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First published online 18 October 2005
doi: 10.1242/jcs.02623

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Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

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

View larger version (153K): [in a new window]   Fig. 1. Bloated X phenotype of salivary gland polytene chromosomes from Su(var)3-7 or Su(var)2-5 mutant males. (A) Su(var)3-77.1A homozygote mutant. (B) Su(var)3-79/Su(var)3-714 mutant. (C) Su(var)2-504/Su(var)2-505 mutant. (D) Wild-type control male. Chromosomes are stained with orcein. X indicates the X chromosome.

View larger version (165K): [in a new window]   Fig. 2. Loose chromocenter morphology of salivary gland polytene chromosomes of Su(var)3-7 or Su(var)2-5 mutants. (A) Severe Su(var)3-714/Su(var)3-79 mutant, and (B) less-affected Su(var)3-714 homozygote. (C) Wild-type control. (D) HP1 mutant: Su(var)2-504/Su(var)2-505. Chromosomes are stained with orcein. Arrows point at granules and dotted arrows at fibers.

View larger version (56K): [in a new window]   Fig. 3. Immunolocalization of Su(var)3-7 and HP1 on polytene chromosomes. Gray, DAPI; red, HP1; green, Su(var)3-7. (A-C) Detection of Su(var)3-7 protein on Su(var)2-504/Su(var)2-505 mutant (arrow) with heterozygote control Su(var)2-504 or 05/CyO on the same slide (dotted arrow). (D,E) Detection of HP1 protein on Su(var)3-79/Su(var)3-714 mutant.

View larger version (79K): [in a new window]   Fig. 4. Immunolocalization of Su(var)3-7 and HP1 on mitotic chromosomes from larval brains. Gray, DAPI; green, Su(var)3-7; red, HP1. (A-C) Wild-type mitotic chromosomes. (D-F) Su(var)2-504/Su(var)2-505 mitotic chromosomes. (G-I) Su(var)3-77.1A homozygote mitotic chromosomes.

View larger version (124K): [in a new window]   Fig. 5. Developmental defects shared by larvae mutant for either Su(var)3-7 or Su(var)2-5. (A-C) Enlarged ring glands. (A) Su(var)3-7R2a8 mutant. (B) Su(var)2-502/Su(var)2-505 mutant. (C) Wild-type control. Arrows point to the ring glands and dotted arrows show the larval brains. (D,E) Polytene chromosomes from the prothoracic gland of Su(var)3-7R2a8 mutant and wild-type control at the same magnification. Stained with orcein. (F) Abnormal gastric ceca, swollen proventriculus and anterior midgut of Su(var)2-502/Su(var)2-505 mutant and wild-type control.

View larger version (81K): [in a new window]   Fig. 6. Bloating of the male X chromosome in Su(var)3-7 mutant requires a functional dosage compensation complex (DCC). (A,B) The DCC associates with the Su(var)3-7 mutant bloated X. (A) DAPI and anti-MSL2 staining of a bloated X from a Su(var)3-714 homozygous male. (B) DAPI and anti-H4-Ac-K16 staining of a bloated X from a Su(var)3-79/Su(var)3-714 male. (C) Male X bloating occurs only with a functional DCC. Bloating of the X is lost in the mle1/mle1; Su(var)3-714/Su(var)3-714 mutant, in contrast to its mle1/CyO; Su(var)3-714/Su(var)3-714 brother. Stained with orcein.

View larger version (16K): [in a new window]   Fig. 7. Targeted mutagenesis of Su(var)3-7. (A) The Su(var)3-7 locus and neighboring genes. (B) Donor construct using 2.6 kb of homology upstream of Su(var)3-7 (red) and 4 kb downstream (blue). (C) Combination of the donor transgenic lines with an I-SceI and FLP-producing strain leads to a double-strand break in the middle of the downstream region (blue) and can give rise to homologous recombination. (D) Use of homologous recombinant bearing a 1033 bp deletion 5' of the I-SceI site displaces the equilibrium towards a reduction in the 5' region of Su(var)3-7. (E,F) Crosses of recombinants with a strain expressing the I-CreI restriction enzyme lead to a double-strand break permitting many different reduction events. (F) Repair of the break in the 5' region of Su(var)3-7 results in deletion of the gene. B, BamHI; H, HindIII; R, EcoRI; X, XbaI.

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