Table 1.
In vivo complementation for DnaJ chaperone activity by dnaJ plasmid chimeras carrying mammalian polyoma virus T antigen T/t common exons in place of the E. coli DnaJ chaperone J-domain
| Plasmid | dnaJ backbone | J-domain |
l-Arabinose inducer
|
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| None
|
66 μM
|
660 μM
|
6.6 mM
|
33 mM
|
|||||||||||||
| 14.5 | 39.5 | 43 | 14.5 | 39.5 | 43 | 14.5 | 39.5 | 43 | 14.5 | 39.5 | 43 | 14.5 | 39.5 | 43 | |||
| pBAD22A vector | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | ||
| pWKG90 | J (71–376) | E. coli J(1-70) | - | - | - | - | 0.14 | - | 0.61 | 0.92 | 0.06 | 0.80 | 1.08 | 0.92 | 0.91 | 1.04 | 0.93 |
| pWKG92 | J (71–376) | SV40 (1-82) T/t | - | - | - | - | - | - | - | - | - | 0.83 | 1.00 | - | 1.17 | 0.03 | 0.03 |
| pWKG93 | J (71–376) | SV40 (1-76) | - | - | - | - | - | - | - | - | - | 0.54 | 0.87 | 0.03 | 0.83 | 0.07 | - |
| pWKG95 | J (71–376) | JCV (1-76) | - | - | - | - | - | - | - | - | - | 0.58 | 0.95 | 0.43 | 0.84 | 0.45 | 0.20 |
| pWKG97 | J (71–376) | BKV (1-76) | - | - | - | - | - | - | - | - | - | 0.11 | 0.94 | - | 0.90 | 0.36 | - |
| pWKG91 | J (71–376) | E. coli (1-70)H33Q | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| pWKG94 | J (71–376) | SV40 (1-76)H42Q | - | - | - | - | - | - | - | - | - | - | - | - | -* | -* | -* |
| pWKG96 | J (71–376) | JCV (1-76)H42Q | - | - | - | - | - | - | - | - | - | - | - | - | -* | -* | -* |
| pWKG100 | J (71–108) | E. coli J (1-70) | - | - | - | - | - | - | - | - | - | 0.65 | 0.66 | - | 0.84 | 0.85 | - |
| pWKG102 | J (71–108) | SV40 (1-82)T/t | - | - | - | - | - | - | - | - | - | 0.03 | - | - | 0.75 | 0.97 | - |
| pWKG103 | J (71–108) | SV40 (1-76) | - | - | - | - | - | - | - | - | - | 0.34 | - | - | 0.67 | 0.89 | - |
| pWKG105 | J (71–108) | JCV (1-76) | - | - | - | - | - | - | - | - | - | - | - | - | 0.57 | 1.03 | - |
| pWKG107 | J (71–108) | BKV (1-76) | - | - | - | - | - | - | - | - | - | - | - | - | 0.008 | 0.07 | - |
| pWKG101 | J (71–108) | E. coli (1-70)H33Q | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| pWKG104 | J (71–108) | SV40 (1-76)H42Q | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
| pWKG106 | J (71–108) | JCV (1-76)H42Q | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Colony formation at both low and high temperatures was measured using the host strain WKG190. WKG190 containing one of the indicated plasmids was grown at 30°C in Luria–Bertani (LB) broth containing 50 μg/ml ampicillin. Serial dilutions were spot tested on LB-ampicillin plates containing none, or the indicated amount of l-arabinose inducer and grown at the indicated temperature (14.5°C, 39.5°C, or 43°C). Cells were counted and efficiencies of plating were normalized to the viable cell titer of the same dilutions determined at 30°C. Plasmids carrying SV40–H42Q or JCV–H42Q mutations showed reduced viability at 30°C at 33 mM arabinose. The (-) sign indicates no detectable complementation [efficiency of plating (eop) < 10−5]. The values reported represent the mean of at least three independent determinations.
For certain plasmids, the addition of arabinose at high concentration resulted in a reduction of cell viability, even at 30°C.