Unanticipated Antigens: Translation Initiation at CUG with Leucine
Figure 1
The CUG Initiation Codon Is Decoded as Leucine Independent of RNA Sequence
(A) The indicated synthetic peptides mixed with extracts from untransfected COS-7 cells were separated by RP-HPLC. Each fraction was tested for BCZ103 T cell-stimulating activity with Kb+B7.2+ L cells as APCs. After overnight incubation, the β-galactosidase induced in activated T cells was measured using the substrate chlorophenol red-β-pyranoside, which yields a colored product with absorbance at 595 nm. The arrows indicate the reproducible peak elution times for the MYL8 and the LYL8 peptides. Injections of buffer alone (Buffer) were carried out under identical conditions, and the fractions were assayed in parallel to ensure absence of cross-contamination between runs.
(B–D) Extracts from COS-7 cells transfected with cDNA encoding Kb and the indicated constructs were separated by RP-HPLC. Fractions were tested for BCZ103 T cell-stimulating activity as in (A).
(E) The indicated synthetic peptides mixed with extracts from untransfected COS-7 cells were separated by RP-HPLC. Each fraction was tested for the specific DBFZ T cell-stimulating activity with Db+B7.2+ L cells as APCs as in (A). The arrows indicate the reproducible peak elution times for the MM9 and the LM9 peptides.
(F and G) Extracts from COS-7 cells transfected with cDNA encoding Db and the indicated constructs were separated by RP-HPLC. Fractions were tested for DBFZ T cell-stimulating activity as in (E).
(H) A range of concentrations of the indicated synthetic peptides was tested for BCZ103 T cell-stimulating activity with Kb+B7.2+ L cells as APCs.
(I) A range of concentrations of the indicated synthetic peptides was tested for DBFZ T cell-stimulating activity with Db+B7.2+ L cells as APCs.