Identification of a novel nuclear-localized adenylate kinase from Drosophila melanogaster

As a step to further understand the role of adenylate kinase (AK) in the energy metabolism network, we identified, purified, and characterized a previously undescribed adenylate kinase in Drosophila melanogaster. The cDNA encodes a 175-amino acid protein, which shows 47.85% identity in 163 amino acids to human AK6. The recombinant protein was successfully expressed in Escherichia coli BL21(DE3) strain. Characterization of this protein by enzyme activity assay showed adenylate kinase activity. AMP and CMP were the preferred substrates, and UMP can also be phosphorylated to some extent, with ATP as the best phosphate donor. Subcellular localization study showed a predominantly nuclear localization. Therefore, based on the substrate specificity, the specific nuclear localization in the cell, and the sequence similarity with human AK6, we named this novel adenylate kinase identified from the fly DAK6.     

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate–mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.     

果蝇miRNA的结构与功能及研究策略

miRNA是一类在动植物基因组中广泛存在的小分子非编码RNA, 作为真核细胞转录后水平上基因表达的关键调控者, 它通过与靶基因mRNA的特定位点结合, 抑制mRNA的翻译或诱导mRNA的降解, 从而介导生物体的许多重要生理活动。本文简要总结了模式昆虫黑腹果蝇Drosophila melanogaster miRNA的鉴定情况, 综述了miRNA的结构特征、生物合成途径和作用机制。miRNA可能同时调节成百上千个靶标, 其生物功能主要体现为: 调节细胞分化与凋亡, 调节器官和神经系统的发育, 控制肌肉分化, 保持能量动态平衡, 调节昆虫变态或综合调节作用。miRNA具有“低丰度、短序列、难富集”的特点, miRNA基因的获得和功能鉴定研究的基本策略是实验生物学和生物信息学方法的有机结合。鉴定新miRNA及其靶标, 深入研究其生物功能和基因进化等可能成为今后一段时间昆虫miRNA研究的重要内容。     

Identification and developmental characterization of a novel Y-box protein from Drosophila melanogaster.

The Y-box proteins are a family of highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this report we have identified a new member of this family from Drosophila melanogaster. Degenerate-PCR was used to identify a conserved region within the highly conserved cold-shock domain (CSD) of Y-box proteins. Subsequently, the cDNA for this gene was sequenced, and the identified open reading frame was named ypsilon schachtel (yps). The expression pattern of yps indicates that this gene is expressed throughout development with the highest level of expression found in adult flies. In situ hybridization shows that the yps mRNA is maternally loaded into the egg cytoplasm. In addition, there appears to be expression of yps mRNA in mesodermal tissue during embryogenesis. YPS, while containing a conserved CSD, is novel in that it completely lacks the alternating acidic and basic regions found in the C-terminus of the other vertebrate eukaryotic Y-box proteins. The CSD of yps was purified and gel-shift analysis showed that this domain can interact with RNA. We predict that YPS would be an RNA-binding protein due to these results and the motifs which have been identified within the amino acid sequence.     

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.     

Characterization of a novel Drosophila melanogaster acylphosphatase

Analysis of the Drosophila melanogaster EST database led to the characterization of a novel acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher K(cat) but lower K(m) with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes.     

Identification and characterization of a novel Drosophila melanogaster glutathione S-transferase-containing FLYWCH zinc finger protein

Glutathione SH-transferase (GST) is a 25-kDa protein and a member of a large family that plays a critical role in the cellular homeostasis of all organisms. In this report, we describe a novel GST-containing protein identified and cloned from Drosophila. This 1045 amino acid protein possesses a zinc finger domain with a tandem array of four FLYWCH zinc finger motifs at its N-terminus and a C-terminal domain that shares a 46% homology with GST. The gene maps to chromosome 3 at position 84C6. Further characterization of this protein shows that it localizes to the cytoplasm of fly cells and is expressed through all stages of fly embryonic development. It binds to glutathione-S agarose beads in vitro. These results indicate that this new protein belongs to the GST family, thus named a Drosophila GST-containing FLYWCH zinc finger protein (dGFZF).     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

Identification and phylogenetic analysis of Drosophila melanogaster myosins

Myosins constitute a superfamily of motor proteins that convert energy from ATP hydrolysis into mechanical movement along the actin filaments. Phylogenetic analysis currently places myosins into 17 classes based on class-specific features of their conserved motor domain. Traditionally, the myosins have been divided into two classes depending on whether they form monomers or dimers. The conventional myosin of muscle and nonmuscle cells forms class II myosins. They are complex molecules of four light chains bound to two heavy chains that form bipolar filaments via interactions between their coiled-coil tails (type II). Class I myosins are smaller monomeric myosins referred to as unconventional myosins. Now, at least 15 other classes of unconventional myosins are known. How many myosins are needed to ensure the proper development and function of eukaryotic organisms? Thus far, three types of myosins were found in budding yeast, six in the nematode Caenorhabditis elegans, and at least 12 in human. Here, we report on the identification and classification of Drosophila melanogaster myosins. Analysis of the Drosophila genome sequence identified 13 myosin genes. Phylogenetic analysis based on the sequence comparison of the myosin motor domains, as well as the presence of the class-specific domains, suggests that Drosophila myosins can be divided into nine major classes. Myosins belonging to previously described classes I, II, III, V, VI, and VII are present. Molecular and phylogenetic analysis indicates that the fruitfly genome contains at least five new myosins. Three of them fall into previously described myosin classes I, VII, and XV. Another myosin is a homolog of the mouse and human PDZ-containing myosins, forming the recently defined class XVIII myosins. PDZ domains are named after the postsynaptic density, disc-large, ZO-1 proteins in which they were first described. The fifth myosin shows a unique domain composition and a low homology to any of the existing classes. We propose that this is classified when similar myosins are identified in other species.     

Identification of chromosome inheritance modifiers in Drosophila melanogaster

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.     

Identification of two novel Drosophila melanogaster histamine-gated chloride channel subunits expressed in the eye.

Histamine has been shown to play a role in arthropod vision; it is the major neurotransmitter of arthropod photoreceptors. Histamine-gated chloride channels have been identified in insect optic lobes. We report the first isolation of cDNA clones encoding histamine-gated chloride channel subunits from the fruit fly Drosophila melanogaster. The encoded proteins, HisCl1 and HisCl2, share 60% amino acid identity with each other. The closest structural homologue is the human glycine alpha3 receptor, which shares 45 and 43% amino acid identity respectively. Northern hybridization analysis suggested that hisCl1 and hisCl2 mRNAs are predominantly expressed in the insect eye. Oocytes injected with in vitro transcribed RNA, encoding either HisCl1 or HisCl2, produced substantial chloride currents in response to histamine but not in response to GABA, glycine, and glutamate. The histamine sensitivity was similar to that observed in insect laminar neurons. Histamine-activated currents were not blocked by picrotoxinin, fipronil, strychnine, or the H2 antagonist cimetidine. Co-injection of both hisCl1 and hisCl2 RNAs resulted in expression of a histamine-gated chloride channel with increased sensitivity to histamine, demonstrating coassembly of the subunits. The insecticide ivermectin reversibly activated homomeric HisCl1 channels and, more potently, HisCl1 and HisCl2 heteromeric channels.     

Identification of common excitatory motoneurons in Drosophila melanogaster larvae

In insects, four types of motoneurons have long been known, including fast motoneurons, slow motoneurons, common inhibitory motoneurons, and DUM neurons. They innervate the same muscle and control its contraction together. Recent studies in Drosophila have suggested the existence of another type of motoneuron, the common excitatory motoneuron. Here, we found that shakB-GAL4 produced by labels this type of motoneuron in Drosophila larvae. We found that Drosophila larvae have two common excitatory motoneurons in each abdominal segment, RP2 for dorsal muscles and MNSNb/d-Is for ventral muscles. They innervate most of the internal longitudinal or oblique muscles on the dorsal or ventral body wall with type-Is terminals and use glutamate as a transmitter. Electrophysiological recording indicated that stimulation of the RP2 axon evoked excitatory junctional potential in a dorsal muscle.     

Dual-tagging gene trap of novel genes in Drosophila melanogaster

A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.     

Nested hierarchal organization of conservation for microRNAs and their putative targets to Drosophila melanogaster

This study examined microRNA network properties traced through taxonomic hierarchy considering both the acquisition of potential network targets and regulators. Primary literature review and database analyses were conducted to establish modules of conserved microRNAs across metazoan taxonomy. A hierarchical schema for the conservation of microRNAs and their putative targets to Drosophila melanogaster was engineered through comprehensive meta-analysis, and conservation history of 90.39% of the total Drosophila dataset could be resolved through this hierarchical sampling regime; tracing from taxonomic order down to empire. The findings presented in this study represent a documentation of Drosophila microRNA regulatory network behavior thorough taxonomic hierarchy. MicroRNA regulatory network properties were found to transect taxonomic hierarchy. Newly acquired microRNAs from novel families reinforce the pre-existing regulatory network, while expanding the target list to include a small number of novel genes. Lineage specific microRNAs were found to exhibit far fewer conserved targets than do the more broadly conserved microRNAs; even when considering only more recently emerged targets. There was a dramatic expansion in network complexity with the expansion of the microRNA repertoire, and this corresponds to the expansion in biological complexity.