Article Figures & Data
Figures
- Figure 1.
Drosophila dCREB2 isoforms. A, Schematic representation of four different dCREB2 isoforms. B, DNA and protein sequence of the predicted full-length open reading frame and hs-dCREB2-a open reading frame in transgenes present in lines C28 and C30. A deletion of a single nucleotide, g (boxed), at amino acid position 38 produces a shift in reading frame to a new stop codon (bold) at amino acid position 79. The DNA sequence illustrated is the sequence with revisions (see Results) except for this single nucleotide g, which is preserved from the original sequence to illustrate the effect of its deletion on the open reading frame. An alternative and downstream start codon is highlighted in bold at position 160. The hypothetical and truncated version predicted by this open reading frame is shown in bold. The PKA-dependent phosphorylation recognition site is boxed and the bZIP domain is underlined.
- Figure 2.
RT-PCR analysis of transgenic and control flies. RT-PCR was performed using total RNA isolated from heads from the hs-dCREB2-b line 17-2, hs-dCREB2-a line C30, and control lines CS, wCS10, and ry. Plasmids containing the dCREB2-a, -b, and -d isoforms were used as PCR templates to synthesize size standards using the same primers used for the RT-PCR. All flies were raised at 22°C, collected within 2 d from eclosion, and kept overnight at 25°C. Those that were treated with heat shock were transferred to empty food vials and placed in a 37°C water bath for 30 min. The flies were rested in food vials at 25°C for 3 hr after heat shock and then RNA was isolated. Heat shock treatment induced synthesis of hs-dCREB2-b and hs-dCREB2-a RNAs in flies carrying the respective transgenes. The level of the three detectable CREB splice variants in the control genotypes is very low. A schematic of the CREB gene exons with the location of the primers used is shown at the bottom of the figure along with the size expected for the PCR product of each CREB variant.
- Figure 3.
Western analysis of dCREB2 isoforms. Total protein was prepared from 10 fly heads from line 17-2 expressing the hs-dCREB2-b transgene, C30 expressing the hs-dCREB-a mutant transgene, control lines wCS10 and ry, and UAS-CREB2-a lines containing the dCREB2-a mutant cassette and the dCREB2-a full-length isoform (line T7.1), expressed from a hs-GAL4 element. The equivalent of two heads was loaded in each well. The blot was probed with a 1:20 dilution of a mouse monoclonal antibody raised against a bacterially expressed full-length dCREB2-b protein. The middle panel is a magnified image of the last four lanes of the top panel. This better illustrates the size difference of dCREB2-a and dCREB-b. To control for loading, the blot was stripped and reprobed with a 1:100 dilution of a mouse monoclonal anti-dynamin antibody (bottom).
- Figure 4.
Induction of hs-dCREB2-a and of UAS-dCREB2-a has no effect on memory performance. A, Performance indices are shown for flies that were conditioned 3 hr after heat shock (+hs) or without heat shock (-hs) using a spaced training protocol consisting of five training trials with 15 min rest intervals or a single training trial. Tests were performed 4 d after training. Performance indexes were calculated as described previously (Pascual and Preat, 2001). ANOVA with Fisher's post hoc tests was used to make several important comparisons: the 5× CS (-hs) group was significantly different from both the 1× CS (-hs) (p = 0.0009) and 1× CS (+hs) (p < 0.0001) groups. The 5× hs-dCREB2-a C30 (-hs) group was significantly different from both the 1× hs-dCREB2-a C30 (-hs) (p = 0.0032) and 1× hs-dCREB2-a C30 (+hs) (p = 0.0028) groups. No significant differences were found between the 5× CS (-hs) and 5× hs-dCREB2-a C30 (-hs) groups (p = 0.2708). No significant differences were found between any of the groups provided with 1× training. These comparisons illustrate the significant effect of 5× spaced over 1× training and the lack of an effect of dCREB2-a induction in the hs-dCREB2-a C30 line on LTM. B, RT-PCR analyses of the transgenic flies used for training (hs-dCREB2-a C30) in A showed the heat shock-dependent induction of the transgene. Plasmids containing the dCREB2-a, -b, and -d isoforms were used as templates for PCR to generate size standards. C, Behavioral analysis of UAS-dCREB2-alines T7.1 and T25.4 containing the corrected dCREB2-a open reading frame and driven by hs-GAL4 showed no improvement in memory performance after 1× training compared with the wild-type control. ANOVA with Fisher's post hoc tests was used to make several comparisons. Most importantly, the 1× UAS-dCREB2-a T7.1 and 1× UAS-dCREB2-a T7.1-hsGAL4 groups were not significantly different (p = 0.2237), and the 1× UAS-dCREB2-a T25.4 and 1× UAS-dCREB2-a T25.4-hsGAL4 groups were not significantly different (p = 0.7323). The 5× hsGAL4 group was significantly different from the 1× hsGAL4 group (p = 0.0005), and the 5× UAS-dCREB2-a T25.4 group was significantly different to the 1× UAS-dCREB2-a T25.4 group (p = 0.0095). No significant differences were found between the 1× and 5× trained groups for UAS-dCREB2-a T7.1, UAS-dCREB2-a T7.1-hsGAL4, and UAS-dCREB-a T25.4-hsGAL4. The reason for failing to observe the effect of 5× spaced training for the latter three groups is unclear. D, Twenty-four hour memory after 5× spaced training with and without heat shock was measured for wild-type flies (CS) and the dCREB2-b repressor line 17-2. ANOVA with Fisher's post hoc tests was used to make several important comparisons; no significant differences were found between the CS (-hs) and CS (+hs) groups (p = 0.1064). The hs-dCREB2-b 17-2 (-hs) group was significantly different from both the CS (-hs) (p < 0.0001) and CS (+hs) (p < 0.0001) groups, showing the effect of basal expression of the transgene. The hs-dCREB2-b 17-2 (+hs) group was significantly different from all groups but most importantly from the hs-dCREB2-b 17-2 (-hs) group (p = 0.0449), showing the effect of induced expression of the transgene. Error bars indicate SEM.
