The Glutamate Dehydrogenase Gene of Drosophila Melanogaster: Molecular Analysis and Expression

Glutamate dehydrogenase is an enzyme that, in addition to its role in the energy metabolism in mitochondria, is involved in neuromuscular transmission. Here we present the structure and sequence of the Gdh gene of Drosophila melanogaster, as well as the analysis of its spatial and temporal pattern of expression. Unlike all other organisms analyzed so far, two forms of the enzyme, differing by the inclusion of 13 extra amino acids, are found in the fruitfly. We show the presence of Gdh mRNA in several tissues of the developing embryo, including the central nervous system, muscles and the alimentary tract. Moreover, we detect the localization of the Gdh protein in specific areas of the muscles, a fact that is consistent both with an involvement in energy metabolism and the role of glutamate as the major neuromuscular transmitter in Drosophila.     

Two Isoforms of Drosophila Dynamin in Wild-Type and Shibirets Neural Tissue: Different Subcellular Localization and Association Mechanisms

The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M_r of 92 kD and 94 kD have been detected in wild-type and shi^ts adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2,000 × g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 × g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65–75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na₂CO₃ (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na₂CO₃ treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi^ts alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis'arrest by GTP-gM-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi^ts mutations and GTP-gM-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).     

The vesicle cycle in motor nerve endings of the mouse diaphragm

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1–2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5–6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 2, pp. 129–141, February, 2008.     

Leaky synapses: regulation of spontaneous neurotransmission in central synapses

The mechanisms underlying spontaneous neurotransmitter release are not well understood. Under physiological as well as pathophysiological circumstances, spontaneous fusion events can set the concentration of ambient levels of neurotransmitter within the synaptic cleft and in the extracellular milieu. In the brain, unregulated release of excitatory neurotransmitters, exacerbated during pathological conditions such as stroke, can lead to neuronal damage and death. In addition, recent findings suggest that under physiological circumstances spontaneous release events can trigger postsynaptic signaling events independent of evoked neurotransmitter release. Therefore, elucidation of mechanisms underlying spontaneous neurotransmission may help us better understand the functional significance of this form of release and provide tools for its selective manipulation. For instance, our recent investigations indicate that the level of cholesterol in the synapse plays a critical role in limiting spontaneous synaptic vesicle fusion. Therefore, alterations in synaptic cholesterol metabolism can be a critical determinant of glutamatergic neurotransmission at rest. This article aims to provide a closer look into our current understanding of the mechanisms underlying spontaneous neurotransmission and the signaling triggered by these unitary release events.     

Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system

Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 μM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 μM) by high K⁺ or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2–30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 μM) as well as nominally zero external Ca²⁺ (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca²⁺]_i increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be ∼27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.     

Generation of a Semi-Dominant Mutation with Temperature Sensitive Effects on Both Locomotion and Phototransduction in Drosophila Melanogaster

A novel Drosophila mutant named Tripped-and-fell (Taf)) was isolated in a F1 screen for dominant temperature sensitive paralytics. Recombination mapping using multiply marked chromosomes and P elements have pinpointed the locus of Taf to polytene band 93 on the right arm of the third chromosome (3R). When exposed to restrictive temperatures, both Taf heterozygotes and homozygotes paralyzed; however, homozygotes paralyzed at lower temperatures and took longer to recover than heterozygotes. There are also positive correlations between recovery time from paralysis and both duration and temperature of exposure. Electroretinograms (ERGs) revealed that both homozygotes and heterozygotes have a grossly normal light response at 22°C, but at 37°C, the ERGs from both homozygotes and heterozygotes are unable to maintain a normal sustained depolarization and have ar educed off-transient potential. The severity of the ERG repolarization phenotype is greater in homozygotes than in heterozygotes.     

Alternative Splicing of Interleukin-6 mRNA in Mice

Expression of mRNA for interleukin-6, interleukin-6Delta3, and interleukin-6Delta5 was detected in placental tissue (second and third trimesters of pregnancy) and spleen of mice immunized with sheep erythrocytes in high dose. We hypothesize that translation of mRNA yields proteins capable of binding to individual subunits of the interleukin-6 receptor and possessing effector functions.     

Vesicle recycling at ribbon synapses in the finely branched axon terminals of mouse retinal bipolar neurons

In retinal bipolar neurons, synaptic ribbons mark the presence of exocytotic active zones in the synaptic terminal. It is unknown, however, where compensatory vesicle retrieval is localized in this cell type and by what mechanism(s) excess membrane is recaptured. To determine whether endocytosis is localized or diffuse in mouse bipolar neurons, we imaged FM4-64 to track vesicles in cells whose synaptic ribbons were tagged with a fluorescent peptide. In synaptic terminals, vesicle retrieval occurred at discrete sites that were spatially consistent over multiple stimuli, indicative of endocytotic “hot spots.” Retrieval sites were spatially correlated with fluorescently labeled synaptic ribbons. Electron microscopy (EM) analysis of bipolar cell terminals after photoconversion of internalized FM dye revealed that almost all of the dye was contained within vesicles ∼30 nm in diameter. Clathrin-coated vesicles were observed budding from the plasma membrane and within the cytosol, and application of dynasore, a dynamin inhibitor, arrested membrane retrieval just after the budding stage. We conclude that synaptic vesicles in the fine branches of mouse bipolar axon terminals are retrieved locally near active zones, at least in part via a clathrin-mediated pathway.     

Chronobiological Analysis of a New Clock Mutant,Toki, in Drosophila Melanogaster

We have isolated a new semidominant clock mutant Toki on the second chromosome in Drosophila melanogaster. This mutant differs from the wild-type Canton-S in several properties as follows. Larger values are obtained in the phase angle difference (ψ, the time from lights-off in a 24-hr light-dark cycle to an activity offset), the ratio of activity time to rest time (α/ρ) and the activity level. The free-running period (τ) is 25.3 hr, one hour longer than in the wild-type. In the phase response curve (PRC), the ratio between the delay and the advance portion is larger and the cross-over point occurs later, although there is no difference in amplitude of the mutant's PRC (Type I). The rhythm is more sensitive to the light intensity, becoming obscure in darker condition. Toki interacts with other clock mutations, per^S, per^L and And, in such a way that τ s associated with these three X-linked mutations are lengthened and ψ values become smaller.     

Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a

Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.     

Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.     

Molecular and Behavioral Analysis of Sex-Linked Courtship Song Variation in a Natural Population of Drosophila Melanogaster

Genes controlling the “lovesong” in Drosophila are particularly interesting under a evolutionary point of view as they could be involved in the reproductive isolation between closely related species and, as a consequence, in the speciation process. We carried out a survey of sex-linked molecular and behavioral courtship song variation in 27 lines derived from a natural population of Drosophila melanogaster in Italy. We sequenced a 983 bp fragment of cacophony(cac), a calcium channel gene controlling aspects of the courtship song. The same region was also sequenced in a D. simulans strain. Only 5 non-coding sites were polymorphic among the D. melanogaster lines, and no aminoacid substitutions were found between the two species. Statistical tests applied to the data did not reveal any significant deviations from a neutral model. Using the same lines we also carried out an analysis of three different song parameters which are known to be affected by the cac^S song mutation: interpulse-interval (IPI), pulse amplitude (PA) and cycles per pulse (CPP). We found significant differences among the lines in IPI and PA, and for the latter a significant association with one of the polymorphic sites of cac.     

选择性剪接在低氧应答中的调控作用

细胞为适应低氧环境,其相关基因的表达方式发生了改变,其中选择性剪接在低氧应答调控过程中起到了重要的作用.低氧诱导因子介导的低氧应答信号通路在机体适应低氧环境过程中起到了十分重要的作用,低氧诱导因子剪接体通过此通路调控红细胞生成、血管生成、糖酵解等过程.而抑制性PAS蛋白质、脯氨酸羟化酶、促血管生长因子、芳香羟受体核转运蛋白剪接体则通过其它通路进行调控.选择性剪接不仅在低氧应答中起重要作用,而且与阿尔茨海默病、动脉粥样硬化、癌症等常见人类疾病相关.     

肿瘤细胞中基因选择性剪接的研究进展

选择性剪接是高等真核细胞在转录后水平调控基因表达以及产生蛋白质组多样性的重要机制。选择性剪接过程受多种顺式作用元件和反式作用因子相互作用调节。肿瘤癌基因、抑癌基因、肿瘤转移抑制基因可发生选择性剪接，与肿瘤发生发展关系密切，其蛋白异构体参与基因转录、细胞周期和凋亡等生命过程，对肿瘤生长有一定作用。以选择性剪接蛋白异构体为靶点或干预选择性剪接过程，可望进行肿瘤的分子治疗。