RECOMBINATION DURING TRANSFORMATION AS A SOURCE OF CHIMERIC MAMMALIANARTIFICIAL CHROMOSOMES IN YEAST (YACS)

Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) freque ntly are chimeras formed between noncontiguous DNAs. Using pairs of hu man and mouse YACs we examined the contribution of recombination durin g transformation or subsequent mitotic growth to chimeric YAC formatio n. The DNA from pairs of yeast strains containing homologous or hetero logous YACs was transformed into a third strain under conditions typic al for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large mo lecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving t wo homologous YACs, the YACs had undergone recombination. Co-transform ation depends on recombination since it was reduced nearly 10-fold whe n the YACs were heterologous. While mitotic recombination between homo logous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To inve stigate the role of commonly occurring Alu repeats in chimera formatio n, spheroplasts were transformed with various human YACs and an unsele cted DNA fragment containing an Alu at one end and a telomere at the o ther. When unbroken YACs were used, between 1 and 6% of the selected Y ACs could incorporate the fragment as compared to 49% when the YACs we re broken. We propose that Alu's or other commonly occurring repeats c ould be an important source of chimeric YACs. Since the frequency of c himeras formed between YACs or a YAC and an Alu-containing fragment wa s reduced when a rad52 mutant was the recipient and since intra-YAC de letions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development.