An incorrectly predicted initiator methionine can lead to failure to obtain cell-surface expression, or failure to be activated by ligands. Rarely is the initiating methionine defined by experimental N-terminal sequencing studies; thus, there is a clear need to be able to confidently predict the correct one. There are several lines of evidence to suggest the correct one may have been chosen, such as an ATG codon in a favourable context for initiation, or the presence of an upstream in-frame terminator codon, or the prediction of a signal peptide-like sequence at the amino terminus, all of which have some validity (Kozak 1996). The availability of genomic sequence makes the task of identifying an upstream in-frame terminator codon relatively straightforward and any extra sequence evidence from ESTs is also helpful. Comparison with species orthologues, where known or predicted, will also add weight to a correct prediction, since the nucleotide sequence identity decreases going from coding region into the 5' UTR (Makalowski et al. 1996).
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