Fig. 2

Embryonic expression, cellular location and nuclease activity of Nbr. a in situ hybridization of a wild type stage 2 embryo (stages are according to [7]. The transcript is maternally expressed and appears ubiquitously. b in situ hybridization, using the same probe as above, of a stage 11 embryo expressing the transgene UAS-EGFP-nbr-N, driven by prd-GAL4. As expected, the expression of the transgene shows a pattern in 7 stripes. Endogenous nbr transcripts are at low levels at this stage. c salivary glands cells of 3rd instar larvae expressing the EGFP-Nbr-N fusion protein from the cross UAS-EGFP-nbr-N, driven by tub-GAL4. The fusion protein is enriched at the nuage surrounding the nuclei and at lower levels also located in the cytoplasm of the cells. d and e nuclease activity of wild-type and catalytic site-defective recombinant Nbr proteins. Wing discs expressing wild-type EGFP-Nbr (d) and EGFP-Nbr-N (e) under the control of apterous (ap)-GAL4. Nucleic acids are stained with propidium iodine (PI, red). d EGFP-Nbr protein expressed in the proximal compartment (P) is an active nuclease and degrades nuclei, hence PI disappears and only EGFP remains. Note also the extensive shrinking of the P part of the wing upon nuclease activity. The distal compartment (D) is not affected, hence PI is visible. (E) Fusion protein with the disrupted catalytic domain (EGFP-Nbr-N) does not reveal any nuclease activity. Hence, PI remains and merges with EGFP to yellow color. Discs in (d) and (e) are oriented with P to the top and D to the bottom