Molecular biology of myelin basic protein: gene rearrangement and expression of anti-sense RNA in myelin-deficient mutants.

1. Myelin is an important structure for facilitating the conduction of impulses along the axons both in the central nervous system (CNS) and peripheral nervous system (PNS). 2. Myelin basic protein (MBP) is a major protein in CNS myelin. 3. MBP is expressed specifically in the nervous system. 4. The MBP gene has been cloned and characterized. 5. Two mutant mice, Shiverer (shi) and myelin-deficient (mld. shimid), are autosomal recessive mutants that show severe symptoms such as intentional tremor. They have been found to have a mutation in the MBP gene that results in poor myelination in the central nervous system. 6. It was found that rearrangement within the MBP gene results in low expression of the gene. 7. In Shiverer, the MBP gene is partially deleted (from exons 3 to 7), and in mld, the gene is duplicated tandemly and a large portion of the duplication is inverted upstream of the intact copy. 8. In mld, anti-sense RNA complementary to exons 3-7, which correspond to the inverted segment, was detected by RNase protection studies, and presumed to be responsible for the reduced expressions of MBP. 9. The mechanism of gene rearrangement in MBP was also characterized. 10. This article reviews the recent progress in the study of the MBP gene, especially the rearrangement of the gene and its expression in mutant mice.     

Expression of myelin protein genes in the developing brain

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.     

Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.     

Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.     

Developmental expression of the myelin gene MOBP in the rat nervous system

The myelin-associated/oligodendrocyte basic proteins (MOBPs) are recently discovered constituents of myelin and are small, cytoplasmic, and highly basic proteins exclusively expressed postnatally by oligodendrocytes. Due to a clustering of positively charged amino acids observed in the most abundant MOBP isoform similar to myelin basic protein (MBP) and P0, it was speculated that MOBP could function in myelin sheath compaction. The present report strongly supports this view. A direct comparison of MBP and proteolipid protein (PLP) gene expression with that of MOBP by in situ hybridization revealed a very similar regional distribution. It was found that MOBP expression was abundant in the rat CNS at postnatal day 15 (P 15) but is restricted to densely myelinated regions. In contrast to MBP and PLP, expression of MOBP was undetectable in the peripheral nervous system during the entire development. Interestingly, MOBP mRNA was localized in oligodendrocyte processes even at early postnatal stages and throughout development. MOBP showed a very specific timing of expression: in spinal cord and brain, MOBP gene expression occurred significantly later (2–3 days) than that of MBP and PLP, but slightly earlier than myelin oligodendrocyte glycoprotein gene expression. MOBP proteins appeared in spinal cord and brain stem also after MBP protein, suggesting that the MOBPs functionally act after the structural myelin proteins MBP and PLP. Our findings imply a function of MOBP during the late steps of myelin formation, presumably at the initiation of sheath compaction.     

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.     

Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization

The developmental program controlling the expression of myelin basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A 35S-labeled cDNA clone of mouse MBP encoding an amino acid sequence present in all four of the major forms of rodent MBP was used. The probe hybridized to the tracts of white matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5- and 7-day-old rats and was confined to the tectospinal tracts, fibers of the seventh cranial nerve, and the spinocerebellar tracts. By 12 days the amount of MBP mRNA had decreased in these areas. In the cerebrum, the greatest amount of MBP mRNA was observed in 17-day-old rats in the radiations of the corpus callosum. Thereafter, the levels decreased but could still be observed in the adult animals. Thus, using this technique, we have been able to demonstrate that the level of MBP-specific mRNA correlates closely with the development of myelin in different regions of the brain.     

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.