Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA.
Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA.
复制标题
二甲基亚硝胺体内甲基化的非随机性质以及随后从大鼠肝脏染色质 DNA 中去除甲基化产物。
作者：
R. Ramanathan;S. Rajalakshmi;D. Sarma;E. Farber
这项研究旨在研究二甲基亚硝胺(DMN)对肝染色质DNA的甲基化以及随后体内DNA结合甲基化产物的去除是否是随机的。肝染色质DNA在注射[~3H]DMN(0.5 mg/250Muci/100g体重)后4h分离为核酸酶可消化和不可消化物质。用胰腺DNase I或微球菌核酸酶消化这种甲基化的肝染色质，并对核酸酶消化的酸溶产物进行分析，发现释放的放射性(72%)与260 nm处的吸光度(50%)不一致。以甲基化后的染色质总DNA为底物，代替核酸酶反应中的染色质DNA，消除了这种差异，在260 nm处的放射性和吸光度的释放速度和程度是相同的。这些结果，再加上用核酸酶消化分离的两种染色质的DNA中鸟嘌呤含量相同的事实，表明肝染色质DNA核酸酶可及区的甲基化程度相对大于不可及区。对染色质DNA可及区域中甲基化产物去除的研究进一步表明，在4小时存在的甲基化产物中，62%在3天内丢失，87%在1周内丢失，94%在2周内丢失。然而，染色质DNA核酸酶不可及区域的丢失在3天后仅为27%，1周后为49%，2周后为86%，这表明与染色质DNA核酸酶可及区域相比，该区域甲基化产物的去除相对较慢。因此，这项研究的结果表明：(A)在染色质DNA的核酸酶可及区域，DMN诱导的甲基化产物的去除速度更快，甲基化程度更高；(B)染色质DNA的核酸酶不能到达的区域，甲基化程度降低，去除速度更慢。结果表明，DMN诱导的甲基化产物在肝染色质DNA中的分布和去除是非随机的。
This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.