Recovery of Yeast Artificial Chromosomes in Circularized Form

基于酵母人工染色体（YACs）等大插入克隆系统的物理图谱是基因组研究的宝贵资源。每个克隆可容纳数百个数千个碱基的DNA，因此，基于YACs并使用序列标记位点（STSs）格式化的物理图谱为新基因的定位提供了框架，并为下游分析提供了克隆。然而，与基于细菌的克隆相比，从酵母中分离没有基因组DNA污染的YACs涉及繁琐的技术，并且至少20%的酵母基因组DNA仍然是最终制剂的污染物。为了利用YACs作为进一步分析的合适底物，我们设计了一种策略，使用细菌人工染色体为基础的载体来循环通常是线性的YACs。经圆化处理后，BAC/YAC的电泳迁移率发生了急剧变化，并且圆形结构在纯化过程中更能抵抗剪切力。这些特点有助于分离完整的纯克隆。此外，BAC/YAC可以通过转化转移到细菌中。这提供了一种从纯化到同质性的替代途径。我们构建的基于bac的载体包含基因组插入物附近的YAC中发现的钩序列。这些YAC序列是通过与传入的矢量片段重组而圆化的目标。该载体还含有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，并通过脉冲场凝胶电泳检测YAC圆化。在转移到细菌之前和之后的几个案例中测试了BAC/YAC的完整性。最多300 kb的克隆可以完整地转移到细菌中；较大的克隆转移效率低得多，而且通常是截短的形式，但可以从酵母中完整地纯化。

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.