Effects of extra copies of the TEF3 , TEF4 and TEF5 genes on temperature sensitivity of the sup45-sl23tsand sup35-168tsmutants. (A) Growth of transformants of the 33G-D373-sl23 strain with the sup45-sl23tsmutation, bearing the centromeric or multicopy empty vectors pRS315 (C-empty) or YEplac181 (M-empty), respectively (negative controls), or centromeric plasmid pRS315-SUP45 (SUP45-CEN) (positive control), was compared with growth of transformants, bearing one of the multicopy plasmids: YEplac181-TEF2 (TEF2↑), YEplac181-TEF3 (TEF3↑), YEplac181-TEF4Δi (TEF4-Δi↑) or YEplac181-TEF5Δi (TEF5-Δi↑). Transformants were grown as described in legend to Figure 1B, except cell suspensions were spotted on C-Leu without doxycycline and incubated at 30°C or 35°C. (B) The strain BY4741-ΔSUP35 with disrupted SUP35 contained the URA3 SUP35-C centromeric plasmid pRG416-SUP35C, which was shuffled for either the centromeric LEU2 pRS315-SUP35 or pRS315-sup35-168 plasmids bearing the wild type SUP35 or mutant sup35-168tsalleles, respectively. Shuffling for the sup35-168tsplasmid produced transformants unable to grow at 37°C. Then, the strains with either the SUP35 or sup35-168tsplasmids were transformed with one of the multicopy plasmids YEplac195 (M-empty), YEplac195-TEF2 (TEF2↑), YEplac195-TEF5Δi (TEF5-Δi↑) or YEplac195-SUP45 (SUP45↑). The transformants were grown in liquid C-Ura medium and diluted to OD600 of 1.0. Ten-fold serial dilutions were spotted onto C-Ura medium and grown at 30°C or 37°C for 4 days.