Figure 5.

Yeast Complementation Studies.

(A) Complementation test with the yeast speΔ3 mutant. The growth of the speΔ3 yeast strain carrying the Arabidopsis SPDS expression vectors YEpACT-SPDS1, YEpACT-SPDS2, and YEpACT-SPMS, and the empty control vector YEpACT, was monitored after diluting the cultures 1:500 every 24 h and measuring OD₆₀₀ as described (Alabadí and Carbonell, 1999). SPE3 strain 2602 was used as a control. After 4 days in culture, 100 μM spermidine was added to the medium of YEpACT and YEpACT-SPMS transformants. The OD₆₀₀ values correspond to the mean of three independent assays.

(B) Complementation test with the yeast speΔ4 mutant. Putrescine, spermidine, and spermine contents were determined, as described in Methods, in the extracts of the yeast wild type (strain 2602), the spe4 mutant (Y504), and the speΔ4 mutant transformed with the Arabidopsis SPMS cDNA under the control of the yeast ADH1 promoter.

(C) Conversion of ¹⁴C-spermidine into ¹⁴C-spermine in the yeast speΔ4 mutant transformed with the Arabidopsis SPMS cDNA under the control of the yeast ADH1 promoter. Yeast cells were incubated with ¹⁴C-spermidine, and radioactivity was quantified in fractions after HPLC separation of dansylated polyamines. The inset shows the radioactivity of the HPLC fractions collected every 1 min during the interval 11 to 18 min. HD, 1,6-diamine hexane; spd, spermidine; spm, spermine.