Unknown,Transcriptomics,Genomics,Proteomics

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DamID from Drosophila CNS with Dam fused FruMA, FruMB, FruMC.

ABSTRACT: In Drosophila,male courtship behaviour serves as an excellent paradigm to study how innate behaviors are controlled by the nervous system. These behaviors are in large part regulated by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behaior by controlling the development of sexually-dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation. To characterize the roles of fru sex-specific isoforms in specifying male behavior, we generated novel isoform-specific mutants, and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specificed by either a single, or combination, of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genoic enhancers, and novel downstream regulators of male-sexual behavior. 3 replicates per condition, one dye swap; per condition: one control, one experimental per replicate. No processed data for GSM1261882 and GSM1261883 are available.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Megan Goodwin

PROVIDER: E-GEOD-52247 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Stem cells reside in a specialized microenvironment, called niche, which provides essential signals controlling stem cell behavior. Proper niche architecture is a key for normal stem cell function, yet only few upstream regulators are known. Here we report that the Hox transcription factor Abd-B, active in pre-meiotic spermatocytes, affects niche positioning in the Drosophila testis by regulating integrin localization in differentiated somatic cyst cells. Loss of Abd-B results in cell non-autonomous effects within the niche including centrosome misorientation in germline stem cells (GSCs) and reduced GSC divisions in larval testis, leading to a dramatic reduction of pre-meiotic stages in adult testes. By identifying Abd-B binding regions throughout the genome, we find that Abd-B mediates its effects on niche function by directly controlling at multiple levels the localization and thus signaling activity of the Sevenless (Sev) ligand, Bride of Sevenless (Boss), via its direct targets src42A and sec63. In sum, our data show for the first time that Abd-B through local signaling provides positional cues for integrin localization, which is critical for niche localization and architecture, and ensures proper niche function and GSC activity. DamID (DNA adenine methyltransferase identification) method was used to identify direct Abd-B target genes in the Drosophila 3rd instar larval testis Dam was fused to the N terminus of Abd-B and transgenic flies were generated. For identifying Abd-B targets in the Drosophila testis the fusion protein was expressed from the uninduced minimal Hsp70 promoter of the UAS vector pUAST (Brand and Perrimon, 1993). As a control for nonspecific Dam activity, transgenic flies expressing the Dam alone were used (Choksi et al., 2006). Subsequently, genomic DNA was extracted from 3rd instar larval testes, expressing either the Dam-Abd-B fusion protein or the Dam protein alone using a specific protocol (Tolhuis et al., 2011); and van Steensel personal communication). Two individual replicates, for Dam-AbdB and Dam alone, have been generated. Following a methylation-sensitive DNA digestion and PCR amplification, DNA fragments from Dam-Abd-B and control DNA were labeled and hybridized to genomic Affymetrix arrays in duplicates (Protocol available at M-bM-^@M-^\www.flychip.org.ukM-bM-^@M-^]).

Project description:Radix bupleuri is a traditional Chinese crude drug and it is still widely used in contemporary medicine. Bupleurum chinense DC. and B. scorzonerifolium Willd. are officially recorded as the source species of Radix bupleuri in Pharmacopoeia of the PeopleM-bM-^@M-^Ys Republic of China. The obvious difference in chemical active components and pharmacological action of Radix bupleuri derived from the two species has caused attention. But to date, no definite criterions and regulates are issued based on plant species sources to direct the standard uses of Radix bupleuri for their extracts and traditional decoction pieces in medical market. Using 454-FLX pyrosequencing with one half-plate run for each cDNA library, a total of 419,576 and 416,112 reads, among those 313,483 and 342,263 high quality ESTs were obtained for B. chinense and B. scorzonerifolium, respectively. The average read length was 279 bp and 310 bp, respectively. The assembled 28,897 and 30,912 unigenes for B. chinense and B. scorzonerifolium were successively annotated based on sequence similarity by major public nucleotide/protein databases, Nt, Nr, SwissProt, KEGG, COG, Interpro and GO. Based on the alignments, 71.9% (20,787) and 75.8% (23,426) of the total unigenes for B. chinense and B. scorzonerifolium could be matched to nucleotides from NCBI Nt database. Certain percent from 27.1% (7,820) to 70.8% (20,461) of the total unigenes for B. chinense, and from 26.4% (8,145) to 72.1% (22,289) of the total unigenes for B. scorzonerifolium, could be matched to proteins from different databases. For the same database, similar percent of the total unigenes for B. chinense and B. scorzonerifolium were annotated. A total of 17,117 (59.2%) and 19,416 (62.8%) unigenes for B. chinense and B. scorzonerifolium could be found homologous genes in opposite dataset. According to KEGG annotation, 3,637 and 3,821 unigenes were assigned relating to metabolism in datasets of B. chinense and B. scorzonerifolium, respectively. Carbohydrate metabolism, energy metabolism and amino acid metabolism were the highest three groups in metabolism category and followed by lipid metabolism group for B. chinense. In comparison, lipid metabolism group had most unigenes for B. scorzonerifolium and it had much more unigenes than the same group for B. chinense (827 to 562). A total of 70 and 52 unigenes were related to metabolism of terpenoids and polyketides in two separate datasets, which seems agree with the situation of higher content of terpenoid saikosaponins in B. chinense than B. scorzonerifolium in some extent. To compare the biosynthesis of main bioactive components in roots of B. chinense and B. scorzonerifolium, putative genes which may participate in the biosynthesis of terpenoid, triterpenoid, sterol, ligan and flavonoid were searched according to unigenes annotation. Most lignan and flavonoid biosynthesis related genes were found in both datasets of B. chinense and B. scorzonerifolium, while some terpenoid or triterpenoid related genes were dominantly found in the dataset of B. chinense. This reflected in some extent that these genes expressed in low levels and relative metabolites were biosynthesized in lower quantities in roots of B. chinense than those of B. scorzonerifolium. Besides abovementioned comparison on two transcriptome, the expression of some genes involved in biosyntheses of main medicinal secondary metabolites were analyzed by qRT-PCR in roots of the two species. These molecular biological data will make a useful clue and arouse more studies on the reasonable application of the two Bupleurum species. The fine roots of two-year old plants of B. chinense and B. scorzonerifolium planted in the same place at the same time were collected at the usual harvest time of medicinal materials of Bupleurum. Those roots from the two species plants were used for 454 sequencing.

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DamID from Drosophila CNS with Dam fused FruMA, FruMB, FruMC.

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