Interactions between purine synthesis and cell death pathways. Denise V. Clark, Ashley M. DiPasquale. Dept of Biology, Univ New Brunswick, Fredericton, NB, Canada.

   Mutations affecting de novo synthesis of purine nucleotides have a pleiotropic phenotype. Surviving adults have wing and leg defects, reduced bristles, and reduced red eye pigmentation. More severe mutations cause arrest in prepupal and pupal stages, often with development of necrosis in wing and leg discs. We are interested in determining the pathways that lead to the development of this phenotype. We previously found that the pupal lethality and necrosis phenotypes have a link with apoptosis, since they are dependent on caspase activity, and since prepupal wing discs show apoptotic nuclei prior to development of the necrosis. These phenotypes do not appear dependent on p53 or the apoptosis effector region containing the reaper, grim and hid genes [1]. To explore further this link between reduced purine synthesis, lethality, and apoptosis, we examined other apoptosis pathway genes for interactions with reduced purine synthesis. To reduce purine synthesis, we focused on the ade2 gene, which encodes the 4th step in the purine de novo synthesis pathway, and a deletion allele ade2^1-6. For apoptosis, we focused on Drosophila inhibitor of apoptosis protein 1 (DIAP1, or thread). Counter to our predictions, over-expression of DIAP1 enhances the early pupal arrest and degree of necrosis, whereas under-expression in thread mutant heterozygotes partially rescues both of these phenotypes. This result is leading us to explore the role of other cell death pathways in the development of the purine synthesis phenotype. In addition to DIAP1, we also explored the role of HtrA2, a mitochondrial serine protease that can activate DIAP1, in the purine syndrome phenotype. HtrA2 RNAi did not suppress pupal necrosis; however, it suppressed the lethality of ade2^1-6. Further characterization of the lethal phenotype of ade2^1-6 showed death of at least half of the mutants in third instar larvae. Our results suggest that HtrA2 may have a role in mediating the response to reduced purine synthesis during larval development. [1] Holland et al (2011) Genetics Jun;188(2):359-67.