Worm Breeder's Gazette 15(5): 26 (February 1, 1999)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
The mitochondrial respiratory chain (MRC) is composed of 5 protein complexes (I-V) and functions to generate energy in the form of ATP. The biogenesis of the MRC is complex, involving the coordinate expression of genes from both nuclear and mitochondrial genomes. In humans, mutations in MRC genes in either genome result in a wide variety of neuromuscular or endocrine disorders. We intend to use C. elegans as a model system to characterize MRC gene mutations. Nuclear MRC mutation Previously [WBG 15(3):20], we isolated and cloned two deletion mutants. The first is a 1.2 kb deletion in the nuo-1 gene (CO9H10.3) encoding the active site subunit of complex I. The second is a 0.7 kb deletion in the atp-2 gene (C34E10.6) encoding the active site subunit of the ATP synthase. Both mutations are homozygous lethal, leading to an L3 arrest phenotype. We hypothesize that a maternal contribution of mRNA is allowing development to the L3 stage. In the absence of maternal mRNA, we predict arrest at an earlier stage of development. Preliminary RNAi experiments with atp-2 dsRNA result in significant embryonic arrest, thus supporting our hypothesis. We will also perform RNAi with nuo-1 dsRNA. Mitochondrial DNA (mtDNA) mutation We isolated and cloned the uaDf5 mutant. This mutant has a 3.1 kb deletion which removes 11 mtDNA encoded genes, including 4 MRC genes and 7 mitochondrial tRNA genes. uaDf5 animals are heteroplasmic; they carry varying proportions of mutant and wildtype mtDNAs. We have demonstrated non-Mendelian (maternal) inheritance of the mtDNA deletion with the following genetic crosses: 1)When a heteroplasmic uaDf5 hermaphrodite is mated to a homoplasmic wildtype male, 100% of the offspring are heteroplasmic. 2) when a heteroplasmic uaDf5 male is mated to a homoplasmic wildtype female, 100% of the offspring are wildtype. We have set up a semiquantitative PCR assay for determining the proportions of mutant and wildtype mtDNAs in a single animal. With this assay, we have investigated the inheritance of the uaDf5 mtDNA. A uaDf5 hermaphrodite with 50% mutant mtDNA gives rise to an F1 brood with a Gaussian distribution of uaDf5 mtDNA centered at 47% and ranging from 12% to 85%. All animals appear to be aphenotypic despite the different proportions of mutant mtDNA. We are attempting to raise the proportion of uaDf5 mtDNA to pathogenic levels. We speculate that once a threshold level is crossed, we will begin to find L3 arrested animals, similar to the nuclear mutations we have investigated. We have also determined the mtDNA copy numbers of each developmental stage in N2 animals. There are 3x104, 2x104, 3x104, 8x104, 7x105, and 9x105 copies of mtDNA in L1, L2, L3, L4, gravid adults, and old adults, respectively. The amplification of mtDNA in progressing from the L3 to the L4 stage may indicate increased energy demands and may be related to the L3 arrest of our atp-2 and nuo-1 mutants.