The cis-regulatory sequences required for expression of the Drosophila melanogaster adult cuticle gene ACP65A.

Post-embryonic development in insects requires successive molts. Molts are triggered by ecdysteroids, and the nature of the molt (larval, pupal or adult) is determined by juvenile hormones. The genes encoding cuticle proteins are targets of both classes of hormones, and therefore are interesting models to study hormone action at the molecular level. The Drosophila ACP65A cuticle gene is expressed exclusively during the synthesis of the adult exoskeleton, in epidermal domains synthesising flexible cuticle. We have examined the cis-regulatory sequences of ACP65A using phylogenetic comparisons and functional analysis, and find that only about 180 bp are essential, including an 81 bp intron. The restriction of ACP65A expression appears to depend on a strong repression mechanism.

The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38.

The regulatory sequences of the Drosophila ACP65A cuticle gene were analyzed in vivo in transgenic flies, using both fusion genes constructs and transposase-mediated deletions within a P element containing ACP65A regulatory sequences fused to the lacZ gene (deletion scanning). The sequences located between -594 and +161 are sufficient to confer both temporal and spatial expression specificities, indicating the presence of tissue-specific enhancers and response elements to hormone-induced factors. In addition, timing of expression and tissue-specificity appear to be controlled by distinct cis-regulatory elements, which suggests the existence of independent hormonal and tissue-specific signaling pathways. Gain and loss of function studies also implicate DHR38, the Drosophila homolog of the vertebrate NGFI-B-type nuclear receptors, as an important activator of the ACP65A gene.

Cloning and characterization of new orphan nuclear receptors and their developmental profiles during Tenebrio metamorphosis.

Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and beta-FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.

Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates.

Autophagy triggered by carbohydrate starvation was characterized at both biochemical and structural levels, with the aim to identify reliable and easily detectable marker(s) and to investigate the factors controlling this process. Incubation of suspension cells in sucrose-free culture medium triggered a marked degradation of the membrane polar lipids, including phospholipids and galactolipids. In contrast, the total amounts of sterols, which are mainly associated with plasmalemma and tonoplast membranes, remained constant. In particular, phosphatidylcholine decreased, whereas phosphodiesters including glycerylphosphorylcholine transiently increased, and phosphorylcholine (P-Cho) steadily accumulated. P-Cho exhibits a remarkable metabolic inertness and therefore can be used as a reliable biochemical marker reflecting the extent of plant cell autophagy. Indeed, whenever P-Cho accumulated, a massive regression of cytoplasm was noticed using EM. Double membrane-bounded vacuoles were formed in the peripheral cytoplasm during sucrose starvation and were eventually expelled into the central vacuole, which increased in volume and squeezed the thin layer of cytoplasm spared by autophagy. The biochemical marker P-Cho was used to investigate the factors controlling autophagy. P-Cho did not accumulate when sucrose was replaced by glycerol or by pyruvate as carbon sources. Both compounds entered the cells and sustained normal rates of respiration. No recycling back to the hexose phosphates was observed, and cells were rapidly depleted in sugars and hexose phosphates, without any sign of autophagy. On the contrary, when pyruvate (or glycerol) was removed from the culture medium, P-Cho accumulated without a lag phase, in correlation with the formation of autophagic vacuoles. These results strongly suggest that the supply of mitochondria with respiratory substrates, and not the decrease of sucrose and hexose phosphates, controls the induction of autophagy in plant cells starved in carbohydrates.