van Leeuwen F, de Kort M, van der Marel G A, van Boom J H, Borst P
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Anal Biochem. 1998 May 1;258(2):223-9. doi: 10.1006/abio.1998.2587.
The hypermodified DNA base beta-D-glucosylhydroxymethyluracil, also called J, is a naturally occurring DNA modification. J was initially detected by 32P-postlabeling in Trypanosoma brucei and was recently also found in several other eukaryotic parasites. To use 32P-postlabeling as a method to quantitate the absolute levels of J in DNA we have tested the postlabeling efficiency of J using various synthesized standard oligonucleotides containing J. It is known that modified nucleotides, especially bulky ones, are often partially recovered by postlabeling and they are poor substrates for some of the enzymes used. We found that on average only 50% of J is recovered, which shows that the amount of J in T. brucei DNA has been twofold underestimated. Experiments with a short oligomer and defined pyrimidine tracts showed that the incomplete recovery of J is caused at least in part by resistance of J-containing DNA to degradation by micrococcal nuclease.
高度修饰的DNA碱基β-D-葡萄糖基羟甲基尿嘧啶，也称为J，是一种天然存在的DNA修饰。J最初是在布氏锥虫中通过32P后标记法检测到的，最近在其他几种真核寄生虫中也被发现。为了将32P后标记法用作定量DNA中J绝对水平的方法，我们使用了各种含有J的合成标准寡核苷酸来测试J的后标记效率。已知修饰的核苷酸，尤其是体积较大的核苷酸，在进行后标记时往往只能部分回收，并且它们是某些所用酶的不良底物。我们发现平均只有50%的J被回收，这表明布氏锥虫DNA中J的含量被低估了两倍。对短寡聚物和特定嘧啶序列的实验表明，J回收不完全至少部分是由于含J的DNA对微球菌核酸酶降解具有抗性所致。
