FIG. 6.

dMi-2 negatively regulates the PCNA gene promoter. (A) Effect of cotransfecting dMi-2-expressing plasmids on luciferase activity directed by the PCNA gene promoter. Schneider cells (1.5 × 10⁵/well) were cotransfected with 50 ng of reporter plasmid (either −168DPCNAluc or −168mutΔ6PCNAluc), 50 to 400 ng of pUAS-HA-dMi-2 and 100 ng of pAct-GAL4 as effector plasmids, and 1 ng of pRL-actin5C as an internal control plasmid. The total amount of DNA in the transfection mixture was adjusted to 1 μg by the addition of pGEM3. Cells were harvested 48 h after transfection. The luciferase assay was carried out by means of the Dual-Glo Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. Averaged values obtained from three independent transfections are shown. (B) Constructs of PCNA-lacZ fusion genes used to establish the transgenic lines analyzed in panels C and D. (C) Half-dose reduction of dMi-2 activates the PCNA gene promoter depending on the presence of DRE. Detection of expression of β-galactosidase in brain lobes from transgenic flies carrying the PCNA-lacZ reporter gene. Male transgenic flies carrying −119PCNAlacZ on the second chromosome were crossed with females with dMi-2¹, dMi-2⁴, and dMi-2⁶ alleles balanced with the TM6C chromosome. Brain lobes were dissected from the third-instar larvae of F1 progeny, fixed, and immunostained with anti-β-galactosidase antibody. (D) Male transgenic flies carrying either −86P3NlacZ or −168ΔPCNAlacZ reporter genes on the second chromosome were crossed with females with the dMi-2⁴ allele balanced with the TM6C chromosome. Brain lobes were dissected from the third-instar larvae of F1 progeny, fixed, and immunostained with anti-β-galactosidase antibody.