Exon shuffling mimicked in cell culture

Undesired side products of DNA transfections are usually discarded. However, here, we show that such products may provide insight into mutational events that are also a major driving force in protein evolution. While studying the small heat-shock protein αA-crystallin, we transfected the hamster αA-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal αA- and alternatively spliced αA(ins)-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant αA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, ≈1.8 kilobases apart in the normal hamster αA-crystallin gene. As a consequence, a tandem-duplicated exon 3 sequence is present in the mature mRNA of this gene, resulting in a 41-residue repeat in the translated proteins. Cells expressing the elongated αA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive αA-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble αA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable αA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.