CONSTRUCTION OF A PILOT HUMAN YAC LIBRARY IN A RECOMBINATION-DEFECTIVE YEAST-STRAIN

Using high-molecular-weight DNA fragments from a human lymphoblastoid cell line, a pilot collection of 2500 YACs was constructed in YKK115, a recombination-deficient strain of Saccharomyces cerevisiae carrying mutations in both the rad51 and rad52 genes. Analysis of 520 clones fr om the current library by pulsed-field gel electrophoresis revealed mo re than 97% single YACs with an insert size averaging 340 kb. Fluoresc ent in situ hybridization (FISH) performed with 37 clones on metaphase chromosomes suggested a high proportion mapping at centromeric (7) or telomeric (4) locations. The results are consistent with the stabiliz ation of YACs in strains disarmed in recombination functions [Kohno, K ., Oshiro, T., Kishine, H., Wada, M., Takeda, H., Ihara, N., Imamoto, F., Kano, Y. and Schlessinger, D. (1997) Human YACs unstable in a rad5 2 single mutant strain become stable in rad51rad52 double mutant. Gene , 000, 000-000 (GENE 10429)], and further suggest that the YACs may in clude regions that have been difficult to clone in other strains.