論文

The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3-week-old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.

P400 protein has been found in the cerebellum and localizes to Purkinje cells. It is a glycoprotein and reacts with plant lectins. Autoradiographic patterns of the protein profiles of mutant mice cerebella after intracranial injections of 14C-leucine were analyzed. P400 protein was one of the dominant proteins labeled in the control cerebellum. No incorporation of 14C-leucine to the position of P400 protein was found in the nervous and pcd mutant mice where no Purkinje cells and no P400 protein are present. P400 protein in weaver cerebellum was labelled more intensely than that in the control. In the staggerer mice, where dendritic arborization of Purkinje cell is poor and no spines are formed on the dendrites, no P400 protein was found and no 14C-leucine incorporation was observed to the position of P400 protein, suggesting that staggerer is a mutation where no P400 protein accumulated in the fractions being studied. P400 protein was phosphorylated independently of the presence of Ca2+. Among the proteins of the isolated Purkinje cells, P400 protein was one of the major proteins phosphorylated.

The twitcher is an autosomal recessive mutant mouse characterized by absence of galactosylceramidase. The twitcher shows clinical and histological features similar to those of human Krabbe-type leukodystrophy. We here present the results of a neurochemical and immunohistochemical analysis of the twitcher. Electrophoretic analysis revealed that in the particulate fraction of the spinal cord, myelin basic proteins (MBP) and proteolipid protein were decreased, and in the sciatic nerve fibers, PO protein, X, Y and MBP were clearly decreased. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) activities of the pallium cerebri, brain stem and spinal cord of the twitcher were about 20% less than those of the control. However, in the sciatic nerve, the activity was half that of the control. Immunohistochemical studies were carried out by means of antisera against MBP and CNPase. There were clear patches indicating both MBP- and CNPase-negative reactions in the white matter of the central nervous system from the twitcher. The reaction on the section of sciatic nerve fibers from the twitcher showed a positive reaction only in a very limited number of fibers with both MBP and CNPase antisera. A clear astrocytic hypertrophy was detected by the antiserum against glial fibrillary acidic protein (GFAP). Even in the grey matter of the cerebral cortex, strong GFAP-positive astrocytes were clearly observed. © 1985 Plenum Publishing Corporation.

A hereditary dysmyelination mutation, named myelin deficient (mld), is considered to be allelic to shiverer, a deletion mutation of the myelin basic protein (MBP) gene. The present study showed that MBP expression is greatly reduced in mld, but that it is still detectable. Northern blot analysis revealed that the pronounced decrease in the MBP level in mld resulted from a reduced mRNA level and was not caused by deletion of a large portion of the MBP gene as in shiverer. Southern blod studies with BamHI-digested chromosomal DNA suggested some part of the MBP gene, at least the 5'-portion, was duplicated in mld. These results indicated that the mld and shiverer mutations were different from each other, even though genetic allelism between the two was reconfirmed. We also examined the developmental pattern of the gene expression of MBP and that of another protein, myelin proteolipid protein (PLP), specifically expressed in the oligodendrocyte, in mld by RNA dot blot study. The mRNA level of MBP in mld was greatly reduced during the active myelination stages, gradually increasing and remaining constant in the later stages. The PLP-mRNA content in mld was almost normal (60-80% that of control) at any stage of development. All these findings imply that the primary defect in mld is due to reduced transcriptional activity of the MBP gene.

The jimpy mouse, an X-linked recessive dysmyelinating and demyelinating mutant, has been shown to contain abnormal myelin proteolipid protein (PLP) mRNA. To understand the molecular basis of the mutation, we analyzed the structure of PLP mRNA by an RNase-mapping procedure, using the probes specific to each exon of the mouse PLP gene. We found that the fifth exon of the PLP gene is not utilized in the jimpy. © 1987.

Myelin deficiency (mld) is an autosomal recessive mutation in mice and is considered to be allelic to the shiverer (shi) mutation. Mld mice are characterized by hypomyelination of the central nervous system (CNS). They show typical symptoms such as tremor, tonic convulsion and ataxic movement. Subcellular fractionation of the CNS revealed that the MBP bands were greatly decreased in the P2A (myelin) fraction and the total content of myelin basic protein (MBP) was much lower than that in the control in all parts of the CNS. Sections from mld mice were examined by immunohistochemical tests with MBP antiserum, and a mosaic expression of MBP was found in the myelin of the mld mice. Since the major dense line is considered to be composed mainly of MBP, we investigated the myelin of mld mice by electron microscopy and found that there were 3 types of myelin: (1) a normal type compact myelin with a major dense line, (2) a shiverer-type myelin with no major dense line, and (3) a mixed-type myelin, in which within a myelin lamella the major dense line abruptly changes to cytoplasm of oligodendrocytes.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.

The first hybrid mice ('shiverer*mld' mice) produced by intercrossing the homozygotes of the shiverer (BALB/c strain) and mld (MDB/Dt strain) were used for investigating the fine structure of the myelin lamellae, immunoreactive pattern for myelin basic proteins (MBP) and Golgi impregnated images of oligodendrocytes, with special reference to the influence of aging. All of the hybrid mice had an intermediate coat color between the white of the shiverer and black-brown of the mld, and revealed the same neurological symptoms, intention tremor, ataxic behavior, etc., as those of the shiverer and mld. The central myelin lamellae of the 'shiverer*mld' mouse exhibited the similar characteristics to the shiverer type rather than the mld type from the standpoint of the infrequent occurrence of major dense lines, although they did display a tendency to increase major dense lines with aging like the mld. Observation of the immunohistochemical preparations for MBP showed that immunopositive myelin sheaths were present in the white matter, although they were far more infrequent than those of the mld mutant, probably reflecting the amount of major dense lines. Thus, in the CNS of the 'shiverer*mld' mouse, the MBP-synthesis was possibly much more disturbed than in the mld mutant, or at least, revealed an intermediate pattern between the mld and shiverer. © 1988.

The mouse proteolipid protein (PLP) gene was cloned into the λ bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region. Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5′-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5′-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system. PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5′-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level. © 1987.

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.

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing. The MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

The myelin-deficient (mld) mouse is an autosomal recessive mutant characterized by hypomyelination of the CNS due to reduced expression of the myelin basic protein (MBP) gene. In the mld mutant, the MBP gene is duplicated in tandem. One gene is intact, but a large portion is inverted upstream of the other copy, and its transcription yields the antisense RNA. This antisense RNA was shown to be localized in the nucleus and to form an RNA:RNA duplex with sense RNA. These findings suggested that inhibition of transport from the nucleus or selective degradation of the duplex is responsible for the reduced expression of the MBP gene in the mld mutant. The mechanism of gene rearrangement at the MBP locus was also characterized. Cosmid clones encompassing whole MBP gene loci from control and mld genomic DNA libraries were isolated. The recombination points indicated that the duplication and inversion observed in mld occurred due to nonhomologous recombination.

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. © 1991.

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 β-pleated sheet.

Expression of inositol 1,4,5-trisphosphate (InsP3) receptor subtypes was determined in mouse brain using oligonucleotide-specific in situ hybridization. All subtypes, except one that deletes 120 bp from the full-length InsP3 receptor cDNA, were expressed in cerebellar Purkinje cells. In hippocampus, various subtypes showed distinct expression patterns. These results suggest that regionally selective expression of InsP3 receptor subtypes may result in the generation of functionally distinct channels.

MAJOR histocompatibility complex (MHC) molecules are not normally expressed in the central nervous system (CNS)1-3. However, aberrant expression has been observed in multiple sclerosis lesions and could contribute to the destruction of myelin or the myelinating cells known as oligodendrocytes4,5. The mechanism of cell damage associated with aberrant MHC molecule expression is unclear: for example, overexpression of class I (ref. 6) and class II (refs 7, 8) MHC molecules in pancreatic β cells in transgenic mice leads to nonimmune destruction of the cells and insulin-dependent diabetes mellitus. We have generated transgenic mice that express class I H-2Kb MHC molecules, under the control of the myelin basic protein promoter, specifically in oligodendrocytes. Homozygous transgenic mice have a shivering phenotype, develop tonic seizures and die at 15-22 days. This phenotype, which we term 'wonky', is due to hypomyelination in the CNS, and not to involvement of the immune system. The primary defect appears to be a shortage of myelinating oligodendrocytes resulting from overexpression of the class I MHC molecules.

Additional subtypes of the inositol 1,4,5-trisphosphate (InsP3) receptor are expressed in a tissue-specific and developmentally specific manner. They differ from the InsP3 receptor structure previously reported in two small variably spliced segments. One segment (SI) is located within the InsP3 binding site, whereas another segment (SII) is located near putative sites for phosphorylation and ATP binding to modulate InsP3 action on Ca2+ flux. Therefore, we speculate that selective use of InsP3 receptor subtypes permits a tissue-specific and developmentally specific expression of functionally distinct channels.