Figure 2.

Generation of S. pombe nda3-F200Y cells and resistance to antimicrotubule benzimidazole agents. (A) Flow chart of PCR cloning, generation of the mutation, and genome replacement of nda3-F200Y at the native nda3⁺ β-tubulin locus of YY105. Homologous recombination generates sequential β-tubulin genes (strain AK102). 5-FOA counterselection regenerates a single allele. Strains AK101 and AK103 contain either nda3-F200Y or nda3⁺ at the native locus, respectively. Primer pairs for PCR experiment in B are shown as asterisks in A, set 1 (^*) and set 2 (^**). HindIII sites used in Southern analysis are represented by vertical bars in A. See MATERIALS AND METHODS for more details to A and B. (B) PCR (lanes 1–6) and Southern analysis (lanes 7–9) of chromosomal DNA from integrants in A. F200Y/AK101, lanes 1, 5, and 7; AK102, lanes 2, 6, and 9; WT/AK103, lanes 3 and 8; and YY105 control, lane 4. DNA molecular weight standards: left bars (for PCR in kilobases): 9.4, 6.6, 4.4, 2.0, and 0.56. Right bars (for Southern in kilobases): 23.1, 9.4, 6.6, 6.1, 4.9, 4.4, 3.6, 2.3, 2.2, 1.2, and 0.49. No DNA band is generated in lanes 1 and 3 (5-FOA^R, ura4-) strains, as expected. Southern confirms removal of a single copy of β-tubulin after 5-FOA treatment of AK102 that has sequential β-tubulin genes. (C and D) MBC and TBZ resistance. (C) Serial dilutions of wild-type (YY105, 972, and WT/AK103) and mutant (F200Y/AK101) cells onto YES plates containing 5, 10, 20, 50, or 100 μg/ml MBC or TBZ grown at 32°C. For the 50 μg/ml concentration, WT/AK103 is omitted. (D) Streaks of cells onto YES plates containing MBC or TBZ. Some growth is indicated at 50 μg/ml MBC with the mutant.