Martin Buess

Summary: Although translation is involved in nearly every aspect of cell biology, our understanding of the detailed role it plays in normal physiology and disease is primitive compared to our knowledge of the role and mechanisms of transcriptional regulation. This is partly due to systematic studies using DNA microarrays which, have expanded and deepened our understanding of how global gene expression, at the level of mRNA abundance, is modulated during numerous cellular and pathological processes. However, insufficient focus on post-transcriptional regulation at the systems level is the main reason we know relatively little about this fundamental cellular mechanism. Competitive hybridization on DNA microarrays has been used to determine the rate of translation of all expressed mRNAs after fractionating polysomes on sucrose density gradients; the mRNA from each fraction, which contains mRNAs that are bound by one or two or three, etc. 80S ribosomal complexes, is hybridized against all expressed mRNAs such that the peak fraction of mRNA abundance describes the relative rate of translation. Because this approach is difficult, time consuming, and requires many DNA microarrays for each sample analyzed, it is limited to a small subset of cellular physiology and pathology. To simplify the method, I propose to develop a more efficient method of polysome isolation which utilizes affinity purification to isolate actively translated mRNAs. This simplified technique requires only one array per sample analyzed and avoids the difficulty in reproducing sucrose density fractionation thereby making systematic investigation of global regulation of gene expression at the translational level practical.

Jason Myers