Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast

We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamb lia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chr omosome (YAC) cassette (a yeast selectable marker and a centromere). The ca ssette allows transferring of BACs into yeast for their further modificatio n. Furthermore, the new hybrid vector provides the opportunity to re-isolat e each DNA insert without construction of a new library of random clones. D igestion of a BAC DNA by an endonuclease that has no recognition site in th e vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allo wing direct gene isolation. Cotransformation of a TAR vector and genomic DN A into yeast spheroplasts, and subsequent recombination between the TAR vec tor's flanking ends and a specific genomic fragment, allows rescue of the f ragment as a circular YAC/BAC molecule. Here we prove a new cloning strateg y by re-isolation of randomly chosen genomic fragments of different size fr om T. brucei cloned in BACs. We conclude that genomic regions of unicellula r eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity.