Functional Analysis

The Organism

Dictyostelium discoideum, a soil-living amoeba, is an excellent organism for the study of the molecular mechanisms underlying differentiation, signal transduction, phagocytosis, cytokinesis, chemotaxis, and cell motility. Many of these processes resemble those seen in mammalian cells and genes involved in any of them can be knocked-out rapidly by targeted homologous recombination. Thus, Dictyostelium is perfectly suited for i) large scale pinpointing differentially regulated genes followed by ii) further analysis using the available armoury of molecular biological techniques.

DNA Microarrays

Biologists today have the possibility to explore the vast and rapidly increasing amount of DNA sequence data provided by genome and EST sequencing projects. However, knowing the entire genome of an organism is only the beginning of a long odyssey to understanding the functions of all its genes. New tools like DNA microarrays have revolutionised molecular biology and are used to take advantage of all the sequence information. With the highly parallel approach of microarray gene expression analysis we can try to unravel and understand the complex regulatory networks of living organisms. The process of creating a new DNA microarray for the entire genome of an organism involves the selection and production of thousands of DNA probes. For spotted DNA microarrays these probes are usually amplified using the polymerase chain reaction.

The D. discoideum Microarray

The ongoing Dictyostelium genome and cDNA projects that are carried out in Germany (Institut für Biochemie Köln, Zentrum Biochemie Cologne in collaboration with the Genome Sequencing Centre Jena), Great Britain (Sanger Institute), the USA (Baylor College of Medicine) and Japan (Tsukuba University) have paved the way for the generation of a Dictyostelium DNA microarray. The 34 Mb genome of this organism is spread across 6 chromosomes and the number of genes is estimated to be around 12,000.

The current Dictyostelium DNA microarray carries partial sequences of 450 known genes, approximately 5,400 non-redundant ESTs from the Dictyostelium cDNA project and appropriate positive and negative controls. We are in the process of incorporating probes representing the app. 4,600 chromosome 1 and 2 genes.

We are using this tool to study genome-wide gene expression patterns of Dictyostelium cells. The experiments are complemented with molecular genetic and cell biological approaches to verify results and to study the most interesting differentially regulated genes in more detail. Three main projects are pursued:

1. Transcriptional regulation in response to environmental stresses in particular hyper- and hypoosmotic conditions.
2. Transcriptional changes during bacterial uptake and multiplication.
3. Analysis of transcriptional regulation in a variety of knock-out mutants in comparison to wild-type cells.

International archives of microarray public data

1. Gene Expression Omnibus: a gene expression/molecular abundance repository supporting MIAME compliant data submissions, and a curated, online resource for gene expression data browsing, query and retrieval.
2. ArrayExpress is a public repository for transcriptomics data, which is aimed at storing MIAME- and MINSEQE- compliant data in accordance with MGED recommendations. The ArrayExpress Warehouse stores gene-indexed expression profiles from a curated subset of experiments in the repository.
3. CIBEX is a public database for microarray data, which is aimed at storing MIAME-compliant data in accordance with MGED Society recommendations.