Characterization of a 1-bp delection in the yE-Crystallin gene leading to a nuclear and zonular cataract in the mouse.

PURPOSE: A previous study had found a mouse mutant to have bilateral nuclear cataract with zonular opacity after paternal irradiation with gamma-rays. The mutation was then demonstrated to be allelic with the Cat2 group of dominant cataract mutations and was referred to as Cat2(nz) in a later study. Because several members of this group have been confirmed as mutations in the gene cluster coding for gamma-crystallins (CRYG:), these genes were now tested as candidates for Cat2(nz). METHODS: All six gamma-crystallin-encoding genes were amplified by polymerase chain reaction (PCR) from cDNA or genomic DNA and sequenced. An antibody against the changed protein was developed and used for Western blot analysis. The mutant was also characterized morphologically. RESULTS: A 1-bp deletion in exon 2 of the gammaE-crystallin-encoding gene CRYGE: was causative of the cataract phenotype. This particular mutation is therefore referred to as CRYGE:(nz). The predicted frameshift after codon 29 led to a changed amino acid sequence of 96 amino acids. The altered 13-kDa protein was expressed in the eye lens as demonstrated by Western blot analysis. Cataracts became visible at day 18.5 of embryonic development and reached the final phenotype at 2 weeks after birth. CONCLUSIONS: The CRYGE:(nz) is the sixth mutation in the mouse that has been reported so far to affect the CRYG: gene cluster, which demonstrates its importance for lens transparency

A 6-bp Deletion in the Crygc Gene Leading to a Nuclear and Radial Cataract in the Mouse.

A mouse mutant expressing a bilateral nuclear and radial cataract was found after paternal treatment with chlorambucil. The purpose of this study was to establish the linkage of the mutation to a particular chromosome to allow molecular characterization. Moreover, the mutants were examined morphologically. METHODS: Isolated lenses were photographed and histologic sections of the eye were analyzed according to standard procedures. The mutation was localized to chromosome 1 by allelism testing with the Cryge(nz) mutation. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced. RESULTS: A novel mouse cataract was characterized by a nuclear and radial opacification of the lens. The lenses of the mutants are smaller than those of the wild type. The histologic analysis demonstrated degeneration of lens fibers in the lens core. Abnormal remnants of cell nuclei are present throughout the entire lens. Genetic analysis revealed allelism to the Cat2 group of dominant cataracts on mouse chromosome 1; therefore, the cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A 6-bp deletion in exon 3 of the gammaC-crystallin encoding gene (Crygc) is causative for the cataract phenotype; the mutation is therefore designated CrygcChl3. The deletion of the bases 420 to 425 leads to a loss of two amino acids, Gly and Arg, in the fourth Greek-key motif. CONCLUSIONS: The CrygcChl3 is the first mutation in the mouse affecting the Crygc gene. Dominant mutations for five of the six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster for lens transparency

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 by 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 similar to15.6 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

Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.

Pax6 is a key regulator of eye development in vertebrates and invertebrates, and heterozygous loss-of-function mutations of the mouse Pax6 gene result in the Small eye phenotype, in which a small lens is a constant feature. To provide an understanding of the mechanisms underlying this haploinsufficient phenotype, we evaluated in Pax6 heterozygous mice the effects of reduced Pax6 gene dosage on the activity of other transcription factors regulating eye formation. We found that Six3 expression was specifically reduced in lenses of Pax6 heterozygous mouse embryos. Interactions between orthologous genes from the Pax and Six families have been identified in Drosophila and vertebrate species, and we examined the control of Pax6 and Six3 gene expression in the developing mouse lens. Using in vitro and transgenic approaches, we found that either transcription factor binds regulatory sequences from the counterpart gene and that both genes mutually activate their expression. These studies define a functional relationship in the lens in which Six3 expression is dosage-dependent on Pax6 and where, conversely, Six3 activates Pax6. Accordingly, we show a rescue of the Pax6 haploinsufficient lens phenotype after lens-specific expression of Six3 in transgenic mice. This phenotypic rescue was accompanied by cell proliferation and activation of the platelet-derived growth factor alpha-R/cyclin D1 signaling pathway. Our findings thus provide a mechanism implicating gene regulatory interactions between Pax6 and Six3 in the tissue-specific defects found in Pax6 heterozygous mice

CrygfRop : The First Mutation in the Crygf Gene Causing a Unique Radial Lens Opacity.

PURPOSE. The Rop (radial opacity) mutation, which was recovered in a mutagenicity screen after paternal treatment with procarbazine, was analyzed to determine phenotype, chromosomal localization, candidate genes, and molecular lesion.METHODS. Native lenses were photographed under a dissecting microscope. Histologic sections of the eye were made according to standard procedures. Fine mapping of the mutation in relation to microsatellite markers for mouse chromosome 1 was performed. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced.RESULTS. The nuclear opacity of the heterozygous mutants showed radial structures, whereas the opacity of the homozygotes was homogenous. The histologic analysis revealed changes in the lens nucleus, which corresponds to the pronounced opacification in lenses of homozygous mutants. The allelism of Rop to the Cat2 group of dominant cataracts on mouse chromosome 1 was confirmed by linkage to microsatellite markers D1Mit156 and D1Mit181. The cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A T-->A exchange in exon 2 of the Crygf gene leads to a Val-->Glu exchange in codon 38 and was considered to be causative for the cataract phenotype; therefore, Crygf(Rop) has been suggested as the designation for the mutation.CONCLUSIONS. Crygf(Rop) is the first mutation affecting the Crygf gene. Dominant cataract mutations for all six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster in lens transparency