Recovery of Yeast Artificial Chromosomes in Circularized Form

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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.