Absolute quantification of chromosome copy numbers in the polyploid macronucleus of Tetrahymena thermophila at the single-cell level
Absolute quantification of chromosome copy numbers in the polyploid macronucleus of Tetrahymena thermophila at the single-cell level
复制标题
在单细胞水平上对嗜热四膜虫多倍体大核中的染色体拷贝数进行绝对定量
DOI：
10.1111/jeu.12907
复制
发表时间：
2022-05-04
影响因子：
2.2
通讯作者：
Wang，Guangying
中科院分区：
文献类型：
--
作者：
Zhou，Yuanyuan;Fu，Lu;Wang，Guangying
无丝分裂在真核生物中广泛存在，但其内在机制却知之甚少。单细胞纤毛虫的多倍体大核（MAC）通过无丝分裂进行分裂，使纤毛虫成为研究这一过程的一个潜在的有价值的模式系统。然而，一种方法来准确地量化MAC染色体的拷贝数尚未建立。在这里，我们使用液滴数字PCR（ddPCR），以量化的MAC染色体的绝对拷贝数在四膜虫嗜热。我们首先证实ddPCR是一种灵敏且可重复的方法，可在单细胞水平上确定准确的染色体拷贝数。然后，我们使用ddPCR通过分析个体T来确定不同MAC染色体的拷贝数。在G1和无丝分裂（AM）期的嗜热细胞。G1期MAC染色体的平均拷贝数为90.9，约为AM期的一半（189.8）。每个MAC染色体的拷贝数在G1期的单个细胞之间变化，并与细胞大小相关，这表明无丝分裂伴随着不平等的胞质分裂导致拷贝数变异。此外，MAC染色体拷贝数在AM期细胞中变化较小的事实表明，拷贝数通过调节DNA复制而标准化。我们还证明了不同MAC染色体之间的拷贝数不同，并且拷贝数的染色体间变异在单个细胞中是一致的。我们的研究结果表明，ddPCR可用于T.可能存在于其他纤毛虫中。
Amitosis is widespread among eukaryotes, but the underlying mechanisms are poorly understood. The polyploid macronucleus (MAC) of unicellular ciliates divides by amitosis, making ciliates a potentially valuable model system to study this process. However, a method to accurately quantify the copy number of MAC chromosomes has not yet been established. Here, we used droplet digital PCR (ddPCR) to quantify the absolute copy number of the MAC chromosomes in Tetrahymena thermophila. We first confirmed that ddPCR is a sensitive and reproducible method to determine accurate chromosome copy numbers at the single‐cell level. We then used ddPCR to determine the copy number of different MAC chromosomes by analyzing individual T. thermophila cells in the G1 and the amitotic (AM) phases. The average copy number of MAC chromosomes was 90.9 at G1 phase, approximately half the number at AM phase (189.8). The copy number of each MAC chromosome varied among individual cells in G1 phase and correlated with cell size, suggesting that amitosis accompanied by unequal cytokinesis causes copy number variability. Furthermore, the fact that MAC chromosome copy number is less variable among AM‐phase cells suggests that the copy number is standardized by regulating DNA replication. We also demonstrated that copy numbers differ among different MAC chromosomes and that interchromosomal variations in copy number are consistent across individual cells. Our findings demonstrate that ddPCR can be used to model amitosis in T. thermophila and possibly in other ciliates.