Mechanistic features of CAG*CTG repeat contractions in cultured cells revealed by a novel genetic assay.
Trinucleotide repeats (TNRs) undergo high frequency mutagenesis to cause at least 15 neurodegenerative diseases. To understand better the molecular mechanisms of TNR instability in cultured cells, a new genetic assay was created using a shuttle vector. The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is dependent on TNR length. The vector harbors the SV40 ori and large T antigen gene, allowing portability between primate cell lines. The shuttle vector is propagated in cultured cells, then recovered and analyzed in yeast using selection for reporter gene expression. We show that (CAG*CTG)25-33 contracts at frequencies as high as 1% in 293T and 293 human cells and in COS-1 monkey cells, provided that the plasmid undergoes replication. Hairpin-forming capacity of the repeat sequence stimulated contractions. Evidence for a threshold was observed between 25 and 33 repeats in COS-1 cells, where contraction frequencies increased sharply (up 720%) over a narrow range of repeat lengths. Expression of the mismatch repair protein Mlh1 does not correlate with repeat instability, suggesting contractions are independent of mismatch repair in our system. Together, these findings recapitulate certain features of human genetics and therefore establish a novel cell culture system to help provide new mechanistic insights into CAG*CTG repeat instability.
Pelletier
Richard
R
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Box 986805, Omaha, NE 68198-6805, USA.
Farrell
Brian T
BT
Miret
Juan José
JJ
Lahue
Robert S
RS
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D018911:Trinucleotide Repeats
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1