Selectable system for monitoring the instability of CTG/CAG triplet repeats in mammalian cells.
Despite substantial progress in understanding the mechanism by which expanded CTG/CAG trinucleotide repeats cause neurodegenerative diseases, little is known about the basis for repeat instability itself. By taking advantage of a novel phenomenon, we have developed a selectable assay to detect contractions of CTG/CAG triplets. When inserted into an intron in the APRT gene or the HPRT minigene, long tracts of CTG/CAG repeats (more than about 33 repeat units) are efficiently incorporated into mRNA as a new exon, thereby rendering the encoded protein nonfunctional, whereas short repeat tracts do not affect the phenotype. Therefore, contractions of long repeats can be monitored in large cell populations, by selecting for HPRT(+) or APRT(+) clones. Using this selectable system, we determined the frequency of spontaneous contractions and showed that treatments with DNA-damaging agents stimulate repeat contractions. The selectable system that we have developed provides a versatile tool for the analysis of CTG/CAG repeat instability in mammalian cells. We also discuss how the effect of long CTG/CAG repeat tracts on splicing may contribute to the progression of polyglutamine diseases.
Gorbunova
Vera
V
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Seluanov
Andrei
A
Dion
Vincent
V
Sandor
Zoltan
Z
Meservy
James L
JL
Wilson
John H
JH
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004249:DNA Damage; D005091:Exons; D005720:Gamma Rays; D017930:Genes, Reporter; D005821:Genetic Techniques; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018911:Trinucleotide Repeats
D010455:Peptides; C097188:polyglutamine; D000228:Adenine Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase