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The PLP gene was assigned to the X-chromosome in both mouse and humans using a bovine cDNA probe (Willard et al. 1985), making it a candidate for X-

linked dysmyelinating conditions of the CNS. Support for this proposal came from the mapping of the jimpy phenotype and the PLP gene to the same region on the X-chromosome and the finding of a defect of the PLP transcript (Dautigny et al. 1986).

It was found that Jimpy mice expressed PLP mRNA at greatly reduced levels and that PLP cDNAs generates from Jimpy PLP mRNA contained a 74bp deletion within the coding region that was present at the genomic level. The mutation was in a splice site that resulted in an aberrant splice event that resulted in the deletion of part of the mRNA (Nave et al. 1987). The predicted protein encoded by this deleted mRNA was altered at the C-terminus, and it was subsequently reported that PLP in Jimpy lacks amino acids 208-232 (Hudson et al. 1987). It has been postulated that the Jimpy mutation leads to a block in oligodendrocyte maturation prior to the completion of the myelination process (Hudson et al. 1987).

A second PLP mutation was identified in mice that was associated with the milder phenotype. In this case the defect was found to be a conservative Alanine-Valine substitution at amino acid number 242 (Gencic et al. 1990). Allelic mutations have since been identified in the md rat (Boison et al. 1989), shaking pup in dog (Nadon et al. 1990), and the pt rabbit (Tosic et al. 1993). Each of these phenotypes includes dysmyelination in the CNS and a reduction in mature oligodendrocytes, and death occurs within the first few weeks of life.

The human gene was mapped between the DNA markers PGK1 and G LA (Buckle et al. 1985). Using in situ hybridisation it was shown that PLP mapped to Xq22 (Mattei et al. 1986) and finer mapping of the gene distal to PGK1 and proximal to PRPS was demonstrated (Willard et al. 1987). The human PLP gene was cloned from a genomic library using a rat PLP cDNA probe and it was found that the gene was made up of seven exons spanning 17kb (Diehl et al. 1986). Human and rat PLP were found to share >97% homology at the amino acid level as predicted from DNA sequence analysis of rat PLP cDNA and human exonic

sequence, indicating a strikingly high degree of sequence conservation in PLP (Diehl et al. 1986) and this degree of homology has been extended to the mouse (Hudson et al. 1987), rat (Dautigny et al. 1985), bovine (Naismith et al. 1985), dog (Nadon et al.1990) and rabbit (Tosic et al.1993) PLP genes.

Using certain protein purification methods, another less abundant CNS specific myelin protein called DM-20 was reported to co-purify with PLP, suggesting that PLP and DM-20 were related. Further investigation revealed that the two proteins shared amino acid sequence homology at the amino- and carboxyl-terminal ends, and were related by immunological cross-reactivity (Lees et al. 1991). It was proposed that DM-20 differed from PLP by the absence of internal amino acid residues (Trifilieff et al. 1986).

A full length DM-20 cDNA clone was isolated in the rat, and it was found to be deleted for 105bp of sequence from exon 3 of the rat PLP gene (Nave et al. 1987). The DM-20 mRNA was found to be generated by an alternative splicing event of the PLP exons, suggesting more than one PLP gene function. This alternative transcript was generated using a 5' splice site which lies 105bp upstream of the 5' splice site in exon 3 that is employed to generate the PLP mRNA (Nave et al. 1987). The predicted protein sequence from the DM-20 mRNA lacked 35 amino acids of PLP that formed a hydrophilic domain and part of a membrane embedded domain.

More recently, other proteolipid forms that exhibit sequence homology to PLP have been detected in bovine CNS (Schindler et al. 1990) and mouse CNS (Ikenaka et al. 1992). One form identified in bovine CNS, called PUB', has the same 31 amino acids at the amino-terminus as PLP and DM-20 (Schindler et al.1990), and it has been postulated that additional alternative splicing events may take place to generate other PLP forms.

Together, PLP and DM-20 have been estimated to make up 60% of the total protein content of the CNS, but the DM-20 mRNA was shown by RNAse

protection studies in the brains of 11 day old and adult to be expressed at only 50% of the level of PLP cDNA (Nave et al. 1987).

The proposal for dual PLP functions was supported by the identification of a different PLP point mutation that gave rise to a mouse phenotype called rumpshaker (GnffWhs et al. 1990). The rumpshaker mice were investigated for the involvement of the PLP locus on account that although they have normal numbers of mature oligodendrocytes and exhibit normal longevity, these mice display hypomyelination restricted to the CNS (Schneider et al. 1992). RNase protection studies in rumpshaker aduii mice demonstrated an increased level of DM-20 transcripts in relation to PLP compared to normal adult mice (Schneider et al. 1992). Thus it was demonstrated that PLP mutations can selectively interfere with oligodendrocyte maturation rather than the structure of myelin, and it was proposed that PLP has a vital function in glial cell development, distinct from its later role in myelin assembly, which correlates with the obsevation of early DM20 expression (Ikenaka et al. 1992).