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. 2005 Jan;166(1):265–274. doi: 10.1016/S0002-9440(10)62250-X

Figure 1.

Figure 1

A: Restriction map of YAC yWME30 containing the Ret proto-oncogene. The arrowheads denote the left and right arms. Ret is ∼50 kb in length and the 5′ end is located ∼80 kb from the left arm of the YAC. The restriction endonucleases are denoted: S, SacI; B, BamHI; SF, SfiI; M, Mlu; N, NotI. B: Site-directed mutagenesis to introduce the 918Met→Thr mutation into Ret within the YAC. ii, The PCR was used to amplify the length of DNA containing Ret exons 15, 16, and 17, with the intervening introns, from DNA obtained from a patient with MEN 2B. The amplified DNA, containing the 918Met→Thr mutation in exon 16 (asterisk), was cloned into the p680 plasma containing the LEU2 gene (for positive selection) and CYH2s gene (for negative selection). iii, The restriction endonuclease BlpI was used to linearize the plasmid, which then allowed targeted transformation of yeast cells containing the YAC by selecting in Leu+ medium for colonies containing the LEU2 gene. iiii, The resulting strains were grown in medium containing cycloheximide to select for the occasional colony in which the p680 plasmid was deleted by homologous recombination, leaving the MEN 2B point mutation in yWME30.