- 作者: Steven W. Granger Hung Fan
- 作者服務機構: Department of Molecular Biology and Biochemistry and Cancer research Insitute, University of Califomia, Irvine, Callf., USA
- 中文摘要: --
- 英文摘要: Our goal was to develop a system to study proteins thatassociate in vivo with the Moloney murine leukemiavirus (M-MuLV) enhancer elements by the isolation ofintact proviral chromatin. The M-MuLV long terminalrepeats (LTRs) contain tandemly repeated transcriptionalenhancer sequences consisting of smaller motifs thatbind cellular DNA-binding proteins implicated in tran-scriptional regulation. The M-MuLV enhancers are alsoimportant for disease specificity and latency of diseaseinduction. To enrich for proviral chromatin containing M-MuLV LTR sequences, an affinity purification schemewas employed that relies on the affinity of bacterial Lacrepressor protein for Lac operator (LacO) DNA se-quences. An infectious M-MuLV recombinant was con-structed that contains bacterial LacO sequences insertedinto a nonessential region downstream from the 5' LTRof the virus (M-M u LV-LacO). Nuclei from M-MuLV-LacO-infected cells were digested with Pvull (which will liber-ate an LTR fragment containing LacO sequences), anddigested chromatin was leached from the nuclei in hypo-tonic buffer. M-MuLV-LacO chromatin was then recov-ered by binding to an affinity matrix consisting of a β-galactosidase-Lac repressor fusion protein anchored toacrylamide beads by an anti-β-galactosidase monoclon-al antibody [7]. Specifically bound chromatin was elutedunder physiological conditions by incubation with thegalactose analog isopropyl-β-D-thiogalactopyranoside.Southern blot analysis confirmed the specific enrich-ment of M-MuLV proviral chromatin by this method.
- 中文關鍵字: --
- 英文關鍵字: Moloney murine leukemia virus. Proviral chromatin. Long terminal repeat. Lac operator