We wish to use a molecular biological approach to study the differential gene expression of the rat glutathione S-transferase isozyme family. We will investigate the multiplicity of the isozyme family in liver, kidney, testis, and lung and establish a molecular basis for the apparent tissue specific expression and the differential introduction by various xenobiotics. We plan to analyze the gene organization for the various tissue specific isozymes and design specific molecular probes obtained through cDNA and genomic DNA cloning to establish a most probable relationship between glutathione S-transferase gene structures and their regulated expressions. With a combination of hybrid-selected in vitro translation, specific immunoprecipitation and two-dimensional gel analyses, we hope to match a GST cDNA structure with a catalytic function. Upon achieving heterospecific expression of rat glutathione S-transferase sequence in E. coli, we can study the catalytic properties of an absolute pure GST which may not be obtainable by conventional biochemical means for this family of highly diversified isozymes. Furthermore, the heterospecific system allows the construction and possible expression of hybrid glutathione S-transferases. This system also allows single amino acid replacement through site specific mutagenesis at the cDNA plasmid level so that we will understand the functional domains of each isozyme more precisely. The high multiplicity of this isozyme family may reflect a very diversified physiological function beyond drug biotransformation and xenobiotics metabolism. Understanding the differential gene expression for GST isozymes not only contributes to a better appreciation of tissue differentiation but also to the possible elucidation of some of their new physiological functions. The plant glutathione S-transferases are essential in mediating resistance to various herbicides. The purified genes from the animal origin may be a valuable source of exotic genetic material for plant genetic engineering in expanding its range of herbicide resistance and possible improvement of crop yield.
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