This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Technology Core Projects 2The goals of this project include the creation and development of genetic technologies that provide direct information on the networks and pathways that proteins belong to. Most of the work will be performed in the yeast Saccharomyces cerevisiae, for which many advanced tools for this work already exist. Using a technology called SLAM (synthetic lethality analyzed by microarray) and variations on this theme, we are providing extensive information on global protein networks. However the methods currently being applied in high throughput currently only apply to those proteins encoded by nonessential genes. We will extend this technology to the analysis of essential genes. We will also drive the technology to new directions such asanalysis of an e xtensive collection of mutant forms of histone proteins. These small proteins, which potentially regulate all genes and are among the most highly post-translationally modified proteins in all of biology. We will also develop analogous technologies for making genetic interaction maps in mammalian cells. All of the technology development will be done in partnership with biologists in the driving biological projects, with a strong intellectual focus on dissecting the biological networks and pathways of lysine modification by acetylation, methylation and ubiquitination, and the removal of those modifications. 1. Develop technologies for creating systematic, tagged, Temperature-sensitive (Ts) and hypomorphic (Hm) alleles of essential genes. Strains carrying these alleles produce altered proteins and can be used to generate genetic interaction maps for essential genes, elucidating the pathways and networks in which they participate, and hence their possible functions.2. Develop technologies for creating genetic interaction maps for essential genes using SLAM (synthetic lethality analysis analyzed by microarray). Two sets of essential genes will be analyzed. The first set involves the set of essential genes in known lysine modification pathways, with the focus on the enzymes that carry out modifications and the proteins that directly interact with them. Secondly, we will analyze the most complex set of acetylation/deacetylation substrates currently known, namely the histone genes. Because of the complexity of both the genes themselves, which are genetically linked and are duplicated, and the fact that there are multiple modification sites in each protein, a special technology is required. Both traditional synthetic lethality and 'dosage' synthetic lethality methods can be used. We have developed a new vector for high copy experiments in yeast that will be applied to the latter.3. Develop technologies for extending SLAM genetic interaction technologies to more complex eukaryotes Two new methodologies will be developed. In the first, we will model the development of a SLAM-like approach based on RNAi in mammalian cells. In the second, we will develop a novel and general technology for identifying the homologs of essential yeast proteins from any organism. 4. Apply the above methods to the analysis of networks and pathways of lysine modifying proteins. We will apply the above technologies to the Driving Biological Projects, including our own ongoing studies of transcriptional silencing, a process controlled in large part by modification of histone lysines. Examples of these collaborative experiments are scattered throughout the prop
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