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Medientyp:
E-Artikel
Titel:
Exon Shuffling Mimicked in Cell Culture
Beteiligte:
de Jong, Wilfried W.;
Bloemendal, Hans
Erschienen:
National Academy of Sciences of the United States of America, 1999
Erschienen in:Proceedings of the National Academy of Sciences of the United States of America
Sprache:
Englisch
ISSN:
0027-8424
Entstehung:
Anmerkungen:
Beschreibung:
<p>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 α A-crystallin, we transfected the hamster α A-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal α A- and alternatively spliced α A<sup>ins</sup>-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant α A-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, ≈ 1.8 kilobases apart in the normal hamster α A-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 α A-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive α A-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble α A-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable α A-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.</p>