Myelin basic protein gene expression in quaking, jimpy, and myelin synthesis-deficient mice

DEVELOPMENTAL

BIOLOGY 106,38-44

(1984)

Myelin Basic Protein Gene Expression in Quaking, Jimpy, and Myelin Synthesis-Deficient Mice THOMAS

B.

CARNOW,*‘t

JOHN

H.

CARSON,*

STEPHEN

W.

BROSTOFF,t

AND EDWARD

L.

HOGAN?

*Department of Biochemistry, University of Gmnecticut Health Center, Farmington, Connecticut 06092, and TDepartment of Neurology, Medical University of South Carolina, Charleston, South Carolina 29&?5 Received December 9, 1983; accepted in revised

form May

21, 1984

Jimpy (jp), myelin synthesis-deficient (jp&), and quaking (qk) are mutations which affect myelination to different degrees in the mouse central nervous system (CNS). Total messenger RNA (mRNA) and myelin basic protein (MBP)specific mRNA from brains of these three mutants have been analyzed by in vitro translation and immunoprecipitation with antibody to MBP. The results indicate that the three mutations do not affect the level of total MBP-specific mRNA in the CNS but do affect the relative proportions of the various MBP-related translation products encoded in vitro. In each case the proportions of 14K and 12K M, MBP-related translation products are reduced and the proportions of 21.5K, 18.5K, and 17K M, MBP-related translation products are increased relative to wild type. This effect is most pronounced in jp, less so in jpd, and least pronounced in qk animals. The MBP-related polypeptides that accumulate in vivo have also been analyzed in the three mutants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody to MBP. The levels of all the major MBPrelated polypeptides that accumulate in wivo are reduced in all three mutations. The reduction is most pronounced in jp, less in jpd, and least pronounced in qk animals. These results indicate that the jp, jpl”‘, and qk mutations exhibit qualitatively similar phenotypic effects on MBP gene expression but the magnitude of the effect is proportional to the extent of hypomyelination in each mutant. 0 1984 Academic press. IN.

21.5K and 17K A& proteins, which is absent from the Myelin is the compact multilamellar membrane 18.5K and 14K ilf, proteins, and a sequence of 40 residues near the carboxyl terminus of the 21.5K and sheath that surrounds the axon, facilitating saltatory nerve conduction. In the central nervous system (CNS),’ 18.5K M, proteins, which is absent from the 17K and the myelin sheath is elaborated as an extension of the 14K iV& proteins. Recently, several additional MBPplasma membrane of the oligodendrocyte. The mor- related polypeptides with molecular weights ranging phogenesis of the myelin sheath is a temporally ordered from 26K up to 1OOKhave been reported (Barbarese process beginning with the initial ensheathment of the et al., 1983; Carson et aL, 1983). There is a complex axon by a single wrapping of oligodendrocyte plasma developmental program of MBP gene expression which results in the temporally ordered accumulation of the membrane, proceeding to the formation of multiple various MBP-related polypeptides in the mouse brain loose wrappings, and eventually to compaction to form (Carson et aZ.,1983). However, the relationship between the mature, multilamellar myelin sheath (Raine, 197’7). MBP polymorphism and myelin morphogenesis is unThis morphogenetic program proceeds pari passu with clear. In this paper we examine the effect on MBP a developmental program of gene expression regulating gene expression of several mutations which perturb the temporal accumulation of the various biochemical myelin morphogenesis at specific stages. components of the myelin sheath. One particular myelin Jimpy (jp) is an X-linked mutation of the mouse constituent, myelin basic protein (MBP), has been that, in hemizygous males, causes severe hypomyelinintensively studied in this regard. ation in the CNS while myelination in the PNS is Mouse CNS myelin contains four structurally related spared (Sidman et al, 1964). The myelin synthesisforms of MBP with molecular weights of 21.5K, 18.5K, deficient mutation (j~“~) is an allele of jimpy that 17K, and 14K (Barbarese et al, 19’7’7).The four procauses less severe hypomyelination (Billings-Gagliardi teins are distinguished by a sequence of approximately 30 residues near the amino terminus of the et al, 1980). Previous studies of MBP expression in jimpy brain have indicated that synthesis of total MBP is unaffected but that accumulation is reduced (Carson i Abbreviations used: CNS, central nervous system; GH, guanidine et uZ.,1975). The 21.5K iV, protein is spared relative to hydrochloride; MBP, myelin basic protein; PAGE, polyacrylamide the other MBPs early in development (Kerner and gel electrophoresis; PB, polysome buffer; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid. Carson, 1984) and is the predominant form of MBP INTRODUCTION

OOE-1606/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

38

CARNOW ET AL

Mgelin

Basic

that accumulates in the myelin fraction from jimpy CNS (Barbarese et al, 1979). Morphological studies of jimpy CNS indicate that myelin morphogenesis is blocked at an early stage (Meier and Bischoff, 1974, 1975). Quaking (qk) is an autosomal recessive mutation on chromosome 17 that causes hypomyelination in both the CNS and PNS (Sidman et UC, 1964). Previous studies have indicated that, as in jimpy, synthesis of total MBP is unaffected in quaking CNS but that accumulation is reduced (Brostoff et aL, 1977). Morphological studies of quaking CNS indicate that myelin morphogenesis proceeds further than in jimpy but is still blocked prior to the formation of mature compact myelin (Hogan, 1977; Nagara and Suzuki, 1981). In the work described here we have analyzed the effects of the jp, jpmsd, and qk mutations on MBP expression in the CNS. By determining how the regulation of MBP gene expression is altered in these mutants which represent a gradation of phenotypic effects with myelination arrested at specific morphogenetic stages, it may be possible to elucidate the relationship between MBP polymorphism and myelin morphogenesis and to establish whether the developmental program of MBP gene expression in the CNS is regulated by morphogenetic signals. MATERIALS

AND

METHODS

Animals

Mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Wild-type mice were C57BL/6J. Jimpy (jp) mice were produced by crossing heterozygous carrier females (BGCBA-A’+‘-j/A-!!‘a jp) with wild-type males. The jp/Y progeny were identified by the presence of axial body tremors. Myelin synthesis-difficient (jp&) mice were produced by crossing heterozygous carrier females B6C3-a/A-% jpwd) with wild-type males. The jpmsd/Y progeny were identified by the presence of axial body tremors. Quaking (qk) mice were produced by crossing heterozygous (C57BL/6J-qk+/+ T’j) mice. The qk/qk progeny were identified by the presence of axial body tremors. Animals were maintained either in the Medical University of South Carolina laboratory breeding colony or in the Center for Laboratory Animal Care at the University of Connecticut Health Center. Animals were sacrificed by cervical dislocation and the cerebral cortex was removed and frozen in liquid nitrogen. Wild-type mice were pooled from several different litters and were sacrificed at 15-25 days after birth. Mutant mice were also pooled from several different litters and sacrificed at 15-25 days after birth. This age range was chosen to allow maximal expression of the mutant phenotype while avoiding the problem

Protein

39

Gene Expression

of premature mortality in jp/Y and jpmd/Y animals. Furthermore, at this age both the level of MBP-specific mRNA and the rate of accumulation of MBP-related polypeptides are maximal in wild-type mouse CNS (Carson et al, 1983). Isolation

and Translation

of Messenger RNA

Polysomes were isolated from frozen mouse brain by the method of Fioretti et al. (1979). Brains from several animals were pooled and rinsed with cold polysome buffer (PB) (250 mM sucrose, 20 mM TrisHCI, pH 7.4, 5 mM MgClz, 25 mM NaCl, 40 pg/ml heparin, 100 pg/ml cycloheximide, 1% Triton X-100, and 1% sodium deoxycholate). The brains were homogenized in PB (lo%, w/v) using a Polytron homogenizer at slow speed for 30 sec. The homogenate was centrifuged (lO,OOOg,10 min) and the supernatant was layered onto a discontinuous sucrose gradient consisting of 5 ml of 0.5 M sucrose in gradient buffer (5 mM MgClz, 25 mM NaCl, 20 mM Tris-HCl, pH 7.4, 40 pg/ml heparin) layered onto 2 ml of 2.5 M sucrose in the same buffer. The gradients were centrifuged in a SW41 rotor (Beckmann) at 40K rpm for 2 hr. The polysomes formed an opalescent band at the interface between the 0.5 and 2.5 M sucrose. The band was removed by puncturing the centrifuge tube with a sterile 23-gauge needle and withdrawing the band into a sterile tuberculin syringe. Polysomes to be used for translation were prepared in PB without heparin or cycloheximide. The polysomes were stored at -80°C. RNA was extracted from the isolated polysomes by the guanidine hydrochloride (GH) method of Deeley et al. (1977). All glassware and solutions except GH were treated with diethyl pyrocarbonate and autoclaved. Polysomes were suspended in 6 M GH, 0.1 M potassium acetate, pH 6, and the RNA was precipitated with 2 vol of absolute ethanol, -2O”C, 30 min. The precipitate was collected by centrifugation and was partially resuspended by vortexing with 6 M GH, 25 mM ethylenediaminetetraacetate (EDTA), 0.1 M potassium acetate, pH 7. The RNA was reprecipitated with ethanol and collected by centrifugation. The pellet was rinsed with ethanol and resuspended in 25 mM EDTA, pH 7, chloroform:butanol (4:l). The aqueous phase was removed and the organic phase was reextracted until there was no detectable precipitate at the interface. The aqueous phases were pooled and the RNA was precipitated with 0.2 Mpotassium acetate, 2 vol absolute ethanol, -20°C. The precipitated RNA was collected by centrifugation, washed three times with 70% ethanol, dissolved in water to a concentration of 1 mg/ml, and stored at -80°C. Poly(A+) RNA was isolated from polysomal RNA by affinity chromatography on poly(U) Sepharose

40

DEVELOPMENTAL

BIOLOGY

(Lindberg and Persson, 1974). The polysomal RNA was dissolved in binding buffer (0.7 M NaCl, 10 mM EDTA, 25% formamide, 50 mM Tris-HCl, pH 7.5) and passed over the column of poly(U) Sepharose several times. The column was washed with 0.5M NaCl, 10 mM EDTA, 50% formamide, 50 mM Tris-HCl, pH 7.5, and the poly(A+) RNA was eluted with 90% formamide, 10 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), 10 mM Tris-HCl, pH 7.5. The RNA was recovered by ethanol precipitation, resuspended in water, and stored at -80°C. Polysomes, polysomal RNA, and poly(A+) RNA were translated in a cell-free in vitro translation system derived from rabbit reticulocyte lysate as described by Pelham and Jackson (1976). The translation system was purchased from New England Nuclear (Boston, Mass.). Subsaturating amounts of mRNA (1 pg) were incubated for 60 min at 30°C in a 25-111reaction mixture containing 30 &i [35S]methionine, 80 mM potassium, and 1 mM magnesium. The reaction was terminated by the addition of ribonuclease (1 mg/ml) and EDTA (5 mM) in order to hydrolyze charged tRNA and to dissociate polysomes. Total incorporation was determined by precipitation with trichloroacetic acid (TCA) and scintillation counting. Total translation products were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) according to Maize1 (1971) followed by fluorography. Immunoprecipitation of MBP-Related Translation Products

Immunoprecipitation of MBP-related translation products was performed as described by Carson et al. (1983). The in vitro translation reaction mixture was diluted with buffer containing 0.4 M NaCl, 0.2 M Trisacetate, pH 7.4,1% Triton X-100, 0.1% aprotinin, 1 mg/ ml bovine serum albumin, 1 mg/ml methionine, and 200 rig/ml 14K M, MBP. Nonspecific binding material was removed by incubating with preimmune serum and protein A. MBP-related translation products were immunoprecipitated with immunoaffinity-purified antibody to mouse 14K M, MBP and protein A. The immunoprecipitated material was analyzed by SDSPAGE according to the method of Swank and Munkres (1971) using a mixture of the four major MBPs from mouse myelin as carrier and as molecular weight markers. The gels were stained with Coomassie blue to visualize the carrier MBPs and then soaked in EN3HANCE (New England Nuclear, Boston, Mass.), dried, and exposed to Kodak XAR-5 film at -70°C to visualize the radioactive translation products. The films were developed after 2 days and after 2 weeks of exposure. Densitometry of the autoradiograms revealed

VOLIJME

106, 1984

the same relative proportions among the different bands for both the short and the long exposure, indicating that the peaks are within the linear response range of the film. Immunoblot

Analysis

of MBP-Related

Polypeptides

Individual brains were homogenized in water (10% w/v) using the Polytron homogenizer. Total protein was determined by the method of Lowry et al. (1951). An aliquot of each homogenate, containing 100 pg protein, was subjected to SDS-PAGE according to Swank and Munkres (1971). The separated proteins were transferred electrophoretically to nitrocellulose according to Towbin et al. (1979) and the MBP-related polypeptides were visualized by indirect immunoperoxidase staining using the same purified anti-MBP antibody that was used for immunoprecipitation. The stained blots were scanned with a densitometer. RESULTS

Polysomes were isolated from brains of 25-day-old wild-type, jp/Y, jpwd/Y, and qk/qk animals. The recovery of polysomes was comparable from the four genotypes (Table 1). The polysomes were incubated in an in vitro translation system to allow run-off completion of nascent polypeptides. Both the total incorporation (Table 1) and the spectrum of completed polypeptides (Fig. 1) were comparable for the four genotypes. Poly(A+) mRNA was isolated from the polysomes and translated in a reticulocyte lysate in vitro translation system. The total recovery of poly(A+) RNA and its total coding capacity in vitro were comparable for the four genotypes. These results indicate that there is no overall defect in protein synthesis in the jp/Y, jpwd/Y, or qk/qk CNS. The MBP-specific coding capacity of the mRNA was determined by in vitro translation and immunoprecipitation of the translation products with antibody to MBP. The amount of MBP-specific incorporation, expressed as a percentage of total incorporation (Table l), was not significantly different from wild type for the jio, jpmd, or qk mRNAs. These results indicate that the total amount of translatable MBP-specific mRNA in the CNS is not significantly affected by the jp, jpmd, and qk mutations. The MBP-related translation products were analyzed by SDS-PAGE and fluorography. As shown in Fig. 2, mRNA from wild-type brain encodes a spectrum of MBP-related translation products with molecular weights ranging from 12K up to 40K. The pattern is quite similar to that reported previously (Carson et ak, 1983) for mRNA isolated by a different procedure from brains of mice at this age. The major difference is the 12K M, immunoprecipitated translation

CARNOW

TABLE ISOLATION

Genotype -t/i jjl/Y w”“/-f qk/qk

0.72 0.75 0.64 0.70

k + k *

0.28 0.27 0.31 0.12

Myelin

Basic

1

AND IN VITRO TRANSLATION FROM WILD-TYPE, jp, jp*,

Recovery of polysomal RNA” (mg/g brain)

ET AL.

OF POLYSOMES AND mRNA AND qk BRAIN

Translational activity of polysomal RNA’ (cpm X 10-“/p(g) 23.1 24.9 26.4 21.9

k t t *

3.4 4.6 4.2 2.6

MBP-specific translational activity’ (% total) 3.5 3.4 3.2 3.6

+ 1.6 t 1.2 -+ 1.5 k 1.0

“Polysomes were isolated from brains of animals 15-25 days of age. Polysomal RNA was isolated by the guanidine hydrochloride method as described under Materials and Methods. The concentration of RNA was determined by measuring the optical density at 260 nm. “Purified polysomal RNA was incubated in a reticulocyte lysate iv rifro translation system and total incorporation was determined by TCA precipitation and scintillation counting. The background incorporation with no added RNA was subtracted. ’ Poly(A+) RNA was purified from the polysomal RNA by affinity chromatography on poly(U) Sepharose as described under Materials and Methods. Poly(A+) RNA was translated in a reticulocyte lysate iv vitro translation system. Total incorporation was determined by TCA precipitation and scintillation counting. The background incorporation with no added RNA was subtracted. MBP-specific translation products were immunoprecipitated with antibody to mouse 14K &I, MBP and protein A as described under Materials and Methods. The immunoprecipitated radioactivity was determined by scintillation counting. The nonspecific counts precipitated in the absence of antibody were subtracted and the specifically immunoprecipitated radioactivity was expressed as a percentage of the total incorporated radioactivity.

product which was not observed in the previous study. The relationship of this polypeptide to the other MBPrelated translation products is not known. The spectrums of MBP-related translation products for jp, jpmd, and qk mRNAs are qualitatively similar to the wildtype spectrum; however, the stoichiometries among the various translation products are significantly different. In each case there is a substantial decrease in the level of 14K and 12K M, translation products with a compensatory increase in the level of 21.5K, 18.5K, and 17K M, MBP-related translation products. The magnitude of this shift in proportions is greatest for jp mRNA, intermediate for jpmd mRNA, the least for qk mRNA. These results indicate that while the jp, jpmTd, and qk mutations have little effect on the level of total MBP-specific mRNA in the CNS, they do have differential effects on the levels of mRNA for individual MBP-related translation products. To determine the effects of the different mutations on the MBP-related polypeptides that accumulate in viva, samples of brain homogenates from the different mutant mice were subjected to SDS-PAGE and im-

Protein

41

Gene Expression

munoblotting with antibody to MBP. The results, shown in Fig. 3, indicate that each of the mutations causes a decrease in the levels of all the MBP-related polypeptides. The decrease is most severe in jp/Y animals, less severe in jpmd/Y animals, and least severe in qk/ qk animals. To facilitate comparison of the levels of the various MBP-related polypeptides that accumulate in vivo with the levels of the various MBP-related translation products that are synthesized in vitro, the peaks in Figs. 2 and 3 were cut out and weighed. The peak weights for each of the mutants, expressed as percentages of the corresponding wild-type peak weights, are given in Table 2. Both in vivo and in vitro the phenotypic effects are qualitatively similar for each of the three mutations. In the case of the 21.5K, 18.5K, and 17K M, MBP-related polypeptides there is a decreased level of accumulation in vivo but increased levels of mRNA translatable in vitro. In the case of the 14K and 12K M, MBP-related polypeptides both the level of accumulation in vivo and the level of mRNA translatable in vitro are decreased. Although each of these pheno-

wild type jimw msd

quaking m.w. x 10-3 FIG. 1. SDS-PAGE fluorography of total translation products from wild-type, jimpy, msd, and quaking polysomes. Polysomes were isolated from 15- to 25-day-old mouse brain as described under Materials and Methods. The isolated polysomes were incubated in a reticulocyte lysate in vitro translation system to allow run-off completion of nascent polypeptides. The translation products were separated by SDS-PAGE as described by Maize1 (1971). The gel was stained with Coomassie blue and then subjected to fluorography to visualize the labeled translation products. The fluorograph was subjected to densitometry. The positions of molecular weight standards were determined from the Coomassie blue-stained gel.

42

DEVELOPMENTAL BIOILXY

wild type

iiwy

msd

VOMJME 106. 1984

mRNAs for higher molecular weight MBPs predominate early in development and mRNAs for lower molecular weight MBPs predominate later; and third, each individual MBP-related polypeptide exhibits a characteristic rate-of-accumulation profile during development in viva which only partially reflects the level of mRNA for the corresponding in vitro translation products. The results presented in this paper indicate that the jr4 jpmd, and qk mutations affect primarily the second and third levels of regulation. That is, all three mutations result in alterations in the relative proportions of mRNAs encoding the different MBP-related in vitro translation products and also decrease the levels of MBP-related polypeptides that accumulate in vivo, but none of the mutations affects the total level of MBPspecific mRNA in the CNS.

quaking r FIG. 2. SDS-PAGE fluorography of MBP-related translation products from wild-type, jimpy, msd, and quaking mRNA. Poly(A+) RNA was purified from isolated polysomes as described under Materials and Methods. The mRNA was translated in vitro in a reticulocyte lysate translation system. The MBP-related translation products were immunoprecipitated with antibody to mouse 14K M, MBP and protein A. The immunoprecipitated material was mixed with carrier MBP from mouse myelin and subjected to SDS-PAGE as described by Swank and Munkres (1971). The gel was stained with Coomassie blue to determine the positions of the four major MBPs in mouse myelin (21.5K, 18.5K, 17K, and 14K Afr) and was then dried and subjected to fluorography to visualize the labeled translation products. The fluorograph was subjected to densitometry.

wild type

jimpy

typic effects on MBP gene expression is exhibited to some degree by each of the three mutants, there is a gradation in the magnitude of the effect which is greatest in jp/Y, intermediate in jpmd/Y, and least in qk/qk animals. This corresponds to the gradation in hypomyelination phenotypes presented by these animals.

msd

DISCUSSION

The results described in this paper concern the effects of various hypomyelinating mutations on MBP gene expression in the mouse CNS. Previous work from this laboratory has established that during normal brain development MBP expression is regulated on three different biosynthetic levels (Carson et aL, 1983). First, the total level of MBP-specific mRNA increases during the period of active myelination and decreases when the rate of myelination declines; second, the relative proportions of mRNAs encoding the various MBP-related in vitro translation products are modulated throughout the period of myelination such that

quaking 2 ZS 9, m.w.xlO-3 (YFIG. 3. Immunoblot analysis of MBP-related polypeptides in wildtype, jimpy, msd, and quaking CNS. Brain homogenates from 15- to 25-day-old animals were subjected to SDS-PAGE as described by Swank and Munkres (1971), and immunoblotting with antibody to mouse 14K M, MBP as described under Materials and Methods. The stained immunoblots were subjected to densitometry. The positions of the four major MBPs in mouse myelin are indicated.

CARNOW ET AL. TABLE

Myelin

Basic

2

COMPARISON OF MBP-RELATED POLYPEPTIDES IN VIVO AND IN VITRO IN WILD-TYPE, JIMPY, MSD, AND QUAKING CNS 21.5K

M,

MBP

Genotype Wild type jp/Y h” “I/Y qk/qk

18.5K/17K M, MBP

14K/12K M, MBP

In

In

In

I?/

I?/

I?/

vivo

vitro

vivo

vitro

vim

v/tro

100 10 26 84

100 138 123 108

100 4 10 53

100 176 115 101

100 9 22 39

100 36 40 58

Note. The peaks on the densitometric scans shown in Figs. 2 and 3 were cut out and weighed. In each case the weight of the wildtype peak was assigned a value of 100 and the weights of the corresponding peaks in the mutant samples were expressed as percentages of the wild-type peak weights. Since the 18.5K and 17K M, peaks and the 14K and 12K M, peaks were not always resolved, the peak weights were pooled in these two regions.

While all three mutations have specific phenotypic effects on MBP gene expression in the CNS, it is unlikely that the genetic lesion in any of the mutations impinges on the structural gene (or genes) for MBP for the following reasons. In the case of jp and jpmsd myelination in the PNS is unaffected by the mutations and Adcock, (Sidman et al., 1964; Billings-Gagliardi 1981). Since the MBP-related polypeptides in the PNS appear to be identical to those in the CNS, a genetic lesion in the gene (or genes) for MBP should affect myelination in both the CNS and PNS. It is possible, however, that the genetic lesion in jp or in jpmd affects a part of the gene which is necessary for expression in CNS but not in PNS. In the case of qk there are pleiotropic effects of the mutation in tissues such as testes where the MBP gene (or genes) are presumably not expressed indicating that the genetic lesion in qk affects a gene other than MBP that is expressed in both neural and nonneural tissues. Finally, there is evidence from restriction mapping of genomic DNA (Roach et ah, 1983) that the structural gene for the 14K M, MBP is closely linked to the shiverer mutation which is unlinked to either jp, jpmd, or qk. If the phenotypic effects of the three mutations on MBP gene expression are not primary effects due to genetic lesions in the gene (or genes) for MBP, then they presumably represent secondary effects related to the perturbations in myelin morphogenesis in the different mutants. The qk and jp, jpmd mutations are independently derived mutations located on different chromosomes and are apparently unrelated except insofar as they affect myelin morphogenesis. Nevertheless, all three mutations have qualitatively similar phenotypic effects on MBP gene expression, the severity

Protein

Gene Expression

43

of which is proportional to the severity of hypomyelination in the three mutants. This implies that myelin morphogenesis and certain aspects of MBP gene expression are interregulated in some manner. Specifically, when myelin morphogenesis is blocked in the different mutants, both the developmental modulation of the levels of various MBP-specific mRNAs and the rate-of-accumulation profiles for the various MBPrelated polypeptides are perturbed. This suggests that regulation of these two aspects of MBP gene expression is mediated through epigenetic signals related to myelin morphogenesis. On the other hand, the regulation of the overall level of MBP-specific mRNA, which presumably reflects the transcriptional activity of the gene (or genes) for MBP, appears to be independent of such morphogenetic signals since it is unaffected in any of the three mutants. The specific ways in which MBP gene expression is perturbed in these mutants have certain implications concerning the relationships among the different forms of MBP and the factors regulating MBP accumulation in the different mutants. The levels of translatable mRNA encoding the 12K/14K M, and the 21.5K/18.5K/ 17K M, MBP-related in vitro translation products, which presumably reflect either differential activities of separate genes or expression of different mRNAs from a single gene, appear to be coordinately regulated in such a way that if the level of the former is decreased the level of the latter is increased. Elucidation of the mechanism for this reciprocal regulation will require a detailed understanding of the molecular basis for MBP gene expression. The level of accumulation of MBP-related polypeptides in each mutant appears to reflect the level of myelin that accumulates rather than the level of MBPspecific mRNA in the CNS. Thus in jp/Y CNS for instance, the level of accumulation of each of the MBPrelated polypeptides in viva is less than 10% of wild type which corresponds to the amount of myelin in jp/ Y CNS, while the level of total MBP-specific mRNA, measured by in vitro translation, is not decreased relative to wild type, and the levels of certain MBPspecific mRNAs are actually increased. Furthermore, the stoichiometries among the different MBP-related polypeptides that do accumulate, while differing somewhat from the stoichiometries in wild-type CNS, do not reflect the stoichiometries of the corresponding mRNAs in the three mutants. There are two possible explanations for these results. Either the mRNA detected in vitro is not translated in vivo, implying a specific effect of the jimpy mutation on translational activity of MBP-specific mRNA, or the mRNA is translated but the translation products fail to accumulate, implying a specific effect of the jimpy mutation on the

44

DEVELOPMENTALBIOLOGY

rates of turnover of certain MBP-related polypeptides. Previous work on metabolism of MBP-related polypeptides in jp/Y CNS supports the latter hypothesis (Carson et aL, 1975). This implies that the accumulation of the various MBP-related polypeptides in vivo is regulated by factors in addition to the levels of mRNA encoding the individual polypeptides. Such factors might include the biochemical prerequisites and constraints imposed by the process of myelin morphogenesis. In summary, the results presented in this paper suggest that certain aspects of MBP gene expression, such as the overall transcriptional activity of the MBP gene (or genes), are unaffected by mutations that interfere with myelin morphogenesis, while other aspects, such as the developmental modulation of MBP polymorphism, both at the mRNA level and the level of accumulation in viva, are regulated by epigenetic factors related to the process of myelin morphogenesis, and are perturbed by mutations that interfere with myelination.

VOLUME106, 1984

CARSON,J. H., HERSCHKOWITZ,N. N., and BRAUN, P. E. (1975). Synthesis and degradation of myelin basic protein in normal and jimpy mouse brain. Trans. Amex Sot Newochem 6,207. CARSON,J. H., NIELSON,M. L., and BARBARESE,E. (1983). Developmental regulation of myelin basic protein expression in mouse brain. Dev. BioL 96, 485-492. DEELEY, R. G., GORDON,J. I., BURNS, A. T., MULLINEX, K. P., BINASTIN, M., and GOLDBERG,R. F. (1977). Primary action of the vitellogenin gene in the rooster. J. BioL Chem. 252, 8310-8319. FIORETTI,W. C., DAVIS, D. F., and LEDFORD,B. E. (1979). Polyribosome size analysis: Measurement of number-average polyribosome sizes. B&him.

Biophys. Acta 664, 79-89.

FRIEDRICH, V. L., JR. (1975). Hyperplasia of oligodendrocytes in quaking mice. Ati. EmbrgoL 147, 259-271. HOGAN, E. L. (1977). Animal models of genetic disorders of myelin. I?z “Myelin” (P. Morrell, ed.), pp, 489-520. Plenum Press, New York. KERNER, A.-L., and CARSON,J. H. (1984). The effect of the jimpy mutation on expression of myelin proteins in heterozygous and hemizygous mouse brain. J. Neurochem, in press. LINDBERG,U., and PERSSON,T. (1974). Messenger RNA isolation with poly(U) agarose. In “Methods in Enzymology” (W. B. Jakoby and M. Wilchek, eds.), Vol. 34, pp. 496-499. Academic Press, New York. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. BioL Chem. 193,265275.

This work was supported by Grants NS15190 and NS12044 from NINCDS and Grant RG1248 from the National Multiple Sclerosis Society. A preliminary report of this work has been presented: T. B. Carnow, J. H. Carson, E. L. Hogan, and S. W. Brostoff (1982) Myelin basic protein gene expression in normal, qk, jp, and msd mice. Trans. Amer. Sot. Neurochem 13, 256. REFERENCES BARBARESE,E., BRAUN,P. E., and CARSON,J. H. (19’77).Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic protein. Proc NatL Acad Sci USA 74,3360-3364.

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