Figure 4

Construction of AIF -deficient strain. A. Map of expression vector, pKoTtAIF, with neomycin resistance cassette (Neo^r-cassette). It consists of 5' and 3' UTR sequences of AIF and part of its 3' ORF. The neomycin resistance is expressed under the control of β-tubulin promoter. Before biolistic bombardment, the plasmid was digested with BamHI. B. Schematic representation of the wild-type (WT) and mutant locus of the AIF together with the targeting plasmid. Replacement of AIF to neomycin-resistant gene (Neo^r) in the macronucleus was confirmed by PCR using 10 ng of genomic DNA from isolated macronuclei as template. Small triangles located in the gene loci indicate specific primer pairs for the PCR amplification. C. Cell growth curve of CU428. Circles and squares indicate cell density of wild-type strain and ΔAIF, respectively. Points and attached bars correspond to the means of four identical measurements and standard deviations. The inset indicates RT-PCR analysis of the expression levels of AIF and Neo^r. β-tubulin (BTU) was used as a control.