Tanaka Tsubasa, Watanabe Tsuyoshi
Graduate School of Life Sciences, Tohoku University, Aoba, Aramaki, Aoba, Sendai, 980-8578, Japan.
Chromosome Res. 2003;11(8):787-95. doi: 10.1023/b:chro.0000005780.77785.5d.
We analyzed the onset and location of replicational and transcriptional activity during the first cell cycle in new macronuclei of the ciliate Paramecium caudatum exconjugants. Synchronous exconjugants were pulse labeled with 5-bromo-2'-deoxyuridine or 5-bromouridine-5'-triphosphate to visualize replication or transcription sites, respectively. The first morphological change after macronuclear determination is the appearance of heterochromatic aggregates. Confocal microscopic examination revealed that DNA replication started at the stage when new macronuclear chromatin was partially decondensed, and that replication sites were located in a large number of small spot-like areas excluding the heterochromatic regions. Transcriptional activation in the new macronuclei also took place in the same developmental stage and in the same region that replication started. As macronuclear development progressed, heterochromatic aggregates disappeared, and replication and transcription sites were scattered throughout the nucleoplasm. Moreover, studies on aphidicolin-treated exconjugants demonstrated that inhibition of the DNA replication did not hinder transcriptional activation in the new macronuclei. On the other hand, replicational and transcriptional activity were also detected in old macronuclear fragments irrespective of their morphology and size, and length and timing of the replication corresponded to those in the new macronuclei.
我们分析了尾草履虫接合后体新大核第一个细胞周期中复制和转录活性的起始及位置。同步接合后体分别用5-溴-2'-脱氧尿苷或5-溴尿苷-5'-三磷酸进行脉冲标记，以分别观察复制或转录位点。大核确定后的第一个形态变化是异染色质聚集体的出现。共聚焦显微镜检查显示，DNA复制始于新大核染色质部分解聚的阶段，且复制位点位于大量小斑点状区域，不包括异染色质区域。新大核中的转录激活也发生在与复制起始相同的发育阶段和相同区域。随着大核发育的进行，异染色质聚集体消失，复制和转录位点分散在整个核质中。此外，对经阿非科林处理的接合后体的研究表明，DNA复制的抑制并不妨碍新大核中的转录激活。另一方面，在旧大核片段中也检测到了复制和转录活性，无论其形态和大小如何，复制的长度和时间与新大核中的一致。
