Studies from this laboratory and by others have described and analyzed two genetic regulatory elements, designated Gus-r and Gus-t, which serve to control the rate of murine glucuronidase (GUS) synthesis. Both elements are either tightly linked to or within the GUs structural gene (Gus-s) on chromosome 5. Gus-r is a cis-active regulator of the androgen-induced rate of GUS synthesis in kidney tubule cells while Gus-t is a trans-active regulator of the basal rates of GUS synthesis in all tissues, with significant tissue-specific effects during postnatal development. Using a Gus-s specific cDNA probe recently developed in our laboratory, attempt will be made, to understand several important features of Gus-t expression. Structural characterization of the GUS cDNA probe (pGUS-1) will continue in order to establish the extent of the 5 prime end relative to GUS mRNA, the 5 prime to 3 prime orientation relative to the GUS polypeptide and the complete DNA sequence. Northern and dot blotting procedures will be determine whether Gus-t and Gus-r regulate GUS synthesis through control of GUS mRNA levels or by altering the translational activity of GUS mRNA. If Gus-r and Gus-t control GUS mRNA levels, isolated nuclei will be used to determine whether such control is exerted at the level of GUS mRNA transcription or at the level of processing and degradation. Experiments will also be performed using primer extension techniques to determine whether or not certain differences in GUS expression are associated with differences in mRNA structure. Analysis of the structure of the Gus-s gene will include isolating genomic clones from each GUS haplotype and determining whether structural differences exist within or flank Gus-s which might be responsible for observed differences in its expression amongst the various GUS haplotypes. Attempts will be made, using DNA-mediated transformation of cultured mouse cells, to determine to what extent the expression of defined Gus-s sequences are capable fo regulating the expression of endogenous Gus-s genes. This will test the possibility that Gus-t resides within the coding region of Gus-s and that GUS influences its own synthesis.
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