Affinity isolation of transcriptionally active murine erythroleukemia cell DNA using a cleavable biotinylated nucleotide analog.
We have developed an affinity technique to obtain active gene domains from murine erythroleukemia cell nuclei, based on the differential sensitivity of potentially active and inactive chromatin to DNase I. Nuclei isolated from potentially active noninduced cells and transcriptionally active induced MEL cells were treated with DNase I at concentrations which did not digest the beta-globin gene, followed by repair using a typical nick translation reaction during which a cleavable biotinylated nucleotide analog, 5-[N-biotinamido)hexanoamido-ethyl-1,3-dithiopropionyl -3-aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), was inserted into DNA sequences. Following purification and digestion with EcoRI restriction endonuclease, biotinylated sequences were affinity isolated by sequential binding to streptavidin and biotincellulose. The streptavidin/biotin-cellulose complex bound up to 80% of the nick-translated DNA, which comprised a small percentage of the total nuclear DNA. Cleavage of the disulfide bond in the linker arm of the biotinylated nucleotide resulted in elution of virtually all of the affinity isolated sequences. Hybridization analysis of this fraction of DNA revealed up to a 16-fold enrichment for the active beta-globin gene, as compared with DNA which did not bind to the biotincellulose. Conversely, the inactive alpha-fetoprotein gene was barely detectable in affinity isolated DNA from noninduced cells and was 2-fold depleted in samples from induced cells.
Dawson
B A
BA
Department of Anatomy and Cellular Biology, Medical College of Wisconsin, Milwaukee 53226.
Herman
T
T
Lough
J
J
D000081:Acetamides; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives*; D002454:Cell Differentiation / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods; D004273:DNA, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism; D003850:Deoxyribonuclease I; D003856:Deoxyuracil Nucleotides; D004900:Erythroblasts / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D005914:Globins / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics* / Q000473:pathology; D051379:Mice; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D019809:Streptavidin; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000081:Acetamides; D001426:Bacterial Proteins; D004273:DNA, Neoplasm; D003856:Deoxyuracil Nucleotides; D015345:Oligonucleotide Probes; C049787:biotin-19-SS-dUTP; D001710:Biotin; D005914:Globins; D019809:Streptavidin; D003850:Deoxyribonuclease I; C014026:hexamethylene bisacetamide