Figure 3. dMi-2 triggers rapid changes in chromosome structure without altering DNA replication.

Third-instar UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4, Gal80^ts larvae were shifted from 18°C to 29°C to activate UAS-dMi-2⁺ expression. (A) Changes in dMi-2 RNA levels were monitored by RT-PCR following the shift from 18° to 29°C using primers that specifically amplify the RNA encoded by UAS-dMi-2⁺ (induced) or both the induced and endogenous dMi-2 RNAs (induced+endogenous). The level of ISWI RNA was assayed as an internal control. (B and C) Live analyses of salivary gland nuclei of UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts maintained at 18°C (B; n = 13) or shifted to 29°C for 27 hours (C; n = 14). (D) Quantification of the volume of salivary gland polytene chromosomes of larvae shown in B and C. (E) Quantification of His2Av-mRFP1 fluorescence in the same larvae shown in B and C. (F) Quantification of chromosome compaction, as assessed by the ratio of volume in µm³ to His2Av-mRFP1 fluorescence per nucleus for the larvae shown in B and C. (G) Scatter plot of the data used to generate D–F. Chromatin compaction vs. DNA content are compared for single nuclei of larvae maintained at 18°C (green triangles) or 29°C (orange circles) for 27 hours. B and C scale bars are 10 µm.