Site-specific mutagenesis in a homogeneous polyglutamine tract: application to spinocerebellar ataxin-3.
In recent years, nine neurodegenerative diseases have been found to be caused by the expansion of a CAG-triplet repeat in the coding region of the respective genes, resulting in lengthening of an otherwise harmless polyglutamine tract in the gene products. To facilitate structural studies of these disease mechanisms, a general protocol is described that allows site-specific mutations to be introduced into the polyglutamine tract. Based on 'cassette mutagenesis', this protocol involves engineering unique restriction sites into the flanking regions of the CAG repeat and subsequently replacing the wild-type CAG repeat with a double-stranded synthetic DNA fragment containing the desired mutations. This method was applied to the spinocerebellar ataxin-3 protein, such that the wild-type amino acid sequence -Q(3)KQ(22)- was replaced by a -Q(9)CQ(9)- sequence. In this case, the incorporated cysteine residue can be exploited for various chemical modifications, lending the host glutamine repeat to many structural and biophysical techniques for the resolution of a specific residue. The method reported here bypasses many problems that can arise from PCR-based mutagenesis methods.
Chen
Yu Wai
YW
Centre for Protein Engineering and Cambridge University Chemical Laboratory, MRC Centre, Hills Road, Cambridge CB2 2QH, UK. ywc@cantab.net
D019943:Amino Acid Substitution; D000067699:Ataxin-3; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D017827:Machado-Joseph Disease / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins; D010455:Peptides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D020824:Spinocerebellar Tracts / Q000378:metabolism*; D018911:Trinucleotide Repeats / Q000235:genetics
D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; C097188:polyglutamine; C092341:ATXN3 protein, human; D000067699:Ataxin-3; D003545:Cysteine