Worm Breeder's Gazette 15(3): 20 (June 1, 1998)

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.

Nematodes with Mitochondrial Diseases

Bernard D. Lemire, William Tsang

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) 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.