ABSTRACT
We describe a simple detection system for DNA based on antibody detection of UV-induced photoproducts. It includes a convenient and inexpensive labeling procedure, which is completed in 5-10 min. The only equipment required is a UV source such as an ordinary transilluminator or a DNA crosslinker. Using a monoclonal antibody specific for thymine dimers, coupled to horseradish peroxidase, we are able to detect subpicogram amounts of UV-irradiated DNA directly, and approximately 10 pg homologues DNA indirectly by hybridization with an irradiated probe.
Subject(s)
DNA/analysis , Immunoenzyme Techniques , Ultraviolet Rays , Antibodies, Monoclonal , Antibody Specificity , Bacteriophage phi X 174/genetics , Cross-Linking Reagents , DNA Probes , DNA, Bacterial/analysis , DNA, Single-Stranded/analysis , DNA, Viral/analysis , Escherichia coli/genetics , Nucleic Acid Hybridization , Pyrimidine Dimers/analysis , Pyrimidine Dimers/immunologyABSTRACT
The Psu (Polarity suppression) protein of satellite bacteriophage P4 was first characterized as an anti-terminator of transcription termination in Escherichia coli. Psu is also a structural component of mature P4 capsids, where it is present as a decoration protein. Psu is located externally on the capsid surface, and it appears to protect the capsid from loss of DNA through the capsid shell. The ability of Psu to specifically bind to the P4 capsid appears not to be dependent on any P4 specific components such as the capsid protein cleavage products h1 and h2, or P4 DNA. We suggest that Psu binds to the P4 capsid as a result of the special structure of the hexamers in the P4 capsid.
Subject(s)
Capsid Proteins , Capsid/metabolism , Coliphages/metabolism , Satellite Viruses/metabolism , Amino Acid Sequence , Capsid/immunology , Capsid/isolation & purification , Capsid/ultrastructure , Coliphages/ultrastructure , DNA, Viral , Microscopy, Immunoelectron , Molecular Sequence Data , Satellite Viruses/ultrastructureABSTRACT
In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid. However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid. By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface. Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.
Subject(s)
Capsid Proteins , Capsid/isolation & purification , Capsid/ultrastructure , Coliphages/ultrastructure , Defective Viruses/ultrastructure , Cryopreservation , Image Processing, Computer-Assisted , Microscopy, Electron , Models, Structural , Mutation , Virion/ultrastructureABSTRACT
We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4's helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.
