Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG
| Autor: | D. A. Zarling, Fred K. Hagen, Thomas M. Jovin |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
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Simian virus 40 Biology General Biochemistry Genetics and Molecular Biology Antibodies Z-DNA chemistry.chemical_compound Ethidium Molecular Biology General Immunology and Microbiology Superhelix General Neuroscience Chromosome Mapping Molecular biology Restriction enzyme Microscopy Electron Cross-Linking Reagents chemistry IgG binding Polynucleotide DNA Viral Nucleic Acid Conformation DNA Circular Ethidium bromide DNA Research Article |
| Zdroj: | The EMBO journal. 4(3) |
| ISSN: | 0261-4189 |
| Popis: | Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density). |
| Databáze: | OpenAIRE |
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