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 alphaA-crystallin, we transfected the hamster alphaA-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal alphaA- and alternatively spliced alphaAins-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant alphaA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, approximately 1.8 kilobases apart in the normal hamster alphaA-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 alphaA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive alphaA-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble alphaA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable alphaA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
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van Rijk
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de Jong
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Bloemendal
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006224:Cricetinae; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D003459:Crystallins; D011994:Recombinant Proteins