Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method.
Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method.
复制标题
通过亲和法从感染细胞中纯化莫洛尼鼠白血病病毒染色质。
DOI：
10.1007/bf02256602
复制
发表时间：
2001
影响因子：
11
通讯作者：
Fan,H
中科院分区：
文献类型：
--
作者：
Granger,SW;Fan,H
我们的目标是开发一个系统来研究蛋白质，在体内与莫洛尼鼠白血病病毒（M-MuLV）增强子元件的分离完整的前病毒染色质。M-MuLV长末端重复序列（LTR）含有串联重复的转录增强子序列，该序列由结合参与转录调控的细胞DNA结合蛋白的较小基序组成。M-MuLV增强子对于疾病特异性和疾病诱导的潜伏期也很重要。为了富集含有M-MuLV LTR序列的前病毒染色质，采用依赖于细菌Lac阻遏蛋白对Lac操纵子（LacO）DNA序列的亲和力的亲和纯化方案。构建了感染性M-MuLV重组体，其含有插入病毒5′ LTR下游非必需区的细菌LacO序列（M-MuLV-LacO）。用Pvull消化来自M-MuLV-LacO感染细胞的细胞核（其将释放含有LacO序列的LTR片段），并在低渗缓冲液中从细胞核中浸出消化的染色质。然后通过与亲和基质结合回收M-MuLV-LacO染色质，所述亲和基质由通过抗β-半乳糖苷酶单克隆抗体锚定到丙烯酰胺珠的β-半乳糖苷酶-Lac阻遏物融合蛋白组成[7]。特异性结合的染色质在生理条件下通过与半乳糖类似物异丙基-β-D-硫代半乳糖吡喃糖苷孵育洗脱。Southern印迹分析证实了该方法对M-MuLV前病毒染色质的特异性富集。
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.