The foremost problem in cell and developmental biology is the mechanism that controls patterns of gene expression in differentiation. Aberrance in this regulation accounts for most developmental abnormalities and, according to some schools, even neoplastic disease. Bone development affords a unique system to study the molecular basis of gene expression patterns. The principal nuclear protein changes during bone development are in the nuclear matrix. We hypothesize that these proteins are essential in establishing differentiation-specific gene expression. We propose to study the role of nuclear matrix proteins in regulating genes expressed in in vitro culture. We will first prepare a complete catalogue of differentiation stage-specific nuclear matrix proteins using 2D gel electrophoresis. We will extend these measurements to cells treated with the differentiation modulators, vitamin D, dexamethasone and TGF-B which will help illuminate agent action and nuclear matrix protein functions. Stage-specific nuclear matrix proteins will be isolated from preparative gel electropherograms and used to generated monoclonal antibodies. These will serve for immuno-colocalization in the nucleus by immunogold stained reinless section electron micrographs and at the level of tissue by immunofluorescence. cDNA clones will be selected with the antibodies for sequencing and studying the regulation of the nuclear matrix protein genes themselves. Immuno-colocalization techniques will be extended to bone tissue developing in utero.
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