Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes
Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes
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DOI：
10.1093/nar/gkg182
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发表时间：
2003-02-01
影响因子：
14.9
通讯作者：
Larionov, V
中科院分区：
文献类型：
--
作者：
Kouprina, N;Ebersole, T;Larionov, V
人类着丝粒仍然是人类基因组中特征不明显的区域，尽管它们对染色体的维持很重要。这部分是由于在基因组文库中克隆高度重复的DNA片段和区分染色体特异性克隆的困难。在这项工作中，我们报告了高选择性的分离人类着丝粒DNA转化相关重组（TAR）克隆。使用具有α DNA单体作为靶向序列的TAR载体从人细胞以及单染色体杂交细胞中分离人染色体2、5、8、11、15、19、21和22的大的着丝粒区域。还从12 Mb人微型染色体DeltaYq 74中分离α DNA阵列，该染色体含有正确染色体分离所需的最少量α DNA。本文报道了不同着丝粒结构分析的初步结果。通过转染人HT 1080细胞来评估克隆的人着丝粒区支持人人工染色体（HAC）形成的能力。来自DeltaYq 74的着丝粒克隆不支持HAC的形成，这表明内源染色体上存在功能性着丝粒的要求和形成从头着丝粒的要求可能是不同的。具有来自22号染色体的α DNA阵列的构建体（没有可检测的CENP-B基序）在不存在药物选择的情况下形成有丝分裂稳定的HAC，而没有可检测的宿主DNA的获得。总之，我们的研究结果表明，TAR克隆是一个有用的工具，调查人类着丝粒组织和HAC载体的形成，可能有一个潜在的治疗应用的结构要求。
Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.