The ability of enzymes to localize within a specific subcellular site is a clearly recognized phenomenon in eukaryotic cells. However, the genetic determinants of such specificities are poorly understood. A useful model for analyzing such interactions is murine Beta-glucuronidase. As much as 50 percent of the product of the Beta-glucuronidase structural gene is found attached to the membranes of the endoplasmic reticulum of liver kidney and lung cells, with the remainder localized in lysosomes. This localization of Beta-glucuronidase within the endoplasmic reticulum requires the presence of a specific glycoprotein, designated egasyn. Egasyn is specified by a single, codominant element on chromosome 8, designated Eg, which is probably the egasyn structural gene. The Eg locus is defined by two alleles, one of which results in a deficiency of egasyn and thus a deficiency of Beta-glucuronidase in the endoplasmic reticulum. Attempts to reconstruct the Beta-glucuronidase-egasyn complex and study the nature of the interaction of egasyn and Beta-glucuronidase have been unsuccessful. However, molecular genetic approaches are now available which may permit identification and characterization of the recognition and binding specificities within each component of egasyn-Beta-glucuronidase complexes. In order to develop molecular reagents necessary for implementation of such approaches, this proposal seeks three years of support for identifying and structurally characterizing mRNA(s) and genomic DNA sequences specific for egasyn in both normal and egasyn-deficient mice. Cloned murine Beta-glucuronidase genomic sequences are already being characterized as part of an ongoing project in the applicant's laboratory. A Lambdagt-11 mouse liver cDNA library will be screened immunochemically for egasyn-specific clones. Specificity of isolates will be confirmed by hybridization-translation analysis, as well as by developmental, physiological, pharmacological and genetic assessments, and by direct comparison of the experimentally-derived amino acid sequence of egasyn with amino acid sequence deduced from experimentally-derived cDNA sequences. Structures of egasyn-specific cDNA clones and the sequence of the egasyn mRNA will be characterized by restriction mapping, DNA sequencing and primer extension analysis. Screening restriction digests of genomic DNA from inbred strains of mice by blot hybridization is proposed to uncover fragment length markers for distinguishing DNAs of normal and egasyn-deficient mice. Analysis of structural features of the egasyn structural gene in both normal and egasyn-deficient mice will be approached by first screening genomic libraries for recombinant DNAs which hybridize with egasyn-specific cDNA probes and then characterizing such clones by restriction mapping, blot hybridization, DNA sequencing and transcriptional runoff assays or S1 mapping. Experiments are also proposed to analyze the molecular defect underlying the genetic deficiency of egasyn, first by comparing the relative levels of egasyn mRNA in normal and mutant mice by blot hybridization with egasyn-specific cDNA probes, and continuing with experimental strategies which will depend on the results of initial comparisons of mRNA levels. Reagents developed in the proposed research project could also provide molecular entry to a cluster of ancestrally-related esterase genes and to the developmentally-significant oligosyndactyly gene, all of which are very closely linked to Eg on chromosome 8.
| Ovnic, M; Tepperman, K; Medda, S et al. (1991) Characterization of a murine cDNA encoding a member of the carboxylesterase multigene family. Genomics 9:344-54 |
| Novak, E K; Baumann, H; Ovnic, M et al. (1991) Expression of egasyn-esterase in mammalian cells. Sequestration in the endoplasmic reticulum and complexation with beta-glucuronidase. J Biol Chem 266:6377-80 |
