The overall aim of this project is the investigation of the molecular mechanism(s) by which myelination occurs. The expression of the myelin proteolipid protein gene will be investigated in normal mice and several mouse dysmyelination mutants. A family of proteolipid mRNAs have been identified in the mouse, which range from 1400 to 4400 nucleotides in length. cDNAs specific to each of the mRNAs will be isolated and characterized to establish the relationships among these mRNAs. Selective probes will be used to screen the proteolipid protein cDNAs to identify cDNAs specific for the DM20, a myelin protein that is very closely related to the major myelin proteolipid protein. These putative DM20 cDNAs will be sequenced to identify their exact relationship to the proteolipid cDNAs. The proteolipid protein gene has been mapped to a region of the human X-chromosome that encompasses the jimpy locus in the mouse genome, and we have demonstrated that proteolipid mRNAs in the jimpy mouse are 100-200 nucleotides shorter than normal proteolipid mRNAs. The current studies will pursue the question of whether there is a structural alteration in the proteolipid protein gene that causes this size differences in the mRNAs. The site within the jimpy proteolipid protein gene that is altered will be determined. Proteolipid protein gene expression will also be studied in the jimpy msd mouse, which is an allele of the jimpy mutation, to establish the effect of this mutation on the synthesis of the family of proteolipid mRNAs, proteolipid and DM20 protein. Proteolipid protein gene expression will be investigated in two other dysmyelination mutants, shiverer and quaking. These two mutations have distinctly different effects on myelin basic protein expression in these animals. The shiverer mouse has a deletion in the myelin basic protein gene and make no myelin basic protein mRNA, while the quaking mouse has close to normal levels of myelin basic protein mRNA. Detailed analyses of the levels of the proteolipid mRNAs and proteins during development in these animals will be conducted. In order to address certain questions on the expression of these myelin genes in the mutant animals, normal and mutant mouse oligodendrocytes in mixed primary glial cell cultures will be characterized, and the expression of the four myelin basic proteins and the two myelin proteolipids (PLP and DM20) and their respective RNAs will be studied in cultured oligodendrocytes, using a battery of antibodies and cDNA probes.
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