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Reduced occurrence of chimeric YACs in recombination-deficient hosts.

Yeast artificial chromosomes (YACs) with inserts averaging hundreds of kilobases in size have proven very useful for the construction of physical maps of large genomes (1). However, YACs are not ideal large-insert clones. For example, the frequent occurrence of chimeric inserts complicates the use of YACs for physical mapping, walking from insert ends, and determination of contiguous DNA sequence. Recombination within the yeast host strain between two YACs or YAC fragments has been suggested as a mechanism to account for the surprisingly high rates of chimerism (40—60%) in most genomic YAC libraries (2). To determine if the genetic background of the yeast host strain contributes to the formation of chimeric YACs, the same YAC ligation mixture was introduced into three isogenic yeast hosts differing only in their recombination abilities. Strain CGY2872 (ATCC9O436) carries a LEU2 insertion at the BglD. site of the RAD52 gene, and strain CGY2897 (ATCC90437) carries the same insertion together with an insertion/replacement of the Clal to Stul region of the RAD1 gene with ADE2 in the parental host strain CGY2570 (ATCC90435) (genotype MA Ta ura3-52 trplA63 lys2-A202 his3-A200 ade2-l(oc) Ieu2-Al ^+) (3). Defects in RAD52 and RAD1 were confirmed by measuring the loss of resistance to methyl methanesulfonate (MMS) and ultraviolet light (UV) respectively (3). Both genes appear to be implicated in mitotic recombination (4, 5). The two genes are in different epistatic groups, and mutations in both genes had been reported to exhibit additive or synergistic effects on several recombinationrelated properties (4).