EXPERIMENTAL FIGURE 6-15 A yeast genomic library can be constructed in a plasmid shuttle vector that can replicate in yeast and in E. coli. (a) Components of a typical plasmid shuttle vector for cloning Saccharomyces genes. The presence of a yeast replication origin (ARS) and a yeast centromere (CEN) allows stable replication and segregation in yeast. Also included is a yeast selectable marker such as URA3, which allows a ura3 mutant to grow on medium lacking uracil. Finally, the vector contains sequences for replication and selection in E. coli (ORI and amp^r) and a polylinker for easy insertion of yeast DNA fragments. (b) Typical protocol for constructing a yeast genomic library. Partial digestion of total yeast genomic DNA with Sau3A is adjusted to generate fragments with an average size of ∼10 kb. The vector is prepared to accept the genomic fragments by digestion with BamHI, which produces the same sticky ends as Sau3A. Each transformed clone of E. coli that grows after selection for ampicillin resistance contains a single type of yeast DNA fragment.