Miller C A, Costa M
Department of Environmental Medicine, New York University School of Medicine, NY 10016.
Mutat Res. 1990 Apr;234(2):97-106. doi: 10.1016/0165-1161(90)90036-n.
Ultraviolet light, formaldehyde, cis-diamminedichloroplatinum(II), chromate (Cr6+), or chromium chloride (Cr3+) under the appropriate conditions caused the formation of DNA-protein crosslinks in intact Chinese hamster ovary (CHO) cells or in cell nuclei. The DNA-protein crosslinks were isolated, applied to nitrocellulose filters, and reacted with antibodies to nuclear proteins. An antiserum to a 97-kD nuclear protein detected p97-DNA complexes in CHO nuclei and cell cultures treated with UV light, cis-Pt and formaldehyde. Exposure to Cr3+ induced p97-DNA crosslinks only in isolated nuclei, while chromate (Cr6+) treatment resulted in significant crosslink formation only in intact cells. Analysis of western blots with the p97 antiserum indicated that crosslinks induced by formaldehyde or ultraviolet light required DNAase I digestion of DNA for migration of the p97 complexes into the gel. In contrast, the 97-kD antigen from the metal-induced crosslinks was released from DNA and resolved in the gel when 2-mercaptoethanol was included in the electrophoresis sample buffer. Assay of slot blots with an antihistone monoclonal antibody indicated that formaldehyde, but not cis-Pt or chromate, crosslinked histones to the DNA. These results illustrate the utility of immuno-slot blots in detecting and characterizing DNA-protein complexes induced by diverse chemical and physical agents.
在适当条件下，紫外线、甲醛、顺二氯二氨合铂（II）、铬酸盐（Cr6+）或氯化铬（Cr3+）会在完整的中国仓鼠卵巢（CHO）细胞或细胞核中导致DNA - 蛋白质交联的形成。分离出DNA - 蛋白质交联物，将其应用于硝酸纤维素滤膜，并与核蛋白抗体反应。一种针对97-kD核蛋白的抗血清在经紫外线、顺铂和甲醛处理的CHO细胞核及细胞培养物中检测到p97 - DNA复合物。暴露于Cr3+仅在分离的细胞核中诱导p97 - DNA交联，而铬酸盐（Cr6+）处理仅在完整细胞中导致显著的交联形成。用p97抗血清对蛋白质免疫印迹分析表明，甲醛或紫外线诱导的交联需要用DNA酶I消化DNA，以使p97复合物迁移到凝胶中。相比之下，当在电泳样品缓冲液中加入2 - 巯基乙醇时，金属诱导交联产生的97-kD抗原从DNA中释放并在凝胶中分离。用抗组蛋白单克隆抗体对狭缝印迹进行检测表明，甲醛而非顺铂或铬酸盐会使组蛋白与DNA交联。这些结果说明了免疫狭缝印迹在检测和表征由不同化学和物理试剂诱导的DNA - 蛋白质复合物方面的实用性。
