The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces postmating responses (PMR) of females via the sex-peptide receptor (SPR) . Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. Here, we developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First we demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next we tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group; therefore, we speculate that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.KEYWORDS sex-peptide; SP receptor; GFP-tag; postmating response T HE mating behavior and physiology of Drosophila melanogaster females are dramatically modified after copulation: they reject courting males by extruding their ovipositor, a behavior not seen in virgin females and start to lay many eggs (Kubli 1992). Sex-peptide (SP), a seminal fluid peptide, has been shown to play a major role in eliciting postmating response (PMR). Injection of SP into the abdominal cavity of virgin females reduces receptivity and stimulates oviposition (Chen et al. 1988) and these phenotypes can be induced by ectopic expression of SP in virgin females (Aigaki et al. 1991). Furthermore, experiments involving an SP null mutant generated by gene targeting and dsRNAi-mediated gene knockdown unambiguously demonstrated that SP is a major component in inducing changes in receptivity, ovulation, and oviposition in mated females (Liu and Kubli 2003;Chapman et al. 2003). In addition, SP stimulates food intake (Hanin et al. 2011) and activates immune response genes (Peng et al. 2005b).An extensive transgenic RNAi screen identified a receptor for sex-peptide (SPR), a G-protein-coupled receptor broadly expressed in the female reproductive tracts and in some neural tissues (Yapici et al. 2008). Mutant females deleted for SPR do not respond to mating, accept repeated mating, and maintain a low level of oviposition (Yapici et al. 2008). It has been demonstrated that SPR expression in a limited number of pickpocket-expressing neurons in the female common oviduct was...

The Drosophila larval neuromuscular system serves as a valuable model for studying the genes required for synaptic development and function. N-Ethylmaleimide sensitive factor (NSF) is a molecule known to be important in vesicular trafficking but neural expression of a dominant negative form of NSF2 induces an unexpected overgrowth of the Drosophila larval neuromuscular synapse. We have taken a genetic approach to understanding this novel phenotype by conducting a gain-of-function modifier screen to isolate genes that interact with the overgrowth phenotype. Our approach was to directly visualize the neuromuscular junction (NMJ) using a GFP transgene and screen for suppressors of NMJ overgrowth using the Gene Search collection of P-element insertions. Of the 3000 lines screened, we identified 99 lines that can partially restore the normal phenotype. Analysis of the GS element insertion sites by inverse PCR and comparison of the flanking DNA sequence to the Drosophila genome sequence revealed nearby genes for all but 10 of the 99 lines. The recovered genes, both known and predicted, include transcription factors, cytoskeletal elements, components of the ubiquitin pathway, and several signaling molecules. This collection of genes that suppress the NSF2 neuromuscular junction overgrowth phenotype is a valuable resource in our efforts to further understand the role of NSF at the synapse.

In context of the semi-sterility exhibited by Drosophila males expressing certain mating-enabling fruitless (fru) mutant genotypes, we examined the transfer of seminal fluid using a transgene that encodes the Sex Peptide (SP) oligopeptide fused to Green Fluorescent Protein (GFP). We found that this fusion construct expresses SP-GFP in a valid manner within accessory glands of the male reproductive system in normal and fru-mutant males. Transfer of SP-GFP to live females was readily detectable during and after copulation. With respect to the pertinent combinations of fru mutations, we demonstrated that these abnormal genotypes cause males to transmit mating-related materials in two aberrant ways: one involving whether any seminal-fluid entities are transferred at all during a given mating; the other revealing an intriguing aspect of these fruitless effects, such that the mutations in question cause males to transfer female-affecting materials in a manner that varies among copulations. In this regard, certain mutant males that do not transfer SP nevertheless are able to transfer sperm: a fru-mated female possessing no GFP who was not fecund initially could produce progeny when seminal-fluid proteins were subsequently supplied by mating with a male that was spermless owing to the effects of a tudor mutation.

N-Ethylmaleimide sensitive factor (NSF) is an ATPase whose activity is important for intracellular trafficking. Previous genetic analysis of Drosophila NSF2 revealed a potential link between NSF and the actin cytoskeleton. The present study was therefore undertaken to specifically examine genetic interactions between the cytoskeleton and NSF. First, we tested for loss-of-function interaction and, indeed, we found that the combination of flies heterozygous for Act5C and NSF2 alleles led to reduced viability. Second, we expanded our gain-of-function approach to include cytoskeletal genes that were not included in our previous screen. Thirteen of 30 genes tested were found to suppress neuromuscular junction (NMJ) overgrowth. Altogether, these data support the idea that diverse NSF2 developmental and physiological phenotypes are related to disruption of the cytoskeleton and the large number of genes which can partially restore NMJ overgrowth and suggests that NSF may function near the top of the actin regulatory pathway.

Spinach leaf ferredoxin and ferredoxin : NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 1 00-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase -Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.Higher-plant chloroplast ferredoxin is a one-electron-carrier protein containing one iron-sulfur cluster (2Fe2S). After being photochemically reduced through photosystem I, it transfers electrons to NADP via a flavoprotein called ferredoxin-NADP reductase, the whole process (NADP photoreduction) being essential to photosynthesis [l]. In addition, plant ferredoxin serves as a substrate for the following enzymes: nitrite reductase, fatty acid desaturase, sulfite reductase, ferredoxin-dependent glutamate synthase (GOGAT) and ferredoxin-thioredoxin reductase, a catalyst involved in the light activation of chloroplastic enzymes 12, 31. Ferredoxin is also the catalyst of cyclic and pseudocyclic photophosphorylations [4]. Ferredoxin thus plays a key role in chloroplast metabolism in dispatching the reducing power to different metabolic pathways. The primary structure of ferredoxin is known in several higher plants and bacteria and serves as a tool for evolutionary studies [S].Mitochondria of certain animal tissues, especially adrenal glands, also contain a peculiar type of ferredoxin more often called adrenodoxin and possessing also one 2Fe2S iron-sulfur center. In adrenal glands, adrenodoxin is reduced by NADPH through the flavoprotein adrenodoxin-NADP reductase, which is the animal counterpart of the plant ferredoxin- NADP reductase. Reduced adrenodoxin can in turn be used by cytochrome P-450 for steroid hydroxylation reactions [6].The similarities and differences between plant and animal ferredoxins and their reductases are summarized below. a) Ferredoxin and adrenodoxin have similar molecular masses (10.5...