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首页> 外文期刊>Molecular and Cellular Biology >Stable episomal maintenance of yeast artificial chromosomes in human cells.
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Stable episomal maintenance of yeast artificial chromosomes in human cells.

机译：人细胞中酵母人工染色体的稳定游离态维持。

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Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.
机译：已显示携带质粒复制的爱泼斯坦-巴尔病毒起源（oriP）的质粒可在潜伏感染的人类细胞中自主复制（J. Yates，N。Warren，D。Reisman和B. Sugden，Proc。Natl。Acad。Sci （美国81：3806-3810，1984）。我们证明此域的添加足以在表达病毒蛋白EBNA-1的人类细胞中稳定地附加维持酵母人工染色体（YAC），最高可达660 kb。为了更好地估计潜伏病毒基因组，在酵母细胞中通过同源重组添加oriP结构域之前，先将YACs环化。最终，将OriPYACs作为染色体外分子进行长期选择。 90 kb的OriPYAC在所有分析的细胞系中均未重排，而660 kb分子的完整形式存在于三个细胞系中的两个中。在没有选择的情况下，分子也相对稳定。这一发现表明，oriP-EBNA-1相互作用足以稳定至少660 kb的游离分子，并且这些元素不会随时间进行重排。荧光原位杂交分析表明，OriPYAC与宿主细胞染色体紧密相关，其中一些成对可见，表明附加体在每个细胞周期复制一次，并且通过附着于宿主染色体实现了稳定性，如病毒所示基因组。 YAC库的广泛可用性，酵母细胞中克隆序列的易操作性以及附加型稳定性使OriPYACs成为研究基因功能和控制基因表达的理想选择。

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