Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
University of Chinese Academy of Sciences, Beijing, China.
J Eukaryot Microbiol. 2022 Jul;69(4):e12907. doi: 10.1111/jeu.12907. Epub 2022 May 4.
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.
有丝分裂在真核生物中广泛存在，但潜在机制仍不清楚。单细胞纤毛虫的多倍体大核（MAC）通过有丝分裂进行分裂，这使得纤毛虫成为研究这一过程的潜在有价值的模型系统。然而，尚未建立一种准确量化 MAC 染色体拷贝数的方法。在这里，我们使用液滴数字 PCR（ddPCR）来量化嗜热四膜虫的 MAC 染色体的绝对拷贝数。我们首先证实 ddPCR 是一种在单细胞水平上确定准确染色体拷贝数的敏感且可重复的方法。然后，我们通过分析处于 G1 期和无丝分裂（AM）期的单个嗜热四膜虫细胞，使用 ddPCR 来确定不同 MAC 染色体的拷贝数。在 G1 期，MAC 染色体的平均拷贝数为 90.9，大约是 AM 期（189.8）的一半。在 G1 期，每个 MAC 染色体的拷贝数在单个细胞中存在差异，并与细胞大小相关，这表明有丝分裂伴随着不均匀的胞质分裂导致了拷贝数的变化。此外，AM 期细胞中 MAC 染色体拷贝数变化较小表明通过调节 DNA 复制使拷贝数标准化。我们还表明，不同 MAC 染色体的拷贝数存在差异，并且跨单个细胞的染色体间拷贝数的变化是一致的。我们的研究结果表明，ddPCR 可用于模拟嗜热四膜虫和其他纤毛虫的有丝分裂。
