The Fields laboratory is interested in developing technologies, especially those to analyze protein function. In the last decade, genome sequences have led to the prediction of large complements of proteins, ranging from a few thousand in bacterial species to more than 20,000 for humans and other mammalian species. However, the determination of protein function remains a difficult task, given the tremendous range of biochemical activities that proteins display, the diverse modifications that a protein can undergo during its lifetime, the multiplicity of proteins potentially encoded by a single gene, and the use of proteins for more than a single function.
For many of our technology efforts, we use the unicellular eukaryote Saccharomyces cerevisiae (baker's yeast) as the host organism for carrying out protein assays. Yeast has a small number of genes, is highly tractable for experimentation, and has been used to derive numerous sets of reagents and high-throughput data. We are using yeast to analyze protein-protein and RNA-protein interactions, to characterize introns on a genomewide basis, to develop approaches to identify substrates of ubiquitin E3 ligases, to profile metabolites, to examine chromosome conformation and chromatin structure, and to identify proteins that promote recombination. We are using in vitro technologies to couple DNA fragments, the mRNA transcribed from this DNA, and the protein translated from this RNA to provide the basis for activity screens and binding selections.
We have also used S. cerevisiae for the analysis of proteins relevant to human disease. Past studies have focused on a human polyglutamine-containing protein implicated in neurodegenerative disease, the human Toll-like receptors that mediate innate immunity, the proteins of the malaria parasite Plasmodium falciparum and yeast proteins that play a role in aging.
Hesselberth, J.R., Chen, X., Zhang, Z., Sabo, P.J., Sandstrom, R., Reynolds, A.P., Thurman, R.E., Neph, S., Kuehn, M.S., Noble, W.S., Fields, S., and Stamatoyannopoulos, J.A. (2009) Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nature Methods 6: 283-289.
Fields, S. (2009) Interactive learning: Lessons from two hybrids over two decades. Proteomics 9: 5209-5213.
Schutz, K., Hesselbeth, J.R. and Fields, S. (2010) Capture and sequence analysis of RNAs with terminal 2',3'-cyclic phosphates. RNA 16: 621-631.
Araya, C.L., Payen, C., Dunham, M.J. and Fields, S. (2010) Whole-genome sequencing of a laboratory-evolved yeast strain. BMC Genomics 11: 88.
Duan, Z., Andronescu, M., Schutz, K., Mcllwain, S., Kim, Y.J., Lee, C., Shendure, J., Fields, S., Blau, C.A. and Noble, W.S. (2010) The three-dimensional architecture of the yeast genome. Nature 465: 363-367.
Cooper, S.J., Finney, G.L., Brown, S., Nelson, S.K., Hesselberth, J., MacCoss, M.J. and Fields, S. (2010) High-throughput profiling of amino acids in strains of the Saccharomyces cerevisiae deletion collection. Genome Research 20: 1288-1296.
Fowler, D.M., Araya, C.L., Fleishman, S.J., Kellogg, E.H., Stephany, J.J., Baker, D. and Fields, S. (2010) High resolution mapping of protein sequence–function relationships. Nature Methods 7: 741-746.
Fowler, D., Cooper, S., Stephany, J., Hendon, N., Nelson, S. and Fields, S. (2011) Suppression by copper and zinc of statin effectiveness in yeast and human cells. Molecular BioSystems 7: 533-544.