Table 1.
Effects of NLS mutant DBTs on locomotor activity in DD.
| DBT | Line | Genotype | Avg Period (h)±SEM (SD) | %Rhythmicity (N) |
|---|---|---|---|---|
| DBTK/R | 31.6 ±0.4 (0.6)*** | 47 (3) | ||
| 1MA | timGAL4>UAS- dbtK/R | 31.9 ±0.5 (2.9) | 48 (58) | |
| 10FB | timGAL4>UAS- dbtK/R | 32 ±0.9 (3.0) | 43 (28) | |
| 4MA | timGAL4>UAS- dbtK/R | 30.9 ±0.8 (2.4) | 50 (16) | |
| >UAS- dbtK/R | 23.5 ±0.1(0.3)* | 100 (21) | ||
| DBTK/R NLS- | 23.8 ±0.1 (0.2)* | 82 (5) | ||
| 1MB | timGAL4>UAS- dbtK/R NLS- | 23.9 ±0.2 (0.7) | 82 (22) | |
| 11MB | timGAL4>UAS- dbtK/R NLS- | 23.6 ±0.1 (0.3) | 75 (16) | |
| 2MB | timGAL4>UAS- dbtK/R NLS- | 23.7 ±0.1 (0.2) | 81 (16) | |
| 3MB | timGAL4>UAS- dbtK/R NLS- | 23.6 ±0.1 (0.2) | 75 (16) | |
| B4 | timGAL4>UAS- dbtK/R NLS- | 24.0 ±0.1 (0.5) | 97 (33) | |
| >UAS- dbtK/R NLS- | 23.5 ±0.1 (0.4)* | 95 (42) | ||
| DBTK/R stNLS | 39.0 ±1.0 (2.0)*** | 34 (4) | ||
| 1MB | timGAL4>UAS- dbtK/R stNLS | 40.3 ±1.5 (4.3) | 32 (28) | |
| 2MB | timGAL4>UAS- dbtK/R stNLS | 38.7 ±1.6 (4.0) | 29 (21) | |
| 10MB | timGAL4>UAS- dbtK/R stNLS | 36.3 ±1.4 (5.2) | 41 (34) | |
| 6FB | timGAL4>UAS- dbtK/R stNLS | 40.6 ±0.7 (1.5) | 33 (15) | |
| >UAS- dbtK/R stNLS | 23.8 ±0.1 (0.5)* | 89 (47) | ||
| DBTWT | 25.0 ±0.1 (0.1)* | 72 (2) | ||
| 6M3B | timGAL4>UAS- dbtWT | 25.1 ±0.5 (1.1) | 44 (9) | |
| 21M1C | timGAL4>UAS- dbtWT | 24.9 ±0.2 (0.9) | 100 (30) | |
| >UAS- dbtWT | 23.5 ±0.1 (0.2)* | 100 (12) | ||
| DBTWT NLS | 20.7 ±0.6 (1.0)** | 83 (3) | ||
| A17 | timGAL4>UAS- dbtWT NLS | 21.8 ±0.2 (0.6) | 85 (13) | |
| A2 | timGAL4>UAS- dbtWT NLS | 20.5 ±0.3 (0.6) | 100 (5) | |
| A7 | timGAL4>UAS- dbtWT NLS | 19.8 ±0.1 (0.5) | 65 (23) | |
| >UAS- dbtWT NLS | 23.7 ±0.1 (0.5)* | 92 (12) | ||
| DBTWT stNLS | 24.1 ±0.03 (0.1)* | 75 (3) | ||
| D2 | timGAL4>UAS- dbtWT stNLS | 24.0 ±0.2 (0.8) | 94 (16) | |
| D3 | timGAL4>UAS- dbtWT stNLS | 24.1 ±0.1(0.5) | 82 (57) | |
| D4 | timGAL4>UAS- dbtWT stNLS | 24.1 ±0.3 (1.0) | 48 (21) | |
| >UAS- dbtWT stNLS | 23.5 ±0.1 (0.2)* | 94 (16) | ||
| DBTK/R stNLS NLS- | 23.9 ±0.03 (0.1) * | 73 (3) | ||
| C7MA | timGAL4>UAS- dbtK/R stNLSNLS- | 24.0 ±0.3(1.0) | 75 (12) | |
| C6M | timGAL4>UAS- dbtK/R stNLSNLS- | 23.9 ±0.2 (0.7) | 80 (10) | |
| B1 | timGAL4>UAS- dbtK/R stNLSNLS- | 23.9 ±0.3 (0.7) | 63 (8) | |
| >UAS- dbtK/R stNLSNLS- | 23.8 ±0.1 (0.2)* | 100 (27) | ||
| DBT+ | ||||
| timGAL4> | 24.0 ±0.1 (0.5)* | 93 (28) | ||
Lines containing the indicated type of insertion were crossed to flies containing the timGAL4 driver, and progeny hemizygous for both the driver and responder were assayed in DD for locomotor activity. Each line contained an independent insertion of the responder UAS-dbt gene, generated at the AttP2 locus (at 68A4) by phiC31-mediated integration, except for the wild type controls. The circadian period was determined by chi-square periodogram analysis. Rhythmic flies produced single strong peaks in the periodogram analysis and rhythmicity that was obvious by inspection of actograms. The mean period ± SEM (SD) and the mean percentage of rhythmicity (N, number of lines for genotype averages and number of flies for line averages; only flies that lived for the entire assay were included in N and the denominator for determination of % rhythmicity). One way ANOVA of all the individual line averages showed a significant effect of genotype on period [F (14,16) = 74.0, p<0.001].
The average periods of lines with these genotypes were significantly different from the average period of all other genotypes with p<0.001 by Tukey HSD.
The average period of lines with this genotype were significantly different from the average periods of all other lines expressing a UAS-dbt transgene with the timGAL4 driver by Tukey HSD (p<0.05).
The average periods of genotypes without the timGAL4 driver, with the timGAL4 driver only, expressing the dbtWT transgene, expressing the dbtWT transgene with a stNLS or expressing the dbtK/R transgene with an NLS mutation did not exhibit statistically significant differences from each other. One way ANOVA of individual fly periods for all the flies also demonstrated a significant effect of line identity on period [F(30, 487) = 142, p<0.0001), but no lines with the same UAS-dbt genotype showed statistically significant differences from each other except UAS-dbtK/R stNLS line 10M1B, which did differ from UAS-dbtK/R stNLS lines 1MB and 6FB by post-hoc Tukey (p< 0.0001).