Rogers J C, Rucinsky T E
Nucleic Acids Res. 1982 Sep 25;10(18):5483-501. doi: 10.1093/nar/10.18.5483.
About 1% of newly synthesized DNA from PHA-stimulated human lymphocytes can be isolated as large (up to 90 kilobase pairs) double stranded fragments that resist sequential alkali and heat denaturation steps but are not closed circular. By electron microscopy about 1% have single-strand hairpin loops at one end and therefore present inverted repetitive sequences (IR-DNA). Most of the remainder have a blunt-appearing double-strand terminus at both ends (78%) or one end (18%). Indirect evidence indicates that these also are inverted complementary structures with terminal hairpin loops too small to be visualized: (1) Treatment with either a 5' or 3' single-strand exonuclease generates essentially only fragments with a single strand at one end; (2) with partial denaturation, the number of fragments with identifiable single-strand hairpin loops increases (to about 20%); (3) after S1 nuclease digestion, greater than 95% can be fully heat denatured. Cot analysis indicates that these fragments are derived from dispersed sites throughout the genome. Up to 25% of DNA released from lymphocytes during growth similarly resists denaturation, and released DNA and IR-DNA are both enriched in the same set of repetitive sequences. Thus at least a portion of IR-DNA appears to be unstable.
从PHA刺激的人淋巴细胞中新合成的DNA中，约1%可被分离为大的（长达90千碱基对）双链片段，这些片段能抵抗连续的碱和热变性步骤，但不是闭环的。通过电子显微镜观察，约1%的片段在一端有单链发夹环，因此呈现反向重复序列（IR-DNA）。其余大部分在两端（78%）或一端（18%）有钝端双链末端。间接证据表明，这些也是反向互补结构，其末端发夹环太小而无法可视化：（1）用5'或3'单链外切核酸酶处理基本上只产生一端有单链的片段；（2）部分变性后，有可识别单链发夹环的片段数量增加（至约20%）；（3）S1核酸酶消化后，超过95%可完全热变性。Cot分析表明，这些片段来自基因组中分散的位点。生长过程中从淋巴细胞释放的DNA中，高达25%同样抵抗变性，并且释放的DNA和IR-DNA都富含同一组重复序列。因此，至少一部分IR-DNA似乎是不稳定的。
