Fig. 7

Two-step mutagenesis. Schematic diagram of the two-step gene replacements and induction of point mutation of the tpz1 gene. List of diagnostic primers is shown in Table 2. a PCR based tpz1 gene deletion by the TKnatCX targeting fragment generated using the tpz1 Top and Bot 100 base primer set (Table 1). 80–100 base of homologous sequences in the 100 base primer set targets 5’ and 3’ UTR regions of tpz1 ⁺ of one of endogenous tpz1 ⁺ alleles. Replacement of tpz1 ⁺ by TKnatCX was confirmed by amplification of DNA fragment using diagnostic PCR primers, nat F900 and tpz1 R400D. b Replacement of the TKnat cassette by the tpz1 mutant gene. The DNA fragment containing the promoter region and the gene of tpz1 ⁺ was amplified using primers tpz1 F800U and R1745 and cloned between EcoO109I and NheI sites of pNX3a-HA3. The tpz1 gene was mutated to generate K75A mutation using the sight-directed mutagenesis method. PvuII and PmeI digested tpz1(K75A)-3xHA:kanMX6 fragment from pTpz1a4-K75A-HA3 can only recombine with TKnatCX deleted tpz1 allele, as right arm homology Ttef sequence is only present in the TKnatCX cassette. c Integration of tpz1-K75A mutation and 3xHA tagging. Correct replacement was confirmed by amplification of DNA fragments using diagnostic PCR primer sets, kan F800 and tpz1 R400D, and tpz1 F842U and Ptef R81 (or kan R276)