TABLE 1.
In vivo effects of PAP
| Plasmid and growth conditions | β-Galactoside activity in the presence ofa:
|
% −1 RFSb | % +1 RFSc | ||
|---|---|---|---|---|---|
| pT125 (0) | pF8 (−1) | pJD104 (+1) | |||
| PSY1 (vector) | |||||
| Raffinosed | 38.8 ± 1.52 | 1.50 ± 0.28 | 0.69f ± 0.15 | 3.9 | 1.8f |
| Galactosed | 38.1 ± 2.37 | 1.61 ± 0.45 | 1.56f ± 0.33 | 4.2 | 4.1f |
| Gal/Raf (% vector control)e | 0.98 | 1.07 | 2.26 | 1.07 (100) | 2.28 (100) |
| pNT123 (active PAP) | |||||
| Raffinose (−PAP) | 43.35 ± 1.48 | 1.26 ± 0.15 | 0.84g ± 0.14 | 2.9 | 1.9g |
| Galactose (+PAP) | 41.54 ± 1.83 | 1.19 ± 0.16 | 0.37g ± 0.05 | 2.9 | 0.89g |
| Gal/Raf (% vector control) | 0.96 | 0.94 | 0.46 | 1.0 (93) | 0.47 (21) |
| pNT123-2 (inactive PAP) | |||||
| Raffinose (−PAP) | 33.60 ± 1.16 | 1.26 ± 0.25 | 0.54f ± 0.06 | 3.8 | 1.6f |
| Galactose (+PAP) | 34.06 ± 2.70 | 1.15 ± 0.29 | 1.16f ± 0.30 | 3.4 | 3.4f |
| Gal/Raf (% vector control) | 1.01 | 0.91 | 2.15 | 0.89 (83) | 2.13 (93) |
β-Galactosidase activities, in arbitrary units, were used as a measure of translational competence. In pT125, lacZ is in the 0 frame. pF8 and pJD104 are the −1 and +1 ribosomal frameshift reporter plasmids, respectively; the reading frames of the lacZ reporter genes are indicated in parentheses.
Percent −1 ribosomal frameshifting (RFS) was calculated by multiplying the ratio of pF8/pT125 β-galactosidase activities by 100.
Percent +1 ribosomal frameshifting was calculated by multiplying the ratio of pJD104/pT125 β-galactosidase activities by 100.
Cells were grown in selective media containing either 2% raffinose (PAP uninduced [−PAP]) or 2% galactose (PAP induced [+PAP]) for 5 h.
The Gal/Raf ratio measures the effect of PAP induction on β-galactosidase activities and programmed ribosomal frameshifting; in percent vector control, the PSY1 Gal/Raf ratio is the denominator and the Gal/Raf ratios of the test plasmids are the numerators.
t < 0.05 (Student’s t test).
t < 0.01 (Student’s t test).