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First published online 13 July 2004
doi: 10.1242/jcs.01225

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Visualization of unconstrained negative supercoils of DNA on polytene chromosomes of Drosophila

¹ Department of Developmental Genetics, National Institute of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuokaken 411-8540, Japan
² Department of Genetics, SOKENDAI, 1111 Yata, Mishima, Shizuokaken 411-8540, Japan

View larger version (38K): [in a new window]   Fig. 1. Detection of negative supercoils of DNA on hsp70 in salivary gland. (A) Genomic organization of the hsp70 genes at 87A. Arrows denote the orientation of transcription. The hsp70 genes are flanked by specialized chromatin structures, scs and scs' (Udvardy et al., 1985). Restriction sites used for Southern analysis are indicated: X, XbaI; E, EcoRI; B, BglII. DNA fragments CR and DDS were used for hybridization probes. (B) Psoralen photocrosslinking to the hsp70 genes at 87A in salivary gland cells. Salivary glands from non-heat-shocked or heat-shocked larvae were incubated with indicated concentrations of psoralen. After photocrosslinking, genomic DNA was isolated, digested with XbaI, denatured, electrophoresed and analyzed by Southern hybridization using CR fragment as a probe. (C) Effect of previous nicking of DNA or inhibition of transcription on the psoralen photocrosslinking. Lanes 1-7: salivary glands from heat-shocked larvae were incubated with (lanes 2-7) or without (lane 1) psoralen and analyzed as in (B). Where indicated, the glands were irradiated with X-rays before (lane 3) or after (lane 5) photocrosslinking. The sample in lane 4 was mock treated without X-ray. The glands were treated with (lane 6) or without (lane 7) -amanitin before incubation with psoralen. Lanes 8 and 9: salivary glands from non-heat-shocked (lane 8) or heat-shocked larvae (lane 9) were incubated with psoralen. Genomic DNA was digested with BglII and EcoRI, and analyzed as in (B) using DDS fragment as a probe.

View larger version (44K): [in a new window]   Fig. 2. Visualization of psoralen signals on polytene chromosomes. (A) Biotinylated psoralen signals were detected with Alexa 488-labeled streptavidin (light green color). (B) DAPI signals (blue color). (C) Merged image. Arrows indicate representative interbands and puffs. In some loci, intense psoralen signals were observed only on the side margins of an interband or puff (arrowheads). (D) Biotinylated psoralen signals disappeared when the photo-crosslinking step was omitted. (E) DAPI image of (D). (F) Biotinylated psoralen signals were resistant to RNase treatment. (G) DAPI image of (F). Bars, 10 µm.

View larger version (69K): [in a new window]   Fig. 3. Bright psoralen signals disappear upon previous nicking of DNA or inhibition of transcription. Salivary glands were irradiated with X-rays for relaxation of DNA by nicking (A-C) or treated with -amanitin for inhibition of transcription (D-F) before photocrosslinking with biotinylated psoralen. (A,D) Biotinylated psoralen. (B,E) DAPI. (C,F) Merged. Representative interbands and puffs are indicated by arrows. Bars, 10 µm.

View larger version (66K): [in a new window]   Fig. 4. Distribution of psoralen signals on polytene chromosomes from heat-shocked larvae. (A) Biotinylated psoralen. (B) DAPI. (C) Merged. Major heat-shock puffs at 87A, 87B and 93D are indicated by arrows. Arrowheads represent interbands with biotinylated psoralen signals that became less prominent after heat shock. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 5. Effect of previous nicking of DNA or inhibition of transcription on psoralen signals on heat-shock puffs. Salivary glands dissected from heat-shocked larvae were irradiated with X-ray (A-C) or treated with -amanitin (D-F) and then photocrosslinked with biotinylated psoralen. (A,D) Biotinylated psoralen. (B,E) DAPI. (C,F) Merged. Arrows indicate heat-shock puffs at 87A and 87B. Bars, 10 µm.

View larger version (85K): [in a new window]   Fig. 6. Colocalization of bright psoralen signals with those of nascent RNA on polytene chromosomes. (A) Biotinylated psoralen. (B) DAPI. (C) Merged image of A and B. (D). Nascent RNA labeled with Br-UTP. (E) Merged image of (B) and (D). (F) Merged image of (A) and (D). Bar, 10 µm.

View larger version (77K): [in a new window]   Fig. 7. Colocalization of bright psoralen and nascent RNA signals on heat-shock puffs. (A) Comparison of psoralen and nascent RNA signals on polytene chromosomes dissected from heat-shocked larvae. (B,C) Detailed inspection on the heat-shock puffs at 87A and 87B. (C) Signal intensities of biotinylated psoralen (green), nascent RNA (pink) and DAPI (blue), and ratio of signal intensity of biotinylated psoralen to DNA (yellow) quantified along the region indicated in (B). Bar, 10 µm.