Structure and Expression of Myelin Basic Protein Gene ... - Europe PMC

Copyright 0 1987 by the Genetics Society of America

Structure and Expression of Myelin Basic Protein Gene Sequences in the mld Mutant Mouse: Reiteration and Rearrangement of the MBP Gene Alfred A. Akowitz,*” Elisa Barbarese,+Kathy Scheld* and John H. Carson* *Department of Biochemistry and TDepartment of Neurology, University of Connecticut Health Center, Farmzngton, Connecticut 06032 Manuscript received January 15, 1987 Revised copy accepted April 4, 1987 ABSTRACT T h e mld mutation on chromosome I8 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 qestriction mapping of genomic DNA. T h e 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. T h e 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. T h e nature of the “revertant” mld oligodendrocytes is not known.

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large number of mutations affecting morphology, behavior, physiology and development in the mouse have been identified (GREEN1981). Many of these have been studied genetically and in some cases the mutant phenotype has been characterized at the biochemical and cellular levels. However in few cases has the genetic lesion been analyzed at the molecular level. T h e nature of the genetic lesions for individual mutations in the mouse is of interest because this provides an indication of the types of mutations to which mammalian genes are susceptible, and also because it may provide insight into the mechanisms of regulation and expression of particular affected genes. Shiverer (shi) is one mutation that has been extensively characterized at the genetic, biochemical, cellular and molecular levels, and for which the genetic lesion has been determined. Shiverer was first identified in the SWV inbred strain (MARCH 1973) as an autosomal recessive behavioral mutant (CHERNOFF, MARCHand MILLER 1974; CHERNOFF1981) causing axial body tremors and occasional convulsions in homozygous animals. T h e clinical manifestations of the mutation are apparently due to the absence of the major forms of myelin basic protein (MBP) in both the central (CNS) and peripheral (PNS) nervous systems of homozygous shi animals (DUPOUEY et al. 1979; KIRSCHNER and GANSER1980; MIKOSHIBA et al.

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Current address: Division of Neuropathology Surgery, Yale University School of Medicine, New Haven, Connecticut 0651 1. Genetics 116: 447-464 (July, 1987)

1981). T h e MBP deficit reflects an absence of MBPspecific mRNA and both aspects of the phenotype are expressed co-dominantly in heterozygous +/shi animals (BARBARESE, NIELSONand CARSON1983). Analysis of shi genomic DNA indicates that a substantial portion of the MBP structural gene is deleted (ROACH et al. 1983, 1985; MOLINEAUX et al. 1986). In situ hybridization (BARNETT et aZ. 1985) and somatic cell genetics (ROACHet al. 1985) indicate that the MBP structural gene is located on the distal end of chromosome 28 in the mouse. Experiments with transgenic et al. 1987) indicate that the clinical, mice (READHEAD morphological and biochemical phenotype of the mutation can be corrected by introducing an intact MBP gene. Myelin deficient (mld), which originally appeared on the MDB/Dt inbred strain, is an independently occurring mutation at the shi locus with a very similar clinical phenotype (DOOLITTLEand SCHWEIKART 1977). Shi and mld are believed to be allelic because complementation test crosses between respective carriers produce heteroallelic progeny which exhibit the “shiverer” phenotype (BOURREet al. 1980; COWEN 1980). Conventional linkage studies indicate that the mld mutation, like the shi mutation, is located at the distal end of chromosome 2 8 (SIDMAN, CONOVER and CARSON1985). Morphological comparisons between shi and mld on the same genetic background (SHENet al. 1986) indicate that the two mutations have very similar phenotypes, characterized by reduced amounts

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of myelin a n d absence of major dense line in t h e myelin sheaths that form. However, in mld, but not in shi, there are occasional clusters of myelin sheaths which have major dense lines and which stain positively for MBP by immunocytochemistry. In this paper we analyze t h e restriction patterns of MBP gene sequences in mld genomic DNA in order t o elucidate t h e nature of t h e genetic lesion a n d t h e possible basis for its biochemical phenotype. We also examine t h e effects of t h e mld a n d shi mutations on steady state levels of MBP polypeptides a n d MBPspecific mRNA, and on t h e n u m b e r of MBP-positive oligodendrocytes in t h e CNS of homozygous, heterozygous and heteroallelic animals in order to compare t h e biochemical phenotypes of t h e two mutations in both whole brain and individual oligodendrocytes and t o determine whether they exhibit co-dominance and allelic additivity. MATERIALS AND METHODS

Animals: Partially congenic (n8) shi and mld mutant mice on the same hybrid C57BL/6J-C3H/HeJ genetic background were obtained from Dr. M. WOLF(University of Massachusetts, Worcester). Inbred mice, C3H/HeJ, C57BL/ 6J, DBA/2J and BALB/cJ, were obtained from the Jackson Laboratory (Bar Harbor, Maine). Partially congenic (n8) mld strains on the C3H/HeJ, C57BL/6J, DBA/2J and BALB/cJ inbred backgrounds were obtained from Dr. R. SIDMAN (Childrens Hospital, Boston). Mice were maintained in the Center for Laboratory Animal Care at the University of Connecticut Health Center. Animals were sacrificed by cervical dislocation. The cerebral hemispheres, livers and spleens were removed, frozen in liquid nitrogen and stored at -80". Primary culture of mouse brain cells: Newborn animals (8- 10 pups per experiment) were sacrificed by decapitation and the brains were removed and freed of meninges. The cerebral hemispheres were dissected out, minced with scissors in a 35-mm dish and transferred to a 15-ml conical tube containing prewarmed (37 ") Dulbecco's modified Eagle medium (DME) containing 10 mM HEPES and 10% fetal calf serum. A cell suspension was obtained by repeated pipetting through a flame-narrowed Pasteur pipet. The suspension was centrifuged at 2000 X g for 7 min at room temperature and the cell pellet was resuspended at a density of 3-5 X I O 6 cells/ml in DME containing 10% fetal calf serum and 100 pg/ml gentamicin. The cells were plated on tissue culture dishes that had been coated with 0.001 % polylysine for 1 hr at room temperature and air-dried. The plates were left undisturbed for 4 days after which the medium was replaced with DME plus serum with no antibiotics. Cultures were maintained at 37" in 6.6% CO2humidifiedatmosphere and medium was changed every 4-5 days. Immunostaining of oligodendrocytes: Cells grown on tissue culture plastic were fixed with formaldehyde (3.7%) in Dulbecco's balanced salt solution (DBSS) for 10 min at room temperature. For MBP staining, the fixed cells were permeabilized in NP40 (0.1 W ) for 4 min at room temperature. The cells were washed several times with DBSS containing normal goat serum (5%) (DBSS/NGS) and then incubated for 90 min at room temperature with monoclonal antibody to either galactocerebroside (RANSCHTet al. 1982) or MBP (SIRESet al. 1981) diluted 1:500 in DBSS/NGS.

The cells were washed several times with DBSS/NGS and incubated with goat anti-mouse IgG conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1:1000 in DBSS/NGS. The cells were washed several times with DBSS and incubated with alkaline phosphatase substrate according to the protocol of BLAKEet al. (1984). The reaction was terminated after 10 min and the cells were photographed. Western blotting of MBPs: Homogenates of cerebral hemispheres from mice of appropriate ages were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)as described by SWANK and MUNKRES(197 1). The separated polypeptides were transferred electrophoretically to nitrocellulose membranes as described by TOWBIN, STAEHELIN and GORDON (1979), and stained with antibody to MBP prepared as described previously (BARBARESE, BRAUN and CARSON1977). Individual stained bands were quantitated by reflectance densitometry as described by (1 984). KERNER and CARSON Analysis of MBP-specific RNA in mouse brain: RNA was isolated from individual mouse brains or from primary cultures of mouse brain by the guanidinium isothiocyanate/ cesium chloride method described by GLISIN,CRKVENJAKOV and BYUS(1974). The yield of RNA was approximately the same for each genotype. Isolated RNA (10 pg/lane) was denatured with glyoxal and dimethylsulfoxide (DMSO) and electrophoresed on agarose gels as described by MCMASTER and CARMICHAEL (1977). The RNA was blotted to chargemodified nylon membrane (GeneScreen Plus, New England Nuclear) and hybridized with appropriate 32P-labeledprobe according to the manufacturer's protocol. Restriction analysis of MBP sequences in genomic DNA: Genomic DNA was isolated from adult mouse liver by the method of BLINand STAFFORD (1976). Isolated DNA (10 pg/lane) was digested with restriction enzyme, electrophoresed on agarose gels and blotted to charge-modified nylon membrane (Zetaprobe, Bio-Rad) as described by REED and MANN(1985). The amount of DNA applied to gel was adjusted slightly so that after ethidium bromide staining the intensity of fluorescence from the smear of digested DNA and from any repetitive DNA bands was equivalent for each genotype. The blots were hybridized with appropriate 32Plabeled probes and washed at a final stringency of 0.1 X SSC, 65", according to the manufacturers protocol. After autoradiography individual bands were quantitated by transmittance densitometry using a Hoefer scanning densitometer interfaced to an Apple I1 computer using Chromatochart software from Interactive Microware Incorporated. Hybridization probes: Exon-specific MBP hybridization probes were prepared from pMBP-I (ROACH et al. 1983) which contains the 5' portion of a cDNA for rat 14-kilodalton MBP. The plasmid DNA was digested with EcoRI and the 1.5 kb fragment containing the cDNA insert was purified by low melt agarose electrophoresis. This fragment contains DNA specific for exon 1 (192 bp), exon 3 (102 bp), exon 4 (36 bp), exon 5 (33 bp) and part of exon 7 (1095 bp) (exons are numbered according to DEFERRA et al. 1985). The homology between the rat MBP sequences and the corresponding mouse MBP sequences is approximately 90%. Two subfragments of the cDNA insert were also prepared by low melt agarose electrophoresis-an EcoRIAlul fragment (362 bp) representing the region from position 1 to 362 in the original insert containing exon 1 (192 bp), exon 3 (102 bp), exon 4 (36 bp) and exon 5 (32 bp), and a BanII-Stul fragment (148 bp) representing the region from position 194 to 342 in the original insert containing 9 bp from exon 1, all of exons 3 and 4, and 5 bp from exon 5. Genomic MBP hybridization probes were prepared from

Myelin Basic Protein Gene in mld cos138 which is a cosmid containing approximately 37 kb of mouse genomic DNA includingthe entire MBP structural gene (TAKAHASHI et al. 1985). The cosmid DNA was digested with BamHI and fragments were isolated by low melt agarose electrophoresis. Probe I is an 8.3-kb BamHI fragment representing the region in the MBP gene from approximately 3 to 11 kb. Probe I1 is a 3.5-kbBanHI fragment representing the region in the MBP gene from approximately 11 to 14.5 kb. Probe 111 is a 4.5-kb BamHI fragment representing the region in the MBP gene from approximately 14.5 to 19 kb. Probe IV is a 14.8-kb fragment representing the region of the MBP gene from approximately 19 kb to the 3’ end plus a small portion of cosmid DNA. A cDNA hybridization probe specific for the Po protein was prepared from pSN63 which containsa full length (13 5 kb) cDNA for rat Po protein (LEMKEand AXEL 1985).The plasmid DNA was digested with EcoRI and the 1.85-kbinsert cDNA fragment was purified by low melt agarose electro-

phoresis. The DNA fragments described above were labeled with “P either by nick translation (RIGBY et al. 1977) or by random oligonucleotide priming (FEINBERGand VOGELSTEIN 1983). RESULTS

The mld mutation is closely linked to the MBP gene: Shi and mld are thought to be alleles because they fail to complement each other in heteroallelic animals, and because both have been localized to the distal end of chromosome 28. However, conventional linkage studies between shi and mld have not been reported. It is not clear whether the genetic lesions in these two mutants affect the same locus or whether they can be separated genetically. T o address this question we have tested for recombination between shi and mld. Heteroallelic shilmld animals were mated and the offspring were scored for the “shiverer” phenotype. The nonrecombinant progeny of this cross will include: homozygous shilshi, homozygous mld/ mld, and heteroallelic shilmld, all of which exhibit the “shiverer” phenotype. If recombination occurs between shi and mld, the possible recombinant chromosomes will include double mutant shi mld and wild type. Animals carrying the former in combination with either shi or mld will presumably exhibit the “shiverer” phenotype; however, animals carrying the latter will appear wild type and will not exhibit the “shiverer” phenotype. We have examined 725 progeny of this cross and all exhibit the “shiverer” phenotype. This means that, within the limits of genetic resolution of the experiment (less than 0.3 centimorgans), the shi and mld mutations are not separable by recombination. Since the genetic lesion in shi is a deletion of the MBP gene, this implies that the mld mutation is within 0.3 cM of the MBP gene. mld contains an intact MBP gene plus reiterated MBP-specific sequences: In order to analyze the structure of the MBP gene in mld we isolated liver genomic DNA from wild type, shilshi and mldlmld

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mice, digested the DNA with different restriction enzymes, and analyzed the patterns of restriction fragments by Southern blotting with an MBP-specific cDNA hybridization probe as described in MATERIALS AND METHODS. The shi and mld mice that were used as a source of DNA were obtained from Dr. M. WOLF and were partially (n8) congenic on a hybrid B6/C3 genetic background. Since the MDB/Dt inbred mouse strain on which the mld mutation originally occurred is extinct (D. P. DOOLITTLE, personal communication), we compared the restriction fragment patterns obtained with the mutant DNA with wild-type DNAs from four different inbred strains of mice (C3H/HeJ, C57BL/6J, DBA/2J and BALB/cJ) in order to determine whether any differences might be attributable to naturally occurring restriction fragment length polymorphism among different inbred strains. T h e wildtype and mutant DNAs were digested with 26 different restriction enzymes (results not shown). No restriction fragment length polymorphisms were found among the four different inbred wild-type DNAs with any of the enzymes tested. With every enzyme tested, mld DNA generated all of the MBP-specific restriction fragments found in wild-type DNA, indicating that mld contains MBP gene sequences which are similar to the wild-type MBP gene. However, with some enzymes, mld DNA generated additional MBP-specific restriction fragments that were not found in wild-type DNA, indicating that mld contains additional MBPrelated sequences. T h e cDNA probe used in screening the different restriction enzymes described above contains sequences from exons 1 , 3 , 4 , 5 and 7 of the MBP gene. However, since exons 3, 4 and 5 are quite small and since the probe is derived from rat cDNA which differs slightly from mouse DNA, under normal stringency conditions this probe hybridizes predominantly to restriction fragments containing exons 1 and 7, representing the 5‘ and 3‘ ends of the MBP gene, respectively. In order to analyze the entire length of the MBP gene and to determine the nature of the additional MBP-specific restriction fragments in mld DNA we have digested wild type (C3H/HeJ) and mld (partially congenic (n8) with C3H/HeJ) DNA with BglII, separated the restriction fragments by agarose gel electrophoresis, and hybridized with a series of probes representing sequential BamHI fragments derived from a cosmid containing the entire mouse MBP gene. As outlined in Figure 1, probe I is an 8.3-kb BamHI fragment representing the region in the MBP gene from approximately 3 to 11 kb. It hybridizes to an 8.1-kb BglII fragment which is present in both wild-type and mld DNA. Probe I1 is a 3.5-kb BamHI fragment representing the region in the MBP gene from approximately 11 to 14.5 kb. It hybridizes to fragments of 9.3 and 0.8 kb in both wild-type and mld

FIGURE1 .-Restriction analysis of MBP specific DNA in wild type and mld. (A) A schematic illustration of the entire wild-type MBP gene is depicted with exons indicated as filled areas, introns as shaded areas and flanking regions as lines. The location of the exons within the gene is accurate but the widths of the filled areas are larger than the exon length in some cases. The length of the MBP gene (in kilobases) is shown beneath the schematic. T h e positions of BamHl and BglII restriction sites within the MBP gene are indicated. The positions of the et al. 1985; DEFERRA el al. 1985). The positions of the BglIl sites have been BamHl sites have been reported previously (TAKAHASHI determined in our laboratory by restriction analysis of both wild-type mouse DNA and cos138 DNA which is a cosmid containing the entire mouse MBP gene with some flanking DNA. Hybridiiation probes were prepared from BamHI fragments of cos138 representing sequential regions within the MBP gene as shown. Probe I is an 8.3-kb BamHl fragment representing the region in the MBP gene from approximately 3 to 1 1 kb. Probe I1 is a 3.5-kb BamHl fragment representing the region in the MBP gene from approximately 1 1 to 14.5 kb. Probe I11 is a 4.5-kb BamHl fragment repiesenting the region in the MBP gene from approximately 14.5 to 19 kb. Probe IV is a 14.8-kb fragment representing the region of the MBP gene from 19 kb to the 3' end plus a small portion of cosmid DNA. (B) Genomic DNA was isolated from wild-type and mfd/mld mice congenic on the C3H/HeJ background as described in MATERIALS AND METHODS. The DNA was digested with BgfIl restriction enzyme and subjected to agarose electrophoresis and Southern blotting as described in MATERIALS AND METHODS. Aliquots of wild-type DNA (lane 2) and mld DNA (lane 3) were electrophoresed in adjacent lanes, with '*P-end labeled Hind111 fragments of X DNA (lane 1) included as size markers. The sizes (in kilobases) of the X fragments are indicated at the left of the first panel. Each of the four panels was hybridized with a different probe as indicated. The arrows indicate restriction fragments that are present in mld DNA but not in wild-type DNA. Similar restriction fragment patterns have been reproducibly observed with DNA from several different animals from separate litters. 450

Myelin Basic Protein Gene in mld DNA, as well as to a fragment of 6 kb which is present only in the mld DNA. Probe 111 is a 4.5-kb BamHI fragment representing the region in the MBP gene from approximately 14.5 to 19 kb. It hybridizes to a fragment of 9.3 kb in both wild-type and mld DNA, as well as to fragments of 6 and 8 kb which are present only in mld DNA. Probe IV is a 14.8-kb fragment representing the region of the MBP gene from approximately 19 kb to the 3’ end plus a small portion of cosmid DNA. It hybridizes to fragments of 14, 9.3 and 6 kb in both wild-type and mld DNA, as well as to a fragment of 8 kb which is present only in mld DNA. These results indicate that mld contains an apparently intact copy of the entire MBP gene, and that the BglII restriction map of the MBP gene in mld is indistinguishable from that of the wild-type MBP gene. In addition, mld contains MBP-specific sequences which share homology with the region in the MBP gene from approximately 11 kb to the 3’ end but which have been altered to generate BglII restriction fragments of 6 and 8 kb which are not found in wild-type DNA. T h e junction between the two extra BglII fragments in mld is within the region of the MBP gene defined by the 4.5-kb BamHI fragment (probe HI), from approximately 14.5 to 19 kb. T h e 6-kb extra BglII fragment extends from this junction toward the 5’ end of the gene, and the 8-kb BglII fragment extends toward the 3‘ end of the gene. The mld phenotype cosegregates with the reiterated MBP-specific gene sequences: The results described above indicate that the mld mutant mouse, in which MBP gene expression is greatly reduced, carries an apparently intact MBP gene plus additional MBPspecific DNA. It is possible that the additional MBPspecific DNA in mld is responsible for the phenotype and somehow blocks expression from the intact MBP gene. Alternatively the apparently intact MBP gene in mld may contain a cryptic genetic lesion which is not detected by restriction mapping but which prevents its expression. In which case the additional MBPspecific DNA in mld may be unrelated to the genetic lesion responsible for the phenotype. In an effort to distinguish between these two hypotheses we have compared the segregation patterns of the mld phenotype and of the additional MBP-specific DNA using two different genetic approaches. First we determined whether the additional MBP-specific DNA was present in five different mld congenic strains. These are strains in which the mld mutation has been repetitively backcrossed onto a defined genetic background, either inbred or hybrid, for several generations. If the unselected additional MBP-specific DNA cosegregates with the selected mld phenotype in these strains, this indicates linkage. If the additional MBP-specific DNA does not cosegregate with the mld phenotype, then it is not linked and cannot be responsible for the muta-

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tion. The limits of linkage resolution by this technique are determined by the number of generations of backcrossing and by the number of strains examined. We obtained from Dr. R. SIDMAN four partially congenic (eight generations of backcrossing) mld strains on C3H/HeJ, C57BL/6J, DBA/2J and BALB/cJ inbred genetic backgrounds, and from Dr. M. WOLF one partially congenic (eight generations of backcrossing) mld strain on a B6/C3 hybrid genetic background. DNA was isolated from each of the congenic mld strains and from each of the corresponding wild-type strains and digested with BglII. T h e MBP-specific fragments were analyzed by Southern blotting as described in MATERIALS AND METHODS. Photographs of representative autoradiograms are shown in Figure 2. Figure 2A shows wild-type (lanes 2-5) and mld (lanes 8- 11) congenic inbred strain DNA, as well as a sample of shi DNA (lane 6), probed with an MBP cDNA that hybridizes to fragments containing exons 1, 3, 4 and 5. The probe hybridizes to fragments of 9.3 kb (containing exons 3 , 4 and 5 ) and 2.8 kb (containing exon 1) in wild-type DNA. shi DNA contains the 2.8-kb fragment but not the 9.3-kb which is derived from the region of the gene that is deleted. mld DNA contains both the 9.3-kb and 2.8-kb fragments as well as a fragment of 8 kb which is present in all of the mld DNAs, but absent from the wild-type DNAs. T h e 6-kb additional fragment that was detected in mld DNA using genomic probes (Figure 1) does not hybridize to the cDNA probe because it does not contains exons 1, 3, 4 or 5. DNA from the congenic mld strain on the hybrid genetic background was not included on the blot shown in Figure 2A but DNA from this congenic strain is analyzed on Figure 2B (described below) and also contains the additional MBPspecific DNA. These results indicate that the additional MBP-specific sequences cosegregate with the mld mutant phenotype through eight generations of backcrossing in five different congenic strains. This means that the additional MBP-specific sequences either represent the genetic lesion responsible for the mld mutant phenotype or are closely linked to it. We also analyzed linkage of the mld mutant phenotype and the additional MBP-specific genomic sequences in conventional Mendelian genetic crosses. Heterozygous +/mld mice on the partially congenic B6/C3 hybrid genetic background were crossed to homozygous shilshi mice on the same partially congenic hybrid genetic background. The shi mutant is being used in this cross as a “null” background for MBP expression on which the activities of the wildtype and mld chromosomes can be distinguished phenotypically since mldlshi animals exhibit the “shiverer” phenotype, while +/shi animals do not. At 20 days of age the progeny were scored as either “shiverer” or wild type, and DNA was isolated, digested with BglII

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FIGURE2.--Cosegregation of the mld phenotype and the additional MBP-specific restriction fragments. (A) Genomic DNA was isolated from four different inbred strains of wild-type mice (CSH/HeJ. C57BL/6J. DBA/2J and BALB/cJ) (lanes 2-5, respectively) and from partially congenic mldlmld strains on the same four inbred genetic backgrounds (lanes 8-1 I , respectively). DNA was also isolated from a shilshi mouse partially congenic on the B6/C3 hybrid genetic background (lane 6).The DNA was digested with BglII and subjected to agaroseelectrophoresis and Southern blotting as described in MATERIALS AND METHODS. The blot was hybridized with a 362 bp EcoRI-Alul subfragment representing nucleotides 1-362 of the rat 14-kilodalton MBP cDNA insert in pMBP-I, prepared as described in MATERIALS AND METHODS. End labeled Hindlll fragments of X DNA were electrophoresed in lanes I , 7 and 12 as molecular size markers. The sizes of the individual fragments in kilobases are indicated at the left of lane 1. (B) Genomic DNA was isolated from individual progeny of a cross between +/mld and shilshi mice. The DNA was digested with Bglll and subjected to agarose electrophoresis and Southern blotting as described in MATERIALS AND METHODS. The lanes designated by an -s” contained DNA from animals that exhibited the characteristic ‘shiverer” clinical phenotype and whose genotype was presumably mldlshi, the lanes with no designation contained DNA from animals that appeared clinically wild type and whose genotype was presumably +/shi. DNA samples from 52 different animals were analyzed on the autoradiogram shown. The blot was hybridized to a 4.5-kb BamHI fragment of cos138 representing the region from approximately 14.5 to 19 kb in the MBP gene (probe 111 in Figure I). End-labeled HindllI fragments of X DNA were electrophoresed in the lanes at the extreme left as molecular size markers. The sizes in kilobases of the different Hind111 fragments are indicated at the left of the panel.

and MBP-specific restriction fragments analyzed by Southern blotting using as a probe the 4.5-kb BamHI fragment representing the region in the MBP gene from 14.5 to 19 kb (probe 111 in Figure 1). Of 93 progeny examined in this way, 46 exhibited the “shiverer“ phenotype, and 47 did not. A photograph of a representative blot on which 52 of the DNAs were analyzed is shown in Figure 2B. T h e lanes marked with an “s” contain DNA from progeny that exhibited the “shiverer“ phenotype, the unmarked lanes contain DNA from progeny that did not. In all of the lanes the probe hybridizes to a 9.3-kb fragment representing the region in the intact MBP gene from approximately 12 to 22 kb (see Figure 1). In all of the lanes marked ‘3,’’ and in none of the unmarked lanes, the probe also hybridizes to two additional fragments of 8 and 6 kb derived from the additional MBP-specific DNA present in mld. These results indicate that the additional MBP-specific sequences cosegregate with the mld mutant phenotype in 93 of 93 animals. This confirms the conclusion from the analysis of the con-

genic mld strains that the additional MBP-specific sequences either represent the genetic lesion responsible for the mid mutant phenotype or are closely linked to it. T h e upper limit of genetic distance between the additional MBP-specific sequences and the mld mutation defined by these genetic experiments is less than 0.3 cM, calculated as described by FLAHERTY (1981). Duplication of the myelin basic protein gene in mld: Our initial analysis indicated that mld genomic DNA contained an intact MBP gene plus additional MBP-specific sequences. We reasoned that this meant that some portion of the MBP gene was reiterated and that the additional MBP-specific restriction fragments in mld were derived from the junction between the reiterated MBP-specific gene sequences and nonMBP DNA. To test this hypothesis we attempted to quantitate the various MBP-specific restriction fragments in mld relative to wild type in order to determine what portion of the MBP gene was reiterated and how many copies were present. Since it was im-

Myelin Basic Protein Gene in mld

portant to correlate the segregation of the reiterated MBP-specific DNA in mld with the segregation of the mld clinical phenotype, we analyzed the animals described in Figure 2B, which are of two types-either +/shi which do not contain additional MBP-specific restriction fragments and do not exhibit the “shiverer” phenotype, or mldlshi which contain additional MBPspecific restriction fragment and also exhibit the “shiverer” phenotype. Five animals of each type were analyzed. T o determine the relative copy number for the various restriction fragments within the MBP gene in wild type and mld, corresponding bands on Southern blots hybridized with MBP-specific probes were quantitated by densitometry. Band intensity on Southern blots is subjected to a number of variables including: partial or overdigestion with restriction enzyme, variation in amount of DNA loaded, variation in transfer efficiency and variation in hybridization efficiency. To control for these variables, an irrelevant, nonMBP hybridization probe was included (we used a cDNA for Po, a major protein of peripheral myelin, reasoning that it represented a single copy gene and was unlikely to be affected by the mld mutation) and the values obtained for MBP-specific restriction fragments were normalized to the control band intensity in each lane. Photographs of representative autoradiograms are shown in Figure 3. The DNA was digested with BglII. Figure 3B shows a blot that was hybridized with a mixture of MBP cDNA and Po cDNA. T h e stringency conditions were such that the MBP cDNA hybridizes only to restriction fragments containing exon 1 (2.9 kb) or exon 7 (14 kb). T h e Po cDNA hybridizes to fragments of 7.6 and 4.8 kb. Lane 1 contains shilshi DNA, lanes 2-6 contain +/shi DNA and lanes 7-1 1 contain mldlshi DNA. Each lane was scanned by transmittance densitometry and the values for the two MBP-specific fragments were normalized to the value for the 7.6-kb Po fragment. In the case of the 14-kb fragment, representing the 3’ end of the MBP gene, the average level in the mldlshi lanes was 1.96 (f0.15) times the average level in the +/shi lanes indicating that the 3’ end of the MBP gene is duplicated in mld. In the case of the 2.9-kb fragment, representing the 5‘ end of the MBP gene, the average level in the mldlshi lanes was 1.46 (kO.12) times the average level in the +/shi lanes. Since this fragment is present in shi DNA, the differential between +/shi and mldlshi suggests that if the former contains two copies of this fragment the latter contains 3 copies, indicating that the 5’ end of the MBP gene is also duplicated in mld. T h e results described above indicate that in mld both the 5’ and 3‘ ends of the MBP gene are duplicated. In order to quantitate a fragment derived from the middle of the gene, the blot shown in Figure 3B was

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stripped and rehybridized with a mixture of probes including: MBP cDNA, PocDNA, and a 4.5-kbBamHI fragment corresponding to the region of the MBP gene from 14.5 to 19 kb (probe I11 in Figure 1). A photograph of the resulting autoradiogram is shown in Figure 3C. T h e two MBP-specific fragments (14 and 2.9 kb) and two POspecific fragments (7.6 and 4.8 kb) seen in Figure 3A are also seen in Figure 3B, however three additional MBP-specific fragments (9.3, 7.8 and 6 kb) are detected by probe 111. T h e 7.8-kb fragment (which is partially obscured by the 7.6-kb Po fragment) and the 6-kb fragment correspond to the additional MBP-specific fragments in mld DNA seen in Figure 1. T h e 9.3-kb fragment represents the region from 12 to 22 kb in the wild-type MBP gene. T h e lanes in Figure 3B were scanned densitometrically and the relative intensities of the 14kb and 9.3-kb bands were determined. T h e values for the 9.3-kb band were normalized assuming that the copy number of the 14-kb fragment was 1 in the +/ shi lanes and 2 in the mldlshi lanes (as determined above). T h e results indicate that the average level of the 9.3-kb fragment in mldlshi is 0.96 (kO.1) times the average level in +/shi, indicating that the number of copies of the 9.3-kb fragment is the same in mZd and in wild type. This data indicates that restriction fragments derived from the 5’ and 3’ ends of the MBP gene are present in twice as many copies in mld as in wild type, while a fragment derived from the middle of the gene is present in the same number of copies in mld and wild type. In order to extend the copy number comparison between mld and wild type to other regions in the MBP gene, the blot shown in Figure 1 was scanned densitometrically and the relative band intensities were compared in the wild-type and mld lanes. T h e values were normalized assuming that the copy number of the 9.3-kb fragment was the same in wild type and mld (as determined above). T h e results are summarized in Figure 3A, where the copies per haploid genome of different BglII restriction fragments in mld relative to wild type is shown in the form of a histogram beneath a schematic representation of the MBP gene. A small region near the 5’ end of the gene was not quantitated because the probes used in Figures 1 and 3 did not overlap this region. Every other BglII restriction fragment in the MBP gene except for the 9.3-kb fragment representing the region from 12 to 22 kb in the gene appears to be duplicated in mld. This suggests that in mld the entire MBP gene is duplicated and that one copy is altered in the region corresponding to 12-22 kb in the wild-type MBP gene so that it does not generate a 9.3-kb BglII fragment like the wild-type gene but instead generates two additional BgZII fragments of 6 and 7.8 kb. From the BglII restriction fragment data in Figure 3 it is appar-

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FIGURE3.-Relative copy number of MBP gene restriction fragments in mld genomic DNA. (A) A schematic illustration of the entire wild-type MBP gene is depicted with exons indicated as filled areas, introns as shaded areas and flanking regions as lines. The location of the exons within the gene is accurate but the widths of the filled areas are larger than the exon length in some cases. The length of the MBP gene in kilobases is shown beneath the schematic. The positions of BgfIl restriction sites within the MBP gene are indicated. The relative copy number per haploid mfd genome for each of the Bglll fragments was determined by densitometry of Southern blots similar to those shown in B and C and in Figure 1. The integrated areas under individual peaks were normalized as described in RESULTS and are plotted as a histogram where the width and position of each bar corresponds to the length and position of a specific BglII restriction fragment within the MBP gene, and where the height of the bar corresponds to the number of copies of that fragment per haploid mld genome. One BgfIl fragment near the 5’ end of the gene was not quantitated because the probes used in Figures 4 and 6 did not overlap this region of the MBP gene. (B) Genomic DNA from homozygous shilshi (lane l), heterozygous +/shi (lanes 2-6). and heterozygous mfdlshi (lanes 7-1 1) mice with the same partially congenic B6/c3 hybrid genetic background was digested with BgflI and subjected to agarose electrophoresis and Southern blotting as described in MATERIALS AND METHODS. End-labeled Hind111 fragments of X DNA were included as size markers and are shown in the leftmost lane. The blot was hybridized with a mixture of MBP and POcDNA probes. The MBP probe is a 1.5-kb rat cDNA containing sequences from exon 1 (192 bp), exon 3 (102 bp), exon 4 (36 bp). exon 5 (33 bp) and part of exon 7 (1095). The Po probe is a 1.85-kb rat cDNA that represents full length Po mRNA. (C) The blot shown in B was stripped and hybridized with the same probes as in B plus a 4.5-kb BnmHl fragment representing the region from 14.5 to 19 kb in mouse genomic DNA (probe 111 in Figure 1).

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Myelin Basic Protein Gene in mld ent that the duplicated region in mld includes approximately 2 kb of flanking DNA at the 3‘ end and approximately 8 kb of flanking DNA at the 5’ end of the MBP gene. In addition, we analyzed the 5‘ and 3‘ termini of the MBP genes in wild type and mld using a variety of different restriction enzymes (data not shown) and both the 5’ and 3’ flanking regions appear to be identical in wild type and mld with every enzyme tested indicating that the duplicated region in mld must include several kilobases of flanking DNA at each end of the MBP gene. mld contains one intact and one rearranged MBP gene: T h e observations described above suggest that the additional MBP gene sequences in mld represent a rearranged MBP gene which is interrupted at a position located within the region of DNA that generates a 9.3-kb BglII restriction fragment in the intact MBP gene, and that the two additional BglII restriction fragments are derived from the newly created junctions at the 5’ and 3’ ends of this interruption. In order to define more precisely the location and the nature of the interruption in the duplicated MBP gene in mld, we have performed more extensive restriction analysis of the region surrounding the interruption. These results are summarized in Figure 4. Wild-type (lanes 1 and 3) and mld (lanes 2 and 4) DNA were digested with three different restriction enzymes (BamHI, BglII and PstI) individually and in double digest combinations. T h e blots were hybridized with two different hybridization probes. The first probe (lanes 1 and 2) was the 4.5-kb BamHI fragment representing the region in the MBP gene between 14.5 and 19 kb (probe 111 in Figure 1). This fragment was used because it hybridizes to both of the additional BglII fragments in mld (see Figure 4) and thus presumably spans the position of the interruption in the duplicated MBP gene. The second probe (lanes 3 and 4) was a 148-bp subfragment of MBP cDNA containing exons 3 and 4. This probe was used because it partially overlaps the first probe (probe 111) and extends in the 3’ direction and thus clarifies the location and orientation of specific restriction fragments. For the sake of discussion, bands derived from the wildtype gene or from the intact gene in mld are labeled with uppercase letters and bands derived from the corresponding region of the interrupted gene in mld are labeled with the corresponding lowercase letter followed by a 1 or a 2 to indicate whether they span the junction on the 5’ or the 3’ side, respectively, of the interruption. Digestion with BamHI generates a 4.5-kb band (designated A) in both the wild-type and mld DNA. mld DNA contains two additional BamHI fragments, one of 8.8 kb (designated a l ) which hybridizes with the first probe but not with the second, indicating it spans the junction on the 5’ side of the interruption, and

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one of 12.5 kb (designated a2) which hybridizes with both probes, indicating that it spans the junction on the 3‘ side of the interruption. This means that the distance between the two junctions in the interrupted gene is greater than 16.8 kb (8.8 12.5 - 4.5). Digestion with BglII generates a 9.3-kb band (designated B) in both wild-type and mld DNA. mld DNA contains two additional BglII fragments, one of 6 kb (designated b l ) which hybridizes with the first probe but not with the second indicating it spans the junction on the 5’ side of the interruption, and one of the 8 kb (designated b2) which hybridizes with both probes indicating it spans the junction on the 3’ side of the interruption. This means that the 5’junction of the interruption in mld is less than 6 kb from the BglII site at 12 kb in the MBP gene and the 3’junction of the interruption is less than 8 kb from the BgZII site at 22 kb in the MBP gene. In other words, the site of the interruption is within the region between 14 and 18 kb of the MBP gene, which is consistent with the observation that the 4.5-kb BamHI fragment representing the region of the MBP gene from 14.5 to 19 kb apparently spans the site of interruption. Double digestion with BamHI and BglII generates a 4.5-kb fragment (which is presumably identical to fragment A in the BamHI digest) in both wild-type and mld DNA. mld DNA contains two additional fragments, one of 5 kb which hybridizes to both probes and presumably represents the region between the 3‘ BamHI site in a2 and the 5’ BglII site in b2, spanning the 3’junction of the interruption, and one of 4 kb which hybridizes to the first probe but not to the second and presumably represents the region between the 5’ BamHI site in a1 and the 3’ BglII site in b l , spanning the 5’ junction of the interruption. These results are entirely consistent with the results from the single digests confirming the positions of BamHI and BglII restriction sites around the interruption. Digestion with PstI generated fragments of 3 kb (designated C) and 3.6 kb (designated D) in both wildtype and mld DNA. mld DNA contains two additional fragments of 5.5 and 6 kb (designated c l and c2) which span the 5‘ and 3’junctions of the interruption. The cDNA probe does not overlap fragment C and thus could not be used to determine which of these two additional bands is derived from the 5’junction and should be designated c l and which is derived from the 3’junction and should be designated c2. Double digestion with PstI and BamHI generated fragments of 3 kb (which is presumably identical to fragment C in the PstI digest) and 0.8 kb (which presumably represents the region from the 5’ PstI site in fragment D to the 3’ BamHI site in fragment A) in both wild-type and mld DNA. mld DNA contains two additional fragments of 5.5 and 6 kb which are pre-

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I FIGURE4.-Restriction analysis of the intact and interrupted MBP gene copies in mld DNA. (A) A schematic illustration of the entire wild-type MBP gene is depicted with exons indicated as filled areas, introns as shaded areas and flanking regions as lines. The locations of the exons within the gene are accurate but the widths of the filled areas are larger than the exon length in some cases. The length of the MBP gene (in kilobases) is shown beneath the schematic. The positions of BumHI, Bglll and f s t l restriction sites within the MBP gene are indicated. The MBP gene sequences in mld are depicted as a linear array for purposes of illustration and does not imply a specific arrangement or orientation of these sequences within the genome. Restriction fragments derived from the wild-type gene or from the intact mld gene are labeled with uppercase letters. Fragments derived from the corresponding region of the interrupted mld gene are labeled with the corresponding lowercase letters followed by a 1 or a 2 to indicate that they span the junction at the 5' or 3' side of the interruption, respectively. (B)Genomic DNA from +/shi (lanes 1 and 3) and mldlshi (lanes 2 and 4) mice with the same partially congenic SS/C3 hybrid genetic background was digested with BumHI, BgllI and Pstl, individually and in double digest combinations. The digested DNA was subjected to agarose electrophoresis and Southern blotting as described in MATERIALS AND METHODS. The blots were hybridized to two different sets of probes. The first probe (lanes 1 and 2) was the 4.5 kb BumHI fragment representing the region from 14.5 to 19 kb in the wild-type MBP gene (probe 111 in Figure 4). The second probe (lanes 3 and 4) was a 148-bp BunllStul subfragment of the rat MBP cDNA specific for exons 3 and 4. The sizes of the individual fragments were determined by comparison to X Hind111 fragments electrophoresed on the same gel (not shown). Similar restriction fragment patterns have been reproducibly observed with DNA from several different animals from separate litters.

sumably identical to fragments c l and c2 in the PstI digest. These results indicate that there is no BamHI site contained within Pstl fragments C, c l or c2, while

there is within fragment D. This means that the site of the interruption is within PstI fragment C or between 15 and 18 kb in the MBP gene.

Myelin Basic Protein Gene in mld Double digestion with PstI and BglII generates fragments of 3 and 3.6 kb (which are presumably identical to fragments C and D in the PstI digest) in wild-type and mld DNA. mld DNA contains two additional fragments of 3.7 and 4.2 kb. These presumably represent the regions from the 5’ PstI site in c l to the 3’ BglII site in b l and from the 5‘ BglII site in b2 to the 3’ PstI site in c2, although we have not established which fragment is derived from which region. These results are entirely consistent with the results from the single digests and confirm the positions of the PstI and BglII sites around the interruption deduced from the single digests. T h e results presented in Figure 4 are entirely consistent with, and provide support for, the hypothesis that mld DNA contains a duplication of the MBP gene, and that one copy of the gene is interrupted at a single site in the region between 15 and 18 kb. T h e interruption could result from insertion of non-MBP sequences at this position or from breakage of the gene at this position and rearrangement of one or both of the two gene fragments. In either case, the distance between the two junctions created by the interruption is greater than 16.8 kb. In the diagram at the top of Figure 4 the two gene fragments and the intact gene in mld are drawn as discontinuous segments of DNA. The three segments are depicted as a linear array. However, it is important to note that this is for clarity of illustration only and is not meant to imply a specific arrangement or orientation of the fragments relative to each other in the mld genome. shi and mld both decrease MBP gene expression in the CNS: Considering the complex nature of the genetic lesion affecting MBP gene sequences in mld, and the apparent allelism of shi and mld, it was important to determine how the two mutations affect MBP gene expression in homozygous, heterozygous and heteroallelic animals. In order to quantify the effects of the shi and mld mutations on MBP expression, and to determine whether the two mutations exhibit codominance and allelic additivity, as would be expected if both affect the MBP gene, aliquots of brain homogenates from homozygous, heterozygous and heteroallelic animals were subjected to SDS-PAGE and immunoblotting with antibody to MBP as described in MATERIALS AND METHODS. A photograph of the stained blot is shown in the upper panel of Figure 5A. As reported previously (CARSON,NIELSONand BARBARESE 1983) wild-type brain (lanes 1-3) contains four major forms of MBP with apparent molecular weights of 21,500, 18,500, 17,000 and 14,000 which are detectable by 10 days of age and accumulate thereafter. There is a band with an apparent molecular weight of approximately 20,000 which is present in every lane. This band is stained nonspecifically with some rabbit antisera and does not appear to be related

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to MBP. In homozygous shilshi brains (lanes 7-9) none of the four major forms of MBP is detected (the limit of detection by this technique is approximately 1’?6 of the wild-type level). Heterozygous +/shi brains (lanes 4-6) contain reduced levels of each of the four major forms of MBP. In homozygous mldlmld brains (lanes 13-15) none of the four major forms of MBP are detected. When the gels are overloaded with protein (data not shown), traces of the four MBPs can be detected in mldlmld samples. Heterozygous +/mld brains (lanes 10- 12) contain the four major forms of MBP, some of which are reduced relative to wild type. In heteroallelic +/shi, mld/+ brains (lanes 16-1 8) none of the four major forms of MBP is detected. These results indicate that both shi and mld cause severe reductions in the steady state levels of all four major forms of MBP. T h e effects of the two mutations on MBP levels are expressed co-dominantly in heterozygous animals, and additively in heteroallelic animals, indicating that both are cis-acting mutations and suggesting that they are allelic. T o facilitate comparison of the levels of the four MBPs at different ages in the wild-type and heterozygous +/shi and +/mld animals, the blot in Figure 5A was scanned by reflectance densitometry and the areas under the appropriate peaks were integrated and expressed as picomole equivalents of MBP per milligram protein. T h e results are shown in Figure 5B. In the case of +/shi, each of the four MBPs is reduced relative to wild type. The differential becomes more pronounced at later ages and the 14kilodalton MBP is the most severely affected. In the case of +/mld, the levels of the 21.5-, 18.5- and 17kilodalton MBPs are comparable to wild type, but the level of the 14-kilodalton MBP is reduced. These results indicate that while both shi and mld are expressed codominantly, the expressivity of mld at the level of MBP accumulation is less severe for some forms of MBP (21.5, 18.5 and 17 kilodaltons) in heterozygous animals than in homozygous or heteroallelic animals. Reduced accumulation of MBPs in shi and mld CNS could reflect reduced amounts of MBP-specific mRNA, reduced translational activity of the mRNA, or increased turnover of the polypeptides. T o distinguish among these possibilities we isolated total RNA from wild type, shi and mld samples and determined the amount of MBP-specific RNA by glyoxal/DMSO agarose electrophoresis followed by Northern blotting and hybridization with MBP cDNA as described in MATERIALS AND METHODS. The results of the Western blots indicated that the expressivity of the mld mutation was less severe in heterozygous animals than in homozygous animals. Since the mld mutation affects oligodendrocyte morphology and differentiation in homozygotes but not in heterozygotes, it is possible

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FIGURE5.-Lkvelopmental accumulation of MBP polypeptides in mice carrying the shi and mld mutations. (A) Cerebral hemispheres from individual animals of different genotypes and ages were homogenized and subjected to SDSPAGE and immunoblotting with antibody to MBP as described in MATERIALS AND METHODS. The same amount of total protein (1 00 pg) was applied to each lane. Lanes 1-3: wild type (10, 20 and 30 days), lanes 4-6: +/shi (6, 15 and 26 days), lanes 7-9: shi/shi ( I O . 20 and 30 days), lanes 10-12: +/mld (6, 15 and 30 days), lanes 13-15: mld/mld ( I O , 20 and 30 days), lanes 16-18: shilmld (10. 20 and 30 days). The mobilities of the four major MBPs (21.5. 18.5. 17 and 14 kilodaltons) are indicated at the left. Qualitatively and quantitatively similar results have been obtained with several different animals from separate litters. (B) Each lane in the immunoblot in Panel A was scanned by reflectance densitometry and the areas under the peaks corresponding to the 21.5-. 18.5-, 17- and 14-kilodalton MBPs were integrated as described in MATERIALS AND METHODS. The area under each peak was converted to picomoles of MBP by comparison to standards. The values for each of the four MBPs in wild type 0,+/ shi (0)and +/mld (0)are plotted. The values for shilshi, mld/mld and shilmld were below the level of detection by this method and are not plotted.

that the phenotypic effect of the mutation on MBP expression is modulated by factors related to the differentiation of the oligodendrocyte. To investigate this possibility, we compared R N A isolated from whole brain, where oligodendrocyte morphology and differentiation are dramatically affected by the "a-

tion (SHEN et al. 1986), and from primary culture, where oligodendrocyte morphology and differentiation are relatively unaffected by the mutation (E. BARBARESE, unpublished results). Photographs of the autoradiograms are shown in Figure 6. The results were comparable for both whole brain and primary

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FIGURE6.-Developmental accumulation of MBP-specific RNA in mice carrying the shi and mld mutations. Total RNA was isolated from either primary culture (A) or cerebral hemispheres (B) of mice of different genotypes and ages. T h e RNA (10 gg per lane) was denatured with glyoxal and dimethyl sulfoxide and subjected to agarose electrophoresis and Northern blotting as described in MATERIALS AND METHODS. Glyoxylated "P-end labeled Hind111 fragments of X DNA were included as size standards and a r e shown in the leftmost lane of A with their sizes in kilobases indicated at the left. T h e blots e r e hybridized with the 1.5-kb insert from pMBP-I (prepared and nick translated as described in MATERIALS AND METHODS) which is a cDNA for rat 14-kilodalton MBP containing DNA specific for MBP exons 1, 3, 4. 5 and part of 7. (A) Lanes 1-3: wild type ( I O . 17 and 2 0 days in culture): lanes 4-5: shi/shi (1 1 and 2 0 days in culture): lanes 6-7: mld/mld (IO and 19 days in culture): lane 8: shilmld (26 days in culture). (B) Lanes 1-3: wild type (1 0 . 2 0 and 30 days); lanes 4-6: shi/shi (1 0.20 and 30 days): lanes 7-9: +/mld ( I 2, 2 0 and 30 days): lanes 10-1 2: mld/mld ( 1 2, 20 and 30 days): lanes 13- 15: shilmld (1 1, 2 I and 30 days).

culture, indicating that, in these two experimental systems at least, the expressivity of the mld mutation a t the level of accumulation of MBP-specific RNA is not affected by the morphological differentiation of the oligodendrocyte. In wild type (lanes 1-3, Figure 6 , A and B) there is a major 2-kb MBP-specific RNA that accumulates to a maximum by 20 days and decreases thereafter. T h e 2-kb MBP-specific RNA rep resents a mixture of mRNAs for each of the four major forms of MBP found in mouse brain. In homozygous shilshi (lanes 3 and 4, Figure 6A; lanes 4-6, Figure 6B) n o MBP-specific RNA is detectable, which is consistent with previous findings (ROACH et al. 1983). In homozygous mldlmld (lanes 6 and 7, Figure 6A; lanes 10-1 2, Figure 6B) 2-kb MBP-specific RNA is detectable albeit a t reduced levels. Densitometry of the autoradiogram indicates that the level of 2-kb MBP-specific RNA is 1-5% of wild type, both in vivo and in culture. Overexposure of the autoradiogram (not shown) did not reveal any additional discrete MBP-specific RNA species in either wild type or mld. In heteroallelic mldlshi (lane 8, Figure 6A; lanes 1315, Figure 6B) 2-kb MBP-specific RNA is detectable, but the levels a r e less than in homozygous mld. In heterozygous +/mid (lanes 7-9, Figure 6B), 2-kb MBP-specific RNA accumulates to approximately 70% of wild type a t 20 days. This level is greater than expected based on strictly additive expression of the wild-type and mld alleles, which is consistent with the

finding from the Western blot analysis of MBP accumulation that the expressivity of mld is less severe in heterozygotes than in homozygotes. T h e results described above indicate that shi and mld both affect MBP gene expression severely. Moreover, both mutations are expressed co-dominantly in heterozygous animals and exhibit additivity of expression in heteroallelic animals, suggesting that they are allelic. However, there is a qualitative difference in their phenotypes. In the case of shi, neither MBPspecific RNA nor MBP polypeptides are detectable in the brain, while in the case of mld both MBP-specific RNA and MBP polypeptides are detectable, albeit a t low levels, in the brain. T h e residual level of MBP expression in mld brain could reflect either a low level of expression in every oligodendrocyte due to "leakiness" of the mutation, or normal expression in a few oligodendrocytes due to occasional "reversion* of the mutation to wild type. In order to distinguish between these two possibilities we analyzed MBP gene expression at the cell-resolved level by immunostaining of individual oligodendrocytes in primary cultures. Primary cultures of newborn brain from wild-type, mldlmld and shilshi mice were immunostained with monoclonal antibody to either galactocerebroside (GC), in order to visualize all the oligodendrocytes, or MBP to visualize those cells expressing MBP. Representative fields a r e shown in Figure 7. T h e immunopositive cells have darkly stained cell bodies with mul-

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FIGURE'I.-Immunostaining of oligodendrocytes in primary cultures of wild-type and mld/mld mouse brain. Primary cultures were established from newborn mouse brain and maintained in culture for 15 days as described in MATERIALS AND METHODS. During this period astrocytes form a confluent layer o n the bottom of the dish and oligodendrocytes appear on the surface of the astrocyte bed. T h e dishes were fixed and stained with either anti-galactocerebroside (A and B) or anti-MBP (C-F) using alkaline phosphatase conjugated second antibody for visualization as described in MATERIALS AND METHODS. A, C and E are wild-type cultures, B. D and F are mld/mld cultures. T h e length of the bar in the lower right corner in A-D corresponds to 60 pm. T h e length of the bar in the lower right corner in E and F corresponds to 30 pm.

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tiple processes, some of which are elaborated into lacy fanlike sheets. This morphology is characteristic of oligodendrocytes in primary culture. T h e total number of GC-positive and MBP-positive oligodendrocytes for each genotype was determined by counting stained cells in ten separate fields on three separate plates from three different litters. T h e number of GCpositive cells is approximately the same in wild-type, mld, and shi cultures (compare Figure 7A (wild type) with B (mld);shi not shown), indicating that the mutations do not affect oligodendrocyte ontogeny o r differentiation. In wild-type cultures the number of MBP-positive cells is approximately the same as the number of GC-positive cells (compare Figure 7C and A) indicating that most wild-type oligodendrocytes are both GC- and MBP-positive. However in mld cultures, the number of MBP-positive oligodendrocytes is 4.7 (+2.3)% of the number of GC-positive cells (compare Figure 7D with B). In shi cultures MBP-positive cells are not detected (not shown). The MBP-positive oligodendrocytes in the mld culture are similar in morphology to wild-type oligodendrocytes. However, in mld cultures a greater proportion of the oligodendrocytes have truncated processes with little elaboration of membrane. This is apparent by comparing Figure 7C which shows wild-type oligodendrocytes most of which have elaborated processes with Figure 7D which shows several mld oligodendrocytes with truncated processes and two with elaborated processes, and at higher magnification by comparing Figure 7E which shows these wild-type oligodendrocytes with elaborated processes with Figure 7F which shows two mld oligodendrocytes, one with truncated processes and one with elaborated processes. The fact that the majority of the oligodendrocytes in mld cultures are MBP-negative by immunostaining, and that the proportion of MBP-positive oligodendrocytes (4.7%) is the same order of magnitude as the level of MBP-specific RNA detected in mld brain (1 5%), suggests that the majority of oligodendrocytes in mld brain exhibit no MBP gene expression, and that the MBP-positive “revertant” oligodendrocytes exhibit wild-type levels of MBP gene expression which accounts for the residual levels of MBP-specific RNA and MBP polypeptides detected in mld brain. Reversion of the shi mutation apparently does not occur, presumably because the genetic lesion is a deletion. If the MBP-positive oligodendrocytes in mld are revertants this means that the mld genome contains a potentially functional MBP gene. DISCUSSION

The results presented in this paper concern the nature of the mld mutation. The major findings can be summarized as follows. T h e mld mutation is closely linked (less than 0.3 cM) to the MBP gene. Mice

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carrying the mld mutation contain reiterated MBP gene sequences. One MBP gene copy appears to be intact and indistinguishable from the wild-type MBP gene. A second MBP gene copy is interrupted by either an insertion or a rearrangement between 15 and 18 kb in the intact MBP gene. Restriction mapping indicates that the distance between the interrupted MBP gene and the intact gene in mld is greater than several kilobases. Linkage studies indicate that the interrupted gene cosegregates with the mld mutant phenotype at a genetic resolution of less than 0.3 cM. T h e mld mutation causes a reduction in MBP gene expression, manifest as reduced steady-state levels of MBP-specific RNA and MBP polypeptides. The mutation is cis-acting and co-dominant. T h e expressivity of the mutation is less severe in heterozygotes than in homozygous o r heteroallelic mice. T h e low levels of MBP-specific RNA and MBP polypeptides that are detected in mld brain are attributable to a small fraction of “revertant” oligodendrocytes which exhibit normal levels of MBP expression. Recent evidence reported by POPKOet al. (1987) confirms the presence of reiterated and rearranged MBP gene copies in mld and also confirms the correlation between the clinical phenotype of the mutation and reduced MBP gene expression. The findings reported here raise several questions concerning the genetic lesion and the mutant phenotype in mld. One question concerns the nature of the mutational event(s) that generated the genetic lesion affecting the MBP gene in mld. There appear to be two components to the genetic alteration. T h e first is a reiteration of a region of greater than 40 kb of DNA containing the entire MBP gene (approximately 33 kb) and several kilobases of flanking DNA on either side. T h e second is either an insertion of nonMBP DNA into one gene copy or a rearrangement involving the 5‘ portion, the 3’ portion, o r both, of one gene copy. In either case, a break is introduced in one MBP gene copy at a position corresponding to the region between 15 and 18 kb in the wild-type gene. This is very close to the position of the deletion in the shi mutation, whose 5’ breakpoint is located at apet proximately 12 kb in the MBP gene (MOLINEAUX al. 1986). T h e breakpoint in mld is not located at exactly the same position as in shi since there is at least on PstI site and one BamHI site separating them. However, the fact that both mutations involve breakpoints within the region of DNA between 12 and 18 kb in the wild-type MBP gene indicates that this may be a “fragile” site or a region particularly susceptible to recombination, as suggested previously (MOLINEAUX et al. 1986). T h e duplication and subsequent insertion or rearrangement events in the MBP gene may have occurred in concert, as the result of a transposition event, or independently as separate mu-

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tations. It is conceivable that the MDB/Dt inbred strain of mouse, on which the mld mutation arose, contained reiterated copies of the MBP gene in its wild-type genome before the mld mutation occurred, and that the mld mutation is an insertion into, or rearrangement of, one gene copy. Since the MDB/Dt inbred line is extinct, this possibility cannot be tested directly. Another question raised by this work concerns the juxtapositions of the intact MBP gene, the interrupted MBP gene and the mld mutation in the mouse genome. Restriction mapping data indicates that several kilobases of 5’ and 3’ flanking regions are identical for the intact and the interrupted MBP genes which means that the two genes are separated by at least several kilobases. Linkage studies failed to demonstrate recombination between the mld mutation and either the normal MBP locus or the interrupted MBP gene. If the mld mutation resulted in extensive DNA rearrangements in the region of the MBP gene this could suppress recombination locally by negative interference. However, if one assumes no distortion of recombination frequency, then the linkage studies reported here indicate that the mld mutation cosegregates with the normal MBP locus, and with the interrupted MBP gene, at a genetic resolution of 0.3 cM. It is estimated that the entire mouse genome consists of approximately 1600 cM of genetic distance, which corresponds to approximately 3 X lo9 bp of DNA. Thus, if the mld mutation, the normal MBP locus, and the interrupted MBP gene are separated from one another by less than 0.3 cM of genetic distance, this means that they are all located within a region of less than 600 kb of DNA. However, the data do not enable us to determine whether the genetic lesion responsible for the mld phenotype is specifically associated with the interrupted MBP gene, the intact MBP gene, both, or neither. The mld genome contains two copies of the MBP gene, one intact and one interrupted. However, studies on the biochemical phenotype indicate that MBP gene expression is greatly reduced in mice carrying the mld mutation. This means that both MBP gene copies in mld are either dysfunctional or have reduced function. A gene can be rendered dysfunctional either directly by changing the DNA sequence within the gene in such a way as to interfere with its expression, or indirectly by positioning the gene within an inactive region of the genome, thereby interfering with its expression through stable position effects. Functionality or dysfunctionality of a gene cannot be determined from the type of restriction analysis reported in this paper. The “intact” MBP gene copy in mld, which is indistinguishable from the wild-type MBP gene by restriction mapping, could nevertheless contain a cryptic change in its DNA sequence or be

located in an inactive region of the genome, and thus be dysfunctional. Similarly, the “interrupted” MBP gene copy in mld could be dysfunctional because of the interruption (which effectively lengthens the intron distance between exons 2 and 3 and thus might interfere with post-transcriptional RNA processing) or because of some other cryptic change in its DNA sequence or its location within the genome. It is also possible that only one MBP gene copy in mld is intrinsically dysfunctional, and that the functional gene copy is somehow inactivated by the presence of the dysfunctional gene copy on the same chromosome. The restriction mapping data indicates that the two MBP gene copies are separated by at least several kilobases, while the linkage studies indicate that they are separated by less than 600 kb. If one gene copy inactivates the other this implies a remote cis interaction since proximate cis-acting regulatory elements such as promoters, enhancers and operators are usually active over only a few kilobases. We were unable, using conventional genetic techniques, to separate the mld mutation from either the intact gene copy or the interrupted gene copy so we cannot determine whether one or both MBP gene copies is(are) intrinsically dysfunctional, o r whether one copy inactivates the other through remote cis interactions. The mechanism responsible for reduced MBP gene expression in mld does not result in an absolute “null” phenotype. Some MBP gene expression does occur in mld. The data on MBP-specific RNA in mld indicates that in homozygous mldlmld mice, a small amount of MBP-specific RNA is detectable (approximately 5% of wild type) and that it is the same size (approximately 2 kb) as wild-type MBP-specific mRNA. This means that in mld, at least one MBP gene copy can be transcribed and processed correctly. Furthermore, in heterozygous +/mid mice the level of accumulation of MBP polypeptides and of MBP-specific RNA is not reduced to the extent expected based on strictly additive expression of the wild-type and mld chromosomes. The decreased severity of expressivity of the mld phenotype in heterozygotes compared to homozygotes could reflect either hyperexpression of the MBP gene on the wild-type chromosome to compensate for reduced expression from the mld chromosome, or increased expression from the mld chromosome in response to differences in the cellular environment in the +/mEd oligodendrocyte compared to the mldlmld oligodendrocyte. The former hypothesis implies the existence of a “feedback” mechanism in the oligodendrocyte for regulating wild-type MBP gene expression. The latter hypothesis implies that at least one MBP gene copy in mld is partially functional in the appropriate cellular environment. T h e data in this paper do not distinguish between these two hypotheses.

Myelin Basic Protein Gene in mld

The nature of the “revertant” MBP-positive oligodendrocytes in mld brain is unclear since the relation between the genetic lesion and the phenotypic effect on MBP expression is not known. Two general explanations are possible. Either the phenotype of the mutation is suppressed in some cells, allowing expression from one or both MBP gene copies, or the mutation reverts in a small fraction of the oligodendrocytes, eliminating the genetic lesion and leaving one intact functional MBP gene. With regard to the latter hypothesis, if the MBP gene sequences are arranged as depicted in Figure 4, a single intrachromosomal recombination event between the 5’ portion of the interrupted MBP gene and the homologous portion of the intact MBP gene would effectively delete the duplicated and rearranged MBP gene from the chromosome, leaving a single intact MBP gene which might now be functional. If the 5 % “revertant” MBPpositive oligodendrocytes in mld arise in this manner, the postulated intrachromosomal homologous recombination between the two MBP gene copies must occur at a significant frequency during oligodendrocyte ontogeny and differentiation in primary culture. An alternative mechanism for reversion might involve elimination of the interruption in the rearranged MBP gene copy. If the mld mutation were due originally to insertion of a transposable element into intron 2 of one MBP gene copy, then excision of the element might restore functionality to this gene copy resulting in “reversion” of the mutant phenotype. Unfortunately, these hypotheses cannot be tested directly by analyzing the MBP gene sequences in the putative “revertant” oligodendrocytes because these cells are identifiable only after they express MBP, by which time they are terminally differentiated and probably postmitotic making it difficult to obtain sufficient DNA for restriction analysis. There are two lines of evidence which suggest that the mld mutation may revert at a significant rate in vivo as well. The first comes from the observation of SHENet al. (1986) that occasional clusters of myelinated axons in mldlmld spinal cord exhibit a morphologically identifiable major dense line and are immunopositive for MBP. This means that MBP gene expression occurs in some oligodendrocytes in mld brain. T h e second comes and from the observation of ROCH, BROWN-LUEDI MATTHIEU (1986) that in mZd/mld mice on the inbred MDB/Dt genetic background, the four major forms of MBP gradually accumulate as the mice grow older. We have not observed this phenomenon in the congenic mld lines used in this study, suggesting that the rate of reversion of the mld mutation may be affected by the genetic background of the mice. T h e significant frequency of “revertant” oligodendrocytes in mld brain is in contrast to the lack of appearance of “revertant” mice. We have never ob-

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served a nonshaking mouse among several thousand offspring of homozygous mldlmld parents. This implies that the reversion frequency of the mld mutation in the germ line (less than 0.05%) is significantly less than in oligodendrocytes (approximately 5%). It is possible that the mld mutation reverts at a significant rate in all somatic cells. However, this phenomenon can be demonstrated only in myelinating cells where the mutation has a characteristic phenotype. In summary, we have demonstrated that mld mutant mice have a complex genetic lesion involving reiteration and rearrangement of the MBP gene. T h e mutation causes a severe deficit in MBP gene expression in oligodendrocytes and exhibits a novel type of tissuespecific reversion in some cells. It is unclear how (or if) the genetic lesion results in the deficit in MBP gene expression. We would like to thank Dr. M. K. WOLF(University of Massachusetts Medical School) for supplying breeding pairs of partially congenic mldlmld and shilshi mice on the B6/C3 hybrid genetic background, Dr. R. L. SIDMAN(Childrens Hospital, Boston) for supplying breeding pairs of partially congenic lines of mldlmld mice on the C3H/HeJ, C57BL/6J, DBA/2J, and BALB/cJ inbred genetic backgrounds, Dr. L. E. HOOD(Caltech, Los Angeles) for providing pMBP-1 and cos138, and Dr. G. LEMKE(Howard Hughes Medical Institute, New York) for providing pSN63. This work was supported by National Institute of Neurological and Communicative Disorders and Stroke grants: 15190 to J.H.C., and 19943 to E.B.

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