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.