TABLE 1.
Complementation analysis of wild-type and mutant Mo-MuLV RNase Ha
| Expression plasmid | Growth of colonies at:
|
Complementation frequency | |
|---|---|---|---|
| 30°C | 42°C | ||
| p6His-3 | + (103) | − (0) | <0.01 |
| pHTRΔP | + (97) | − (0) | <0.01 |
| pBluescript | + (110) | − (0) | <0.01 |
| pAC9-157 | + (98) | + (87) | 0.86 |
| pAC9-175 | + (89) | + (93) | 1.04 |
| pE48A | + (95) | − (0) | <0.01 |
| pE48Q | + (91) | +b (ND)c | ND |
| pD69A | + (74) | +d (ND) | ND |
| pAC2 | + (101) | + (96) | 0.95 |
Mo-MuLV RNase HI dependent growth of E. coli MIC3001. Transformed cells grown in LB-carbenicillin or LB-carbenicillin-choramphenicol were poured onto MLB-agar plates supplemented with 0.1 to 1.0 mM Mn2+. p6His-3 represents a control plasmid lacking an RNase H gene insert. pHTRΔP represents a control plasmid expressing the inactive N-terminal fragment (amino acids 1 to 123) of the T. brucei RNase HI enzyme. pAC-2 (positive control) encodes the full-length active T. brucei type I RNase H. Wild-type Mo-MuLV RNase H is encoded by pAC9-157 and pAC9-175. pE48A, pE48Q, and pD69A encode active site variants of pAC9-157. Growth (+) and failure to grow at the restrictive temperature (−) are indicated. The actual numbers of colonies yielded upon culture dilution and platings are indicated in parentheses. Complementation frequency is calculated by dividing the number of colonies formed at 42°C by the number formed at 30°C.
Microcolony formation after 48 h.
ND, Not determined (colonies too small to count reliably).
Small colony formation after 48 h.