Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Structure and Expression of Myelin Basic Protein Gene Sequences in the mld Mutant Mouse: Reiteration and Rearrangement of the Mbp Gene

The mld mutation on chromosome 18 in the mouse is a putative allele of the shiverer (shi) mutation. We have analyzed the structure of myelin basic protein (MBP) gene sequences in mld DNA by restriction mapping of genomic DNA. The results indicate that the mld chromosome carries two copies of the MBP structural gene, one of which is intact and one of which is interrupted. Genetic analysis indicates that the interrupted gene is close to the intact MBP structural gene and cosegregates with the mld mutation. We have also analyzed the levels of MBP polypeptides and MBP-specific mRNA in wild-type, homozygous and heterozygous shiverer and mld mice and in mice carrying both mutations. The results indicate that both shi and mld are cis-acting codominant mutations that cause severely reduced steady state levels of MBP-specific mRNA and MBP polypeptides in the brain. We have analyzed the total number of oligodendrocytes and the number of MBP-positive oligodendrocytes in mld and shi brain primary cultures. In shi cultures, none of the oligodendrocytes expresses MBP. However, in mld cultures, approximately 5% of the oligodendrocytes express MBP. The nature of the "revertant" mld oligodendrocytes is not known.     

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.     

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.     

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.     

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

Shiverer gene maps near the distal end of chromosome 18 in the house mouse

Several mouse mutations cause unstable locomotion, tremor, seizures, and a reduced lifespan because of deficient myelin formation in the central nervous system. Mutant alleles at the shiverer (shi) locus are the only ones in this series with a selective molecular defect, namely, in myelin basic proteins (MBPs), which are virtually absent in shi homozygotes and 50% reduced in heterozygotes. In the present study, backcross and intercross matings indicate recombination of 21.2 +/- 3.3% between myelin deficient, shimld, and fused phalanges, syfp, a marker near the middle of chromosome 18. Recombination of shimld with twirler (Tw), a marker near the centromere, is 45.7 +/- 4.9%. Thus, the shi locus maps near the distal end of mouse chromosome 18 and is the first available marker for this region. Given the evidence of other workers that an MBP locus maps to the same mouse chromosome, and that part of this chromosome may be syntenic with an MBP-PEPA region on human chromosome 18, it is likely that shi is in or near an MBP gene.     

Expression of myelin basic protein and glial fibrillary acidic protein genes in human glial tumors

Analysis of the expression of genes encoding myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in human glial tumors was carried out for determination of the expression specificity of these genes according to tumor types and their malignancy. Low levels of MBP mRNA in astrocytoma specimens of malignancy grades II-IV and significantly higher levels in perifocal zones adjacent to them have been determined by Northern hybridization. Diffuse astrocytomas and anaplastic astrocytomas are characterized mostly by a low level of MBP gene expression and high level of GFAP gene expression, but distinct subtypes of diffuse and anaplastic astrocytomas with a high level of GFAP gene expression can also be detected that may be the reflection of different oncogenic pathways. Very low levels or even absence of MBP mRNA were revealed in oligodendroglioma and all oligoastrocytomas. Thus, Northern hybridization data are correlated with serial analysis of gene expression (SAGE). Obtained results show that MBP is a nonspecific marker for tumors of oligodendroglial origin, but determination of relative levels of MBP and GFAP mRNAs may be useful for glial tumor recognition. In such a way, these two genes together with YKL-40 and TSC-22, which we found previously, can be included into the gene panel for determination of so-called “gene signatures” of brain tumors. However, strict requirements in relation to a clinical value of these “gene signatures” cannot be formulated without verifying them on a large number of clinical samples of tumors and valid control.     

The Endogenous Lectin Cerebellar Soluble Lectin and Its Ligands in Central Nervous System Myelin of Myelin-Deficient (mld) Mutant Mice

The myelin-deficient (mld) mutation is autosomal recessive mutation in the murine CNS exhibiting severe hypomyelination. The primary defect results in a drastic reduction of myelin basic protein synthesis caused by a duplication of the myelin basic protein gene with partial inversion of the upstream gene copy. The severe deficit of myelin basic protein is responsible for the absence of the major dense line but cannot explain the heterogeneity of myelin compaction found in mld. We have tested the hypothesis that the endogenous cerebellar soluble lectin (CSL) and/or its endogenous glycoprotein ligands could be involved in myelin abnormalities in the dysmyelinating mutant, mld. Immunocytochemical and immunoblotting techniques showed that the CSL level was not reduced significantly in the mld mutant. Furthermore, two ligands of CSL, the myelin-associated glycoprotein and an axonal glycoprotein, with a relative molecular mass of 31 kDa, were not decreased in level in the purified myelin fraction isolated from mld mice. In contrast, three minor glycoprotein ligands of CSL of relative molecular mass of 23, 18, and 16 kDa were greatly reduced in content. The reduced concentration of these low-molecular-mass glycoproteins in mld myelin suggests that they are constituents of compact myelin. Furthermore, the observation that CSL is specifically localized in vivo in regions where mld myelin is more compact and absent from regions devoid of myelin compaction may suggest that the endogenous CSL lectin, as well as its minor glycoprotein ligands, plays a role in the stabilization of the myelin sheath.     

Metabolic properties of normal and mutant mannan-binding proteins in mouse plasma

Human mannan-binding protein (MBP) is a serum lectin involved in innate immunity. MBP activates the complement pathway through its interaction with mannose-rich carbohydrates on various microorganisms and a common opsonic defect has been shown to be associated with a low serum concentration of MBP. This low serum concentration is closely associated with a single base mutation in codon 52, 54 or 57 of the human MBP gene, which results in a change of Arg52 to Cys, Gly54 to Asp, or Gly57 to Gln, respectively, in the collagen-like region of the molecule and prevents the formation of higher oligomers. However, the mechanism underlying the low serum concentration in such patients is completely unknown. The levels of protein synthesis and secretion of the normal and mutant MBPs seem to be similar according to our previous in vitro results. In this study, we examined the plasma clearance of the normal and mutant human (Gly54Asp) MBPs in mice, and found that the half-life of the mutant MBP is about half that of the normal MBP, explaining in part the difference in the plasma levels between the two types of MBP.     

Biochemical Correlates of Myelination in Brain and Spinal Cord of Mice Heterozygous for the Jimpy Gene

Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13-14 days in brains of heterozygous jimpy females but showed normal levels by 31-36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13-14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200-250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.