MOLECULAR-CLONING AND EXPRESSION OF CDNA FOR MURINE GALACTOCEREBROSIDASE AND MUTATION ANALYSIS OF THE TWITCHER MOUSE, A MODEL OF KRABBES DISEASE

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for hum an galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity bet ween the human and murine amino acid sequences was very high, being ca lculated to be 84%. Sequencing of cDNA from liver of the twitcher mous e revealed a nonsense mutation at codon 339 (TGG --> TGA). The most ab undant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, whi ch was not detected in twitcher mice. This suggests that the cDNA (2,2 78 bp) we characterized represents a minor species generated by an alt ernate poly(A) signal and that most of the mRNA has a much longer 3'-u ntranslated region. Genome analysis revealed that this mutation was ho mozygous in the twitcher and heterozygous in the carrier but was not p resent in normal mice. The normal mouse cDNA but not the mutant cDNA o f the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation resul ts in the deficiency of galactocerebrosidase in the twitcher mouse.