Figure 1.

Two-step genetic manipulation of YAC DNA by homologous recombination (a) using YAC amplification vector pYAM4 (b) and YAC insertion vector pYIV3 (c). A YAC contains an insert of genomic DNA between short and long vector arms. In step 1, genomic sequences flanking the desired site of insertion in the target gene are cloned either side of the HA-IRES-lacZ-ADE2 cassette in pYIV3. Integration of the HA-IRES-lacZ-ADE2 cassette into the YAC can be verified by hybridization with an ADE2 probe. In step 2, pYAM4 is used to replace the long vector arm so that the YAC DNA can be amplified when grown in medium containing galactose.