Worm Breeder's Gazette 17(3): 39 (November 1, 2003)
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.
Institut de Génétique et Microbiologie, Université Paris XI, 91405 Orsay cedex, France. emmanuel.culetto@igmors.u-psud.fr
The mitochondria play a critical role in the cellular energy supply and perform other essential roles in a variety of metabolic functions. The mitochondria electron transport respiratory chain (MRC) is typically composed of four multi-subunit enzymes (complexes I to IV) and the ATP synthase (complex V). Complex II is involved in both the tricarboxylic acid (TCA) cycle and the aerobic respiratory chains of mitochondria. It consists of 4 nuclear-encoded polypeptides: the flavoprotein (Fp/ SDHA), an iron-sulphur protein (Ip/ SDHB) and two integral membrane proteins (SDHC and SDHD). Clinically, complex II deficiency associated with SDHA gene mutations can result in myopathy, encephalopathy and isolated cardiomyopathy. Recently, the analysis of the susceptibility gene for familial paraganglioma syndrome revealed germ line mutations in the SDHB, SDHC and SDHD genes. Those genes are therefore considered to be tumor-suppressor genes. One can speculate that oxidative damage or ROS recycling dysfunction from impaired complex II could be important in explaining the damage to muscle and nerve cells. The molecular basis of such MRC enzyme deficiencies in humans remains largely unknown (1).
We aim to generate C. elegans models of these pathologies to identify the underlying mechanisms associated with such deficiencies. We identified by sequence comparison C. elegans homologues of complex II polypeptides (C03G5.1 and C34B2.7 are homologous to SDHA, F42A8.2 is homologue to SDHB, F33A8.5 is SDHD and mev-1 is the structural gene for SDHC (2)). We sequenced several ESTs (thanks to Yuji Kohara) to determine the exon-intron structures of each gene. We used RNAi feeding experiments to study the worm phenotype associated with loss-of-function of worm homologues to the complex II subunits. Our preliminary RNAi experiments, for the anchorage subunits, show a decrease of the progeny numbers that seems to be due to an early arrest of embryo development. The single RNAi experiment for C03G5.1 and C34B2.7 gave no phenotype, whereas the simultaneous RNAi for both genes led to embryonic arrest of development. Thus, the SDHA encoding genes show some functional redundancy. We would like to isolate stable mutants in the genes of interest. Those mutants will be used for complementation experiments with human cDNAs (Wild type and mutants identified in human pathologies). The mel-9 and mel-13 mutants mapped very near the physical localization of SDHB and SDHD respectively. We have started to sequence those genes in the respective genetic background to identify any molecular alteration. We also recently received a SDHA mutant harbouring a 1.7 kb deletion in the C03G5.1 gene (thanks to OMRF KO group). We think that the C. elegans strains expressing Human complex II subunits we will generate, could be used for future drug screening. We also want to identify genes that are functionally linked to complex II activity. As a first approach we are looking for mutants resistant to complex II inhibitors : theonyltrifluoroacetone (TTFA) and 3-nitropropionic acid (3-NP). We already checked the sensitivity of L1 stage worms for the two drugs, and we found lethal doses of 2.2 mM (for TTFA) and 15 mM (for 3-NP). We also tested TTFA effect on L4 worms and found that they are just as sensitive as the L1. We have recently commenced a genetic screen to select mutants resistant to both inhibitors. (1)Rustin P et al. (2002) Eur J Hum Genet., 10, 10289-10291, (2)Ishii N et al. (1998) Nature 394, 694-697