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首页> 外文期刊>Molecular and Cellular Biology >An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells
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An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

机译：在活细胞的中期和后期，人工构建的De Novo人类染色体几乎与自然对等地行为相同

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Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.
机译：人类人工染色体（HAC）是用于分析染色体功能的有前途的试剂。虽然HAC保持稳定，但尚未详细研究HAC的分离机制。为了分析活细胞中的HAC，我们将256拷贝的Lac操纵子整合到了包含α-卫星DNA的前体酵母人工染色体（YAC）中，并在表达GFP-Lac阻遏物融合的HT1080细胞中生成了带有绿色荧光蛋白（GFP）标签的HAC。蛋白。对活的有丝分裂细胞中的GFP-HAC和宿主着丝粒进行时移分析表明，HAC在纺锤体中部正确对齐，并且HAC的姐妹染色单体以与宿主染色体相同的时间间隔分离，并以迁移率相似的方式移至纺锤体极到宿主着丝粒。这些结果表明，由输入α-卫星YAC的多聚体组成的HAC保留了天然染色体上着丝粒的大部分功能。 HAC和宿主染色体之间的唯一区别是，HAC在中期移入纺锤体中区的频率更高，振荡频率更高。但是，这提供了重要的证据，表明单个HAC能够在极对极方向上保持张力平衡，从而稳定其在纺锤中间区周围的位置。

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