Project description:Background: Synaptic transmission is required for functional maturation of the CNS and to induce gene transcription involved in neuronal plasticity. Our aim is to identify genes that are transcriptionally regulated by synaptic transmission during development. cDNA microarrays will be used to compare gene expression in normal embryos and embryos with no synaptic transmission. Using the Gal4 UAS system, the tetanus toxin light fragment (TET) will be expressed throughout the nervous system. The effects of TET are well characterised: the synaptic protein n-Synaptobrevin is cleaved, blocking evokes vesicle fusion in TET expressing neurons and as a result synaptic transmission is blocked. Embryos expressing an inactive, mutant version of TET (iTET) show no detectable phenotype. Thus a comparison of gene expression in iTET expressing embryos and TET expressing embryos focuses our screen cleanly and specifically on genes that are regulated by synaptic transmission. At the end of larval life, during metamorphosis, there is a second phase of nervous system development with the same requirement for neural circuit differentiation and maturation as in the embryo. We will compare gene expression in TET expressing and iTET expressing pupae to identify genes which are regulated by synaptic transmission in this second phase of nervous system development. We will focus our analysis on genes which are similarly regulated in both systems. Plan: elav Gal4 (expressed in all the neurons) for the embryos and Heat Shock Gal4 (which allows a temporally controlled expression) for the pupae are used to drive UAS TET and UAS iTET throughout the nervous system. Crosses are set up with flies homozygous for Gal4 or UAS transgenes, so that all the progeny have the same genotype. Total RNA is extracted from 500 carefully staged embryos that have been collected at the time of hatching. Pupae are heat shocked at 24 hours after puparium formation, and heads are then collected 5 days after puparium formation. TET expressing pupae are paralysed but morphologically normal whereas iTET expressing pupae emerge as normal adults. Total RNA will be extracted from 250 heads of each genotype. Probes made from TET and iTET expressing embryos extracts will be hybridised together to the chip and so will be probes made from TET and iTET expressing pupae extracts. Genes regulated in both systems will be compared together.

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.

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:Drosophila melanogaster males mutant for any meiotic arrest gene have defective spermatogenesis, with cells arresting as primary spermatocytes, and failing to progress to later stages. This phenotype is remarkably similar to meiosis I maturation arrest azoospemia in humans. Drosophila meiotic arrest genes can be categorised as "aly-class" or "can-class". The aly-class (aly, zaa and zab) regulates both meiotic division and male germ line differentiation by controlling transcriptional activation of a large set of target genes in primary spermatocytes. This set includes genes required for cell cycle progression and those required for spermatid differentiation. The can-class control transcription of the same set of differentiation genes, but not cell cycle genes. aly belongs to a protein family conserved from humans to plants. Aly protein locates to chromatin in wild type individuals, and probably controls transcriptional activity of target promoters through interaction with a DNA binding protein. Objectives: So far aly- and can-class target genes have been identified by the small-scale approach of individually testing cDNA clones (total 18). Expression of 7 genes was unaffected by the mutations (group A), transcription of 8 depended on both classes of genes (group B), finally three genes required the aly- but not the can-class for their expression (group C). The aly-class targets (group C) are particularly intriguing, since they include a gene, boule, whose human homologue deleted in azoospermia (DAZ) is essential for male fertility. This project will address the following questions: How many genes in total are regulated by the meiotic arrest genes? What types of proteins do they encode? Are they all testis specific, or are they expressed at other stages of the life cycle? What is their relevance for fertility in other species?<br> <br> Plan: We will prepare samples of wild type, aly, can, zaa and zab testes. Those transcripts expressed in wild type testes will be placed into group A, B or C based on analysis of the competitive hybridisation of wild type samples vs each mutant in turn. Microarrays will also enable us to determine whether there is a group D, consisting of genes whose down-regulation in spermatocytes is dependent on the meiotic arrest genes. We have dissected testes from 0-1 day old males of the relevent genotypes. We estimate to get >20 ug total RNA from 100 flies, so have dissected at least 500 males of each mutant genotype, and 2000 of wild type (red e, the background chromosome for aly and can). In total we have about 5000 flies worth of testes in the freezer. We have been careful to only include testes and seminal vesicles, and have excluded other tissues from the genital tract. All the males are collected from bottles that are emptied daily, to ensure they are all the same age. Because of their age relatively few of the males will have mated. For aly and can we have used well characterised null alleles. For zab we have used the only mutant allele, which we also know is a null. We havn't cloned zaa so have no idea whether the only existing mutant allele is null. The testes were dissected in testis buffer over a period of 30 min. Then they were transfered into a very small drop of testis buffer in an eppendorf and frozen in liquid nitrogen. They have been stored at -80C. Before being sent to Cambridge we will defrost the samples, add trizol, and pool samples to supply 3 or 4 tubes per genotype.

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