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首页> 外文期刊>Molecular and Cellular Biology >Formation of an inverted duplication can be an initial step in gene amplification.
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Formation of an inverted duplication can be an initial step in gene amplification.

机译：反向重复的形成可以是基因扩增的第一步。

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We have developed a gene transfer approach to facilitate the identification and isolation of chromosomal regions which are prone to high-frequency gene amplification. Such regions are identified by assaying for transformants which show high-frequency resistance to PALA and/or methotrexate by amplification of a vector containing the genes which encode the enzyme targets of these antiproliferative agents. We identified 2 of 47 transformants which displayed high-frequency amplification of the transfected genes, and in this report we describe the analysis of one of them (L46). Molecular analysis of the integration site in transformant L46 revealed that the donated genes were at the center of an inverted duplication which spanned more than 70 kilobase pairs and consisted largely of host DNA. The data suggest that integration of the transfected sequences generates a submicroscopic molecule containing the inverted duplication and at least 750 kilobases of additional sequences. The donated sequences and the host sequences were readily amplified and lost in exponentially growing cultures in the absence of drug selection, which suggests that the extrachromosomal elements are acentric. In contrast to the instability of this region following gene insertion, the preinsertion site was maintained at single copy level under growth conditions which produced copy number heterogeneity in L46. The implications of our results for mechanisms of genetic instability and mammalian gene amplification are discussed.
机译：我们已经开发出一种基因转移方法，以促进易于高频基因扩增的染色体区域的识别和分离。通过分析含有编码这些抗增殖剂的酶靶标的基因的载体，通过分析对PALA和/或氨甲蝶呤具有高频抗性的转化体来鉴定这些区域。我们鉴定了47个转化子中的2个，这些转化子显示了转染基因的高频扩增，在本报告中，我们描述了其中一个转化子的分析（L46）。对转化体L46中整合位点的分子分析表明，捐赠的基因位于反向复制的中心，该反向复制跨越70多个碱基对，并且主要由宿主DNA组成。数据表明，转染序列的整合产生了亚显微分子，其包含反向重复和至少750kb的附加序列。在没有药物选择的情况下，捐赠的序列和宿主序列很容易在指数增长的培养物中扩增和丢失，这表明染色体外元件是无心的。与基因插入后该区域的不稳定性相反，在生长条件下在L46中产生拷贝数异质性的预插入位点保持在单拷贝水平。讨论了我们的结果对遗传不稳定和哺乳动物基因扩增机制的影响。

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