A Link Between Impaired Purine Nucleotide Synthesis and Apoptosis in Drosophila melanogaster

Abstract

The biosynthetic pathways and multiple functions of purine nucleotides are well known. However, the pathways that respond to alterations in purine nucleotide synthesis in vivo in an animal model organism have not been identified. We examined the effects of inhibiting purine de novo synthesis in vivo and in cultured cells of Drosophila melanogaster. The purine de novo synthesis gene ade2 encodes phosphoribosylformylglycinamidine synthase (EC 6.3.5.3). An ade2 deletion, generated by P-element transposon excision, causes lethality in early pupal development, with darkening, or necrosis, of leg and wing imaginal disc tissue upon disc eversion. Together with analysis of a previously isolated weaker allele, ade2 4 , and an allele of the Prat gene, which encodes an enzyme for the first step in the pathway, we determined that the lethal arrest and imaginal disc phenotypes involve apoptosis. A transgene expressing the baculovirus caspase inhibitor p35, which suppresses apoptosis caused by other stresses such as DNA damage, suppresses both the imaginal disc tissue darkening and the pupal lethality of all three purine de novo synthesis mutants. Furthermore, we showed the presence of apoptosis at the cellular level in both ade2 and Prat mutants by detecting TUNEL-positive nuclei in wing imaginal discs. Purine de novo synthesis inhibition was also examined in tissue culture by ade2 RNA interference followed by analysis of genome-wide changes in transcript levels. Among the upregulated genes was HtrA2, which encodes an apoptosis effector and is thus a candidate for initiating apoptosis in response to purine depletion.

A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle

“…Concerning PFAS, several mutations have been described in mice, causing a dominant short-face phenotype and most probably recessive embryonic lethality because no homozygous mutant has ever been obtained from mating heterozygous animals (Palmer et al, 2016). In addition, homozygous deficiency of the PFAS ortholog gene ade2 is lethal during pupal development in Drosophila melanogaster (Henikoff et al, 1986;Holland et al, 2011). Taken together, these arguments strongly support a causative role for the g.28511199C>T variant in MH1associated embryonic lethality.…”

Ade2 Functions in the Drosophila Fat Body To Promote Sleep

“…To confirm that the sleep loss phenotype observed with Ade2 knockdown was not due to off-target effects of RNAi, we assayed sleep in Ade2 mutant Drosophila . Two Ade2 mutants, Ade2 3 -20 and Ade2 1-6 , have been generated by P -element excision ( Holland et al 2011 ). For Ade2 1-6 , the deletion includes the transcription start site and part of the first coding exon resulting in a null allele ( Holland et al 2011 ).…”

“…Two Ade2 mutants, Ade2 3 -20 and Ade2 1-6 , have been generated by P -element excision ( Holland et al 2011 ). For Ade2 1-6 , the deletion includes the transcription start site and part of the first coding exon resulting in a null allele ( Holland et al 2011 ). While both alleles are homozygous lethal, heterozygous flies are viable.…”

“…Mutations in Ade2 , or other components of the de novo purine biosynthesis pathway, have been implicated in the arrest of cell growth as well as reduced fertility and lifespan, suggesting broad biological functions of this gene ( Tiong and Nash 1990 ; Malmanche and Clark 2004 ). For example, flies heterozygous for Ade2 mutations develop necrosis as pupae, suggesting haploinsufficiency may result in physiological abnormalities ( Holland et al 2011 ). The possibility that targeted disruption of Ade2 in the fat body disrupts development of this organ, or its function in energy storage, is supported by our findings that whole-body triglycerides and/or free glucose levels are reduced in Ade2 -deficient flies.…”

“…The following fly strains were ordered from Bloomington Stock Center, w 1118 (5905; Levis et al 1985 ), CG-GAL4 (7011; Asha et al 2003 ), r 4 -GAL4 (33832; Lee and Park 2004 ), and hmlΔ3-GAL4 (30141; Goto et al 2003 ). Ade2 3-20 , Ade2 1-6 , and UAS-Ade2 were obtained from D. Clark and have been previously characterized ( Holland et al 2011 ). Drosophila lines used in the RNAi screen originate from the TRiP collection ( Brand and Perrimon 1993 ; Ni et al 2009 ) and are described in ( Table 1 ).…”