Figure 1.

Key features of the in vitro assay for expansions. (A) Assay procedure. A 22-repeat shuttle vector was incubated with cell-free extract, then recovered and transformed into yeast. Plasmids containing an expanded (≥ 26) repeat confer canavanine resistance. The repeat region is amplified by PCR and the expansion is confirmed on polyacrylamide gels. (B) Expansion selection²². At 22 repeats, CAN1 is expressed, causing canavanine sensitivity. Expansions adding ≥ 4 repeats cause a change in the transcriptional start site, such that an out-of-frame initiator codon is incorporated into the mRNA. Translation from this initiator codon is out-of-frame with respect to the CAN1 structural gene, causing canavanine resistance²². (C) Shuttle vectors. Plasmids are based on those used by Claassen and Lahue³⁵, modified with CAN1 selection cassette²². Briefly, each plasmid is a three-way shuttle vector capable of replicating in Saccharomyces cerevisiae, E. coli, and human cells or cell-free extracts supplemented with SV40 large T antigen. The TNR is usually (CTG)₂₂, although other sequences can be substituted. P, adh1 promoter for expression in yeast.