Background A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila . These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. Results We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora , and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. Conclusion The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

Huntington's disease (HD) is caused by an extended polyglutamine (polyQ) tract in the Huntingtin protein. Neuronal and glial dysfunction precedes the neurodegeneration and appears to be the primary cause for the early symptoms in HD. In recent years, development of Drosophila models of polyQ-related diseases facilitated research of candidate rescuer genes. In most cases, analysis in Drosophila was performed by assessing toxicity on retinal and/or brain neurons. However, none of the potential rescuers were evaluated on glial alterations. Here we used a genetic approach in Drosophila to characterize the phenotypic effects of mutant Huntingtin (mHtt) expressed in neurons or different glia subsets and we established a sensitive assay for evaluating modifiers of glial alterations. We determined the level of cell protection ensured by activation of the AKT and ERK anti-apoptotic kinases in the retina as well as in neurons and glia of the fly brain, compared with the rescuing effects of the HSP70 chaperone. We found that both AKT and HSP70 alleviated mHtt-induced toxicity in the retina. In contrast, their protective effects differed in the brain. HSP70 rescued neurodegeneration, locomotor defects and early lethality of flies expressing mHtt in neurons or glia. AKT failed to prevent brain neuronal death and lethality of flies, but significantly improved their locomotor performance when co-expressed with mHtt in glia. ERK had no beneficial effects in the retina or brain. These results indicate that mHtt activates distinct pathways of toxicity in Drosophila, either sensitive to AKT in retinal photoreceptors and glia, or independent in brain neurons.

Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and size selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV (NRX IV) are required for the formation of SJs. In this study, we report the genetic, molecular and biochemical characterization of the Drosophila homolog of vertebrate contactin, CONT. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that CONT is required for organization of SJs and paracellular barrier function. We show that CONT, Neuroglian (NRG) (Drosophila homolog of NF-155) and NRX IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, our data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules.

Calpains are neutral Ca2+-dependent cysteine proteases. In this study, we utilized casein zymography to detect such a proteolytic activity in Drosophila melanogaster extracts throughout the life of this organism. One calpain-like activity that was sensitive to the general cysteine protease inhibitors, E64 and calpain inhibitor I, but insensitive to the human calpain-specific inhibitor, calpastatin, is demonstrated. The relevance of this finding is discussed with respect to the absence of a corresponding Drosophila gene, homologous to the vertebrate calpastatin genes, as concluded from our unsuccessful attempts to clone such a gene and our Blast searches using the FlyBase. The mechanisms of Drosophila calpain regulation require further investigation. However, we suggest that single chain, non-heterodimeric calpains may be insensitive to calpastatin and that Drosophila cystatin-like molecules may play a role in negatively regulating Drosophila calpain.

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

We have previously shown the presence, in the amplified DNA of a Drosophila cell line resistant to N-phosphonacetyl-L-aspartate (PALA), of two units of 150 kb and 120 kb respectively duplicated and amplified. The two joints (J1 and J2) linking these units as well as their respective wild-type counterparts have been sequenced. Sequence analysis indicates that a region of the Drosophila genome which corresponds to the proximal boundary of the 150 kb unit is common to both joints. In addition to this common region, the J1 junction possesses a 26-nucleotide sequence belonging to the J2 junction. This indicates that the J2 junction was the first formed, and that J1, therefore, results from recombination between J2 and a region of the wild-type genome 120 kb distal to J2. Sequence analysis also reveals that the joints result from illegitimate recombination between unrelated regions. AT-rich sequences, strand bias composition and putative topoisomerase I and II sites were found in at least one of the two parental sequences involved in the formation of the joints. On the basis of these results we can hypothesize that after two illegitimate recombinations between sister chromatids, leading first to J2 and then to J1, the amplification may have arisen by a series of homologous (unequal crossing-over) or illegitimate recombinations, or by an intrachromosomal rolling circle.

Two ecdysone-response elements from the hsp27 (hsp27 EcRE) and the Fbp1 (D EcRE) genes of Drosophila melanogaster were used as probes in a gel shift assay to investigate the interactions of the ecdysone receptor (EcR) with its cognate DNA response element. The source of EcR was a nuclear extract from the late third-larval instar fat body. The hsp27 and D EcREs share a sequence similarity at 12 positions over a 15bp region including an imperfect palindromic structure consisting of two pentamer half-sites separated by a single intervening nucleotide. We have shown that a short oligonucleotide containing this 11bp imperfect palindrome of the hsp27 EcRE and three flanking bp on each side is an efficient EcR binding site. Mutational analysis confirms that the integrity of both these half-sites as well as their 1bp spacing are critical for binding of the ecdysone receptor. The D EcRE behaved as a much weaker EcR binding sapacity equivalent to that of the hsp27 EcRE. These results have led us to propose the sequence PuG(G/T)T(C/G)A(N)TG(C/A)(C/A)(C/t)Py as a revised version of a previously proposed EcRE consensus sequence.

Transcription of the D. melanogaster Fat-body-protein-1 (Fbp1) gene is induced by the steroid hormone 20-hydroxyecdysone and is restricted to the fat body tissue at the end of the third larval instar. The location and functional properties of the Fbp1 cis-acting regulatory sequences contained in the region from - 1386 to + 80 relative to the transcription start were examined by transformation using hybrid constructs with the Adh or lacZ genes as reporters. Regulatory element(s) required for the full level of transcription of the Fbp1 gene were located between positions - 1386 and - 138. Sequences between - 138 and - 68 were able to drive transcription from a heterologous minimal promoter in the fat body of late third instar larvae. Remarkably, these sequences also conferred 20-hydroxyecdysone inducibility and behaved as an enhancer-like element. These results provide the first functional characterization, at the level of the whole organism, using a direct in vivo ecdysone induction assay, of a discrete ecdysone response element.

We have used 160 kilobases of cloned Drosophila genomic DNA from the rudimentary (r) region to examine the organization of amplified DNA in Drosophila cells resistant to 10 mM N-(phosphonacetyl)-l-aspartate (PALAr cells) obtained by stepwise selection. Evidence for the direct tandem linkage of the amplified sequences is presented. The pattern and intensity of amplified bands as well as the presence of novel junctions in the DNA sequence of PALAr cells indicate that there are two types of units of 150 and 120 kilobases long. The sequence of the smaller unit is entirely included within the larger one. The longer of the two units is present twice while the shorter one is amplified eightfold as compared to the level of the relevant DNA sequences in the wild-type. These data are consistent with a model in which successive crossing-over events over several cell cycles lead to amplification of the selected r gene and flanking sequences. However, these data can also be accounted for by a totally different mechanism in which multiple copies of DNA are generated by rolling circle replication. Transcription units other than the r gene are present within the 150 kilobase region of amplified DNA. These are found to be overexpressed in PALA' cells, though some transcripts are underrepresented relative to the copy number of the corresponding coding sequences.

Drosophila cells were treated in vitro with N-phosphonacetyl-L-aspartate (PALA) which is a specific inhibitor of aspartate transcarbamylase, the second enzyme of the pyrimidine biosynthetic pathway. By stepwise selection using increasing amounts of this inhibitor, PALA-resistant (PALAr) stable clones have been isolated. Enzymatic activities of aspartate transcarbamylase, carbamyl phosphate synthetase and dihydro-orotase, borne by the same multifunctional protein, CAD, are increased 6-12-fold in these resistant clones compared with parental cells. The aspartate transcarbamylase in PALAr cells is shown by physical, kinetic and immunological criteria to be normal. The data from immunotitration and immunoblotting experiments indicate that the increased enzyme activities result from the overproduction of CAD.

In Drosophila melanogaster the rudimentary locus encodes for a multifunctional protein catalyzing the first three enzymatic activities of pyrimidine biosynthesis. Cell lines were selected which were resistant to PALA (N‐(phosphonoacetyl)‐L‐aspartate), a specific inhibitor of aspartate transcarbamylase, the second enzyme of this pathway. In a cell line where the enzyme production is increased 5 times, Southern blot analyses show that the rudimentary gene and surrounding regions are amplified about 5 times. In this case gene amplification could therefore account for the observed enzyme overproduction.