Related Experiment Videos
Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method
1Department of Molecular Biology and Biochemistry and Cancer Research Institute, University of California, Irvine, Calif., USA.
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 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.
Insights
Researchers developed a novel method to isolate Moloney murine leukemia virus (M-MuLV) chromatin using Lac operator DNA sequences. This technique enables the study of proteins interacting with M-MuLV enhancer elements in vivo.
Area of Science:
- Molecular Biology
- Virology
- Chromatin Biology
Background:
- Moloney murine leukemia virus (M-MuLV) enhancers regulate viral transcription and are crucial for disease specificity and latency.
- Understanding protein interactions with M-MuLV enhancer elements requires studying intact proviral chromatin.
- Existing methods may not efficiently isolate specific viral chromatin for in vivo protein association studies.
Purpose of the Study:
- To develop a system for isolating intact proviral chromatin associated with M-MuLV enhancer elements.
- To enable the study of cellular proteins that bind to M-MuLV enhancer regions in vivo.
- To enrich for M-MuLV chromatin containing long terminal repeat (LTR) sequences for further analysis.
Main Methods:
- Construction of an M-MuLV recombinant virus (M-MuLV-LacO) containing bacterial Lac operator (LacO) sequences.
- Infection of cells with M-MuLV-LacO and subsequent PvuII digestion to release LTR fragments with LacO sequences.
- Affinity purification of M-MuLV-LacO chromatin using a Lac repressor-beta-galactosidase fusion protein matrix.
Main Results:
- Successfully constructed M-MuLV-LacO and isolated chromatin fragments containing the viral LTR and LacO sequences.
- Developed an affinity purification strategy to specifically enrich M-MuLV proviral chromatin.
- Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin using the described method.
Conclusions:
- The developed system effectively isolates intact M-MuLV proviral chromatin.
- This method provides a valuable tool for studying in vivo protein-DNA interactions at viral enhancer elements.
- The technique facilitates research into M-MuLV transcriptional regulation and disease mechanisms.