Stewart B W, Huang P H, Brian M J
Biochem J. 1979 May 1;179(2):341-52. doi: 10.1042/bj1790341.
Rat liver DNA may be separated into two fractions by stepwise elution from benzoylated-DEAE-cellulose with NaCl and caffeine solutions respectively. Other studies using bacterical and yeast DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. In the present experiments, chromatography of DNA was monitored by labelling in vivo with [methyl-3H]thymidine in rats previously subjected to partial hepatectomy. In animals killed up to 1 h after thymidine injection, radioactivity eluted in the second fraction was inversely related to the incorporation time, being greatest when animals were killed 10 min after radioisotope injection. However, for most experiments, animals were allowed to survive 2-4 weeks after surgery before use, analysis being made on non-dividing DNA. Under these conditions, the proportion of caffeine-eluted DNA was decreased by subjecting the preparation to shear, before chromatography. A procedure that resulted in 12% of the recovered radioactivity being eluted with caffeine was adopted for experiments involving comparisons of the two DNA fractions. Under these conditions, cross-contamination could be detected by rechromatography, but this did not preclude distinction being made between the two fractions in terms of DNA structure. NaCl-eluted DNA did not bind to nitrocellulose filters. Caffeine-eluted DNA was retained by the filters and released by washing with 3mM-Tris/HCl,pH9.4. The fractions did not differ in terms of isopycnic centrifugation in CsCl. The NaCl-eluted fraction migrated as a single band in polyacrylamide gels, and this pattern was not modified by prior digestion with Neurospora crassa endonuclease. In contrast, caffeine-eluted DNA contained a minor component having a wide molecular-weight distribution and was subject to limited digestion by the endonuclease. The kinetics of denaturation of NaCi-eluted DNA in the presence of formaldehyde, in common with unfractionated DNA, were consistent with double-stranded structure. The same analysis of caffeine-eluted DNA revealed structural abnormality equivalent to two defects per 10000 base-pairs. The data are consistent with the minor fraction of rat liver DNA, separated by using benzoylated-DEAE-cellulose, containing regions of local denaturation. We previously showed that administration of the hepatocarcinogen dimethylnitrosamine is associated with an increase in the proportion of caffeine-eluted DNA. In terms of most analysis, differences between DNA fraction from nitrosamine-treated rats were similar to differences exhibited by preparations from control animals. However, structural analysis using denaturation kinetics indicated defects in both the NaCl- and caffeine-eluted DNA isolated from nitrosamine-treated rats. The two fractions differed from each other in that caffeine-eluted DNA exhibited a degree of structural damage far greater than that detected in any preparation from control animals...
大鼠肝脏DNA可以通过分别用NaCl和咖啡因溶液从苯甲酰化DEAE - 纤维素上逐步洗脱而分成两个组分。其他使用细菌和酵母DNA的研究表明，第一组分包含天然DNA，而第二组分可能表现出一定程度的单链特征。在本实验中，通过在部分肝切除的大鼠体内用[甲基 - 3H]胸苷进行体内标记来监测DNA的色谱分离。在胸苷注射后1小时内处死的动物中，第二组分中洗脱的放射性与掺入时间呈负相关，在放射性同位素注射后10分钟处死动物时最高。然而，对于大多数实验，在使用前让动物在手术后存活2 - 4周，对非分裂DNA进行分析。在这些条件下，在色谱分离前对样品进行剪切处理会使咖啡因洗脱的DNA比例降低。对于涉及两种DNA组分比较的实验，采用了一种导致12%回收放射性用咖啡因洗脱的方法。在这些条件下，可以通过再色谱分离检测到交叉污染，但这并不妨碍在DNA结构方面区分这两个组分。NaCl洗脱的DNA不与硝酸纤维素滤膜结合。咖啡因洗脱的DNA被滤膜保留，并通过用3mM - Tris/HCl，pH9.4洗涤而释放。在CsCl中的等密度离心方面，这两个组分没有差异。NaCl洗脱的组分在聚丙烯酰胺凝胶中迁移为单一带，并且这种模式不会因先用粗糙脉孢菌内切酶消化而改变。相比之下，咖啡因洗脱的DNA含有一个分子量分布较宽的次要组分，并且受到内切酶的有限消化。在甲醛存在下，NaCl洗脱的DNA的变性动力学与未分级的DNA一样，与双链结构一致。对咖啡因洗脱的DNA进行相同分析显示，其结构异常相当于每10000个碱基对有两个缺陷。这些数据与通过使用苯甲酰化DEAE - 纤维素分离的大鼠肝脏DNA的次要组分含有局部变性区域一致。我们之前表明，给予肝癌致癌物二甲基亚硝胺与咖啡因洗脱的DNA比例增加有关。在大多数分析中，来自亚硝胺处理大鼠的DNA组分之间的差异与来自对照动物样品所表现出的差异相似。然而，使用变性动力学的结构分析表明，从亚硝胺处理大鼠分离的NaCl洗脱和咖啡因洗脱的DNA都存在缺陷。这两个组分彼此不同之处在于，咖啡因洗脱的DNA表现出的结构损伤程度远大于在来自对照动物任何样品中检测到的损伤程度……
