The long-term goal of this research is directed at understanding the genetic basis and resulting physiological processes that produce a mutant mouse phenotype known as mitg. Numerous mutations at the mouse microphthalmia (mi) locus have been described. Generally, the phenotypes are complex and consist of a lack of pigmentation, small eyes, a mast cell defect, and bone abnormalities. Through backcrossing and linkage studies, the locus for this allele has been assigned to the mouse chromosome 6. In many separate isolates of mutant mice, the defect invariably segregates as a single Mendelian trait. A single gene defect that produces such a pleiotropic effect involving several cell types suggests some involvement at a control point in development or differentiation. An understanding of the basis of this mutant phenotype may therefore provide information regarding the normal cellular interactions and development for the affected systems. The particular mi mutant line that we are characterizing arose as an insertional mutant from the production of a transgenic mouse used in an unrelated study. The presence of these transgenic sequences thus serve as a flag for the precise location of genetic material responsible for the mi phenotype. Preliminary data indicates presence of an expressed gene that is interrupted by this insertional event. This investigation involves the identification of the gene producing the microphthalmia phenotype and an initial characterization of its expression.
The specific aims of this proposal are: (1) to isolate and characterize the cDNA encoding the transcription factor from the mitg locus. This will be accomplished by screening mast cell and skin cDNA libraries with junction fragments from the integration site of the transgene. (2) to provide evidence that the disrupted gene in the mi locus represents the basic defect in our mitg line and in other allelic mi lines. Evidence for a defect in expression levels or transcript size will be sought by comparing RNA from the tissues of wild type and mutant animals. In addition, antibody generated against the mi protein will be used in Western blot analyses of tissue extracts. Studies to functionally correct various aspects of the mi phenotype with the transcription factor gene will also be performed. (3) to isolate and characterize the mi gene. Cosmid clones spanning the transgene insertion site will be isolated by screening genomic libraries with the junction fragments or the isolated cDNA. (4) to investigate the tissue and developmental expression of the mi gene. In situ hybridization to sectioned mouse embryos will be performed as well as standard Northern blot hybridization analyses from various tissues and developmental timepoints. (5) to further investigate the functional characteristics of the mitg mutant mice.
