Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.
A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.
GSF-National Research Center for Environment and Health, Institute of Mammalian Genetics, D-85764 Neuherberg, Germany. graw@gsf.de
Jochen
J
Graw
Angelika
A
Neuhäuser-Klaus
Jana
J
Löster
Norman
N
Klopp
Jack
J
Favor
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002386:Cataract / Q000235:genetics*; D002874:Chromosome Mapping; D003459:Crystallins / Q000235:genetics*; D005038:Ethylnitrosourea / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D012326:RNA Splicing / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D038222:gamma-Crystallins
D000477:Alkylating Agents; D003459:Crystallins; D011993:Recombinant Fusion Proteins; C107256:crystallin gammaE; D038222:gamma-Crystallins; D005038:Ethylnitrosourea