Expression of human plasminogen in Drosophila Schneider S2 cells.

The cDNA that encodes full-length human plasminogen (Glu1-hPg) has been expressed in Drosophila Schneider S2 cells under the influence of the Drosophila BiP protein signal sequence, which allowed the protein to be secreted into the medium. A procedure was devised for clonal selection of high-expressing cells, which were then used for large-scale expression of 10-15 mg/liter of the protein in the culture medium. The protein produced using this system was extensively characterized and contained full-length recombinant (r) Glu1-hPg plasminogen. As with human plasma Glu1-hPg, the S2-expressed protein underwent the Cl--induced transition to the tight conformation, which resulted in a weakly activatable zymogen. The addition of the ligand, epsilon-amino caproic acid, induced the relaxed conformation of r-Glu1-hPg, which was highly activatable, again in agreement with similar data for human plasma Glu1-hPg. The thermal stability of the S2-expressed r-Glu1-hPg also correlated well with that of human plasma hPg. These studies show that intact r-Glu1-hPg can be produced in high yield in Drosophila Schneider S2 cells, which possesses similar properties to its human plasma counterpart.     

Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair.

P-element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8,000 bp of nonhomologous sequence could be copied from an ectopic template at essentially the same rate as a single-base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila melanogaster, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as a template. This synthesis-dependent strand annealing (SDSA) model as a possible general mechanism in complex organisms is discussed.     

A Proton Spin-Echo Study of the State of Water in Frog Nerves

A proton spin-echo study of the water in the sciatic nerve trunk of the bullfrog reveals two distinct types of water as distinguished by their spin-spin relaxation times. The relative concentrations of these two types were determined for nerves which had been treated with normal Ringer's solution and also for nerves which had been treated with potassium-doped (0.15 M) Ringer's solution. In both cases the relative concentrations are the same as those previously determined through signal integration of high resolution proton magnetic resonance spectra obtained for the same systems, although the high resolution studies were performed on nerves doped with paramagnetic ions and the spin-echo studies were performed on undoped nerves. The doping procedure would appear to be a valid and very useful way of studying water in this neural system.     

Stochastic segmentation models for array-based comparative genomic hybridization data analysis

Array-based comparative genomic hybridization (array-CGH) is a high throughput, high resolution technique for studying the genetics of cancer. Analysis of array-CGH data typically involves estimation of the underlying chromosome copy numbers from the log fluorescence ratios and segmenting the chromosome into regions with the same copy number at each location. We propose for the analysis of array-CGH data, a new stochastic segmentation model and an associated estimation procedure that has attractive statistical and computational properties. An important benefit of this Bayesian segmentation model is that it yields explicit formulas for posterior means, which can be used to estimate the signal directly without performing segmentation. Other quantities relating to the posterior distribution that are useful for providing confidence assessments of any given segmentation can also be estimated by using our method. We propose an approximation method whose computation time is linear in sequence length which makes our method practically applicable to the new higher density arrays. Simulation studies and applications to real array-CGH data illustrate the advantages of the proposed approach.     

Monomeric red fluorescent protein variants used for imaging studies in different species

Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation and intracellular fluorescence. mRFPmars, a red fluorescent protein designed especially for the use in Dictyostelium, has been employed to tag different proteins for live-cell investigations in Dictyostelium. mRFPruby, which differs in sequence from mRFPmars in four amino acids, has a codon usage optimised for the application in mammalian cells. Here, we show that both mRFP variants can also be applied for localisation studies in other organisms. mRFPmars was expressed in Hydra and fused to the Bcl-2 family protein Bax. mRFPruby in combination with histone 2B was expressed in Drosophila S2 cells to monitor mitosis. Using mouse cell lines, mRFPruby fused to beta-actin was assayed with high spatial resolution to study details of actin cytoskeleton dynamics. In addition, we demonstrate that both mRFP variants are also suitable for dual-colour microscopy in the different species.     

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.     

影响果蝇心脏发育的基因突变

最近的研究表明,果蝇与脊椎动物及人的心脏早期发育具有极为相似的基因控制机理,果蝇已成为研究人体心脏早期发育基因控制的理想模式动物。利用化学诱变剂甲磺酸乙酯大规模地诱变影响果蝇心脏发育的基因,利用心脏特异性抗体染色进行筛选,获得了112个有心脏突变表型的致死系,其中32个致死系的心脏畸变表型有别于目前已知心脏发育基因的突变表型。细胞遗传学定位研究表明在多线染色体的13个带纹区的某些隐性致死突变基因是目前未知的,其功能可能与发育有关的基因。     

A strategy for mapping the heterochromatin of chromosome 2 of Drosophila melanogaster

The heterochromatin of chromosome 2 of Drosophila melanogaster has been among the best characterized models for functional studies of heterochromatin owing to its abundance of genetic markers. To determine whether it might also provide a favorable system for mapping extended regions of heterochromatin, we undertook a project to molecularly map the heterochromatin of the left arm of chromosome 2 (2Lh). In this paper, we describe a strategy that used clones and sequence information available from the Drosophila Genome Project and chromosome rearrangements to construct a map of the distal most portion of 2Lh. We also describe studies that used fluorescent in situ hybridization (FISH) to examine the resolution of this technique for cytologically resolving heterochromatic sequences on mitotic chromosomes. We discuss how these mapping studies can be extended to more proximal regions of the heterochromatin to determine the structural patterns and physical dimensions of 2Lh and the relationship of structure to function.     

Purification of aspartate transcarbamylase from Drosophila melanogaster.

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of Drosophila melanogaster as part of a high-molecular-weight enzyme complex. The complex is shown to contain several polypeptides. An antiserum directed against the complex enzyme inhibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihydro-orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220 000-molecular-weight protein in addition to the polypeptides present in the complex. A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro-orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high-molecular-weight complex of cultured cells.     

Native purification of protein and RNA-protein complexes using a novel affinity procedure

Genetic studies in invertebrate model organisms such as Drosophila melanogaster have been a fundament of cell and developmental biology for more than one century. It is mainly the lack of an efficient purification strategy which has hampered biochemical and proteomic analyses of gene products. We describe a novel affinity-tag, termed TagIt-epitope specifically designed for affinity-purifications of multiprotein complexes from Drosophila. TagIt-fusion proteins can be efficiently purified using a monoclonal antibody and eluted under native conditions by competition with synthetic peptide encompassing the epitope. We demonstrate that this tag is suitable for the purification of proteinaceous assemblies such as the PRMT5-complex and RNA-protein complexes such as snoRNPs from Drosophila Schneider2 cells. Furthermore, we describe a novel approach by which this tag can be used to affinity-purify RNA-binding proteins from cell extracts. Therefore, the TagIt-technique or modifications thereof will be of great value in analyzing macromolecular complexes in Drosophila and also other invertebrates by biochemical means. In addition, RNA-peptide hybrid molecules may become a novel tool to purify RNA binding proteins.     

Guidance of primordial germ cell migration

Primordial germ cells (PGCs), the progenitors of the gametes, migrate from the position where they are specified towards the region where the gonad develops. To reach their target, the PGCs obtain directional cues from cells positioned along their migration path. One such cue, the chemokine SDF-1, has recently been found to be critical for proper PGC migration in zebrafish and in mice. In Drosophila, too, a molecule that is structurally related to chemokine receptors and is important for PGC migration has been identified. The ability to visualize chemokine-guided migration at a high resolution in vivo in these model organisms provides a unique opportunity to study this process, which is relevant for many events in normal development and disease.     

High calorie diet augments age-associated sleep impairment in Drosophila

The fruit fly, Drosophila melanogaster is an established model used for aging and longevity studies and more recently for sleep studies. Mammals and Drosophila share various physiological, pathological, pharmacological and genetic similarities in these processes. In particular, sleep is essential for survival in both species and both have age-associated sleep quality alterations. Here we report that a high calorie diet, which accelerates the aging process and reduces lifespan across species, also accelerates age-associated sleep changes in Drosophila. These changes are more evident in the dopamine transporter mutant, fumin, that displays a short sleep phenotype due to enhanced dopaminergic signaling. With normal food, fumin mutants sleep for only one third of the time that the control flies do, but still show equivalent longevity. However, when on a mildly high calorie diet, their sleep length shows a marked decrease and they have a reduced longevity. These data indicate that the age-associated change in sleep in Drosophila is a physiologically regulated aging process that is tightly linked to calorie intake and that the dopamine level plays an important role. In addition, this provides another evidence that sleep is essential for the longevity of Drosophila.     

Mutagenesis by imprecise excision of the piggyBac transposon in Drosophila melanogaster

Mutagenesis by transposon-mediated imprecise excision is the most extensively used technique for mutagenesis in Drosophila. Although P-element is the most widely used transposon in Drosophila to generate deletion mutants, it is limited by the insertion coldspots in the genome where P-elements are rarely found. The piggyBac transposon was developed as an alternative mutagenic vector for mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac transposon can more randomly integrate into the genome. Previous studies suggested that the piggyBac transposon always excises precisely from the insertion site without initiating a deletion or leaving behind an additional footprint. This unique characteristic of the piggyBac transposon facilitates reversible gene-transfer in several studies, such as the generation of induced pluripotent stem (iPS) cells from fibroblasts. However, it also raised a potential limitation of its utility in generating deletion mutants in Drosophila. In this study, we report multiple imprecise excisions of the piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila. Through imprecise excision of the piggyBac transposon inserted in the 5'-UTR of the SR gene, we generated a hypomorphic mutant allele of the SR gene which showed markedly decreased levels of SR expression. Our finding suggests that it is possible to generate deletion mutants by piggyBac transposon-mediated imprecise excision in Drosophila. However, it also suggests a limitation of piggyBac transposon-mediated reversible gene transfer for the generation of induced pluripotent stem (iPS) cells.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Glial interactions with neurons during Drosophila embryogenesis

A monoclonal antibody (Mab5B12) demonstrating specificity for glial cells within the central and peripheral nervous systems of Drosophila has been used in combination with neural-specific antibodies to study the early organization of the Drosophila embryo. The embryonic central nervous system of Drosophila contains cells within the ventral midline that are recognized by monoclonal antibody 5B12. These cells are not recognized by either a polyclonal antiserum to horse radish peroxidase, which recognizes several antigens on the surface of Drosophila neurons, or Mab22C10, which recognizes an antigen specific to the peripheral nervous system. Mab5B12-positive cells lie dorsal both to the developing anterior and posterior commissures in each thoracic and abdominal segment and to the supraoesophageal commissure. They ensheath these commissures in later stage embryos. Other Mab5B12-positive cells lie dorsolateral to the CNS and send processes laterally to the lateral sensilla during axonogenesis in the PNS. These cells surround the axons of the intersegmental and segmental nerves. Other cells that line the advancing ectoderm during dorsal closure and surround the anal pads also express the Mab5B12 antigen. Neuronal cell cultures derived from Drosophila gastrulae contain cells expressing the Mab5B12 antigen. These cells can be found separate or in close association with neuronal clusters and their axons.     

Laser microdissection of semi-thin sections from plastic-embedded tissue for studying plant–organism developmental processes at single-cell resolution

The ability to analyze the gene activity occurring within a single cell has ushered in a new understanding of complex biological processes. Furthermore, this capability has established the prerequisite technologies for the analysis of cells involved in complex pathogenic and/or symbiotic interactions. Collectively, the identification of biological models permitting the analysis of individual cells and improvements in histological technology are allowing for analyses of cells positioned within tissues and involved in complex cellular interactions at unprecedented resolution. Here, a plastic embedding procedure is used for laser microdissection of plant tissues infected with a pathogen. This technique enabled the acquisition of nucleic acids from semi-thin sections that can be used for downstream biological studies of host–pathogen interaction at the single-cell resolution.     

A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

The Drosophila heart has become an exciting model for elucidating the molecular basis for cardiac function in higher organisms. To complement the genetic approaches that have recently identified an array of genes essential for cardiac function, we developed a method to obtain optimal semi-thin cross sections of embryonic, larval, and adult fly hearts in a desired orientation. A procedure for fluorescent labeling of these sections with multiple markers has also been developed, allowing the detection of proteins at high subcellular resolution. Sections obtained by our method reveal changes in cell shape between embryonic heart and aorta cardioblasts and elucidate the morphology of the adult heart. Analysis of the adult heart reveals the precise cardiac tube morphology, differential distribution of the extracellular matrix protein Laminin within the cardiac tube, as well as individual hand-positive, and Held Out Wings (HOW)-positive luminal cells that might represent blood cells. In summary, our method enables visualization of cross sections of the embryonic and adult hearts at high resolution while maintaining the ability to co-label the sections with multiple markers, thereby facilitating the analysis of cardiac tube formation and maintenance at different developmental stages.     

Light,heat, action: neural control of fruit fly behaviour

The fruit fly Drosophila melanogaster has emerged as a popular model to investigate fundamental principles of neural circuit operation. The sophisticated genetics and small brain permit a cellular resolution understanding of innate and learned behavioural processes. Relatively recent genetic and technical advances provide the means to specifically and reproducibly manipulate the function of many fly neurons with temporal resolution. The same cellular precision can also be exploited to express genetically encoded reporters of neural activity and cell-signalling pathways. Combining these approaches in living behaving animals has great potential to generate a holistic view of behavioural control that transcends the usual molecular, cellular and systems boundaries. In this review, we discuss these approaches with particular emphasis on the pioneering studies and those involving learning and memory.