The overall focus of this grant is to study the regulation of mouse metallothionein (MT) genes in developmental, physiological and molecular terms. We propose to isolate and characterize the mouse MT-II gene with particular emphasis on comparing its transcriptional regulation in response to various metal and steroid inducers to that of the MT-I gene. The regulatory regions of these genes will be defined by in vitro mutagenesis coupled with expression assays following injection into mouse eggs or transfection into tissue culture cells. We also propose to study the binding of purified glucocorticoid and heavy metal receptors to the regulatory regions of these MT genes. Mutations in the structural genes will be created to test: (a) the effects of introns on mRNA processing, (b) the role of untranslated regions on MRNA stability and function and (c) the importance of amino acid sequence on MT function and stability. The molecular basis of MT gene commitment will be explored by trying to define critical sites of DNA methylation that affect MT gene inducibility. Changes in MT gene commitment and expression will be correlated with changes in chromatin structure, especially with the appearance of a nuclease hypersensitive site that lies 5' of the transcription start site. The MT promoter/regulatory region will be fused to other structural genes and these fusion genes will be introduced into mice by injecting them into fertilized eggs. This system allows analysis of the effects of chromosome location on tissue specific gene expression, the inheritance of foreign genes and changes in gene expression that occur from one generation to the next. The regulation of these fusion genes by zinc permits testing the biological consequences of altering the level of expression of a particular gene product. In addition, this technique affords an opportunity to correct certain gentic defects.
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