The objective of this project is to define the regulatory signals that control myelination, the event where oligodendrocytes and Schwann cells extend processes that enwrap and ensheath axons. We have focused on the expression of the major protein in myelin, proteolipid protein (PLP), which we previously cloned and reported on the gene structure and mode of alternative splicing. During the current reporting period, we have identified several sequences in the promoter region of the PLP gene which are conserved among the myelin-specific genes and which act as binding sites for nuclear proteins isolated from brain. The contracts that these putative regulatory proteins make with the conserved motifs have been precisely defined by footprinting techniques. In addition, the function of these conserved cis elements has been approached by two methods. First, plasmid constructions containing the various cis elements next to a reporter gene have been assayed in cultured glial cells. Second, one construction has been introduced into the germ line of mice to assay tissue specificity and developmental regulation in an in vivo system. PLP is an extremely conserved protein, as might be expected for a protein that provides the underpinning of the myelin sheath. The rigid conservation of PLP between species predicts that little diversity in the sequence of this structural protein would be tolerated. Indeed, we have traced the defect in a number of dysmyelinating to point mutations in the PLP gene which resulted in amino acid substitutions, at least one of which was a conservative amino acid replacement. We have analyzed the structure of PLP in the oligodendrocyte plasma membrane, the precursor of the myelin membrane. Our results demonstrate that PLP is a transmembrane protein with the great majority of the molecule located on the extracellular face of the membrane. This observation, along with the potential for interactions between extracellular domains of PLP, suggests a structural role for PLP in supplying and maintaining the lamellar structure of myelin.
