TABLE 5.
The half-life of ADH2 mRNA made in glucose-containing medium is affected by the activatora
Plasmid | Activator | Half-life (min)b | Mean relative abundance (SD) of ADH2 mRNA atc: |
|
---|---|---|---|---|
25°C | 36.5°C | |||
pKD16 | Adr1 | 65 | 0.74 (0.36) | 0.33 (0.13) |
pKD17 | oeAdr1 | 56 | 21 (7.5) | 10 (8.3) |
pAG11 | Adr1-Gal11 | 10 | 28 (19) | 0.5 (0.14) |
Strain KBY108 (relevant genotype, rpb1-1 trp1) was transformed with the TRP1-containing plasmids pKD16, pKD17, and pAG11. Three transformants of each type were grown in synthetic complete (SC) medium lacking tryptophan and containing 5% glucose at 25°C to an A600 of 0.8. Samples were taken for RNA isolation and analysis, and the temperature of the cultures was raised to 36.5°C by adding an equal volume of 50°C medium. After 60 min of vigorous shaking, the cultures were collected for RNA isolation and analysis as described in Materials and Methods.
The apparent half-life of ADH2 mRNA was estimated as described in the legend to Table 3 from a graph of log(ADH2 mRNA/18S rRNA) versus time using the 0- and 60-min time points.
The abundance of ADH2 mRNA and 18s rRNA was determined by RT-qPCR as described in Materials and Methods. Each value represents the mean ADH2/18S RNA level (multiplied by 1,000) and standard deviation from three replicate cultures.