The goal of the proposed research is to determine the mechanism of bacteriophage T7 DNA packaging at the molecular level. Intermediates in T7 morphogenesis are being isolated after gentle cell lysis from T7-infected E. coli and are being characterized using: a) buoyant and velocity ultracentrifugation, b) non-denaturing electrophoresis in agarose gels, c) electron microscopy, d) SDS polyacrylamide gel electrophoresis, e) electrophoresis of DNA sequence-specific restriction enzyme fragments. Kinetic labeling experiments will be used to observe the time course of intermediates isolated. Mutational alteration of the T7 morphogenetic pathway will be used to isolate intermediates that are transient or unstable during wild-type infections. Attempts will be made to have purified intermediates reenter the pathway in a cell-free system. To obtain the data described above, new electrophoretic and ultracentrifugal techniques have and will be developed and characterizing intermediates made of DNA, supramolecular complexes of protein and DNA-protein complexes. New techniques for preparing such intermediates for electron microscopy will also be developed. Techniques developed should be useful for the study of other viruses and for the study of cells.
NIH ExPorter
NIH ExPorter