Figure 4. The singletons and paired genes (expressed simultaneously) are able to complement the absence of their yeast ortholog (Gdt1p).

The different strains were precultured in minimal SD-UL medium to an OD₆₀₀ of 0.3, and then serial tenfold dilutions were dropped onto solid SD-UL medium supplemented with 500 mM CaCl₂ and incubated at 28°C for 10 days. All of the genetic constructions were in a pRS316 or pRS315 plasmid under the control of the pTPI promoter, and transformed into the pmr1Δ/gdt1Δ mutant. Φ represents the empty plasmid(s). This complementation assay demonstrates that a singleton gene (Dma) expressed alone or paired genes (Ter1a and Ter2b) expressed together are able to restore the growth of the pmr1Δ/gdt1Δ mutant in the presence of high Ca²⁺ concentrations. The paired genes expressed separately do not complement the absence of Gdt1p, confirming interdependency.