Krokan H, Wist E, Prydz H
Biochim Biophys Acta. 1977 Apr 19;475(4):553-61. doi: 10.1016/0005-2787(77)90316-1.
Replicative intermediates have been studied in intact HeLa cells and in nuclei isolated from such cells. In whole cells the smallest DNA (primary DNA pieces) observed after pulse labelling with [3H]thymidine were 90-160 nucleotides long, and the size of the molecules in this class of DNA did not increase with increasing pulse length. Some increase in size was, however, observed when cells were pulse labelled at 25 degrees C instead of 37 degrees C. Chase experiments using nuclei from pulse-labelled cells suggested that the primary DNA pieces could be chased rapidly into DNA of high molecular weight (30-70 S, corresponding to a molecular weight of 0.7 - 10(7)-6.4-10(7)). Longer chases showed that the label eventually accumulated in DNA with s values greater than 150 S. In isolated nuclei the primary DNA pieces after a 1 min pulse at 37 degrees C were approximately 200 nucleotides long. Primary pieces of this size were also rapidly chased into the 30-70 S region. However, during longer pulses in vitro a fraction of the primary DNA pieces grew beyond their normal size to reach a size of up to 2000-3000 nucleotides before being attached to the 30-70 S molecules.
已经在完整的HeLa细胞以及从这类细胞中分离出的细胞核中对复制中间体进行了研究。在用[3H]胸腺嘧啶脉冲标记后，在完整细胞中观察到的最小DNA（初级DNA片段）长度为90 - 160个核苷酸，并且这类DNA分子的大小不会随着脉冲长度的增加而增大。然而，当细胞在25℃而非37℃下进行脉冲标记时，观察到了大小的一些增加。使用来自脉冲标记细胞的细胞核进行的追踪实验表明，初级DNA片段能够迅速追踪到高分子量的DNA（30 - 70 S，对应分子量为0.7 - 10(7)-6.4-10(7)）中。更长时间的追踪表明，标记最终积累在s值大于150 S的DNA中。在分离的细胞核中，在37℃下脉冲1分钟后的初级DNA片段长度约为200个核苷酸。这种大小的初级片段也迅速追踪到30 - 70 S区域。然而，在体外更长时间的脉冲期间，一部分初级DNA片段在连接到30 - 70 S分子之前，其大小增长超过正常大小，达到高达2000 - 3000个核苷酸的大小。
