Fig. 7. Schematic strategy of yeast-based gap repair assay by homologous recombination. Three different plasmids were used as gap vectors. The first gap vector was pSS16 (1). The other two vectors were newly constructed by removing the 5'-side (p53 NcoI) or the 3'-side (p53 StuI) of the p53 open reading frame, and the deleted cDNA was introduced into the pLSX plasmid (2). (a) Plasmid pSS16 was linearized by HindIII and StuI digestion and was cotransformed with the second PCR product with full-length p53 cDNA derived from with mutant oligonucleotides in 96-well microtiter plates labeled #1, #2, #5-#16 (Fig. 6). (b) Plasmid p53NcoI was linearized by BamHI and NcoI digestion and was cotransformed with the second PCR product with the 5'-half of p53 cDNA derived from with mutant oligonucleotides in 96-well microtiter plates labeled #17-#25 (Fig. 6). (c) Plasmid p53StuI was linearized by HindIII digestion, and was cotransformed the second PCR product with the 3'-half of p53 cDNA derived from with mutant oligonucleotides in 96-well microtiter plates labeled #26-#28 (Fig. 6). The yeast strain used for transformation was YPH499 harboring one of the reporter plasmids (see Materials and Methods in the main text). The second PCR product was homologously recombinated into the gap vector in the yeast cells. The resulting circular plasmid was identical with the wild-type p53 expression vector except for specific missense mutation (red box).

1. Ishioka, C., Frebourg, T., Yan, Y., Vidal, M., Friend, S., Schmidt, S. & Iggo, R. (1993) Nat. Genet. 5, 124-129.

2. Ishioka, C., Englert, C., Winge, P., Yan, Y. X., Engelstein, M. & Friend, S. H. (1995)

Oncogene 10, 1485-1492.