News and Publications

Parallel Functional Genomics of Microbes

E.A. Winzeler, K.G. Le Roch, B. Grunenfelder

As the causative agent of the most severe form of malaria in humans, Plasmodium falciparum is the source of marked global morbidity and mortality. Despite the importance of this pathogen in public health, little is known about how the parasite functions at the molecular level. Knowing which genes are associated with particular biochemical pathways and stages of the life cycle would provide a wealth of new drug targets for the treatment of malaria and would advance our understanding of the parasite. In addition, identifying genetic regulatory pathways in P falciparum would facilitate genetic manipulation of the organism. Because only blood stages of P falciparum can be maintained in culture, the organism is resistant to classical genetic approaches for determining gene function. Furthermore, no genetically tractable model systems are available, such as the mouse is for humans.

In the past few years, expression profiling has emerged as an important new technology for determining gene function in genetically intractable organisms. In many instances, genes that have similar expression patterns when enough conditions are examined often function in similar biochemical pathways. Making this technology work requires generating high-quality data for a large number of conditions for every gene in the genome. This approach most likely would work well for P falciparum, because the genome sequence of the organism is now virtually complete.

Therefore, we created a high-density oligonucleotide array that contains probes (~500,000) to every region in the P falciparum genome as well to a large number of the predicted genes in the Plasmodium yoelli genome. If we assume that 26 Mb of coding sequence (because both strands potentially can code) exists, then there is one 25-base oligonucleotide for every 100 bases for expression and one 25-base probe for every 50 bases for genome analysis (50% coverage). We determined that measurements of gene expression obtained by using this array accurately reflect the abundance of transcripts. We are now collecting expression profiles for a large number of growth stages of P falciparum, including during gametogenesis and during synchronized asexual stages. Our overall goals are to determine which genes are expressed at particular stages of the parasite's life cycle and to assign genes to specific regulatory networks.

In addition, we showed that this technology can be used to identify regions of the genome that are under selective pressure either from the host's immune system or from drugs, as indicated by a higher frequency of allelic variability in worldwide isolates in such genes.

PUBLICATIONS

Karlyshev, V., Oyston, P.C., Williams, K., Clark, G.C., Titball, R.W., Winzeler, E.A., Wren, B.W. Application of high-density array-based signature-tagged mutagenesis to discover novel Yersinia virulence-associated genes. Infect. Immun. 69:7810, 2002.

Raghuraman, M.K., Winzeler, E.A., Collingwood, D., Hunt, S., Wodicka, L., Conway, A., Lockhart, D.J., Davis, R.W., Brewer, B.J., Fangman, W.L. Replication dynamics of the yeast genome. Science 294:115, 2001.