Figure 5

Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES-tauEGFP-cI-spc and IRES-tauLacZ into MOR42-3 and MOR42-2, respectively, and the assumed deletion at the IRES-tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA. The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.