TABLE 2.
Characterization of double and triple mutants of stem 2
| Pseudoknot mutation | Colony phenotypea | TER levelb (% of WT) | Telomere length (% of WT) |
Tm (°C)d at pH:
|
Dimer formation (% of total signal)e at:
|
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 4°C, pH 6.25 | 20°C
|
30°C
|
|||||||||
| 5 | 6 | 7 | pH 5 | pH 7 | pH 5 | pH 7 | |||||
| WT | 1 | 100 | 100 | 54.5 | 55 | 50 | + | 100 | 100 | 100 | 100 |
| S2.0+0′ | 2-3 | 50 | 55 | 55 | 55 | 49 | 40 | 52 | 28 | ||
| S2.1+1′ | 1 | 100 | + | ||||||||
| S2.2+2′ | 3-4 | Rec-40c | − | ||||||||
| S2.3+3′ | 1-2 | 80 | 60-80 | 57 | 57 | 56 | −/+ | 53 | 27 | 37 | 14 |
| S2.4+4′ | 3 | 40 | |||||||||
| S2.0+0′+L.a | 4 | 30 | |||||||||
| S2.0+0′+L.b | 1-2 | 65 | 65 | 55 | 55 | 100 | 57 | 100 | 42 | ||
| S2.2+2′+L.d | 4 | Rec | |||||||||
| S2.3+3′+L.b | 4 | 57 | Rec | 66 | 43 | 56 | 37 | ||||
| S2.3+3′+L.c | 1-2 | 82 | 60-80 | 71 | 63 | 55 | + | 100 | 51 | 38 | |
Colony phenotypes are rated according to the severity of the phenotype from 1 (the smooth appearance of WT strains) to 4 (the severe, rough phenotype observed in a terΔ strain).
TER levels were measured by Northern analysis, quantified with a phosphorimager, and corrected to the signal measured for 18S rRNA, as described previously (32).
Rec, deregulated telomere length typical of the alternative, telomerase-independent recombination pathway. Such a phenotype is observed in a terΔ strain after its telomeres shorten to a critical length. Rec-40 indicates variability among different clones. Some clones show short but stable telomeres (40% of the WT length), and some reveal deregulated telomere length typical of the recombination pathway.
Determined by UV melting experiments.
Determined by nondenaturing PAGE. Indicated are the portions (percentage) of the dimer form out of the total radioactive signal. +, formation of dimers; −, no dimer formation; −/+, partial dimer formation.