TRP gating is linked to the metabolic state and maintenance of the Drosophila photoreceptor cells

The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.     

The TRP channel and phospholipase C-mediated signaling

Drosophila photoreceptors use a phospholipase C-mediated signaling for phototransduction. This pathway begins by light activation of a G-protein-coupled photopigment and ends by activation of the TRP and TRPL channels. The Drosophila TRP protein is essential for the high Ca²⁺ permeability and constitutes the major component of the light-induced current, thereby affecting both excitation and adaptation of the photoreceptor cell. TRP is the prototype of a large and diverse multigene family whose members are sharing a structure, which is conserved through evolution from the worm Caenorhabditis elegans to humans. TRP-related channel proteins are found in a variety of cells and tissues and show a large functional diversity although the gating mechanism of Drosophila TRP and of other TRP-related channels is still unknown.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Metabolic pools differ among ecologically diverse Drosophila species

Studies of the genetic mechanisms underlying metabolic storage have focused on a few model organisms. Although very fruitful, these studies have not allowed for the examination of mechanisms across a phylogenetic spectrum. The exploration of natural patterns of metabolic pool size variation across species will help us to better understand the genetics of metabolic adaptation.We examined the metabolic pools size (triglyceride, glycogen and protein) at two ages in 12 Drosophila species with distinctly different ecologies for which complete genome sequences (for 11 of the 12 species) are known. Overall, there were significant differences across species for all three pools, while age and sex appear to affect some metabolic pools more than others. After correcting for the phylogenetic relatedness of the species used, we observed no association between triglyceride and glycogen content. Although within species these two pools sometimes are correlated, at a larger phylogenetic scale control of triglyceride and glycogen contents may have been shaped independently by natural selection.     

Retention of autonomous replicating plasmids in cultured Drosophila cells

Summary Six kinds of autonomously replicating sequences (ARSs) derived from Drosophila or tobacco were inserted into the vector pDSV, constructed with pSV2-gpt and the copia long terminal repeat (LTR). The resulting ARS-containing plasmids, pDSV-ARSs, were transfected into the cultured Drosophila cells of GM1 S1cl1. Most of the plasmids remained for about 2 weeks and some for about 1 month in these cells. The retention time of the plasmid was not directly correlated with autonomously replicating activity of ARSs detected in the yeast. Two plasmids, one carrying ARS of Drosophila nuclear DNA and the other carrying tobacco DNA, showed the longest retention time in transformed cells and replication was confirmed in these cells. Some of these long lived plasmids were recovered, however, as modified forms. Other plasmids had disappeared 1 month after transfection. Two months following transfection, none of plasmids were recovered but they were detected in nuclear DNA as the integrated form. The integration patterns in all the cells transformed by different kinds of ARS-containing plasmids were similar to each other, and to the distribution pattern of copia LTR in the genome. These results suggest that copia LTR sequences contained in the pDSV-ARSs may participate in the integration process of these plasmids into Drosophila DNA.     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

RNA干扰技术在果蝇中的应用

RNA干扰是双链RNA特异诱导的转录后期基因沉默.该技术随着不断完善而越来越被广泛地运用于果蝇的功能基因组研究上,双链RNA已经成为果蝇中功能基因的一个十分有效的抑制子,势必使RNA干扰技术成为研究果蝇体内基因功能的强有力的反向遗传学研究技术.     

Postembryonic Hematopoiesis in Drosophila

We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte.     

以果蝇模型研究人类神经退行性疾病

人类神经退行性疾病是一类以神经元退行性病变导致个体行为异常乃至死亡为主要特征的疾病.果蝇以其独特的分子遗传学优势成为研究人类神经退行性疾病的理想模型.通过对果蝇模型的研究不仅可以揭示神经退行性疾病发生的细胞分子通路,还可以研究对疾病有调控作用的基因及其表达产物,寻找可能的药物作用靶点并进行药物筛选,为防治人类神经退行性疾病提供新的方法和有效的药物.     

TRP channels in Drosophila photoreceptors: the lipid connection

The light-sensitive current in Drosophila photoreceptors is mediated by transient receptor potential (TRP) channels, at least two members of which (TRP and TRPL) are activated downstream of phospholipase C (PLC) in response to light. Recent evidence is reviewed suggesting that Drosophila TRP channels are activated by one or more lipid products of PLC activity: namely diacylglycerol (DAG), its metabolites (polyunsaturated fatty acids) or the reduction in phosphatidylinositol 4,5-bisphosphate (PIP₂). The most compelling evidence for this view comes from analysis of rdgA mutants which are unable to effectively metabolise DAG due to a defect in DAG kinase. The rdgA mutation leads to constitutive activation of both TRP and TRPL channels and dramatically increases sensitivity to light in hypomorphic mutations of PLC and G protein.     

果蝇学习记忆行为的分子机制

分子遗传学技术的应用一方面发展了新的神经组织学方法,使果蝇脑中的细微结构得以展示;另一方面,对记忆从形成到提取过程中信息处理的研究,表明蘑菇体可能在形成长时程记忆方面起重要作用,而一对背内侧核团（dorsal paired medial cells）与蘑菇体之间的信息传递对于记忆的“提取(retrieval)”是至关重要的.行为功能检测为视觉信号整和的研究提供了新的实验依据,从而使果蝇蘑菇体的高级脑中枢功能逐渐被揭示出来.     

A mitochondrial nuclease is modified in Drosophila mutants (mus308) that are hypersensitive to DNA crosslinking agents

Summary The mus308 mutants of Drosophila have previously been demonstrated to be defective in an enzyme that is designated Nuclease 3 (Boyd et al. 1990b). In this study that enzyme is shown to be present in mitochondria of both wild-type flies and embryos. Since the mus308 mutants are hypersensitive to DNA crosslinking agents, Nuclease 3 is potentially required for resistance of the mitochondrial genome to such agents. In support of this hypothesis, electron microscopic studies of mus308 mutant flies that had been exposed to nitrogen mustard revealed an increased frequency of mitochondrial abnormalities. Further investigation of the defect at the enzymological level revealed that the mutants possess a new nuclease activity that is apparently a modified form of the wild-type protein. In the earlier study, enzyme extracts from mus308 mutants were found to lack an enzyme with a pl of approximately 6.2. More precisely defined assay conditions in this study revealed the appearance of a new nuclease activity with a higher pI in extracts from mutants. This observation, together with the finding that only the normal enzyme form is present in heterozygous individuals, supports the hypothesis that the mus308 locus is not the structural gene for the enzyme. Rather, the mus308 gene product is necessary for Nuclease 3 to assume the lower pI. Nuclease 3 has been partially purified and characterized from wild-type embryos. Its activity is stimulated by Mg⁺⁺ and ATP. Optimum activity is found at a pH of 5.5 and a NaCl concentration of 50–100 mM. Nuclease 3 exhibits a temperature optimum of 42°C and is insensitive to N-ethylmaleimide. The enzyme is probably membrane-associated because it exhibits a strong tendency to aggregate and detergent is required for full solubilization.     

Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K⁺ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.     

Genotoxicity is modulated by ascorbic acid: Studies using the wing spot test in Drosophila

The ability of ascorbic acid (Vitamin C) to modulate the genotoxic action of several mutagens was investigated in the wing spot test of Drosophila melanogaster. In this assay, 3-day-old transheterozygous larvae for the multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) genes were treated with three reference mutagenic compounds, namely cobalt chloride (CoCl₂), 4-nitroquinoline 1-oxide (4-NQO) and potassium dichromate (K₂Cr₂O₇). The results obtained show that the three reference mutagens tested were clearly genotoxic in the Drosophila wing somatic mutation and recombination test (SMART). None of the three concentrations tested of ascorbic acid (25, 75 and 250 mM) induced significant increases in the frequency of the mutant clones recorded. When co-treatment experiments with ascorbic acid were carried out, different results were found. Thus, ascorbic acid was effective in reducing the genotoxicity of K₂Cr₂O₇ virtually to the control level; on the contrary, it did not show any antigenotoxic effect on the genotoxicity of 4-NQO. Finally, co-treatments with CoCl₂ and ascorbic acid show a significant increase in the frequency of mutant clones over the values obtained with CoCl₂ alone.     

JNK protects Drosophila from oxidative stress by trancriptionally activating autophagy

JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, we investigated the role of autophagy as such a JNK-regulated protective mechanism. We show that oxidative stress can induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms.     

Cell proliferation, survival, and death in the Drosophila eye

The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

Normal prion protein in Drosophila enhances the toxicity of pathogenic polyglutamine proteins and alters susceptibility to oxidative and autophagy signaling modulators

To investigate the in vivo functions of normal prion protein (PrP) in Drosophila, we utilized characterized transgenic flies expressing 3F4-tagged mouse PrP (Mo-PrP^3F4). The neurotoxicity of pathogenic Machado-Joseph Disease (MJD) glutamine (Q) 78 and 127Q proteins were enhanced by the co-expression of Mo-PrP^3F4 in the fly eyes, while the eyes of controls flies and flies expressing Mo-PrP^3F4 alone or together with MJD-Q27 or 20Q proteins did not show any defect. Susceptibilities to H₂O₂, paraquat, and Dithiothreitol (DTT) were altered in Mo-PrP^3F4 flies. In addition, Mo-PrP^3F4 flies were significantly more susceptible to the perturbation of autophagy signaling by an autophagy inhibitor, 3-methyladenine (3-MA), and inducer, LiCl. Taken together, our data suggest that Mo-PrP^3F4 may enhance the neurotoxicity of pathogenic Poly-Q proteins by perturbing oxidative and autophagy signaling.     

黑腹果蝇dCAF-1-p55突变引起果蝇发育迟缓和染色体不稳定性

在真核生物中高度保守的染色质装配因子1(chromatin assembly factor 1,CAF-1)是染色质装配过程中的组蛋白分子伴侣之一.dCAF-1-p55是果蝇中CAF-1复合物中的最小亚基,它与另外两个亚基dCAF-1-p180及dCAF-1-p105一起负责将组蛋白H3/H4组装到新合成的DNA上.除了CAF-1复合物,dCAF-1-p55还参与其他多个复合物的形成,如NURF、PRC2及Sin3-HDAC1.dCAF-1-p55的这一广泛参与性提示了其功能的多样性和重要性.为了研究dCAF-1-p55的体内功能,我们利用基因靶向敲除技术制备了果蝇dCAF-1-p55突变体.实验结果表明,dCAF-1-p55的缺失导致果蝇发育迟缓并且最终致死.进一步研究发现,在dCAF-1-p55突变细胞中,中期染色体较为松散,姐妹染色单体连接异常,后期染色体不能正常分离.这些缺陷都是与癌症发生密切相关的染色体不稳定性(chromosome instability,CIN)的典型特征.综上所述,我们的研究表明了dCAF-1-p55在果蝇发育过程及维持染色体稳定性方面的重要作用,同时提示该基因具有保护细胞免遭CIN和癌变的潜在功能.