No abstract

The disulfide reducing enzymes glutathione reductase and thioredoxin reductase are highly conserved among bacteria, fungi, worms, and mammals. These proteins maintain intracellular redox homeostasis to protect the organism from oxidative damage. Here we demonstrate the absence of glutathione reductase in Drosophila melanogaster, identify a new type of thioredoxin reductase, and provide evidence that a thioredoxin system supports GSSG reduction. Our data suggest that antioxidant defense in Drosophila, and probably in related insects, differs fundamentally from that in other organisms.

We describe a collection of P-element insertions that have considerable utility for generating custom chromosomal aberrations in Drosophila melanogaster. We have mobilized a pair of engineered P elements, p{RS3} and p{RS5}, to collect 3243 lines unambiguously mapped to the Drosophila genome sequence. The collection contains, on average, an element every 35 kb. We demonstrate the utility of the collection for generating custom chromosomal deletions that have their end points mapped, with base-pair resolution, to the genome sequence. The collection was generated in an isogenic strain, thus affording a uniform background for screens where sensitivity to genetic background is high. The entire collection, along with a computational and genetic toolbox for designing and generating custom deletions, is publicly available. Using the collection it is theoretically possible to generate Ͼ12,000 deletions between 1 bp and 1 Mb in size by simple eye color selection. In addition, a further 37,000 deletions, selectable by molecular screening, may be generated. We are now using the collection to generate a second-generation deficiency kit that is precisely mapped to the genome sequence. G ENETICALLY tractable model organisms are valufor components that function in particular pathways and characterize how individual genes participate in able research tools for uncovering basic biological such pathways. principles that are conserved through evolution. ManyThe fruit fly, Drosophila melanogaster, is one such tractamolecular pathways, such as signaling cascades, gene ble model that has been used extensively to elucidate regulatory pathways, and cell cycle control circuits, were many conserved genetic hierarchies. One particularly first characterized genetically in model systems. The powerful approach with Drosophila is the ability to rapsubsequent molecular cloning of the genes involved in idly carry out focused genome-wide screens for pathsuch pathways has shown how evolution has utilized way components by identifying loci that modify specific basic molecular building blocks to control a wide variety phenotypes (see St. Johnston 2002 for review). In this of biological processes. Key to the success of such apapproach, a sensitized genetic background, most comproaches has been the ability to carry out genetic screens monly exhibiting an easily scored adult phenotype such as rough eyes or a wing defect, is used to search for mutations in genes that make the phenotype more se- sensitized background and the phenotype is assessed. specific recombinase (FRT site) placed within intron one. In the case of RS3, a second FRT site is placed Importantly, the mutagenized chromosome is heterozygous, allowing genetic interactions between the sensiupstream of the first of the mini-white exons; in the case of RS5 the second FRT site is located downstream of tized background and mutations that are homozygous lethal to be detected. Particularly useful tools for such the mini-white exons. Golic and Golic demonstrated how a pair of RS3 and RS5 e...

Genetic and molecular studies on the expression of Antennapedia (Antp) have suggested that this gene specifies mainly the second thoracic segment. On the basis of our molecular analysis of dominant gain-of-function mutants we have postulated that the transformation of antennae into second legs is due to the ectopic overexpression of the Antp+ protein. This hypothesis was tested by inserting the complementary DNA encoding the normal Antp protein into a heat-shock expression vector and subsequent germ-line transformation. As predicted, heat induction at defined larval stages leads to the transformation of antennae into second legs. The dorsal part of the head can also be transformed into second thoracic structures (scutum) indicating that Antp indeed specifies the second thoracic segment. By ectopic overexpression of the Antp protein the body plan of the fruit fly can be altered in a predictable way.

Circadian rhythms can be entrained by light to follow the daily solar cycle. In Drosophila melanogaster a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Krüppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. We thus demonstrate that eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LN(v)), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues in eyelet. We compared action spectra of entrainment in different fly strains: in flies lacking compound eyes but retaining eyelet (so(1)), lacking both compound eyes and eyelet (so(1);gl(60j)), and retaining eyelet but lacking compound eyes as well as cryptochrome (so(1);cry(b)). Responses to phase shifts suggest that, in the absence of compound eyes, eyelet together with cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring.

To study the function of the neuropeptide pigment-dispersing factor (PDF) in the circadian system of Drosophila, we misexpressed the pdf gene from the grasshopper Romalea in the CNS of Drosophila and investigated the effect of this on behavioral rhythmicity. pdf was either ectopically expressed in different numbers of neurons in the brain or the thoracical nervous system or overexpressed in the pacemaker neurons alone. We found severe alterations in the activity and eclosion rhythm of several but not all lines with ectopic pdf expression. Only ectopic pdf expression in neurons that projected into the dorsal central brain severely influenced activity rhythms. Therefore, we conclude that PDF acts as a neuromodulator in the dorsal central brain that is involved in the rhythmic control of behavior. Overexpression of pdf in the pacemaker neurons alone or in the other neurons that express the clock genes period (per) and timeless (tim) did not disturb the activity rhythm. Such flies still showed a rhythm in PDF accumulation in the central brain terminals. This rhythm was absent in the terminals of neurons that expressed PDF ectopically. Probably, PDF is rhythmically processed, transported, or secreted in neurons expressing per and tim, and additional PDF expression in these cells does not influence this rhythmic process. In neurons lacking per and tim, PDF appears to be continuously processed, leading to a constant PDF secretion at their nerve terminals. This may lead to conflicting signals in the rhythmic output pathway and result in a severely altered rhythmic behavior.

Friedreich ataxia (FA), the most common form of hereditary ataxia, is caused by a deficit in the mitochondrial protein frataxin. While several hypotheses have been suggested, frataxin function is not well understood. Oxidative stress has been suggested to play a role in the pathophysiology of FA, but this view has been recently questioned, and its link to frataxin is unclear. Here, we report the use of RNA interference (RNAi) to suppress the Drosophila frataxin gene (fh) expression. This model system parallels the situation in FA patients, namely a moderate systemic reduction of frataxin levels compatible with normal embryonic development. Under these conditions, fh-RNAi flies showed a shortened life span, reduced climbing abilities, and enhanced sensitivity to oxidative stress. Under hyperoxia, fh-RNAi flies also showed a dramatic reduction of aconitase activity that seriously impairs the mitochondrial respiration while the activities of succinate dehydrogenase, respiratory complex I and II, and indirectly complex III and IV are normal. Remarkably, frataxin overexpression also induced the oxidative-mediated inactivation of mitochondrial aconitase. This work demonstrates, for the first time, the essential function of frataxin in protecting aconitase from oxidative stress-dependent inactivation in a multicellular organism. Moreover our data support an important role of oxidative stress in the progression of FA and suggest a tissue-dependent sensitivity to frataxin imbalance. We propose that in FA, the oxidative mediated inactivation of aconitase, which occurs normally during the aging process, is enhanced due to the lack of frataxin.

Friedreich's ataxia (FRDA) is the most common form of autosomal recessive ataxia caused by a deficit in the mitochondrial protein frataxin. Although demyelination is a common symptom in FRDA patients, no multicellular model has yet been developed to study the involvement of glial cells in FRDA. Using the recently established RNAi lines for targeted suppression of frataxin in Drosophila, we were able to study the effects of general versus glial-specific frataxin downregulation. In particular, we wanted to study the interplay between lowered frataxin content, lipid accumulation and peroxidation and the consequences of these effects on the sensitivity to oxidative stress and fly fitness. Interestingly, ubiquitous frataxin reduction leads to an increase in fatty acids catalyzing an enhancement of lipid peroxidation levels, elevating the intracellular toxic potential. Specific loss of frataxin in glial cells triggers a similar phenotype which can be visualized by accumulating lipid droplets in glial cells. This phenotype is associated with a reduced lifespan, an increased sensitivity to oxidative insult, neurodegenerative effects and a serious impairment of locomotor activity. These symptoms fit very well with our observation of an increase in intracellular toxicity by lipid peroxides. Interestingly, co-expression of a Drosophila apolipoprotein D ortholog (glial lazarillo) has a strong protective effect in our frataxin models, mainly by controlling the level of lipid peroxidation. Our results clearly support a strong involvement of glial cells and lipid peroxidation in the generation of FRDA-like symptoms.