The broad goal of this research remains the definition of the mechanisms by which acid hydrolases are transported to lysosomes. The program has become progressively more molecular to take advantage of our progress in cloning the human cDNA and genomic sequences specifying beta-glucuronidase, in cloning the rat cDNA for beta-glucuronidase, in cloning the 8.5 kb cDNA for the """"""""215K"""""""" Man 6-P receptor gene, and in discovering a new mouse mutant model for beta-glucuronidase deficiency mucopolysaccharidosis.
The specific aims are: 1) Complete studies defining the genomic organization of the human beta-glucuronidase gene. 2) Define the minimum 5' flanking sequences required for expression of the human beta-glucuronidase gene in mammalian cells. 3) Study the cellular and molecular biology of beta-glucuronidase transport in cultured mammalian cells. 4) Study expression of the human beta-glucuronidase gene in E. coli. 5) Study the expression and transport of the products of the E. coli beta-glucuronidase gene and of E. coli/human beta- glucuronidase chimeric genes in mammalian cells. 6) Study expression and transport of the human beta-glucuronidase gene in transgenic mice. 7) Study the molecular biology and molecular genetics of the cloned """"""""215K"""""""" Man 6-P receptor gene. A variety of biochemical, cell biological. immunological, molecular biological and genetic approaches are applied to the problems of enzyme transport and receptor function. The answers sought have fundamental significance, and may provide information leading to novel therapeutic approaches for lysosomal storage diseases.
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