RESUMEN
The murine autosomal dominant cataract mutants created in mutagenesis experiments have proven to be a powerful resource for modelling the biological processes involved in cataractogenesis. We report a mutant which in the heterozygous state exhibits mild pulverulent cataract named 'opaque flecks in lens', symbol Ofl. By molecular mapping, followed by a candidate gene approach, the mutant was shown to be allelic with a knockout of the bZIP transcription factor, Maf. Homozygotes for Ofl and for Maf null mutations are similar but a new effect, renal tubular nephritis, was found in Ofl homozygotes surviving beyond 4 weeks, which may contribute to early lethality. Sequencing identified the mutation as a G-->A change, leading to the amino-acid substitution mutation R291Q in the basic region of the DNA-binding domain. Since mice heterozygous for knockouts of Maf show no cataracts, this suggests that the Ofl R291Q mutant protein has a dominant effect. We have demonstrated that this mutation results in a selective alteration in DNA binding affinities to target oligonucleotides containing variations in the core CRE and TRE elements. This implies that arginine 291 is important for core element binding and suggests that the mutant protein may exert a differential downstream effect amongst its binding targets. The cataracts seen in Ofl heterozygotes and human MAF mutations are similar to one another, implying that Ofl may be a model of human pulverulent cortical cataract. Furthermore, when bred onto a different genetic background Ofl heterozygotes also show anterior segment abnormalities. The Ofl mutant therefore provides a valuable model system for the study of Maf, and its interacting factors, in normal and abnormal lens and anterior segment development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Catarata/genética , Genes Dominantes , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Arginina/química , Catarata/metabolismo , Cruzamientos Genéticos , ADN/metabolismo , Análisis Mutacional de ADN , Heterocigoto , Homocigoto , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fenotipo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8-12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.