Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana

Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of meta bolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having > 40% i dentity, and P450s of the same subfamily having > 55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabido psis, and its genome is estimated to contain as many as 286. Genome-wide DN A microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within the se families of closely related genes may lead to cross-hybridization. We de signed experiments to systematically evaluate the specificity of P450 micro arrays, and showed that conditions could be optimized to provide a very hig h degree of hybridization specificity. Under these conditions, and employin g a 20% intensity value of maximum hybridization intensity as a cut-off, la beled P450 genes exhibited essentially no cross-hybridization between famil ies and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from vario us Arabidopsis tissue as the target. Many of the P450 genes displayed tissu e-specific expression, leading to hypotheses as to the function of individu al genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone. (C) 2001 Elsevier Science B.V. All rights reserved.