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BACKGROUND: In the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved. RESULTS: We found that roX RNAs colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. We were able to immunoprecipitate roX2 RNA from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicated an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. We also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis. CONCLUSIONS: Our results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome.

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Animals that have XX females and XY or XO males have differing doses of X-linked genes in each sex. Overcoming this is the most immediate and vital aspect of sexual differentiation. A number of systems that accurately compensate for sex-chromosome dosage have evolved independently: silencing a single X chromosome in female mammals, downregulating both X chromosomes in hermaphrodite Caenorhabditis elegans and upregulating the X chromosome in male Drosophila all equalize X-linked gene expression. Each organism uses a largely non-overlapping set of molecules to achieve the same outcome: 1X = 2X.

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The multisubunit MSL dosage compensation complex binds to hundreds of sites along the Drosophila single male X chromosome, mediating its hypertranscription. The male X chromosome is also coated with noncoding roX RNAs. When either msl3, mle, or mof is mutant, a partial MSL complex is bound at only approximately 35 unusual sites distributed along the X. We show that two of these sites are the roX1 and roX2 genes and postulate that one of their functions is to provide entry sites for the MSL complex to recognize the X chromosome. The roX1 gene provides a nucleation site for extensive spreading of the MSL complex into flanking chromatin even when moved to an autosome. The spreading can occur in cis or in trans between paired homologs. We present a model for how the dosage compensation complex recognizes X chromatin.

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The Drosophila roX1 gene is X-linked and produces RNAs that are male-specific, somatic, and preferentially expressed in the central nervous system. These RNAs are retained in the nucleus and lack any significant open reading frame. Although all sexually dimorphic characteristics in Drosophila were thought to be controlled by the sex determination pathway through the gene transformer (tra), the expression of roX1 is independent of tra activity. Instead, the dosage compensation system is necessary and sufficient for the expression of roX1. Consistent with a potential function in dosage compensation, roX1 RNAs localize specifically to the male X chromosome. This localization occurs even when roX1 RNAs are expressed from autosomal locations in X-to-autosome translocations. The novel regulation and subnuclear localization of roX1 RNAs makes them candidates for an RNA component of the dosage compensation machinery.

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Recent advances in the study of learning in insects are examined with an emphasis on two of the most powerful model systems, the honeybee (Apis mellifera) and the fruit fly (Drosophila melanogaster). The honeybee exhibits easily manipulated feeding behavior coupled with extremely high mnemonic fidelity. The size of the honeybee brain has allowed for electrophysiological analysis of the neural correlates of behavior, sometimes with single cell resolution, as well as identification of critical brain regions. Drosophila has proved to be invaluable in the genetic dissection of learning. Through analysis of learning and memory mutants the biochemistry of critical steps has been elucidated and the temporal phases of memory in the fly have been described. Two regions of brain neurophil are essential for olfactory learning in these species: the antennal lobes and the mushroom bodies. In spite of similarities, temporal, and possibly biochemical aspects of learning differ markedly between these organisms.

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Domain-specific anti-Drosophila DNA topoisomerase II antibodies were generated, affinity purified and used for confocal laser scanning immunofluorescence microscopy. Except for the nucleolus, DNA topoisomerase II is distributed throughout interphase nuclei. In adult accessory glands as well as third instar larval neural ganglion and imaginal disk nuclei, DNA topoisomerase II shows areas of co-localization with chromatin adjacent to areas of extrachromosomal distribution. These observations made in a variety of tissues under different fixation conditions and with a number of molecular probes support the notion that DNA topoisomerase II is a component of a substantially extrachromosomal network that functions to organize interphase chromatin within nuclei.

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Monoclonal antibodies were used to localize the putative Drosophila homolog of mammalian nuclear pore complex glycoprotein, gp210, to Drosophila nuclear pore complexes. Both immunofluorescence and immunoelectron microscopy were performed. To the best of our knowledge, this establishes Drosophila gp210 as the first invertebrate gp210 homolog. Results of developmental studies demonstrated that like nuclear lamin and DNA topoisomerase II, gp210 is found abundantly in nonnuclear form early in embryogenesis where it presumably fuels the rapid assembly of new nuclei. Unlike thse other two proteins, gp210 levels are maximal early after wich they decrease significantly; in addition, nonnuclear gp210 found in early Drosophila embryos is apparently associated with membrane vesicles. These results have implications for understanding the regulation of higher eukaryotic nuclear pore complex behavior through development as well as for determining gp210 function genetically.

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The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.

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Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).

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The occurrence, structure and function of the alpha and beta subunits of GTP-binding proteins (G proteins) were investigated in the Manduca sexta prothoracic gland, a tissue which possesses a hormonally regulated adenylate cyclase. Subunit-specific antibodies were utilized in immunoblotting studies of tissue from Manduca prothoracic glands, brain, eyes and antennae, and compared to the substrates present in the heads of Drosophila, as well as in a mammalian cell line. All Manduca tissues examined showed putative G beta subunits of 37 and 38 kDa, an unidentified alpha subunit of 41 kDa, in addition to an eye specific alpha subunit of 42 kDa. Manduca tissues also produced putative Gs alpha subunits of 48 and 51 kDa which were coupled to prothoracic gland adenylate cyclase as demonstrated by immunoprecipitation. Prothoracic gland G proteins have a definite and limited quaternary structure, consistent with a heterotrimeric model, as demonstrated by crosslinking of prothoracic gland membrane preparations followed by immunoblotting. These studies also yielded data on relative titers of alpha subunits, and suggest that Gs alpha is present in lower amounts than other alpha subunits. The G protein subunits studied in the prothoracic gland appear strikingly similar in molecular weight, function and structure to their mammalian counterparts.

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The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.

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High levels of cyclic AMP were found in the corpora allata of adult female Diploptera punctata. The adenylate cyclase activator forskolin caused a rapid, reversible and dose-dependent accumulation of cAMP in the corpora allata in vitro. The sensitivity of the corpora allata to forskolin was low when juvenile hormone (JH) synthetic activity was high, and vice versa. Incubation of corpora allata with compounds which cause or mimic elevated intracellular cAMP levels (forskolin, 3-isobutyl-1-methylxanthine, 8-bromo-cAMP) led to a rapid and dose-dependent inhibition of juvenile hormone synthesis. Glands from day 5 virgin females were more sensitive to forskolin than glands from mated females of the same age. The results suggest that a cAMP second messenger system may be responsible for the intracellular transduction of inhibitory signals to the corpora allata of D. punctata.

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Maize (Zea mays L.) stem is thought to function alternately as a net importing and net exporting organ during ontogeny, depending on whole plant photosynthetic source and sink status. The [(14)C]sucrose and [(14)C]glucose uptake capacity of stem tissues was investigated to increase our understanding of the transport factors which may influence sink status.Uptake from solutions containing up to 200 millimolar radiolabeled sugar showed that d-glucose uptake consisted of saturable and nonsaturable components, while sucrose uptake was primarily nonsaturable during the kernel-fill stages. l-Glucose uptake lacked the saturable component but both d and l isomers apparently had similar slopes for the nonsaturable component. Uptake was sensitive to inhibitors and temperature, and was increased slightly by lowered pH.The seasonal chronology for saturable uptake by isolated vascular bundles and associated pith revealed highest rates between anthesis and early kernel growth, corresponding with the stage when net sugar accumulation rates were highest. For isolated pith, the rates increased at the final stages of plant development.The rate of labeled l-glucose movement from vascular bundles into pith in isolated stem segments was greater at the silking stage than at later developmental stages, suggesting a lower resistance to diffusive transport from vascular bundles into pith at silking. Studies with stem plus ear explants showed that the capability for sugar transport from pith to vascular bundles and for phloem loading and export from the stem region was present throughout the developmental period from early kernel fill (milk) to late kernel fill (dent).