Nuclease; D027383:Papillomaviridae / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*
D004274:DNA, Recombinant; D003852:Deoxyribonucleoproteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease