Stable episomal maintenance of yeast artificial chromosomes in human cells
Stable episomal maintenance of yeast artificial chromosomes in human cells
复制标题
人体细胞中酵母人工染色体的稳定游离维持
DOI：
10.1128/mcb.16.9.5117
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发表时间：
1996
影响因子：
5.3
通讯作者：
Clare Huxley
中科院分区：
文献类型：
--
作者：
Kaetrin Simpson;A. McGuigan;Clare Huxley
携带Epstein-Barr病毒起源的质粒复制(ORIP)的质粒已被证明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。娜塔莉。阿卡德。SCI。美国81：3806-3810,1984)。我们证明，在表达病毒蛋白EBNA-1的人类细胞中，添加这个结构域足以稳定地维持酵母人工染色体(YAC)的至少660kb的上体。为了更好地接近潜伏的病毒基因组，YAC在酵母细胞中通过同源重组在添加ORIP结构域之前被环化。所得到的OriPYAC在长时间的选择中以染色体外分子的形式存在；在所有被分析的细胞系中，90kb的OriPYAC没有重排，而在三个细胞系中有两个存在660kb的完整分子形式。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660kb的异构体分子，并且这些元件不会随着时间的推移而发生重排。荧光原位杂交分析表明，OriPYAC与宿主细胞染色体密切相关，其中一些可见成对，这表明附着体在每个细胞周期复制一次，稳定是通过附着在宿主染色体上实现的，正如病毒基因组所建议的那样。YAC文库的广泛可获得性、酵母细胞克隆序列的易操作性以及表型的稳定性使OriPYAC成为研究基因功能和基因表达调控的理想工具。
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.
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影响因子：
--
作者：
G. Pfeifer;A. Riggs
通讯作者：
G. Pfeifer;A. Riggs
克隆为 YAC 的哺乳动物 DNA 的修饰和操作。
DOI：
10.1016/0735-0651(90)90015-8
发表时间：
1990
期刊：
Genetic analysis, techniques and applications
影响因子：
--
作者：
Reeves,RH;Pavan,WJ;Hieter,P
通讯作者：
Hieter,P
酵母人工染色体上的人类 HPRT 基因通过细胞融合转移到小鼠细胞后发挥功能。
影响因子：
4.4
作者：
Huxley,C;Hagino,Y;Schlessinger,D;Olson,MV
通讯作者：
Olson,MV
影响因子：
2.9
作者：
BURGERS, PMJ;PERCIVAL, KJ
通讯作者：
PERCIVAL, KJ
DOI：
10.1073/pnas.81.12.3806
发表时间：
1984-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
YATES, J;WARREN, N;SUGDEN, B
通讯作者：
SUGDEN, B