Recovery of Yeast Artificial Chromosomes in Circularized Form

基于大插入克隆系统的物理图谱，如酵母人工染色体(YAC)，是基因组研究的宝贵资源。它们每个克隆可容纳数百个千碱基的DNA，因此基于YAC和用序列标记位点(STSS)格式化的物理图谱为新基因的放置提供了框架，并为下游分析提供了克隆。然而，与细菌克隆相比，从酵母中分离出没有基因组DNA污染的YAC需要繁琐的技术，至少20%的酵母基因组DNA在最终准备过程中仍是污染物。为了利用YAC作为进一步分析的合适底物，我们设计了一种策略，使用基于细菌人工染色体的载体使通常线性的YAC循环。环化后，BAC/YAC的电泳迁移率发生了急剧的变化，环状结构在纯化过程中更能抵抗剪切力。这些特征有助于分离完整的纯克隆。此外，BAC/YAC还可以通过转化转化为细菌。这提供了从纯化到同质性的另一种途径。我们构建的基于BAC的载体将YAC中发现的钩状序列整合到基因组插入物附近。这些YAC序列是通过与输入的载体片段重组而环化的靶序列。该载体还包含G418r或HIS5等位基因，以便在酵母细胞中进行选择。将含有YAC的AB1380菌株转化后，分离G418r或HIS5+菌落，用脉冲场凝胶电泳法检测YAC环化情况。BAC/YAC的完整性在转移到细菌之前和之后的几个案例中进行了测试。最大300kb的克隆可以原封不动地转移给细菌；更大的克隆转移的效率要低得多，而且通常是截短的形式，但可以从酵母中原封不动地进行纯化。

Physical maps based on large-insert cloning systems such as yeast artificial chromosomes (YACs) are invaluable resources for genomic studies. They accommodate hundreds of kilobases of DNA per clone, so that physical maps based on YACs and formatted with sequence-tagged sites (STSs) provide frameworks for the placement of new genes and provide clones for downstream analysis. However, in contrast to bacterial-based clones, isolation of YACs free of genomic DNA contamination from yeast has involved tedious techniques, and at least 20% yeast genomic DNA remains as a contaminant of final preparations. In order to make use of YACs as suitable substrates for further analysis, we have devised a strategy to circularize the usually linear YACs using a bacterial artificial chromosome-based vector. Upon circularization the electrophoretic mobility of the BAC/YAC changes sharply, and the circular structure is more resistant to shear forces during purification. These features facilitate the isolation of intact pure clones. In addition, the BAC/YAC can be transferred into bacteria by transformation. This provides an alternative route to purification to homogeneity. The BAC-based vector we have constructed incorporates hook sequences found in the YAC adjacent to genomic insert. Those YAC sequences are targets for circularization by recombination with the incoming vector fragment. The vector also contains G418r or HIS5 alleles to allow for selection in yeast cells. After transformation of a YAC-containing AB1380 strain, G418r or HIS5+ colonies are isolated and tested for YAC circularization by pulse-field gel electrophoresis. The integrity of the BAC/YAC has been tested in several cases before and after transfer into bacteria. Clones of up to 300 kb can be transferred intact to bacteria; larger clones are transferred much less efficiently, and often in truncated form, but can be purified intact from yeast.