Biomedical subjects

E Barbarese

Publications and source records attributed to E Barbarese.

At least 37 records · Page 2Linked to original sources

PubMed1996

Brain cytoplasmic and flagellar outer arm dyneins share a highly conserved Mr 8,000 light chain.

Sequence comparisons with the Mr 8,000 light chain from Chlamydomonas outer arm dynein revealed the presence of highly conserved homologues (up to 90% identity) in the expressed sequence tag data base (King, S. M. & Patel-King, R. S. (1995a) J. Biol. Chem. 270, 11445-11452). Several of these homologous sequences were derived from organisms and/or tissues that lack motile cilia/flagella, suggesting that these proteins may function in the cytoplasm. In Drosophila, lack of the homologous protein results in embryonic lethality (Dick, T., Ray, K., Salz, H. K. & Chia, W.(1996) Mol. Cell. Biol., 16, 1966-1977). Fractionation of mammalian brain homogenates reveals three distinct cytosolic pools of the homologous protein, one of which specifically copurifies with cytoplasmic dynein following both ATP-sensitive microtubule affinity/sucrose density gradient centrifugation and immunoprecipitation with a monoclonal antibody specific for the 74-kDa intermediate chain (IC74). Quantitative densitometry indicates that there is one copy of the Mr 8,000 polypeptide per IC74. Dual channel confocal immunofluorescent microscopy revealed that the Mr 8,000 protein is significantly colocalized with cytoplasmic dynein but not with kinesin in punctate structures (many of which are associated with microtubules) within mammalian oligodendrocytes. Thus, it appears that flagellar outer arm and brain cytoplasmic dyneins share a highly conserved light chain polypeptide that, at least in Drosophila, is essential for viability.

Amino Acid Sequence↗

PubMed1996

Regional heterogeneity in the response of astrocytes following traumatic brain injury in the adult rat.

The regional distribution and temporal appearance of astrocytes expressing glial fibrillary acidic protein (GFAP), S100 protein, and vimentin were determined in a nonpenetrating lateral fluid percussion (LFP) brain injury model. Following injury, reactive astrocytes were observed in the subcortical white matter tracts as early as 1 day, in the hippocampus and injured cortex by 3 days, and in the thalamus by 1 week. Reactive astrocytes in the injured cortex, subcortical white matter tracts, and CA3 region of the hippocampus were all vimentin positive at 1 month post-injury. These astrocytes had a distinct morphology characterized by an enlarged cell body and long intertwined processes. In contrast, reactive astrocytes in the thalamic nuclei never expressed vimentin, and displayed an enlarged cell body with thick shortened processes. An increase in S100 protein was detected in all reactive astrocytes following LFP brain injury. Quantitative assessment of GFAP, S100, and vimentin polypeptides confirmed the immunohistochemical evaluation. Our data indicate that although astrogliosis mirrors the spatial pattern of post-traumatic neuronal cell loss, the expression of vimentin and the cellular morphology of the cells were regionally distinct, suggesting that astrogliosis may be modulated by factors present in the post-traumatic brain.

Animals↗

PubMed1996

Morphological organization of oligodendrocyte processes during development in culture and in vivo.

In order to meet the requirements for myelin biogenesis, the processes of oligodendrocytes must differentiate into specialized cellular compartments. For translation of myelin basic protein mRNA to occur in the oligodendrocyte processes or the myelin membrane, these structures must contain ribosomes and other components of the protein translation machinery. Light microscopy and electron microscopy (EM) were used to explore this possibility by analyzing the cytoskeletal organization and the organelle population of the processes of oligodendrocytes during development in vivo and in culture. Microtubules (MTs) were few and bundled in the younger processes of oligodendrocytes in culture. Some of these processes were characterized by the additional presence of serpentine MTs. EM revealed the presence of clusters of ribosomes in both immature and mature oligodendrocyte processes. In the former these clusters were frequently found at regular intervals along the processes. A similar interval characterized the distribution of myelin-like figures along the processes of mature cells in culture. Both in vivo and in culture, the processes became enriched in endoplasmic reticulum cisternae and mitochondria as the cells matured. The spatial arrangement of cellular organelles is compatible with protein and lipid synthesis occurring in the processes, at or near the site of myelin assembly.

Aging↗

PubMed1995

Internalization of microbubbles by tumor cells in vivo and in vitro.

Lipid-coated microbubbles (LCM) administered intravenously (i.v.) to rats bearing brain tumor, specifically enhance tumor visualization by ultrasound [1]. In order to understand the basis for this observation, we have examined the interactions of LCM with glioblastoma (C6) and gliosarcoma (9L) tumor cells in vivo and in vitro. LCM and LCM labeled with the fluorescent lipophilic dye 3,3'-dioctadecyloxacarbocyanine perchlorate (diO) were administered to rats bearing brain tumor. LCM and diO-labeled LCM were found principally at the tumor site with no evidence of label in the surrounding normal brain tissue. Analysis of the tumor by confocal laser scanning microscopy revealed that labeled LCM were inside the tumor cells. Similar analysis of LCM interactions with C6 and 9L cells in culture showed that LCM first adsorb at the surface of the cells, and with time became localized inside the cells. Binding and internalization proceeded faster at 37 degrees C than at room temperature (RT). Staining of live cells with N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl) methylamine dihydrochloride (DAMP), a dye that recognizes acidic compartments, showed that the majority of internalized LCM was associated with compartments containing DAMP. If the same uptake mechanism were operative in vivo, it would indicate that a portion of LCM bypasses the reticuloendothelial system and become endocytosed directly by tumor cells.

Animals↗

PubMed1995

The number of Schmidt-Lanterman incisures is more than doubled in shiverer PNS myelin sheaths.

In the PNS, myelin basic protein (MBP) appears not to be essential for myelination, for in shiverer (shi) and mld mutant mice peripheral nerves, where MBP is not or only poorly expressed, myelination occurs normally. Only a few morphological abnormalities, i.e. reduction in axon calibre and myelin sheath thickness, and aberrant Schwann cell-axon contacts, have been reported. Here, we document a consistent difference between shi and wild type (wt) myelinated sciatic nerve fibres. The number of Schmidt-Lanterman incisures seen in longitudinally and transversely-sectioned sciatic nerves, or in teased fibres stained for the presence of F-actin, is dramatically increased in homozygous shi mice. With both methods, a twofold increase in Schmidt-Lanterman incisure number is seen in 15-day-old mice, the earliest time examined. The increase is slightly greater in nerve fibres from 30- and 90-day-old mice. The overproduction of Schmidt-Lanterman incisures in shi occurs in spite of the fact that the mean diameter of myelinated fibres in shi sciatic nerves is smaller than in wt sciatic nerves. These results lead us to suggest that the increase in Schmidt-Lanterman incisure density in shi compensates for a defect in Schwann cell-axon communication.

Actins↗

PubMed1995

Protein translation components are colocalized in granules in oligodendrocytes.

The intracellular distribution of various components of the protein translational machinery was visualized in mouse oligodendrocytes in culture using high resolution fluorescence in situ hybridization and immunofluorescence in conjunction with dual channel confocal laser scanning microscopy. Arginyl-tRNA synthetase, elongation factor 1a, ribosomal RNA, and myelin basic protein mRNA were all co-localized in granules in the processes, veins and membrane sheets of the cell. Colocalization was evaluated by dual channel cross correlation analysis to determine the correlation index (% colocalization) and correlation distance (granule radius), and by single granule ratiometric analysis to determine the distribution of the different components in individual granules. Most granules contained synthetase, elongation factor, ribosomal RNA and myelin basic protein mRNA. These results indicate that several different components of the protein synthetic machinery, including aminoacyl-tRNA synthetases, elongation factors, ribosomes and mRNAs, are colocalized in granules in oligodendrocytes. We propose that these granules are supramolecular complexes containing all of the necessary macromolecular components for protein translation and that they represent a heretofore undescribed subcellular organization of the protein synthetic machinery. This spatial organization may increase the efficiency of protein synthesis and may also provide a vehicle for transport and localization of specific mRNAs within the cell.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts.

The capacity of oligodendrocytes (OLs) and their progenitors to migrate, proliferate, and differentiate in vivo was evaluated by transplanting highly enriched populations of sequential stages of the OL lineage (A2B5+O4-, O4+GalC-, and GalC+) into the telencephalon of the hypomyelinating mouse, shiverer. The shiverer mouse neither expresses the major myelin basic protein (MBP) nor makes normal myelin due to a large deletion in the gene for MBP. Thirty days after transplantation, serial 225 micron sections of the host brain were immunostained with antiserum to MBP and analyzed by confocal microscopy. The presence of MBP+ patches of myelin in the otherwise MBP- host brain allowed a retrospective analysis of the myelinogenic activity of the transplanted progenitors cells. Both the extent of MBP+ myelin and the location of MBP+ structures relative to the initial site of cell deposition were highly dependent on the developmental stage of the transplanted cells. Specifically, A2B5+O4- OL progenitors migrated distances of > or = 600 microns and produced MBP+ patches in nearly every slice of the host brain. An average of over 250 separate patches were found per host brain, some of which had cross-sectional areas of > 250,000 microns2 containing as many as 60 MBP+ OL cell bodies, and with densities of myelination rivaling that of normal brain. In marked contrast, transplantation of O4+GalC- cells produced only small (1,000-25,000 microns2), scattered (25-40 per brain) patches of MBP+ myelin containing one to five cell bodies, all of which were within 50 microns of the needle track or the nearest ventricular surface. GalC+ cells produced MBP+ myelin at a level similar to that of O4+CalC- cells. These data suggest that the developmental transition of OL progenitors from the O4- to the O4+ phenotype is accompanied by a dramatic reduction in the innate capacity of the cells to migrate and survive in vivo. The use of developmentally identified, enriched populations of OL progenitor cells offers the opportunity for more precise analyses of transplantation and remyelination behavior, and relates to clinically relevant studies indicating that contaminant cell types can seriously interfere with the stable integration of donor tissue into the host.

Animals↗

PubMed1993

Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Animals↗

PubMed1992

Comparison of CNS homing pattern among murine TH cell lines responsive to myelin basic protein.

A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).

Animals↗

PubMed1992

Molecular genetic analysis of the mldr mouse: a spontaneous revertant at the mld locus containing a recombinant myelin basic protein gene.

The mld mutation is a complex genetic lesion affecting the myelin basic protein (MBP) locus in the mouse. The mutation consists of a variety of DNA rearrangements including: tandem duplication of the MBP structural gene, partial inversion of the 3' end of the upstream gene copy, duplication of a region flanking the rearrangement junction in the upstream copy and insertion between the two gene copies of a segment of extraneous DNA not associated with the wild-type MBP locus. The net result of the mutation is a dysfunctional MBP locus. Homozygous mld/mld mice produce very little MBP and consequently very little myelin. They exhibit a clinical phenotype characteristic of hypomyelination (shaking, convulsions). We have discovered a revertant mld mouse which does not exhibit clinical symptoms of hypomyelination. Genetic analysis indicates that the reversion is allelic to mld. We have designated the revertant locus mldr. Restriction analysis of mldr genomic DNA indicates that there is a single intact MBP gene. Analysis of various junction regions using the polymerase chain reaction indicates that the single MBP gene in mldr is derived by recombination from the 5' end of the upstream gene and the 3' end of the downstream gene. Studies on MBP expression in mldr mice indicate that the developmental regulation, level of expression and pattern of post-transcriptional processing of MBP gene products in mldr are similar to wild type. These results indicate that the recombinant MBP gene in mldr is fully functional. From this we infer that the MBP-deficient phenotype of the original mld mutant is attributable to the complex rearrangements in the upstream gene copy which render the locus dysfunctional.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1992

Stage specific, (O4+GalC-) isolated oligodendrocyte progenitors produce MBP+ myelin in vivo.

O4+/A007+GalC- proligodendroblasts represent a distinct stage of development in the oligodendrocyte lineage, occurring just prior to the appearance of postmitotic GalC+ oligodendrocytes. These cells, isolated directly from postnatal rat telencephalon by an immunopanning procedure, can terminally differentiate and myelinate axons when transplanted back into an in vivo environment. Specifically, after 30 days in the brain of newborn shiverer mouse hosts, O4+GalC- oligodendrocyte progenitors produced myelin basic protein positive (MBP+) patches. These MBP+ patches, examined by both light and confocal microscopy, contained oligodendrocyte cell bodies and ensheathed host shiverer axons morphologically similar to those found in normal rat brain at an analogous age. These results suggest that isolated O4+GalC- cells can become biochemically mature oligodendrocytes with the capacity to elaborate myelin sheaths, and further define the period of development during which oligodendrocytes retain their capacity to myelinate axons when given a receptive environment.

Animals↗

PubMed1992

Visualizing cells in three dimensions using confocal microscopy, image reconstruction and isosurface rendering: application to glial cells in mouse central nervous system.

This paper describes a general method for visualizing individual cells in intact tissue in three dimensions. The method involves immunostaining intact tissue to label specific cells, "optical sectioning" the stained tissue by laser scanning confocal microscopy, computationally reconstructing a three dimensional image data set from the digitized confocal optical sections, delineating isosurfaces of specific intensity within the reconstructed image by a "marching cubes" algorithm to generate polygon meshes defining boundaries of cells, and displaying individual cells, identified as three dimensional objects enclosed by contiguous polygon meshes, using computer graphics techniques. Each of the components of this method has been described previously in conjunction with other applications. However the combination of these techniques to visualize a variety of different individual cell types in three dimensions in intact tissue represents a new approach. To illustrate the application of this method, we have visualized three different glial cell types in mouse CNS tissue. Oligodendrocytes, specifically stained with antibody to myelin basic protein, were used as an example of cells labelled with an internal membrane antigen. Astrocytes, specifically stained with antibody to glial fibrillary acidic protein, were used as an example of cells labelled with a cytoplasmic antigen. Microglia, specifically stained with Mac.1 antibody, were used as an example of cells labelled with an external membrane antigen. The images that are generated contain remarkably detailed volumetric and textural information that is not obtainable by conventional imaging techniques.

Algorithms↗

PubMed1991

Diversification of glial lineages: a novel method to clone brain cells in vitro on nitrocellulose substratum.

We have developed a novel in vitro method to analyze the diversification of glial cells during development. The primary advantage of the approach is that glial lineages are formed in discrete clones on a nitrocellulose substratum where the relationship of the progeny is strictly defined. This method facilitates the comparison of a large complement of astrocyte and oligodendrocyte lineages under controlled conditions. Clones were formed by plating a brain dissociate on nitrocellulose at very low density (5,000-40,000 cells/154 mm2). However, growth depended on diffusible factors produced by brain cells growing under the nitrocellulose support at high density (feeder layer). The cloning efficiency of cells from mouse forebrain (P0) was 1-3%. This means we can detect 100,000 to 300,000 clonal progenitors in the dissociate (10(7) cells per forebrain) using the clonal culture technique. Cell phenotypes were determined by immunocytochemical staining with anti-glial fibrillary acidic protein (GFAP) to label astrocytes and anti-galactocerebroside (GC) and anti-myelin basic protein (MBP) to label oligodendrocytes. There was a remarkable diversity of glia represented in different lineages. The number of astrocyte clones was greater than the number of oligodendrocyte clones but combined their total was 90%. Clone sizes were distributed over a wide range, which indicated that growth rates varied. Clones appeared compact or dispersed but astrocyte clones exhibited three different morphologies-fibroblast-like, stellate, and elongated. Oligodendrocytes had different morphologies distinct from astrocytes. Although there were different glial lineages the cells in most clones were homogeneous, indicating the progeny had the same fate. However, a small number of the clones, approximately 2%, were heterogeneous and contained both astrocytes and oligodendrocytes. The application of this technique to glial lineages demonstrates that intrinsic factors have a role in determining cell fate since different clones formed under the same external conditions. Finally, these results are consistent with the existence of multiple glial progenitors or the continued presence of multipotential progenitors at the time of birth.

Animals↗

PubMed1991

Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture.

In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.

Animals↗

PubMed1989

Radiation sensitivity of glial cells in primary culture.

Primary cultures from newborn mouse brain were subjected to ionizing radiation to determine the sensitivity of galactocerebroside-positive oligodendrocytes and A2B5-positive progenitor cells during development. The radiosensitivity or D0 of the 2 cell populations was determined by measuring the fraction of surviving cells after various doses of x-rays. When the cultures were exposed to a single dose of x-rays at 6 days, oligodendrocytes showed a D0 of 1.4 Gy, and A2B5-positive cells a D0 of 4.5 Gy. When the cultures were exposed to a single dose of x-rays at 12 days, oligodendrocytes showed a D0 of 4.4 Gy, indicating that they have become more radioresistant. Cultures exposed to a dose of 1.5 Gy and monitored from 9 h to 2 weeks after irradiation showed a reduced but constant number of galactocerebroside-positive cells at all time points. Since galactocerebroside-positive oligodendrocytes are non-dividing or slowly dividing cells, the radiosensitivity of young oligodendrocytes was attributed to interphase cell death. These results indicate that young differentiated oligodendrocytes are more radiosensitive than other glial cell types, and that there is no significant replacement of lost oligodendrocytes either through proliferation or through differentiation of progenitors after irradiation in the culture system.

Animals↗

PubMed1988

Expression of myelin basic protein mRNA and polypeptides in mouse oligodendrocytes in culture: differential regulation by genetic and epigenetic factors.

We have analyzed the effects of genetic and epigenetic factors on the steady-state levels of myelin basic protein mRNA and polypeptides during development of mouse oligodendrocytes in culture. Oligodendrocytes were characterized by immunofluorescent staining with antibodies for the following markers: galactocerebroside, myelin basic protein, proteolipid protein, myelin-associated glycoprotein and 2',3'-cyclic nucleotide phosphohydrolase. Oligodendrocytes expressing one or more of these markers first appeared at 3 days in culture and increased to a maximum of 1.5 X 10(5) per brain around 6 days, after which the number remained constant up to 31 days. In medium containing fetal calf serum, accumulation of myelin basic protein polypeptides was delayed relative to in vivo in cultures derived from C57BL/6J, BALB/cJ and DBA/2J inbred mice, but not in cultures derived from C3H/HeJ and AKR/J inbred mice. In medium containing serum from other species or in serum substitute, the temporal expression of myelin basic protein polypeptides in cultures from all the inbred strains was contemporaneous with that in brain. Northern hybridization analysis indicated that the steady-state level of myelin basic protein-specific mRNA in all cultures was regulated similarly to in vivo suggesting that the delayed expression of myelin basic protein polypeptides in some cultures was due to translational and/or post-translational regulation. Analysis of myelin basic protein expression in cultures from informative hybrid and recombinant inbred strains indicated that translational or post-translational expression of myelin basic protein requires trans-acting factors, the inducibility of which is controlled by multiple genetic determinants which segregate independently and are expressed additively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Expression and localization of myelin basic protein in oligodendrocytes and transfected fibroblasts.

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

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.

Animals↗