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Proposal for a Cocoa Gene Expression Microarray Consortium
American Cocoa Research Institute, Program in the Molecular Biology of Cacao, The Pennsylvania State University, University Park, PA, 16802
Microarrays are miniaturised systems which allow the simultaneous measurement of the expression levels of thousands of genes. This is a modernised version of northern blots or dot blots, where RNA or various gene fragments are immobilised and then hybridised to labelled probes, to measure gene expression levels. With the advent of robotic printing and processing systems, 10,000 or more spots of DNA, each representing a different gene, can be placed on a microscope slide in a space the size of a postage stamp. The microarray can then be hybridised to fluorescently labelled cONA probes, revealing gene expression levels for each of the genes in a few hours. This is equivalent to years of work by molecular biologists using conventional methods. These methods open whole new worlds of possibilities for molecular researchers, enabling monitoring of gene expression profiles in a global sense (all or many genes) during growth and development and in response to biotic and abiotic stresses. This can provide leads to understanding basic molecular mechanisms, for example which pathways are up regulated in response to a pathogen, and which are turned off. Microarrays can also be used to assess differences in gene expression between individuals in a population, different genotypes, or even different cell types. For example, it has been shown that different types of cancer cell lines can be differentiated on the basis of expression profiles. In turn, this type of research may lead to identification of potential candidate genes, i.e. genes that are potentially causative of a trait such as cancer or in plants, disease resistance. Identification of such genes can then lead to enhanced breeding through molecular markers or genetic engineering. To learn more about how micnoanrays are made, hybridised and analysed, you can go to the excellent web site by Alan Robinson (1).
An example of micnoarray gene expression profiling is the work of Nancy Eckardt and Nina Fedenoff at The Pennsylvania State University. The full text of this work can be viewed at (2). In this study, these researchers examined the effects of ozone treatment on gene expression profiles of Arabidopsis plants, during the time course of exposure. Plants were exposed to ozone in growth chambers and leaf samples were periodically taken for RNA analysis. At each time point, a no ozone control was also taken. Then the expression patterns of severa~ hundred Arabidopsis genes were measured by hybridising the fluorescently labelled RNAs to the microarrays containing the various genes (Figurel). The raw data were processed using sophisticated software, to reveal the patterns of gene expression over time (Figure 2), red representing gene induction, and green, gene repression. The genes were then clustered according to the pattern of expression. A number of interesting genes were induced by ozone, and thus these are candidate genes for mechanisms of ozone resistance. For example, two k˝iases were turned on in response to ozone, and these are amongst a family of known signal transduction molecules involved in signalling within and between cells. Also, a transcription factor erebp-1 thought to be involved in the activation of genes during stress responses is also activated by ozone. Subsequent detailed studies on these putative candidate genes are now underway by these researchers. They hope to understand the mechanisms of ozone response, in an effort to protect plants from ozone in the future.
The need for collaborative network
To date, only very few cocoa genes have been isolated, so there has not been a need for a cocoa microarray effort, however, this picture is changing rapidly. Several groups are beginning to isolate genes at an accelerating pace, and several major efforts are now underway on being planned in multiple laboratories. Microarray analysis will clearly be an important technology to the future of cocoa research. However, with limited resources, it is also clear that we can proceed most effectively by working together to build a microanray consortium. The consortium would work together to combine all cocoa genes isolated into a large cone collection, to produce microarrays and provide them to cocoa researchers worldwide. In this way, each lab could test a larger number of genes, and all the datasets would be comparable. Eventually a large publicly available data set would begin to accrue. Similar datasets are already available for other species, combining data from many labonatonies and used by even more (3). Besides the fact that this consortium would save resources, it would also put the technology within reach of cocoa laboratories which otherwise could not use it. This is particularly important for a plant like cocoa, which is grown in areas which often do not have sufficient resources to use cutting edge technologies, but which have so much to contribute to cocoa research. It is exactly this type of synergy between cutting edge technology and breeders and researchers in producing countries that will move cocoa research ahead most rapidly.
An organising centre could be formed which would have the responsibility to coordinate the consortium activities. The consortium will be open to all cocoa researchers worldwide, all results will be released to the public domain. The proposal also includes:
Participating groups submit all cocoa cDNAs and genes to co-ordinating facility. The co-ordinating facility will conduct microarray fabrication using all clones in neplicate, with controls, New versions will be developed over time as new clones come in. Microanray distribution will be at cost. Hybnidisation and detection services will be provided upon request at cost. Training and hosting of visiting scientists, to learn methods or carry out research will be made available as space and time permit, at cost.
One potential centre is at The Pennsylvania State University. Our University has invested in the development of a service facility for researchers campus wide (4). It is University subsidised, and is run by a director and technician. The facility already has a proven track record, fabricating and using microarrays from yeast, human, Arabidopsis etc. The facility can provide all the services mentioned above, at very low costs (5). Finally, because The Pennsylvania State University Cacao program is also located near the facility, it could act as a facilitator for the access of the resource to the cocoa research community. Our programme is permanently funded through an industry endowment, and thus can commit to a long-term support role of this effort with existing funds. Interested? You are invited to contact Mark Guiltinan via email, mip9@psu,edu.
The author would like to thank the American Cocoa Research Institute and its member companies for support and advice over the years.