The long-term goal of this project is the study of the fundamental biological processes which control the inheritance, growth, and replication of mitochondria in mammalian cells. These studies should also provide information concerning the interrelationship between the nucleus and the mitochondria as well as answers to questions about the informational content of mitochondrial DNA. In the shorter-term we hope to establish conditions and procedures for bringing about the transfer of mitochondria and/or mitochondrial genetic material from one cell to a cell of another species and for their establishment in the recipient cell with the production of a stable transformed clone. Specifically we will determine how many and which human chromosomes are required to maintain human mitochondria using interspecific cell fusions. The resulting hybrids will be examined by enzymatic, isozyme and karyotype analysis. They would also be examined for human, mouse and recombinant mitochondrial DNA. We will correlate the retention of human mtDNA and the retention of specific human chromosomes. The mitochondrial DNA of the hybrids will be examined using restriction endonuclears and molecular hybridization procedures to obtain evidence for recombinational events. New mutants of mouse L cells and human HeLa cells will be sought which are resitant to inhibitors of mitchondrial protein synthesis or oxidative phosphorylation. These will be tested for cytoplasmic inheritance of their resistance characteristics by the methods established in this laboratory for the cytoplasmically inherited chloramphenicol-resistant mutants. The molecular entity altered by each cytoplasmically inherited chloramphenicol-resistant mutants. The molecular entity altered by each cytoplasmic mutation will be investigated. Bifactorial recombination experiments between chloramphenicol- and other antibiotic resistant mutants will be performed in both cis and trans configurations. The same crosses with one parent enucleated will detect any nuclear influences on cytoplasmic genetic behavior. All crosses can be intraspecific or interspecific and can be beween cells of similar or different tissue origin. These techniques can be extended to multifactorial crosses.