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 even ts that are also a major driving force in protein evolution. While studying the small heat-shock protein alpha A-crystaIlin, we transfected the hamste r alpha A-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal alpha A- and alternatively spliced alpha A(ins)- crystallins, two slightly larger , immunologically crossreacting proteins, These proteins were found to be e ncoded by a mutant alpha A-crystallin gene with a large intragenic duplicat ion, arisen by illegitimate recombination at two CCCAT homologies, approxim ate to 1.8 kilobases apart in the normal hamster alpha A-crystallin gene. A s a consequence, a tandem-duplicated exon 3 sequence is present in the matu re mRNA of this gene, resulting in a 41-residue repeat in the translated pr oteins. Cells expressing the elongated alpha A-crystallins have normal grow th characteristics and the usual diffuse cytoplasmic distribution of immuno reactive alpha A-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the norma l large water-soluble alpha A-crystallin complexes, showing that the insert does not disturb the integrity of these complexes, This viable alpha A-cry stallin mutant thus-mimics the origins and effects of exon duplication, whi ch is a common consequence of exon shuffling in mammalian genome evolution.