We are isolating and characterizing nuclear and mitochondrial DNA (mtDNA) mutants with an impaired mitochondrial respiratory chain (MRC) in the nematode, Caenorhabditis elegans. Mitochondria are the major source of ATP for many highly oxidative tissues such as neurons, skeletal muscle, and endocrine organs and thus, defective mitochondrial energy production is increasingly being recognized as a contributor to many diseases including encephalomyopathies, diabetes, and heart disease. Expression of a functional MRC relies on the integrity of two genomes, the nuclear and the mitochondrial genomes. Thus, the genetic, physiological, and biochemical features of mitochondrial diseases are very complex. There is currently no suitable model system for studying mitochondrial diseases. We are developing the nematode as our model system because it is anatomically simple and because a wealth of genetic information and technology is available. The C. elegans MRC and mtDNA display many similarities to their mammalian counterparts. We have constructed a library of about 2.7 x 105 ethyl methanesulfonate (EMS) mutagenized animals and have used target selected mutagenesis using nested PCR to isolate deletion mutations in nuclear MRC genes. We have identified 4 deletion mutations: in the
nuo-1 (C09H10.3), and the
nuo-2 genes of the NADH-ubiquinone oxidoreductase or complex I, in the
atp-2 (C34E10.6) gene of the ATP synthase or complex V, and in the
cox-6 gene of cytochrome c oxidase or complex IV. For the
nuo-1 and the
atp-2 mutations, we have cloned and outcrossed strains bearing the deletion alleles. The
nuo-1 (
ua1) allele is a 1.2-kb deletion partially or completely removing 4 of the 6 exons. The
atp-2 (
ua2) allele is a 0.8-kb deletion removing the first 3 of 6 exons. Both genes are essential since homozygous mutants are inviable. Both homozygous mutants hatch, develop through 2 larval stages, and arrest at the L3 stage, suggesting that a common energy requiring step in development is blocked by complex I and complex V deficiencies. Interestingly, the arrested animals survive for many days, but do not develop further. We are currently determining the levels of respiratory activity in the mutants and pursuing this approach to isolate deletion mutants in all 5 MRC complexes. Many human mitochondrial diseases arise from mtDNA mutations and we have also obtained such mutants with the target selected mutagenesis approach. We have identified two bona fide mtDNA deletions in our library and are currently outcrossing the strains. The 3.1 (
ua5) and 3.2-kb (
ua6) deletions remove all or part of the ND-1 and ND-2 genes encoding complex I subunits, the ATP-6 gene of complex V, and the CYT-b of complex III. We will determine the level of mutant mitochondrial DNA in the outcrossed strains. In addition to the biochemical and phenotypic characterization of mitochondrial DNA mutants, we will investigate the role of heteroplasmy in the inheritance and expression of energy deficit related phenotypes.