Poster: Regulation of gene expression
Abs #
777: One-step purification of polyribosomes for global characterization of cell-type specific gene expression in Arabidopsis
Two significant deficiencies of routine mRNA expression profiling studies are, (1) the abundance of an individual mRNA may not reflect the level of its translation, and (2) the abundance of an mRNA in specific cell-types cannot be gleaned from organ extracts. To circumvent these challenges and augment the elucidation of developmental processes and environmental responses, we are developing a means to isolate polyribosomes for global characterization of cell-specific gene expression in Arabidopsis thaliana. The basis of this technology is the use of cell-type specific promoters to drive the expression of an epitope-tagged ribosomal protein (r-protein). Polyribosomes are affinity purified and the associated mRNAs are extracted, amplified and hybridized to DNA microarrays to analyze mRNA expression. Transgenic lines over-expressing individual His6-FLAG-tagged versions of five 60S subunit (RPL5, RPL7, RPL12, RPL18 and RPL23) and two 40S subunit r-proteins (RPS9 and RPS20) were generated. The accumulation of these proteins into ribosomes, from the tagged subunit to large polyribosome complexes, was confirmed by immuno blot analyses. Two His6-FLAG-tagged 60S r-proteins allowed for an efficient one-step immuno-purification of 80S ribosomes from crude leaf homogenates. Sucrose gradient fractionation of the purified complexes then confirmed the isolation of polyribosomes, and RT-PCR demonstrated the presence of intact mRNAs. Experiments are underway to express these epitope-tagged r-proteins under the control of cell-type specific promoters. These tools will be used for a quantitative assessment of nuclear and polyribosomal mRNA populations of plant cells. Research supported by a grant from NSF-Plant Genome Program to J.B.-S. and D. Galbraith (DBI 0211857).
