An astounding number of novel techniques are currently being developed that will shape biomedical research in the post-genomic era. Many of these approaches fall into the area of genomics and will allow the prediction of function of genes using sophisticated comparisons of protein sequence and structure between various organisms. Additional techniques will emphasize functional genomics and will take advantage of DNA microarrays to probe the expression of the estimated 100,000 mammalian genes in various cells and tissues from both physiological and diseased states. Finally, great technological advances in instrumentation have given rise to the field of proteomics, where proteins and protein modifications are studied on a large scale in the context of genomic information. This proposal describes the development of a novel technique at the interface between functional genomics and proteomics. This technique involves the physical attachment of expression vector DNA to surfaces on which cells can be grown in culture. Cells in culture are then applied to the treated surfaces and those cells applied to the """"""""spots"""""""" containing expression vector are transfected, expressing the encoded protein. The STEP (Surface Transfection and Expression Protocol) has the potential to study the function of tens of thousands of proteins derived from cDNA expression vectors on a single microscope slide. STEP transfection can be readily adapted to DNA microarray formats for generation of printed slides and for quantitation of protein function. The research proposed is organized under three specific aims. All of these Specific Aims represent instances where the efficacy of STEP can be developed in a scientifically meaningful context. The first Specific Aim is to develop the STEP technique to the point where it can be used to screen a mutational library of a protein kinase in order to define the structural domains of the kinase. The second Specific Aim will use STEP to optimize an antisense oligonucleotide strategy to reduce the enzyme activity of a protein kinase thought to play a central role in cell proliferation. The third Specific Aim will define the interactions of a small, defined set of protein kinases, transcription factors and gene regulatory elements in functional STEP assays. Upon completion of these three Specific Aims, the STEP technique will be sufficiently developed for the scientific community to apply it to related research problems.
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