Purification and physical properties of the male and female double sex proteins of Drosophila.
The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.
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Department of Biochemistry and The Rosenstiel Center, Brandeis University, Waltham, MA 02254-9110, USA.
Wensink
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry*; D005260:Female; D007301:Insect Hormones / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins; D018411:Spodoptera; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D007301:Insect Hormones; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors