Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila [RNA-seq]
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Bas van Steensel
PROVIDER: E-GEOD-75833 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. We determined DamID scores for Polycomb, normalized by Dam only control, for Drosophila larval central brain, larval fat bodies and repo+ glial cells of larval central brain. All samples were performed with 2 biological replicates. In case of Dam only control for larval central brain, each biological replicate was performed with 3 technical replicates.
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Project description:Sex chromosomes and more particularely the X chromosomes are known to have a major effect on hybrid male sterility. In this experiment by making use of the reciprocal hybrids between D. simulans and D. sechellia, we are showing the effect of these chromosomes on gene expression in male hybrids Keywords: X chromosome, hybrid Testes from for days old individuals (D. simulans, D. sechellia, hybrid D. simulans female x D. sechellia male, hybrid D. sechellia female x D. simulans male) were dissected and RNA was extracted and hybridized along with a reference RNA from the whole body of 4 days old D. melanogaster male. Gene expression in hybrids were compared to parental gene expression in order to isolate misexpressed genes in each hybrids. In order to reveal the cross effect misexpressed genes in hybrids were compared to identify genes commonly misexpressed and genes genes misexpressed in only one hybrid.
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Project description:Males hybrids from the crosses between species of the D. simulans clade are steriles as the females are fertiles. Hybrid male sterility is due to severe defects in spermatogenesis and phenotypic differences are observed between the different hybrids involving D. simulans, D. sechellia and D. mauritiana. In this study we are comparing gene expression in the testes of hybrids involving the female D. simulans and the males D. melanogaster, D. mauritiana, or D. sechellia to the gene expression in species testes. Keywords: Comparative genomic hybridization 4 species (D. melanogaster, D. simulans, D. sechellia, D. mauritiana) and 3 different hybrids were used in this study. RNA extracted from whole D. melanogaster males was used as a reference. Hybridizations were perfomed using RNA extraxted from a pool of 200 testes from a sample with RNA extracted from whole D. melanogaster males. At least three independent replicates per hybridizations were performed
2006-06-11 | E-GEOD-3673 | biostudies-arrayexpress
Project description:Background: The early stages of D. melanogaster embryogenesis involve cell migration and pattern formation, and lead to the formation of three germ layers (the ectoderm, mesoderm and endoderm). These developmental events are controlled by differential gene activity. In the current study we used a suppressive subtractive hybridization (SSH) procedure to identify a group of genes potentially involved in D. melanogaster early embryogenesis and to study the temporal activity of developmentally regulated genes at five different intervals covering 12 stages of embryogenesis. Results: Macroarrays were constructed to confirm induction of expression and determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes that significantly increased their expression levels at least at one developmental interval compared with the reference interval. 53.4% of them have a phenotype and/or molecular function reported in the literature, whereas 46.6% are essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 30 of the transcripts and of these, 19 (63%) proved to have restricted expression patterns. 11 of the uncharacterized genes that exhibited temporal and spatially restricted patterns of expression in developing embryos encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in S2R+ cells and analyze the subcellular distribution of recombinant proteins. Conclusions: Our data provides a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis, including novel information on the temporal and spatial expression patterns of several previously uncharacterized genes. In particular, we recovered a significant number of novel genes encoding putative secreted and transmembrane proteins, suggesting new components of signalling pathways that might be incorporated within the existing regulatory networks controlling Drosophila embryogenesis; they are also good candidates for more functional targeted analyses. Keywords: Drosophila melanogaster, gastrulation, macroarray, developmental time course We generated a subtracted stage specific cDNA library representing genes differentially expressed between Drosophila melanogaster synciyial blastoderm and gastrula stages. Clones were spotted onto nylon membranes and hybridized against cDNA probes representing mRNA from embryos at five different developmental intervals, in order to obtain time course expression data covering the first 12 stages of Drosophila development. We designed macroarrays containing either 302 non-redundant cDNAs, including protein-coding sequences, ncRNA, transposons, introns and intergenic regions (membranes A, B and C) and the complete subtracted library (579 cDNAs, membrane D). Each clone was spotted in duplicate. The following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T7 and SP6 universal primers; (2) several spots of DMSO 50% (solvent used); (3) PCR amplified fragment of genes serendipity α, twist, tinman, fog and snail as positive controls, and (4) actin, tubulin, and RP49 as housekeeping, and (5) four dilutions of a PCR amplified fragment from a B. subtilis dap cDNA (ATCC; number 87486), which hybridizes to an in vitro synthesized poly(A)-RNA that was added to the embryo RNA samples (dilution 1/200) prior to the labelling process and used as spike mRNA (Kane et al., 2000).
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