ABSTRACT
X-raymicroscopy is applied to detect specific proteins in whole cell nuclei of Drosophila melanogaster using immunogold labeling and silver enhancement. As a model for a small subnuclear structure the Drosophila dosage compensation protein MSL-1 was chosen. It associates with a number of other proteins to form a hetero-multiprotein complex, which elevates the transcriptional activity of the single X chromosome in males. This phenomenon is known as dosage compensation and is essential for the survival of male flies. The distribution of the Drosophila dosage compensation complex was studied by X-ray microscopy, because though the complex is expected to function by remodeling the structure of chromatin, its exact mode of action is not yet known. Many similar protein complexes are associated with different aspects of chromatin-mediated gene regulation in all eucaryotic organisms and can also be studied with the approach presented in this work. The distribution of MSL-1 protein in the nuclei of fixed D. melanogaster culture cells is visualized using the Göttingen X-ray microscope at the electron storage ring BESSY I. In addition to conventional and confocal laserscan fluorescence microscopy, X-ray microscopic investigations were performed at room as well as at cryogenic temperatures. The label can clearly be identified in the X-ray micrographs and shows detailed structure in the cell nuclei. Currently, X-ray micrographs show details in the cell nuclei about five times smaller than those in visible light micrographs.
Subject(s)
Dosage Compensation, Genetic , Drosophila Proteins , Drosophila/genetics , Microscopy, Electron, Scanning Transmission , Nuclear Proteins/chemistry , Transcription Factors/chemistry , Animals , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , Chromatin/ultrastructure , Drosophila/cytology , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Immunohistochemistry/methods , Male , Microscopy, Fluorescence , Nuclear Proteins/metabolism , Temperature , Transcription Factors/metabolism , X Chromosome/genetics , X Chromosome/ultrastructureABSTRACT
The dipteran insects Chironomus and Drosophila have high mobility group (HMG) 1 proteins that are similar to mammalian HMG1 but contain one instead of two HMG1 boxes. The interaction of the Chironomus HMG1 proteins cHMG1a and cHMG1b with double-stranded and four-way junction DNA was analyzed by investigating the accessibility of defined sequences of the HMG1 box to specific antibodies within the DNA-protein complex in vitro. Antibody epitopes located in the three helices and in the turn between helices 1 and 2 of the HMG1 box were identified on a membrane onto which 90 decapeptides with overlapping sequences spanning the entire sequence of cHMG1a had been bound. Monospecific antibodies directed against selected epitopes were purified from a polyclonal antiserum by affinity chromatography. Helices 1 and 3 as well as the turn between helices 1 and 2 were found to be accessible to antibodies in the complex. In contrast, antibodies recognizing an epitope on putative helix 2 of cHMG1a and cHMG1b were unable to produce supershifts on gels of the DNA-protein complexes with both DNAs. These data suggest that helix 2 of the HMG1 box of proteins cHMG1a and cHMG1b is essentially responsible for contacts with DNA.
Subject(s)
Chironomidae/metabolism , DNA/metabolism , Drosophila/metabolism , High Mobility Group Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/isolation & purification , DNA-Binding Proteins/immunology , High Mobility Group Proteins/genetics , High Mobility Group Proteins/immunology , Molecular Sequence Data , RatsABSTRACT
12-O-Tetradecanoylphorbol-13-acetate (TPA) binds specifically to histone H1 prepared from HeLa cells and to calf histone H1 with dissociation constants of 4.3 x 10(-7) and 9.3 x 10(-7) M, respectively. The TPA-binding to histone H1 was confirmed immunologically using a monoclonal antibody directed against an evolutionally conserved epitope of histone H1. This binding would influence DNA structure and the basal level of transcription, and may, in turn, account for the cell growth-regulatory activity of TPA.
