The Role of Kinetoplast DNA-Binding Proteins in the Struc- ture and Organization of the Kinetoplast in the Trypanosome Crithidia Fasciculata

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Abstract 9404955 Kinetoplastid protozoa contain a novel form of mitochondrial DNA (kinetoplast DNA or kDNA) consisting of thousands of minicircles and 20-30 maxicircles catenated into a single sheet or network. These kDNA networks are condensed in vivo, forming a highly organized disk-like structure about 1 um in diameter and 0.4 um thick. Previous studies have focused almost entirely on the novel DNA components of the kinetoplast and their organization into a catenated network. The research proposed here addresses the role of small histone-like proteins in the structural organization of the kinetoplast. Prior work in our laboratory has identified five small lysine-rich proteins associated with kDNA in vivo. Genes encoding three of these have already been cloned. The proposed research will lead to the cloning of the remaining genes and their use in determining the possible roles of these proteins in the structural organization of the kinetoplast. Each of these histone-like proteins will be produced in large amounts by expression in bacteria and used for producing antibodies and for biochemical studies of the interactions of each protein with DNA. These studies will determine the DNA-binding specificity of each protein and its ability to bend DNA, to induce supercoiling and to condense kDNA networks. Antibodies against these proteins will provide essential reagents for localizing each of the proteins within the cell using both immunofluorescence microscopy and immuno-electron microscopy. Through the use of this range of experimental approaches we expect to gain an understanding of the possible roles of histone-like proteins in organizing the kinetoplast structure. %%% Genetic information is encoded in living cells in extremely long strands of DNA that must be organized or "packaged" in some highly condensed and regular manner. This research program is aimed at identifying and analyzing some of the factors involved in condensing DNA within living cells. One of the m ost highly condensed forms of cellular DNA in nature is the DNA found in tropical parasitic organisms called trypanosomes. These organisms are widespread parasites of both plants and animals. One of their characteristic features is the presence of a large body of DNA called kinetoplast DNA (kDNA) which is located outside of the cell nucleus where chromosomes are found. Like the DNA of chromosomes, kDNA condensation appears to involve association with a group of small positively charged proteins. Methods have been developed for isolating and characterizing these proteins. Through the study of the interactions of these proteins with DNA, it may be possible to determine how these proteins bind to DNA and how this binding contributes to the folding and organizing of the DNA into a compact structure. These studies are made possible by the many developments in biotechnology including DNA cloning and sequence analysis. An important aspect of this research is the training of future scientists in these areas of biotechnology and assisting them to acquire the necessary skills to contribute to the growth of an important area of technology in this country. ***