Worm Breeder's Gazette 16(1): 34 (October 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.
MRC Group on the Molecular Biology of Membrane Proteins, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
We are isolating and characterizing nuclear and mitochondrial
DNA (mtDNA) Caenorhabditis elegans mutants with an impaired
mitochondrial respiratory chain (MRC). The MRC is made up of 5 protein
complexes, and its biogenesis requires the coordinate expression of
genes from both the nuclear and the mitochondrial genomes. The MRC is
the major source of ATP and thus, defective mitochondrial energy
production is increasingly being recognized as a contributor to many
human diseases including diabetes, myopathies, neuromuscular, and heart
diseases.
We have identified and cloned 3 MRC mutations. The first
mutation is a 1.2-kb deletion that removes 4 of the 6 exons in the nuo-1
gene encoding the active site subunit of complex I. This mutation is
homozygous lethal, leading to an L3 arrest phenotype.
The second mutation is a 0.7-kb deletion that removes 3 of the 6
exons in the atp-2 gene encoding the active site subunit of complex V.
This mutation is also homozygous lethal, leading to an L3 arrest
phenotype. Despite the developmental arrest, atp-2/atp-2 animals have a
similar lifespan compared to that of N2. We believe that the mutation
is a null mutation, since both atp-2/atp-2 and atp-2/sDf121 animals
(sDf121 is a deficiency that covers atp-2) have the same phenotype. RNA
interference experiments with atp-2 dsRNA result in significant levels
of embryonic arrest. We speculate that the more severe phenotype is due
to the absence of a maternal contribution of mRNA which allows animals
to develop to the L3 stage. We are currently attempting to measure the
levels of respiratory activity in this mutant.
The third mutation (uaDf5) is a 3.1-kb mtDNA deletion that
removes 4 MRC and 7 tRNA genes. uaDf5 animals are heteroplasmic,
having varying proportions of wildtype and mutant mtDNAs. We have
maintained the heteroplasmy for over 50 generations and have
demonstrated maternal inheritance of the mtDNA deletion. In addition,
we have shown a stochastic inheritance pattern for the uaDf5 mtDNA. All
uaDf5 animals appear to be aphenotypic despite high (>80%) proportions
of mutant mtDNA. We are currently attempting to elevate the proportion
of uaDf5 mtDNA in hope of crossing a threshold level where the mutation
would become pathogenic.
We have examined the effect of ethidium bromide (EtBr) on N2
worms. EtBr is a DNA-intercalating dye known to inhibit mtDNA
replication and to deplete mtDNA in yeast and in mammalian cell lines.
Interestingly, when N2 gravid adults are exposed to EtBr, L3 arrested F1
progeny are observed. The concentration of EtBr at which 50% of the
animals arrest at L3 is about 35 ug/ml. Despite the developmental
arrest, these animals appear to have a lifespan similar to control N2.
Furthermore, the arrested animals exhibit a progressive decrease in the
steady-state level of mtDNA with age. The observations that both
nuclear mutants and the mtDNA-depleted animals arrest at L3 lead us to
speculate that pathogenic uaDf5 animals are likely to have an L3 arrest
phenotype as well.
Finally, we speculate that the L3 arrest phenotype is a
consequence of impaired energy production. In support of this
hypothesis, we have determined that development from L3 to L4 is
associated with a 3-fold increase in mtDNA copy number, as well as a
significant increase in the levels of ATP-2.