Project description:Background: We would like to use Drosophila cell lines to investigate the transcriptional responses to Nitric Oxide donors and inhibitors. As a pilot to this we would like to compare the transcriptional profile of 3 cell lines we are using in our lab (S3, Kc and S3). This study is being done in parallel to an in vivo genetic screen to indentify required genes. This work is funded by the BBSRC. Plan: Pairwise comparisons of each cell line to the other two. Cells will be grown at low density in identical conditions. Cells will be harvested and RNA prepared in Trizol as recommended.
Project description:The C-line contains additional copies of chalcone synthase (CHS) and these result in silencing of the endogenous copy at tt4. The clv1-2 mutation was introduced into this line as a second marker. The line was mutagenised and several revertants obtained which show reduced silencing. The best characterised of these is hog1. The hog1 mutant shows reduced DNA methylation and more intact CHS transcript in Northern blots. I have been funded by the BBSRC gene flow initiative to look for downstream targets of DNA methylation by looking for genes which are up or down regulated by hog1. Three week old plants of the genotype C/C clv1-2 with or without the hog1 mutation will be used to prepare RNA for the study. I have set the sample size at two slides for each sample.
Project description:The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. !Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC. Experimenter name = Stuart Casson and Matthew Spencer Experimenter phone = 0191 374 7356 Experimenter fax = 0191 374 2417 Experimenter institute = Durham University Experimenter address = Integrative Cell Biology Laboratory Experimenter address = School of Biological Sciences Experimenter address = Durham University Experimenter address = South Road Experimenter address = Durham Experimenter zip/postal_code = DH1 3LE Experimenter country = UK Keywords: organism_part_comparison_design; development_or_differentiation_design
Project description:Aims We aim to use transcriptome analysis to establish on a genome-wide scale the identity and regulatory clusters of genes that specify microgametogenesis from the haploid microspore to mature functional pollen in Arabidopsis. Background Pollen as the haploid male gametophyte plays a vital role in plant fertility and crop production through the ability to deliver the male gametes in fertilisation. Despite the obvious importance for plant fertility and crop production we have a very limited understanding of the regulatory mechanisms that have evolved to specify male gametophyte development and functions and less than 150 genes have been identified that are gametophytically expressed in the anther.The availability of functional genomic resources now provides the opportunity to undertake a comprehensive approach to describing cellular development in terms of the transcriptome. This approach is particularly powerful where the complete transcriptome of a single developing cell can be analysed. The male gametophyte is a uniquely accessible cell type for such studies, enabling RNA analysis from distinct purified cell populations during development.The proposed experiments are designed to support a current application (P19208, Twell) to investigate the gametophytic transcriptome and transcription factor networks. The results obtained will extend our knowledge of the contribution of haploid gene expression to anther development and will be used directly to extend BBSRC funded work (P15086, Wilson) to investigate the role and targets the MALE STERILE 1 gene (MS1). In particular the data will be used in collaboration to extract haploid gene expression from datasets of transcriptome analysis of staged flower buds of wild type (Ler) and ms1. This work will also complement BBSRC funded work on sporogenesis (G13338, Dickinson and Scott) and meiosis (G15941, Franklin and Jones) that are focussed on earlier steps in anther development. Biological material and methods. Isolated microspores and pollen at 4 different developmental stages will be analysed. We will isolate spores from developmentally staged buds of Ler grown under defined growth conditions. Buds from several batches of 100 plants will be rapidly sorted into 4 groups according to developmental age, uninucleate microspores (UM), bicellular pollen (BP) tricellular pollen (TP) and mature pollen. Spores will be released by gentle mechanical tissue disruption and purified by filtration and purification of spores. We are confident that our spore isolation procedures are rigorous since we could not detect even trace expression of highly abundant sporophytic transcripts such RbcS and Cab transcripts in microarray data from pollen RNA. Keywords: development_or_differentiation_design