The object of this research is to gain a better understanding of how specific proteins which can be assembled into membrane associated bacterial structures are synthesized, interact with, and are assembled within the bacterial membrane. Three basic approaches will be used. The first is a continuation of our studies on the membrane events associated with the assembly of the filamentous bacteriophage f1. The phage specific proteins which have not been identified in the bacteria, will be isolated, characterized and their location within the bacteria determined using genetic, biochemical and immunological techniques. Attempts will be made to isolate membrane associated intermediates in bacteriophage assembly and characterize them using biochemical and electron microscope techniques. We will try to define an in vitro system in which DNA from the gene V-DAN intermediate will be assembled into a mature phage particle. The second approach will be to examine F-like sex pili using some of the technique we have developed in the study of the f1 bacteriophage. F pili will be analyzed for the presence of a specific protein or structure at the tip which specifically interacts with f1 phage or F- cells. Attempts will be made to isolate the protein and use it to examine the receptor on the F- cells. The orientation of F pilin will be analyzed in the inner membrane and the intact pilus as was done for the major coat protein of the f1 phage. Last, different group specific reagents will be tested for their ability to label proteins on the outside of spheroplasts. Promising techniques will be used to ascertain the orientation of various inner membrane proteins.
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