The proposed project involves the use of low energy (0.5 keV - 5.0 keV) Ar+ beams to study the structure of bacteriophage lambda, the large (50S) subunit of E. coli ribosomes and equine herpes virus type 1 (EHV-1). The experimental strategy is to employ ion beams with limited (5.0nm - 10nm) penetrating power to etch or erode the particle surface progressively from the outside toward the inside. At various stages of the etching process, virions will be examined in the electron microscope to identify internal structural features revealed when more external layers are eroded away. Biochemical analyses will also be carried out with irradiated particles to identify exposed proteins or regions of the nucleic acid that have been damaged during etching. Studies of lambda phage will be devoted to clarifying the way DNA is arranged in the mature phage head. The goal is to test our recent suggestion that the first DNA to enter the prohead (the left end on the standard genetic map) should be found at the center of the overall DNA condensate with the last-packaged DNA at the periphery. Experimentally, this will involve etching intact phage and then examining the DNA (by Southern hybridization to sequence-specific probes) to identify the sites of damage. The proposed analysis of E. coli 50S ribosomal subunits is intended to identify those proteins and regions of the 23S RNA nearest the particle surface. Intact 50S subunits will be etched for various periods and the remaining protein components analyzed by two- dimensional SDS-polyacrylamide gel electrophoresis. Sites of damage to the 23S RNA will be identified by Southern hybridization experiments involving chemically-synthesized DNA probes corresponding to specific regions of the rRNA sequence. Etching experiments with intact EHV-1 are designed to clarify the structure of the tegument layer and its polypeptide composition. Analyses of EHV-1 nucleocapsids should illuminate the arrangement of DNA in these structures and allow one to determine the polypeptide composition of the core found in the center of the DNA-containing torroid.
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