ARS | Publication request: Validation of DNA Microarrays Derived from the Fusarium Verticillioides Gene Index

Submitted to: Fungal Genetics Conference Proceedings
Publication Type: Abstract
Publication Acceptance Date: December 19, 2006
Publication Date: March 20, 2007
Citation: Butchko, R.A., Brown, D.W., Proctor, R. 2007. Validation of DNA microarrays derived from the Fusarium verticillioides gene index [abstract]. Fungal Genetics Conference. p. 82.

Technical Abstract: Fusarium verticillioides is a pathogen of maize and it can produce the toxic polyketide derived secondary metabolites called fumonisins. Fumonisins have been shown to cause animal diseases and are epidemiologically correlated to esophageal cancer and neural tube defects in humans. In an effort to identify genes involved in regulation of fumonisin production and in pathogenicity on maize, a large scale EST sequencing project was conducted in collaboration with The Institute for Genomic Research. The Fusarium verticillioides Gene Index (FvGI) contains up to 11,126 sequences including 7,198 tentative consensus (TC) and 3,921 singleton EST sequences. DNA microarrays were designed based on the FvGI and were constructed by NimbleGen Systems, Madison, WI. Each DNA microarray contains 12 different 24-bp oligonucleotide probes specific to each of the unique sequences in the FvGI. Here, we present data from an experiment where total RNA from a six-point time course of F. verticillioides cultured in a liquid medium conducive to fumonisin production was used to hybridize to the microarrays. Analysis of FUM gene expression patterns over time were used to validate the microarray. The expression of FUM genes determined by the microarray analysis is consistent with expression patterns determined by Northern analysis and RT-PCR. To help determine the orientation of singleton ESTs whose direction could not be determined from sequence data or BLAST analysis, probe sets to both plus and minus strand sequences were included for all of the singletons as well as TCs containing two open reading frames. Microarray analysis successfully resolved the orientation of a number of the singletons for which the orientation was not obvious, and demonstrated the existence of two open reading frames in some TCs. Global patterns of gene expression under these conditions are also presented.