Purification and physical properties of the male and female double sex proteus of Drosophila
Purification and physical properties of the male and female double sex proteus of Drosophila
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DOI：
10.1073/pnas.93.5.2043
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发表时间：
1996-03-05
影响因子：
11.1
通讯作者：
Wensink, PC
中科院分区：
文献类型：
--
作者：
Cho, SY;Wensink, PC
双性基因（dsx）编码两种蛋白质，dsx (M)和dsx (F)，它们调节果蝇的性别特异性转录。这些蛋白结合DNA中的靶位点，雄性特异性DSX(M)抑制靶位点，雌性特异性DSX(F)激活卵黄蛋白（Yp）基因的转录。我们研究了这些DSX蛋白的物理性质，它们在氨基末端397个残基上是相同的，但在羧基末端序列上完全不同(DSX(F)， 30个氨基酸；DSX(M)， 152个氨基酸)。DSX(M)和DSX(F)在培养的昆虫细胞中过表达并纯化至接近均匀性，凝胶过滤层析和甘油梯度沉降表明，在低浓度下，这两种蛋白都是高度不对称形状的二聚体，轴比接近18:1 （DSX(M)， 860 x 48埃）。DSX(F)， 735 x 43埃)，在较高的浓度下，蛋白质形成四聚体，通过使用一种新的双交联试验（蛋白质-DNA加蛋白质-蛋白质），我们证明了DNA调控位点与DSX二聚体的两个单体结合，并且仅与四聚体的两个单体结合，此外，将另一个DNA分子结合到我们认为是四聚体中第二个相同的位点显著地将平衡从四聚体转变为二聚体。这些寡聚化和DNA结合特性在男性和女性蛋白质之间是无法区分的。
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 approximate to 18:1 (DSX(M), 860 x 48 Angstrom: DSX(F), 735 x 43 Angstrom), 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.