DETERMINING THE EVOLUTIONARY POTENTIAL OF A GENE

In addition to information for current functions, the sequence of a ge ne includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia co li encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene f or growth on the beta-galactoside sugars lactose and lactulose. Experi mental evidence, which has suggested that the evolutionary potential o f Ebg enzyme is limited to two specific amino acid replacements, is li mited to examining the consequences of single base-substitutions. Thir teen beta-galactusidases homologous with the Ebg beta-galactosidase ar e widely dispersed, being found in gram-negative and gram-positive eub acteria and in a eukaryote. A comparison of Ebg beta-galactosidase wit h those 13 beta-galactosidases shows that Ebg is part of an ancient cl ade that diverged from the paralogous lacZ beta-galactosidase over 2 b illion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for f ull Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the onl y acceptable amino acids at those positions, or all of the single-base -substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitu te a deep selective valley that has not been traversed in over 2 billi on years. The evolutionary potential of Ebg is thus limited to those t wo replacements.