Figure 2

TAR cloning of genomic regions lacking ARS sequences. The TAR vector carries a yeast centromere (CEN6), a yeast positive selectable marker (HIS3), a negative-selectable marker (URA3), and two gene-specific targeting hooks. URA3 is a hybrid gene containing the open reading frame of the S. cerevisiae URA3 gene and the promoter of the S. pombe ADH1 gene. The promoter tolerates the insertion of up to a 130-bp sequence between the TATA box and the transcription initiation site. Further increase of the distance between the TATA box and the transcription initiation site inactivates URA3 expression. The reason the targeting hooks inserted into the ADH1 promoter do not disrupt URA3 expression is that their combined length is less than 130 bp. This configuration allows selection of TAR cloning events against the vector re-circularization. (A) Homologous recombination between the gene-specific targeting hooks and the genomic fragment containing the gene of interest leads to insertion of the genomic fragment between the TATA box and the transcription initiation site. Such clones can be selected by their ability to grow on media containing 5-FOA, which abolishes URA3 expression. (B) Nonhomologous end-joining forms a circular vector. The URA3 marker is normally expressed in these clones, so they do not grow on media containing 5-FOA. (R = resistant, S = sensitive to 5-FOA, DBS = double strand break.)