Journal of Biomedical Science

, Volume 8, Issue 3, pp 278–289 | Cite as

Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method

  • Steven W. Granger
  • Hung Fan
Original Paper

Abstract

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 withPvull (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 β-galactosidase-Lac repressor fusion protein anchored to acrylamide beads by an anti-β-galactosidase monoclonal antibody [7]. Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog isopropyl-β-D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method.

Key Words

Moloney murine leukemia virus Proviral chromatin Long terminal repeat Lac operator 

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Copyright information

© National Science Council 2001

Authors and Affiliations

  • Steven W. Granger
    • 1
  • Hung Fan
    • 1
  1. 1.Hung Fan Department of Molecular Biology and Biochemistry Cancer Research InstituteUniversity of CaliforniaIrvineUSA

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