A single-base-pair deletion in the beta-glucuronidase gene accounts for the phenotype of murine mucopolysaccharidosis type VII

Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6567-71. doi: 10.1073/pnas.90.14.6567.

Abstract

Murine mucopolysaccharidosis type VII is a heritable disease caused by a spontaneous mutation, gus(mps), closely linked to the beta-glucuronidase structural gene on chromosome 5. Mice homozygous for the mutation have a > 200-fold decrease in beta-glucuronidase mRNA levels and virtually no enzyme activity detectable by a sensitive fluorometric assay. Approximately 20 kb of genomic DNA containing the beta-glucuronidase gene Gus and > 2 kb of 5' and 3' flanking sequences were cloned from both a gus(mps)/gus(mps) mouse and a +/+ mouse of the progenitor strain. Restriction enzyme digests containing DNA fragments 20-400 bp in length were generated from each of the two Gus alleles and then compared by using nondenaturing polyacrylamide DNA-sequencing gels. This method rapidly identified a large number of restriction sites and was sensitive enough to detect a restriction fragment length variation resulting from a 1-bp deletion in the gus(mps) allele. DNA-sequence analysis of the mutant genomic fragment showed that the 1-bp deletion created a frameshift mutation within exon 10. Insertion of the deleted nucleotide by oligonucleotide site-directed mutagenesis restored function to the corrected mutant gene when transfected into gus(mps)/gus(mps) fibroblasts. We concluded that the frameshift mutation, which introduces a premature stop codon at codon 497 in exon 10, accounts for the molecular, biochemical, and pathological abnormalities associated with the gus(mps) phenotype.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Exons / genetics
  • Frameshift Mutation / genetics*
  • Genome
  • Glucuronidase / genetics*
  • Mice
  • Mice, Mutant Strains
  • Molecular Sequence Data
  • Mucopolysaccharidosis VII / enzymology
  • Mucopolysaccharidosis VII / genetics*
  • Mucopolysaccharidosis VII / veterinary*
  • Mutagenesis, Site-Directed
  • Phenotype
  • Polymorphism, Restriction Fragment Length
  • Restriction Mapping
  • Rodent Diseases / genetics*
  • Sequence Analysis, DNA
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Sequence Homology, Nucleic Acid
  • Transfection

Substances

  • Glucuronidase