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Contains fulltext : 252430.pdf (Publisher’s version ) (Closed access) 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.

Contains fulltext : 122335.pdf (Publisher’s version ) (Closed access) We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous system occurred. Homozygous transgenic mice developed hind limb paralysis after 8 weeks of age and displayed progressive loss of myelin and axonal dystrophy in both the central and peripheral nervous system with ongoing age. Pathologically the phenotype resembled, to a certain extent, neuroaxonal dystrophy. The biochemical findings presented in this paper (activity of the enzymes superoxide dismutase, catalase and transglutamase, myelin protein zero expression levels and blood sugar levels) confirm this pathology and exclude other putative pathologies like Amyothrophic Lateral Sclerosis and Hereditary Motor and Sensory Neuropathy. Consequently, an excessive cytoplasmic accumulation of the transgenic protein or a disturbance of the normal metabolism are considered to cause the observed neuropathology. Therefore, extra-ocular alphaA-crystallin-expressing transgenic mice may serve as a useful animal model to study neuroaxonal dystrophy.

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