Mutations in the genes for the myelin structural proteins MBP and PLP produce defects in oligodendrocyte development and morphology as well as in levels of the respective proteins. In double mutant mice, the morphological defects produced by these mutations are often altered separately from the protein levels, suggesting that: a. The MBP gene has at least two separately regulated functions. b. The PLP gene has at least three such independent functions. c. When major myelin proteins are lacking, the oligodendrocyte may make myelin-like sheaths using other proteins - minor, transient, or non-specific. d. Some function of the MBP gene may be toxic to oligodendrocytes unless it is complemented by the proper function of the PLP gene. To test these ideas, we propose an integrated, multidisciplinary analysis of the molecular, biochemical, and quantitative morphological phenotypes of specific sets of double mutant combinations selected on the basis of their predicted value. Genes from which these combinations will be engineered include the familiar MBP and PLP mutations (shi, shi(mld), jp, and jp(msd); the MBP(1) transgene; rsh, a new and exciting PLP mutation; and Tr, which affects PNS myelin. All mice will be studied on the unique, genetically comparable B6C3Fl-based stocks prepared in our laboratory. This is essential to validate quantitative comparisons. Genotypes will be confirmed by Southern blots and restriction enzyme analysis of PCR-amplified DNA fragments; steady-state mRNA and protein levels will be determined by Northerns and immunoblots; proteins will be localized by immunocytochemistry; and myelin structure, including the newly-described stitches at the cytoplasmic apposition of MBP mutant myelin, and oligodendrocyte development will be analyzed by quantitative morphology. Methods for high resolution in situ hybridization newly refined in our laboratories will be used to study the detailed distributions and ultrastructural cellular associations of myelin-specific mRNAs in wild-type, jp, and ultimately double mutant oligodendrocytes.
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