Gene expression profiling in Arabidopsis using DNA microarrays

Abstract

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with highly variable multisystemic manifestations. The mutation underlying DM is an unstable (CTG)n expansion in the 3´ UTR of the myotonic dystrophy protein kinase gene (DMPK). The pathophysiological mechanism(s) of the expanded (CTG)n repeat remains unclear. Various effects have been proposed, most recently a gain of function for mutant DMPK transcripts that results in a generalized RNA metabolism defect, mediated through one or several trans-acting proteins involved in RNA processing. This would in turn lead to the loss of function of multiple genes by qualitatively or quantitatively affecting post-transcriptional RNA processing, splicing or nuclear export of their transcripts. To test these hypotheses, we examined global mRNA expression changes between DM patients and normal controls by comprehensively and simultaneously profiling more than 6,800 human genes with oligo-based Genechip microarrays (Affymetrix). Total, nuclear and cytoplasmic RNA fractions of DM patient lymphoblastoid cell lines (four adult-onset, one congenital) as well as primary undifferentiated myoblasts and differentiated myotubes (one adult-onset, one congenital) were profiled. DM myoblasts in culture showed a reduced differentiation rate to myotubes and a tendency to dedifferentiate, suggesting a general block in or reprogramming of differentiation. Expression profiles of DM cell lines differed considerably from controls. Between the different DM cell lines profiled, many of the more than 6,800 genes assayed showed dysregulation. Moreover, comparison of nuclear and cytoplasmic fractions suggested a defect in the nuclear export of some processed transcripts. The number of genes dysregulated and the degree of dysregulation correlated with expansion size. Functions of the dysregulated genes were highly varied. In conclusion, DNA microarray expression profiling identified several novel DM phenotype effector candidate genes that may explain the complex pathogenesis of DM.Incyte has developed a new gene expression microarray (GEM) for analysis of the plant functional genomic system Arabidopsis thaliana. This GEM features over 7,000 array elements from a non-redundant cDNA collection; nearly 30% of the estimated 25,000 genes in this organism. Over 2,000 of these clones represent genes that have functional annotation in public databases. We are offering hybridization and analysis services for the Arabidopsis GEM via Incyte and Genome Systems. Arabidopsis is the primary model system for the study of dicot plant genomics. Academic and commercial groups are using the combination of genomic and genetic tools available for Arabidopsis to study complex problems in plant biology. We have performed a few biological studies using the Arabidopsis GEM to validate the platform and provide examples of possible applications. We will present sample data on these studies.The microarray facility at MSU is part of the Arabidopsis Functional Genomics Consortium (AFGC), a collaboration recently f...