Figure 2

(A) Growth retardation by overexpression of NAB6 and PAP1. (Left) Empty pGP564 vector, pGP564-NAB6, pGP564-PAP1, and pGP564-PAP1-NAB6 were separately transformed into the wild-type W303-1A strain (upper; marked NAB6) or the nab6Δ strain (lower; marked nab6Δ). Two days after inoculation on SD-leucine medium at 30 °C, the colonies were observed. (Right) Optical density (OD) was measured every 30 min from 0 to 24 h after reinoculation at OD₆₆₀ = 0.1. The averages of three replicate experiments from 6 to 24 h are indicated. The nab6Δ/DIT1t strain (gold) and the NAB6/DIT1t strains harboring pGP564 (white), pGP564-NAB6 (gray), pGP564-NAB6 (blue), or pGP564-PAP1-NAB6 (green) are indicated. (B) Identification of the strongest terminator derived from DIT1t. (a) Activation of strong DIT1t-derived terminators by overexpression of NAB6 and PAP1. The fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard. These experiments were conducted independently of those in Fig. 1. (b) Comparison of terminator activities among DIT1t strains and the standard PGK1t strain in various growth phases. The fluorescence intensities of terminator strains with the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. Sampling times were 6, 12, 24, and 30 h after reinoculation at OD₆₆₀ = 0.1. (c) Characterization of the strongest DIT1t-d22 terminator. (Left) Effects of promoter and reporter gene exchange on 3′-UTR activity. These strains expressed either the GFP gene under the control of ACT1pro (left) or the mKO2 gene under the control of TDH3pro (right), as indicated. (Right) Effect of the host strain on 3′-UTR activity. Two wild-type Saccharomyces cerevisiae strains (A451 and TDO2) were transformed individually. The fluorescence intensities of the terminator strains containing the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. The terminator strains examined were PGK1t (purple), DIT1t (orange). DIT1t-m22 (yellow), DIT1t-d7 (gray), DIT1t-d21 (dark gray), and DIT1t-d22 (pink). Data are means ± 1 SD of at least three independent experiments. n.s., not significant; *p < 0.01; **p < 0.05. See also Supplementary Table 5.