Once the nucleotide or amino acid sequences of molecules from different organisms have been determined, they can be compared. Homologous positions can be identified only if we first pinpoint the locations of deletions and insertions that have occurred in the molecules of interest in the time since the organisms diverged from a common ancestor. A simple hypothetical example illustrates this sequence alignment technique. In Figure 23.1 we compare two amino acid sequences from homologous proteins in different organisms. The two sequences at first appear to differ in both the number and identity of their amino acids. If we insert a gap after the first amino acid in sequence 2 (after leucine), however, the similarities in the two sequences become obvious. This gap represents the occurrence of one of two evolutionary events: an insertion of an amino acid in the longer protein or a deletion of an amino acid in the shorter protein. Having adjusted for this insertion or deletion event, we can see that the two sequences differ by only one amino acid at position 6 (serine or phenylalanine).

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Adding a single gap—that is, identifying a deletion or an insertion—aligns the two sequences in Figure 23.1. Additional sequences can now be added to the alignment in a similar manner. Longer amino acid sequences, and those that have diverged more extensively, require more elaborate adjustments. Explicit models (incorporated into computer algorithms) have been developed to account for the relative probabilities of deletions, insertions, and particular amino acid replacements.

Having aligned the sequences, we can compare them by counting the number of nucleotides or amino acids that differ between them. Summing the numbers of the same and different amino acids in each pair of sequences allows us to construct a similarity matrix, which gives us a measure of the minimum number of changes that have occurred since the divergence of each pair of organisms (see Figure 23.1).