FIG. 7.

Phenotypic complementation of an S. cerevisiae trx1 trx2 double mutant (EMY63) by expression of Chlamydomonas Trxh1. (A) Expression of Trxh1, under the control of a galactose-inducible promoter in a centromeric plasmid restored the tolerance of the mutant to MMS to the same extent that transformation with S. cerevisiae Trx1 did (left panel). In contrast, Trxh1 did not complement the hypersensitivity to H₂O₂ (right panel). Yeast cells were transformed with the empty plasmid (YCpGal2), with S. cerevisiae Trx1 (Yeast Trx1), with Chlamydomonas Trxh1 (CrTrxh1), or with a mutant form of Trxh1 lacking a catalytically active cysteine (CrTrxh1 C36S). Cells were plated, forming a lawn, and exposed to a gradient of MMS or H₂O₂ concentrations, established by diffusion from a centrally placed disk containing 1.36 M MMS or 500 mM H₂O₂. The size of the halo where cell growth was suppressed was used as an estimation of the sensitivity to genotoxic agents. The results show the averages (± standard deviations) of three to five independent experiments. (B) Relative DNA content in asynchronous cultures of transformed trx1 trx2 mutant cells. Yeast cells of the indicated transformants were grown to logarithmic phase, stained with PI, and analyzed by flow cytometry. Relative fluorescence intensity (DNA content) is plotted against the number of counted events (Nuclei counts). Relative DNA contents corresponding to 1N (G₁) and 2N (G₂) are indicated.