Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ChIP-chip of Drosophila polycomb group protein Ph and of GlcNAc in larvae

ABSTRACT: Analysis of binding profile of the Polycomb group protein Ph and of GlcNAc sites in Drosophila melanogaster larvae.

Note some of the protocols for this experiment were changed in May 2010.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Charles Girardot

PROVIDER: E-TABM-697 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CP190 is a Drosophila protein of unknown function that exhibits cell cycle-specific localisation to the centrosome during mitosis and the nucleus during the interphase. CP190 associates with multiple sites on polytene chromosomes and its predicted amino acid sequence identifies four C2h2 zinc finger motifs along with an N-terminal BTB/POZ domain (similar to GAGA factor). Experimental studies have identified a 128 amino acid domain that direct centrosome association and a bipartite NLS. Interest in the function of CP190 was initially driven by an interest in centrosome function. Is is also fascinating to know why a protein that forms a core component of the centrosome also shares the attributes and distribution expected of a regulator of gene expression. CP190 mutants (EMS generated) were recently isolated whose recessive lethal phenotype indicates that CP190 is essential. The two best-characterised isolates, CP190[1] and CP190[2], are either nulls or severe hypomorphs (CP190 protein is undetectable on Western blots of total protein from homozygous mutatant third instar larvae). Both mutants can be rescued as homozygotes by expression of wild type CP190 from a transgenic second chromosome carrying a CP190 cDNA under the control of the polyubiquitin promoter. Stocks homozygous or hemizygous for either of these mutant alleles die as pharate adults that show no obvious externally visible defects. Mutant larvae show a slight developmental delay compared to their heterozygous siblings, but their brains and discs are of normal size and brain squash preparation show no obvious mitotic phenotype. Since CP190 is a chromatin-associated protein and has motifs that are typical of proteins involved in regulating gene expression, it seems likely that the gene expression profile of CP190 mutants will be aberrant. This experiment hopes to identify genes that exhibit aberrant expression in the mutant compared to the wild type. Target RNA were isolated from: homozygous CP190[1] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous CP190[2] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous red[1] e[1] 0 h white prepupae (source from which the mutated chromosome was obtained) For each CP190 mutant, total RNA preparations were made from three separate groups of 100 prepupae selected from independent cultures, similarly, six independent RNA samples were prepared from red[1] e[1] stock as 'wild type' controls, giving a total of 12 target RNA samples. Prepupae were collected from bottles every 30 min and frozen directly in liquid N2. Frozen samples were pooled and ground to a powder in liquid N2 before resuspension in TRIZOL and shipment to Flychip facility at the University of Cambridge, UK.

Project description:The Drosophila-Wolbachia system is being used to study the molecular nature of the interactions between a host and a symbiont. This system offers a unique opportunity for such a study since the Drosophila genome sequence is available, several Wolbachi genomes will also be available soon and there are at least three known Wolbachia strains infecting Drosophila: a) mod+ strain that induces cytoplasmic incompatibility, b) mod- strain that cannot induce cytoplasmic incompatibility, and c) popcorn strain, a virulent strain which reduces in half the adult lifespan of Drosophila due to its massive proliferation in adult brain. The Drosophila-Wolbachia interaction manifests itself in 3 main ways; first, destruction of the CNS in infected adults, second, induction of some kind of modification or imprinting in the male germ-line resulting in an early failure in embryonic development, (cytoplasmic incompatability (CI)) and third, modification of the female germ-line resulting in resistance to modified sperm. We are interested in identifying Drosophila genes with changes in expression due to Wolbachia infection. We have generated a series of isogenic fly lines (those being used in the IGF P-element project) which we have infected with Wolbachia strains, infection is readily cured by growth on medium containing tetracycline. Thus, we have equivalent genetic background with and without the parasite. We have tested all of the transgenic lines for the level of CI and find strain-specific levels ranging from 0-50%. We also have a strain of D. simulans that shows over 95% CI. Plan: For our initial experiments we wish to make 4 comparisons, in all cases 2 day old males will be collected and for each comparison we will isolate 3 independent biological replicates: Melanogaster no CI [tet] x Melanogaster no CI [+wol] Melanogaster high CI [tet] x Melanogaster high CI [+wol] We will therefore identify genes with changed expression levels in the male upon Wolbachia infection by comparing the melanogaster strains with high or no CI in the presence of tetracycline and Wolbachia. We also hope to identify similar genes in simulans (where we expect the magnitude of the effect to be larger), differences between melanogaster and simulans are controlled for in the mel v sim comparison.

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ChIP-chip of Drosophila polycomb group protein Ph and of GlcNAc in larvae

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