The Genome of Dictyostelium discoideum Adam Kuspa and William F Loomis

The Dictyostelium discoideum genome has been sequenced, assembled and annotated to a high degree of reliability. The parts-list of proteins and RNA encoded by the six chromosomes can now be accessed and analyzed. One of the initial surprises was the remarkably large number of genes that are shared with plants, animals, and fungi that must have been present in their common progenitor over a billion years ago. The genome encodes a total of about 10,300 proteins including protein families involved...

Contributors

Adley Department of Biology and Wolfson Institute for Biomedical Research, University College London, WC1 E6BT, UK Sarah J. Annesley Department of Microbiology, La Trobe University, Bundoora, VIC 3086, Australia Laurence Aubry Laboratoire de Biochimie et Biophysique des Syst mes Int gr s (UMR5092), D partement R ponse et Dynamique Cellulaires, CNRS-CEA-UJF, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 09, France Vincent Blancheteau Department of Biological Sciences, Sir...

Humana Press Totowa New Jersey

2006 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s),...

Ludwig Eichinger Francisco Rivero

DictyBase and the Dicty Stock Center Petra Fey, Pascale Gaudet, Karen E. Pilcher, Jakob Franke, and Rex L. Chisholm dictyBase is the model organism database that houses all the sequence and associated data for Dictyostelium discoideum, including literature, researchers, and strains from the Dicty Stock Center. The database makes it possible to connect genes, proteins, and publications and is designed to address the needs of biologists and bioinformaticists alike. We provide tools for retrieving...

Cultivation Spore Production and Mating

Dictyostelium discoideum proliferates as solitary amoebae, constitutes multicellular structures called fruiting bodies, and mates to form macrocysts depending on environmental conditions. All of these processes can be easily induced in the laboratory. The amoebae are normally cultured with food bacteria, but the strains with mutations in axe loci can proliferate in nutrient media without bacteria. The strains can be stored either as spores or amoebae. Synchronous development of fruiting bodies...

Dictyostelium Discoideum Microscope Image

Fig. 2 (continued) fluorescence images were deconvoluted (Leica Deblur software, Blind Deconvolution, 20 iterations) and processed to projections using ImageJ V1.32j. The specimens were prepared and treated as follows Cells were grown on coverslips (A-C, grade 0 D-I, grade 1), and three different fixation protocols were applied. For images A-C, coverslips were plunged into ultracold methanol (see Subheading 3.3.) in images D-F, cells were fixed using the paraformaldehyde (PAF) fixative as...

RNA Interference and Antisense Mediated Gene Silencing in Dictyostelium

Markus Kuhlmann, Blagovesta Popova, and Wolfgang Nellen Summary Knockouts by homologous recombination are frequently used to investigate the function of genes in Dictyostelium and other organisms. Antisense-mediated gene silencing and RNA interference (RNAi) are convenient alternatives to reduce gene expression to different levels and to silence multigene families. We describe here the methods for efficient RNA interference in Dictyostelium and some useful mutant strains that enhance the...

Info

A spore-storage ampoule (up) and a stock of freeze-dried spores (down). Fig. 1. A spore-storage ampoule (up) and a stock of freeze-dried spores (down). 3. Silica gel white fine granules. We use 28-200 mesh from Wako Pure Chemical Industries (Tokyo, Japan). 4. Glass ampoules these are commercially available, but we make our own for fun. Cut and flame-seal the tip of a Pasteur pipet and elongate the middle part to create a narrow portion. Insert five to six glass beads ( 2-3 mm) to...

Investigating Gene Expression

In Situ Hybridization and Reporter Genes Ricardo Escalante and Leandro Sastre Summary Coordinated cell type differentiation is essential for morphogenesis during Dictyostelium development. The specification of different cell types and the regulation of temporal and spatial patterns of expression of cell type-specific genes are important problems currently being addressed in many laboratories. Besides, determination of gene expression patterns provides significant information in the...

Under Agarose Chemotaxis of Dictyostelium discoideum

Dictyostelium Under Microscope

In the vegetative state, Dictyostelium amoebae are chemotactic toward pterins released by bacteria, whereas during multicellular development, they become chemotac-tic to endogenously produced cAMP. A variety of assays have been used to visualize and quantify chemotactic movement. Under-agarose chemotaxis provides a simple and flexible assay that permits high-resolution imaging and quantification of the motility behavior of individual cells and populations by both transmitted light and...

The Secret Lives of Dictyostelium

Dictyostelium Discoideum

Research on Dictyostelium has a long history, the trend of which has been to add cell biology, natural products, and evolutionary biology research to the traditional studies of development. The methods presented here and the recent publication of the genome present vast new opportunities. Key Words Cellular slime mold natural products soil ameba amoeboid genome. In 1991, the archives of the Soviet Union and its satellites began to open and to inform us in the West about events that we had...

Optimized Fixation and Immunofluorescence Staining Methods for Dictyostelium Cells

Draw The Setup For Fixation

Neuhaus, and Thierry Soldati Summary Recent years have seen a powerful revival of fluorescence microscopy techniques, both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme. Therefore, it is essential to improve in parallel the quality of the structural and antigenic preservation during fixation and immunostaining. Chemical fixations are broadly...

Application of 2D and 3D DIAS to Motion Analysis of Live Cells in Transmission and Confocal Microscopy Imaging

Deborah Wessels, Spencer Kuhl, and David R. Soll Summary The chemotactic signal in Dictyostelium is a cAMP wave that is relayed over relatively large distances through a cell population during aggregation. Cells exhibit unique behaviors in response to the different spatial, temporal, and concentration components of the cAMP wave, suggesting that distinct signal transduction pathways are evoked in each of the various phases of the wave. For this reason, we designed a set of experimental...

Generation of Multiple Knockout Mutants Using the CreloxP System

Streak Plate Gene Knocouts

Dictyostelium discoideum is an exceptionally powerful system by which to study various aspects of modern cell and developmental biology. The completion of the genome sequencing project together with a high-efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, as a result of many gene products with overlapping functions, there is great need to produce mutants carrying mutations in multiple genes. We have, therefore, developed a...

Restriction Enzyme Mediated Integration REMI Mutagenesis

A method for the integration of linear DNA into the Dictyostelium genome is described. Restriction enzyme-mediated integration, or REMI, involves the transformation of cells with a mixture of plasmid DNA, linearized with a restriction enzyme, along with a restriction enzyme that is capable of generating compatible cohesive ends in the genome. The enzyme stimulates integration of the DNA into cognate restriction sites in the chromosomes, usually as a single-copy insertion event and with little...

Proteomic Analysis of Dictyostelium discoideum

Dictyostelium Discoideum

Udo Roth, Stefan M ller, and Franz-Georg Hanisch The social amoeba Dictyostelium discoideum is already known as a model organism for a variety of cellular and molecular studies. Now that the genome sequencing project has been completed and different tools with which to overexpress or knock out genes are available, this species has also moved into the spotlight of functional genomics studies. Consequently, this genomic sequence information can now be exploited to realize D. discoideum proteomics...