Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method.
Our goal was to develop a system to study proteins that associate in vivo with the Moloney murine leukemia virus (M-MuLV) enhancer elements by the isolation of intact proviral chromatin. The M-MuLV long terminal repeats (LTRs) contain tandemly repeated transcriptional enhancer sequences consisting of smaller motifs that bind cellular DNA-binding proteins implicated in transcriptional regulation. The M-MuLV enhancers are also important for disease specificity and latency of disease induction. To enrich for proviral chromatin containing M-MuLV LTR sequences, an affinity purification scheme was employed that relies on the affinity of bacterial Lac repressor protein for Lac operator (LacO) DNA sequences. An infectious M-MuLV recombinant was constructed that contains bacterial LacO sequences inserted into a nonessential region downstream from the 5' LTR of the virus (M-MuLV-LacO). Nuclei from M-MuLV-LacO-infected cells were digested with PvuII (which will liberate an LTR fragment containing LacO sequences), and digested chromatin was leached from the nuclei in hypotonic buffer. M-MuLV-LacO chromatin was then recovered by binding to an affinity matrix consisting of a beta-galactosidase-Lac repressor fusion protein anchored to acrylamide beads by an anti-beta-galactosidase monoclonal antibody [7]. Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog isopropyl-beta-D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method.
Granger
S W
SW
Department of Molecular Biology and Biochemistry and Cancer Research Institute, University of California, Irvine, Calif., USA.
Fan
H
H
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000821:virology; D002843:Chromatin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016679:Genome, Viral; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics; D051381:Rats; D013997:Time Factors
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific