The work carried out during the current grant period has identified a new mitochondrial D-loop encoded protein of about 6 kDa, that binds to common sequence motifs on both mitochondrial and nuclear genes. The results of protein purification, Western blot analysis and immunocytochemical analyses demonstrate that the mitochondrial protein is transported to the nucleus as a possible regulatory signal. A PCR based screening of an array of mouse tumors has identified three tumor lines with mutations affecting the sequence of this newly identified protein. Thus, the present proposal is based on the hypothesis that the mitochondrial protein is involved in the regulation of a number of nuclear genes, and it will address some of the long standing questions on the regulation of mitochondrial biogenesis and mechanisms of nuclear-mitochondrial gene interaction, and how these regulatory controls are altered in chemically transformed and established tumor cells as follows: 1) The mouse mitochondrial D-loop encoded protein will be functionally characterized with respect to its DNA binding specificity and its role in transcription stimulation, or down-regulation using in vivo (transient expression in cells) and in vitro transcription systems. Experiments will be carried out to determine if the 6 kDA protein functions as a homodimer or a heterodimer with other nuclear proteins. In the latter case, the protein partner(s) involved in the protein-protein interaction will be characterized. 2) The mode of transport of the D-loop encoded protein to the nuclear compartment and the nature of the transport signal(s) will be studied by in vitro mutagenesis and expression of cDNA constructs in COS cells or human P cells, which lack the mitochondrial DNA. 3) The structure-function relationships and also the possible patho- physiological consequences of the mitochondrial DNA mutations will be determined by analyzing the mutant proteins found in some of the tumor cells and also those derived by in vitro mutagenesis. The effects of in vivo expression of wild type and mutated D-loop proteins in human P cells, and also in cells carrying known mutations on the transcriptional regulation of some of the well characterized nuclear genes will be studied. 4) The nature and types of nuclear genes under the regulatory control of the D-loop encoded mitochondrial protein will be studied by screening the mouse genomic library by in situ protein binding with 32P labeled protein and partial characterization of positive clones by sequence analysis.
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