Exon shuffling mimicked in cell culture.

Abstract:

: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.

authors

van Rijk AA,de Jong WW,Bloemendal H

doi

10.1073/pnas.96.14.8074

keywords:

subject

Has Abstract

pub_date

1999-07-06 00:00:00

pages

8074-9

issue

14

eissn

0027-8424

issn

1091-6490

journal_volume

96

pub_type

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