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Functional dissection of the Drosophila modifier of variegation Su(var)3-7

Department of Zoology and Animal Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

View larger version (46K): [in a new window]   Fig. 1. Tagged Su(var)3-7 constructs. (A) Alignment of the tagged Su(var)3-7 constructs. (B) Western blot of crude protein extracts from heat shocked and non-heat shocked adult flies expressing different tagged constructs. Detection with an anti-HA antibody. (Left) 8% polyacrylamide gel; (right) 10% polyacrylamide gel.

View larger version (69K): [in a new window]   Fig. 2. Chromosomal localisation and effect on PEV of the N- and C-terminal parts of Su(var)3-7. (A) Polytene chromosomes from salivary glands of transgenic larvae expressing the HA:FL, HA:1-6 or HA:7-Ct constructs. Staining with an anti-HA antibody detected with a Cy3-labelled secondary antibody (red) and DAPI (blue). HA:FL and HA:1-6 did not require heat-shock induction for detectable expression. (B) Banding pattern of polytene chromosomes. HA:FL was heat shocked to detect a banding pattern. This was not necessary for HA:1-6. Staining is as in A. (C) Effect on Heidi variegation of the different tagged constructs. Eyes of heat shocked males from crosses between w; Heidi/CyO females and yw/Y; P{y+, HA:Su(var)3-7} males. Corresponding eye pigment measurements, normalised to the control Heidi/+:Heidi/+ = 100±5.4; Heidi/+; HA:FL/+ = 49±4.5; Heidi/HA:1-6 = 94±6.4; Heidi/HA:7-Ct = 240±13.

View larger version (9K): [in a new window]   Fig. 3. Su(var)3-7 interacts with itself in the yeast two-hybrid assay. 21 is a fragment of Su(var)3-7 that uses Su(var)3-7 itself as bait in a two-hybrid screening of a Drosophila embryonic cDNA library. A schematic representation of 21 is given above the wild-type protein. A series of Su(var)3-7 deletion constructs have been tested to precisely map the domain of interaction of Su(var)3-7 with itself. /, impossible to test (Delattre et al., 2000); +, interaction with 21; –, no interaction. Grey boxes: zinc fingers; black bars: tryptophan boxes.

View larger version (92K): [in a new window]   Fig. 4. The C-terminal part of Su(var)3-7 recruits the endogenous protein and suppresses PEV. (A) Double staining of polytene chromosomes from larvae expressing the HA:7-Ct protein with an anti-HA antibody (red) and anti-Su(var)3-7 antibody (green). The anti-Su(var)3-7 antibody was raised against a N-terminal fragment of the protein, and permits the detection of the endogenous protein but not of the HA:7-Ct protein. hs, heat shocked (B) Effect of different tagged constructs on Heidi variegation. Corresponding eye pigment measurements normalised to the control Heidi/+: Heidi/+ = 100±5.3; Heidi/+; HA:7-B/+ = 881±103; Heidi/+; HA:B-Ct/+ = 88±4.4.

View larger version (59K): [in a new window]   Fig. 5. The Su(var)3-7 wild type protein recruits the HA:7-Ct construct. Double staining of polytene chromosomes using an anti-HA antibody (red), an anti-Su(var)3-7 antibody (green), and DAPI (blue). Top: chromosomes of homozygous yw; HA:7-Ct larvae; bottom: chromosomes from heterozygous yw; HA:7-Ct/+; P{HS-Su(var)3-7}/+ larvae. Control and test chromosomes were squashed on the same slide in this experiment to allow for semi-quantitative evaluation.

View larger version (12K): [in a new window]   Fig. 6. Localisation and effect on PEV of tagged constructs containing different sets of zinc fingers. Localisation was determined by immunostaining of polytene chromosomes. –, no binding on euchromatic arms: +, discrete number of bands on euchromatic arms; +++, band-interband pattern on euchromatic arms. Effect of constructs on PEV were determined by crosses with the Heidi variegating line. Black box, HA tag; grey boxes, zinc fingers; light grey boxes, incomplete zinc finger; black bars, tryptophan boxes.

View larger version (52K): [in a new window]   Fig. 7. Delocalisation of endogenous Su(var)3-7 by tagged constructs does not modify endogenous HP1 localisation. Staining of polytene chromosomes with an anti-HP1 antibody (red), an anti-Su(var)3-7 antibody (green) and DAPI (blue). (Top) Chromosomes from control yw larvae; (bottom) chromosomes from yw;HA:7-B larvae.