A Buoyancy-Based Screen of Drosophila Larvae for Fat-Storage Mutants Reveals a Role for Sir2 in Coupling Fat Storage to Nutrient Availability

Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet

Identification of a proctolin preprohormone gene (Proct) of Drosophila melanogaster: Expression and predicted prohormone processing

Proctolin was the first insect neuropeptide to be sequenced and has been the subject of many physiological and pharmacological studies in insects and crustaceans. We have identified a Drosophila gene (CG7105, Proct) encoding a precursor protein containing the neuropeptide proctolin (RYLPT). In situ hybridization with a riboprobe to the Proct gene revealed a distribution of transcript in neurons of the larval central nervous system (CNS) matching that seen with antiserum to proctolin. An antiserum raised to a sequence in the precursor downstream of proctolin labeled the same neurons as those seen with the antiproctolin antisera. The predicted protein encoded by Proct has a single copy of the RYLPT sequence that directly follows the predicted signal peptidase cleavage point and precedes a consensus recognition site for a furinlike processing endoprotease. Ectopic expression of Proct in the CNS and midgut via the GAL4-UAS system, using an Actin5C-GAL4 driver, confirmed that the predicted preproproctolin can be processed to generate immunoreactive proctolin peptide. Pupae over-expressing Proct displayed a 14% increase in heart rate, providing evidence in support of a cardioacceleratory endocrine function for proctolin in Drosophila. The distribution of proctolin suggests roles as a neuromodulator in motoneurons and interneurons, and as a neurohormone that could be released from brain neurosecretory cells with terminations in the ring gland. (C) 2003 Wiley Periodicals, Inc. J Neurobiol 58: 379-391, 2004

Functional genomics of ozone stress in Arabidopsis.

Ozone is an important pollutant that has significant and frequently deleterious effects on plant health resulting in a reduction in cropy ields. This is due largely to oxidative damage caused either directly by ozone or by the reactive oxygen species generated by ozone. Plants possess a range of antioxidant defences to protect them against oxidative stress such as that caused by ozone and the expression of a wide-range of genes, including genes encoding important antioxidants, is altered by exposure to ozone. However, little is known about the signal transduction pathways linking the perception of ozone stress to gene expression. We have used a genomics approach to identify novel genes that respond to acute ozone stress in Arabidopsis thaliana. Initial transcript profiling studies using microarray analysis identified twenty genes which were highly up-regulated and one gene that was down-regulated by ozone treatment. Real Time RT-PCR and additional microarray analysis have subsequently confirmed this result. We have analyzed the expression of five of these genes in detail. Our studies show that the induction of all of these genes in response to oxidative stress was dependent on an increase in cytosolic free calcium and was specific to ozone. Importantly, they were not induced by other calcium-mobilizing oxidative stresses including hydrogen peroxide and cold. These data raise important questions about the mechanism by which these genes are induced by ozone and how stimulus specificity is encoded in the ozone calcium signature

Control of imaginal cell development by the escargot gene of Drosophila.

Mutations in the escargot (esg) locus, which codes for a zinc-finger-containing protein with similarity to the product of the snail gene, cause a variety of defects in adult structures such as loss of abdominal cuticle and malformation of the wings and legs. esg RNA is expressed in wing, haltere, leg and genital imaginal discs and in abdominal histoblast nests in the embryo. Expression in imaginal tissues is also found in third instar larvae. In esg mutant larvae, normally diploid abdominal histoblasts replicate their DNA without cell division and become similar in appearance to the polytene larval epidermal cells. A similar phenotype was also found in imaginal discs of larvae mutant for both esg and the Drosophila raf gene. These results suggest that one of the normal functions of esg may be the maintenance of diploidy in imaginal cells