Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

<p>Abstract</p> <p>Background</p> <p>Core promoters are <it>cis</it>-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the <it>BhC4-1 </it>gene which is located in DNA puff C4 of the sciarid fly <it>Bradysia hygida</it>. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity.</p> <p>Results</p> <p>Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic <it>Drosophila</it>. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized <it>Fbp1 </it>enhancer or downstream of the UAS element, we showed that the <it>BhC4-1 </it>core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the <it>BhC4-1 </it>core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system.</p> <p>Conclusions</p> <p>Our results from functional analysis in transgenic <it>Drosophila </it>show that the <it>BhC4-1 </it>core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in <it>Drosophila </it>were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid <it>BhC4-1 </it>core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.</p

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.Deutsche Forschungsgemeinschaft - DFG[He 1623/19]CAPES/(DAAD) Deutscher Akademischer Austausch Dienst, Germany[261/07]FAPESPDSMA by CNP

ISOLAMENTO, IDENTIFICAÇÃO E CARACTERIZAÇÃO DE MICROALGAS DE ÁGUA DOCE COMO FONTE POTENCIAL PARA A EXTRAÇÃO DE ÓLEO

A limitação das reservas de petróleo e seu uso e de seus derivados tem causado grandes preocupações no cenário econômico, social e ambiental. Fontes de energia renováveis, como biocombustíveis, tem sido vistas como uma ótima alternativa sustentável. O biodiesel é um biocombustível que tem ganhado grande atenção, sendo produzido no brasil a partir de diferentes espécies oleaginosas como mamona, canola, girassol, o amendoim e principalmente a soja. Entretanto, há várias limitações concernentes ao plantio e utilização dessas plantas. Uma promissora opção à essas oleaginosas são as microalgas, pois apresentam baixos custos de colheita e transporte, menor gasto de água e área de implantação menor comparado aos cultivos de plantas, além de poder ser realizado em condições não adequadas para a produção de outras culturas. Além do biodiesel, o óleo extraído das microalgas tem outros potenciais, entre eles a indústria farmacêutica, cosméticos, suplementos alimentares, entre outros. No presente projeto, amostras de microalgas de água doce do lago do centro de Matão-SP foram crescidas em meios de cultura com condições que estimularam o seu crescimento, isoladas e identificadas, e seu óleo foi caracterizado como fonte em potencial para futura produção de biodiesel. PALAVRAS-CHAVE: biodiesel; microalgas; óleo; energia renováveis; sustentabilidad

Transcript levels of ten caste-related genes in adult diploid males of Melipona quadrifasciata (Hymenoptera, Apidae) -A comparison with haploid males, queens and workers

Abstract In Hymenoptera, homozygosity at the sex locus results in the production of diploid males. In social species, these pose a double burden by having low fitness and drawing resources normally spent for increasing the work force of a colony. Yet, diploid males are of academic interest as they can elucidate effects of ploidy (normal males are haploid, whereas the female castes, the queens and workers, are diploid) on morphology and life history. Herein we investigated expression levels of ten caste-related genes in the stingless bee Melipona quadrifasciata, comparing newly emerged and 5-day-old diploid males with haploid males, queens and workers. In diploid males, transcript levels for dunce and paramyosin were increased during the first five days of adult life, while those for diacylglycerol kinase and the transcriptional co-repressor groucho diminished. Two general trends were apparent, (i) gene expression patterns in diploid males were overall more similar to haploid ones and workers than to queens, and (ii) in queens and workers, more genes were up-regulated after emergence until day five, whereas in diploid and especially so in haploid males more genes were down-regulated. This difference between the sexes may be related to longevity, which is much longer in females than in males

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Transcript levels of ten caste-related genes in adult diploid males of Melipona quadrifasciata (Hymenoptera, Apidae) - A comparison with haploid males, queens and workers

In Hymenoptera, homozygosity at the sex locus results in the production of diploid males. In social species, these pose a double burden by having low fitness and drawing resources normally spent for increasing the work force of a colony. Yet, diploid males are of academic interest as they can elucidate effects of ploidy (normal males are haploid, whereas the female castes, the queens and workers, are diploid) on morphology and life history. Herein we investigated expression levels of ten caste-related genes in the stingless bee Melipona quadrifasciata, comparing newly emerged and 5-day-old diploid males with haploid males, queens and workers. In diploid males, transcript levels for dunce and paramyosin were increased during the first five days of adult life, while those for diacylglycerol kinase and the transcriptional co-repressor groucho diminished. Two general trends were apparent, (i) gene expression patterns in diploid males were overall more similar to haploid ones and workers than to queens, and (ii) in queens and workers, more genes were up-regulated after emergence until day five, whereas in diploid and especially so in haploid males more genes were down-regulated. This difference between the sexes may be related to longevity, which is much longer in females than in males

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[05/039926-5]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[08/07321-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/06251]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)[305378/2007-4

Schematic representation of the <i>lncov1</i> and <i>lncov2</i> gene positions in the chromosome 11 region containing a QTL for transgressive ovary size in workers.

<p>The dotted and dashed LOD score lines represent the 95% and 99% QTL thresholds, respectively, above which ovariole number is significantly influenced <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078915#pone.0078915-Linksvayer1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078915#pone.0078915-Graham1" target="_blank">[6]</a>. The orange bar indicates the location of Group 11.31 scaffold and the green one the Group 11.35 scaffold (genome version 4.0). Black vertical lines indicate the respective positions of <i>lncov1</i> and <i>lncov2</i> within these scaffolds.</p

Fluorescence <i>in situ</i> hybridization (FISH) revealing the localization of <i>lncov1</i> transcripts in ovaries of fifth instar worker larvae.

<p>(A) Detection of an Alexa 594-labeled <i>lncov1</i> probe (red) in ovary whole mounts; nuclei are counterstained with DAPI (blue). White arrows indicate some of the <i>lncov1</i> agglomerates, showing their distribution throughout the developing ovarioles. (B) High resolution image of (A) evidencing <i>lncov1</i> RNA agglomerates in perinuclear positions. The images were captured by confocal microscopy. Scale bars represent 20 and 5 µm, respectively.</p

Full length cDNAs of honey bee <i>lncov1</i> and <i>lncov2</i> and their genomic mapping.

<p>(A) Southern Blot showing probe hybridizations with full-length cDNAs of <i>lncov1</i> and <i>lncov2</i> produced by 3′5′RACE reactions. The <i>lncov1</i> probe labelled a cDNA of 1367 bp, the one for <i>lncov2</i> hybridized to a 684 bp transcript. (B) Full length mRNA sequence of <i>lncov1</i>. Coverage with the genome sequence is shown on grey background, the tandem repeat sequence lacking in the genome is shown on white background. (C) Genomic mapping of <i>lncov1</i>; <i>lncov1</i> (arrow) is located in the sense strand to the fifth intron of the predicted protein LOC726407. (D) <i>lncov2</i> (arrow) maps into the fourth intron of <i>fringe</i>, also in sense direction.</p