Gilmour D S, Lis J T
Proc Natl Acad Sci U S A. 1984 Jul;81(14):4275-9. doi: 10.1073/pnas.81.14.4275.
We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.
我们提出了一种确定蛋白质在染色体DNA特定片段上体内分布的方法。首先，通过用紫外线照射完整细胞，使蛋白质与DNA共价连接。其次，用去污剂破坏这些细胞，并从裂解物中免疫沉淀出特定蛋白质。第三，纯化沉淀中与蛋白质共价连接的DNA，并通过杂交进行检测。为了测试这种方法，我们检测了大肠杆菌中RNA聚合酶与组成型表达的λcI基因以及未诱导和异丙基-β-D-硫代半乳糖苷（IPTG）诱导的乳糖操纵子的交联情况。正如预期的那样，免疫沉淀中组成型表达基因的回收率取决于细胞的照射以及RNA聚合酶抗血清的添加。乳糖操纵子DNA的回收率在交联步骤之前也需要用IPTG进行转录激活。在这些初步测试之后，我们检测了野生型、弱化子突变体和启动子突变体菌株中沙门氏菌亮氨酸操纵子上RNA聚合酶的分布。我们的体内数据与调控的衰减模型预测完全一致。从这些以及其他实验中，我们讨论了这些方法的分辨率、灵敏度和通用性。
