IMMUNODETECTION OF DNA PROTEIN CROSS-LINKS BY SLOT BLOTTING

被引：14
作者：
MILLER, CA [1 ]
COSTA, M [1 ]
机构：
[1] NYU,SCH MED,DEPT ENVIRONM MED,NEW YORK,NY 10016
来源：
MUTATION RESEARCH | 1990年 / 234卷 / 02期
关键词：
Chromium; cis-Diamminedichloroplatinum(II); DNA damage; DNA-protein crosslinks; Formaldehyde; Slot blotting;
D O I：
10.1016/0165-1161(90)90036-N
中图分类号：
Q3 [遗传学];
学科分类号：
071007 ; 090102 ;
摘要：
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. © 1990.
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页码：97 / 106
页数：10
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