Anthony T. CampagnoniS, Thomas M. PribylS, Celia W. CampagnoniS, Kathy ...... Molineaux, S., En h H , deFerra, F., Hudson, L., and Lazzarini, R. A. (1986).
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 268, No. 7, Issue of March 5, pp. 4930-4938, 1993 Prrnted m U.S.A.
Structure and Developmental Regulationof GoZZi-mbp, a 105-Kilobase Gene That Encompasses the Myelin Basic Protein Gene andIs Expressed in Cells in the OligodendrocyteLineage in the Brain* (Received for publication, September 9, 1992)
Anthony T. CampagnoniS, ThomasM. PribylS, Celia W. CampagnoniS, Kathy KampfS,Shashi Amur-UmarjeeS, Charles F. LandryS, VanceW. HandleyS, Sammye L. Newmane,Bertrand Garbayq, and Kunio Kitamurall From the $.Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, California 90024, the $Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Richmond, Virginia 23298, the VZnstitut de Biochemie Celluluire et Neurochimie du Centre National de la Recherche Scientifique, 1 rue Camille St. Saens, 33077 Bordeaux Cedex, France, and the 11 Department of Physiology, Saitama Medical School, 38 Moroyama, Zrumn-gun, Saitamn 350-04, Japan
We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cellsof the oligodendrocyte lineage atprogressive stages of differentiation. Thus, the MBP gene is a part of a morecomplex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Gollimbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.
The myelin basic protein(MBP)’ gene anditsprotein products have been of intense interest to neurobiologists and immunologists because MBP is ( a ) a major structural protein of the myelin membrane in the nervous system and (b) a potent autoimmunogen. Injection of MBP into animals produces an autoimmune demyelination, termed experimental allergic encephalomyelitis, which has been studied as a model for autoimmune diseases and for multiple sclerosis (Bell and Steinman, 1991). In the central nervous system, myelination is a developmentally regulated event that requires the precise coordination of expression of a number of myelin proteins and lipids (Campagnoni and Macklin, 1988). During late embryonic and neonatal murine brain development, oligodendrocyte precursor cells differentiate intoimmature oligodendrocytes (see Cameron and Rakic (1991) for review). These cells are characterized by the expression of certain myelin/oligodendrocytespecific lipids and surface components, such as galactocerebrosides and sulfatides. During the first postnatalweek, these
* This work wassupported inpart by National Institutes of Health Grants NS23022, NS23322,and HD25831 and Grant RG 2233A1 (to A. T. C.) from the National Multiple Sclerosis Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs) reported in this paper has been submitted to theGenBankTM/EMBL Data Bankwith accession numberfs) u)7503-L07509. ‘The abbreviations used are: MBP, myelin basic protein; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction.
immature oligodendrocytes differentiate further into mature oligodendrocytes and begin to express the myelin-specific proteinsMBPand proteolipid protein. Expression of the genes encoding these proteins is accompanied by massive synthesis of myelin-specific lipids followed rapidly by elaboration of the myelin sheath by the oligodendrocytes. The “classic” MBP gene consists of seven exons distributed over a 32-kb stretch of chromosome 18 in mouse (deFerra et al., 1985; Roach et al., 1985). At least seven transcripts are expressed from this gene in the mouse through alternative splicing of exons 2,5A, 5B, and 6 (deFerraet al., 1985; Newman et al., 1987; Aruga et al., 1991). The alternatively spliced MBP mRNAs encode MBP isoforms that range in molecular mass from14 to 21 kDa. The splicing of the transcripts is developmentally regulated. For example, those containing exon 2 (i.e. encoding the 17- and 21.5-kDa isoforms) appear early in myelination and decline with development (Campagnoni, 1988). Recently, we reported the existence of M41-MBP mRNA, which we believe is produced from a transcription start site upstream of the classic MBP gene transcription start site (Kitamura et al., 1990). The M41-MBP mRNA is 2.3-2.4 kb in length, encodes a 14-kDa MBP, and contains a longer 5’untranslated region than the “usual” MBP mRNAs. The 5’untranslated region on the M41-MBP mRNA derives from two exons, i.e. exon 0, which is 32 kb upstream of exon 1 of the MBP gene, and exon lA, which is immediately upstream of the classic MBP gene transcription start site and which is contiguous with exon 1 of the MBP gene. Developmental expression of M41-MBP mRNAs is the same as that of the major class of MBP mRNAs. In addition to the M41-MBP cDNA, we isolated several other MBP gene-related clones that contained exon 1A sequences, but that were quite different in their 5”sequences from the M41-MBP cDNA. In thispaper, we report the results of the analyses of these clones through which we have identified a new gene that utilizes exons of the MBP gene. This gene contains three unique exons that span a region 73 kb upstream of the classic MBP transcription start site. It produces at least three alternatively spliced products, all of which are expressed much earlier in development than MBP in the brain and which are expressed in oligodendrocytes and their precursor cells. Hence, we propose the name Golli-mbp to signify that most of the products of this gene are expressed in the oligodendrocyte lineage in the brain and that it encompasses the MBP gene.
4930
Structure and Expression of Murine Golli-mbp Gene
4931
radioactive band was purified after electrophoresis on a 1%alkaline agarose gel (50 mM NaOH, 1mM EDTA). Probe (-2 X lo6 cpm) was Northern Blots and RNA Isolation-RNAwas isolated by the mixed with 3 pgof poly(A+) RNA and ethanol-precipitated with guanidinium thiocyanate method and purified through acesium chlo- sodium acetate, pH 5.2. Hybridization and S1 nuclease digestions ride gradient (MacDonald et al., 1987). Poly(A+) RNA was selected were performed essentially as described by Ausubel et al. (1991). Both by means of an oligo(dT)-cellulose column (Jacobson, 1987). North- steps were carried out at25 "Cand S1nuclease concentrations of 15ern blots were prepared by the methods detailed by Kitamura et al. 235 units/ml. The digestion products were sized on a 6% sequencing (1990). gel adjacent to a sequencing ladder that was generated using the same Library Preparation and Screening-cDNA libraries were prepared oligonucleotideprimer as thatused to prime the probe reaction. as described by Kitamura et al. (1990) using either Moloney murine Combined in Situ Hybridization/Zmmunocytochemistry of Cell Culleukemia virus reverse transcriptaseoraSuperscript RNase Htures-Primary cultures were prepared as described by Amur-Umarsystem (GIBCO/BRL). The blunt-ended cDNA was cloned into hgtll jee et al. (1990a), and enriched oligodendrocytes were purified by the with EcoRI linkers. The libraries were screened as previously de- method of Suzumura et al. (1984). DNA probes were labeled with scribed (Kitamuraet al., 1990). The mousegenomic library was digoxigenin as previously described (Amur-Umarjee et aZ., 1990b). A prepared from a partialMboI digest of purified liver DNA cloned into combination of nonradioactive in situ hybridization and immunofluthe BamHI site of XJ1. The average insert size was 15-20 kb. hJ1 was orescence techniques (Amur-Umajee et al., 1990a) was employed to derived from XK47.1 by Dr. Jim Mullins and was the gift of Dr. J. N. localize Golli-mbpgene transcripts with markers of cells in the oligoHansen. dendroglial lineage. Cells were fixed with 4% paraformaldehyde soSequenceAnalysis-This was performed on nested deletions (Hen- lution for 1 h and permeabilized with 0.1% Triton X-100 when ikoff, 1984) by the dideoxy method of Sanger et al. (1977) using the necessary. The cells were then incubated with the primary antibody Sequenase version 2.0 system (U. S. Biochemical Corp.). Both strands at room temperature for 1 h and washed threetimes with Triswere sequenced; and in areasof compression, the dGTPsequence was buffered saline, pH 7.5. In situ hybridization was then carried out, compared with sequences in which dITP and7-deaza-GTP had been after which the secondary antibody conjugated to fluorescein isothisubstituted for this nucleotide. ocyanate or rhodamine was added. After 1 h of incubation at room In Vitro Translations-In vitro translations in rabbit reticulocyte temperature, the cells were washed three times and mounted on lysates were performed exactly as described previously by Verdi and aqueous mounting medium. A confocal imaging system (Bio-Rad Campagnoni (1990). Capped cRNA transcripts were prepared and MRC-500) was utilized to enhance the fluorescence images and to purified as described previously (Verdi and Campagnoni, 1990). reverse the phase contrast of the cells labeled by in situ hybridization. Reverse Transcription PCR-Poly(A+) RNA (1 pg)was reverse- Antibodies used in these studies were generously provided by Dr. transcribed a t 45 "C using the Superscript RNase H- system and Kari Stefansson (A007) and Dr. Joyce Benjamins (A2B5 and antioligo(dT) as a primer. For PCR amplification, a 50-pl reaction was galactocerebroside). assembled containing one-tenth of the cDNA reaction (2 pl),40 pmol I n Situ Hybridization to Tissue Sections-Normal (Balb/cByJ) and of each primer, 2.4 mM MgC12, 10 mM Tris-C1, pH 8.3, 50 mM KCl, shi mouse brains were collected, prepared, and sectioned as described 250 mM dNTPs, and 5 units of Amplitaq (Perkin-Elmer Cetus In- by Verity and Campagnoni (1988). Hybridization was at 50 "C in struments). The cycling conditions were as follows: (a) 4 min at 94 "C buffer containing 50% formamide (Landry et al., 1989) using 100 ng for 1 cycle; ( b ) 90 s at 94 "C, 90 s a t 44 "C, and 135 s at 72 "C for 7 of digoxigenin-labeledriboprobe/slide. Following hybridization, slides cycles; (c) 90 s at 94 "C,75 s at 55 "C, and 150 s at 72 "C for 35 cycles; were incubated in 20 pg/ml RNase A (37 "C for 15 min) and washed and (d) 420 s a t 72 "C for 1 cycle. The 5'-primer was ggccacgcgtat a final stringency of 0.1 X SSC (60 "C for 1 h). Anti-digoxigenin GGCCTCCGAGCAGCAGCCAGCAC. The 3'-primer was ggccacgcg antibody detection and color development were performed as recomt T T A A G G C A G T T A T A T T A A G A A G C (the lower-case letters mended by Boehringer Mannheim. Digoxigenin-labeled riboprobes denote an unrelated leader sequence containing an MZuI site that was were prepared using digoxigenin-labeled UTP as described by the added for use in cloning). manufacturer (Boehringer Mannheim) and transcribed from either Puked-field Gel Electrophoresis-Mouse lymphocytes were pre- the T7or T3 RNA polymerase promoter to produce sense or antisense pared and resuspended in phosphate-buffered saline at a concentra- riboprobes. tion of 5 X lo' cells/ml. A volume of cells was mixed with an equal volume of 1.4% InCert agarose (FMC) prepared in phosphate-buffRESULTS ered saline, and proteinase K was added to a final concentration of Structure and Sequence Analysis of Novel MBP Gene-re200 pg/ml. The cell suspension was mixed and cast into blocks, which were then suspended in lysis buffer (1%Sarkosyl, 250 mM EDTA, luted cDNAs-As part of an extensive screen of several cDNA 500 mM NaCl, 50 mM Tris-C1, pH 8.0) containing 200 pg/ml protein- libraries of mouse brains ranging in age from 2 to 15 days, we ase K and incubated for 24-36 h a t 37 "C. The blocks were then isolated a number of MBP gene-related cDNAs with 3'-ends rinsed extensively with wash buffer (10 mM Tris-C1, 100 mM EDTA, pH 8.0) and stored at 4 "C. Restriction enzyme digestion was per- derived from the MBP gene, but with 5'-ends that did not formed under the conditions recommended by the manufacturer (New correspond to known sequences in the MBP gene. The seEngland BioLabs, Inc.) with 8-15 units of enzyme/pg of DNA and quences of two of these clones are shown in Fig. 1. These incubation at therecommended temperature for 12-24 h. In the case clones (537 and BG21) shared the same 5'-ends and exon 1A of double restriction enzyme digestions, the first digestion was ter1B of the MBPgene, but they had different3"ends. Clone minated by adding phenylmethylsulfonyl fluoride (2 p~ final concen- 537 contained MBP exons 1A 1B, 3, 4, and 7, terminating tration), and the blocks were rinsed extensively with wash buffer at the normal polyadenylation site in exon 7 (see genomic followed by digestion with the second enzyme. Pulsed-field gel electrophoresis was performed using the CHEF-DR I1 system (Bio-Rad). map in Fig. 2). Clone BG21 also contained MBP exon 1A between MBP exons All pulsed-field gels were 1%FastLane agarose (FMC) prepared in lB, butcontinued 462 bp into the intron 0.5 X Tris/borate/EDTA, and electrophoresis was performed at 12 "C. 1 and 2, ending at anA-rich region within the intron (shown Conditions for separation of DNA were as follows: ( a ) from 5 to 150 in boldface type in Fig. 2, Exon 5 C ) . Subclones of a portion of kb, 1-5-9 ramp/l3 h/200 V followed by 5-8-s ramp/3.5 h/200 V; and the 5'-end of 537 and BG21 were used to screen a X genomic (b) from 5 to 350 kb, 12-20-9 ramp/23 h/200 V. After electrophoresis, library, and threeunique nonoverlapping genomic clones were the gels were transferred to positively charged nylon (MSI) under alkaline conditions (0.5 M NaOH, 1.5 M NaC1). Following transfer, isolated and characterized. Mapping, subcloning, and sequencing of these genomic regions demonstrated that the 5'the blot was neutralized and UV-cross-linked. SI Nuclease Analysis-A uniformly labeled 410-bp DNAprobe and ends of 537 and BG21 were derived from three new exons. a 580-bp DNAprobe were prepared using 7 pmol of primer and 18 pg The identification, mapping, and analysis of these three new of a denatured plasmid genomic DNA and Sequenase (version 2.0). exons indicated that they were part of a larger gene, i.e. the The reaction was initiated with 6.6 p~ dCTP containing 4 pmol of Golli-mbp gene. The positions of these exons relative to the [3ZP]dCTP(3000 Ci/mmol) and 80 p~ each dATP, dGTP, andTTP. After 12 min a t room temperature, the dCTP concentration was MBP gene as well as theirsequences and surrounding genomic raised to 12 pM, and incubation was continued for an additional 10 regions are shown in Fig. 2. The numbering system of the min. Labeled fragments with a homogeneous 3'-end were produced Golli-mbp exons is included along with that of the MBPgene by digestion with Hind111 (Bethesda Research Laboratories). The numbering system in Fig. 2 to facilitate comparison. MATERIALS ANDMETHODS
+
+
+
Structure and Expressionof Murine Golli-mbp Gene
4932
Golli-mbp EXONS 1
537 Golli exon 161 121
AGCACTGGGAGGCGGGGCGGGAGCGGGGTGCGCGCCCGTCCCCGGAGCAGCCGCTGTCAG TCACCGCCGCTGCGCGCCAGGGAGGACCAGCCTCTGGCTC CCCCGGAACATAGTATCCGCGCCCCAGCGACCCCTAGCCCAGCGTTGGGTCCCATCCGCC
181
GGGATCCCGGGATCCTGGGCCCCTCTTGGCCTCCGAGCAGCAGCCAGCACCA CTCTTGAA
1
2
5 6
34
A 0
481
.........
MBP EXONS
A0
1
0
..........
11
ID
....
AB
2
345 6
7
Exon 1 cgtgacaaca tcccagaaca tccggtggtt ctgaagcaat
ggcgtcctag cagagatcca ctgggcgatg aaagtccaga
gccctccagc cctcaggcct ccatttagac gagcagcggt
cagcacctgt agtcggggac ctctcttgcc gcgggagcgt
ggtctcagct aggttccagg aggtcctgct agggcggtga
241 301 361 421 481 541
cccgccccac AGTGGCGCGC CCGTCCCCGG GGCTCAGGCG AGCCCAGCGT AGCAGCAGCC
cgctccgagg GTTTACCTGC AGCAGCCGCT GCCTGAGCTT TGGGTCCCAT AGCACCAgta
gctgccctgc TTTCAAGCAC GTCAGTCACC CACGACCCCG CCGCCGGGAT agtgatcatt
cccctgggtc TGGGAGGCGG GCCGCTGCGC GAACATAGTA CCCGGGATCC
CCCgtGTCCC GGCGGGAGCG GCCAGGGAGG TCCGCGCCCC TGGGCCCCTC
Golli exon 1 -
GCGTTTACCTGCTTTCAAGCACTGGGAGGCGGGGCGGGAGCGGGGTGCGCGCCCGTCCCC GTTGGGTCCCATCCGCCGGGATCCCGGGATCCTGGGCCCCTCTTGGCCTCCGAGCAGCAG -Golli I 7 [Golli 2-
241 CCAGCACCA CTCTTGAACACCCCATTGGGTCGCCATGG-CCACTCTGGAAAGAGAGA -Golli 27 rGolli 3 -
301 ATTATCTGCTGAGAAGGCCAGTMG GATGGAGAGATTCACCGAGGAGAGGCTG-GAA G o l l i 37 r m p IA361 GAGAAGCGTGGGCMGCTTTCTCAGACGGCCTCAGAGGACAGTGATGTGTTTG
GGGAGGC AGATGCGATCCAGAACMTGGGACCTCGGCTGAGGACACGGCGGTGACAGACTCCMGCA
4 8 1 CACAGCAGACCCRMGAATMCTGGCMGGCGCCCACCCAGCTGACCCAGGGAACCGCCC 5 4 1 CCACTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGAAGGGAGGACMCACCTTCRMGA ”BP 1 ~ I7 m p IB601 CAGGCCCTCAGAGTCCGACGAGCTT CAGACCATCCAAGAAGACCCCACAGCAGCTTCCGG 661 AGGCCTGGATGTGATGGCATCACAGAAGAGACCCTCACAGCGATCCMGTACCTGGCCAC 1 2 1 AGCMGTAC~TGGACCATGCCAGGCATGGCTTCCTCCCAAGGCACAGAGACACGGGCAT 7 8 1 CCTTGACTCCATCGGGCGCTTCTTTAGCGGTGACAGGGGTGCGCCCAAGCGGGOCTCTGG “BP I 8 7 rGollf X 841 CAAG GTGAGCTCCGAGCCGTAGAGAAGCTGTGGGTTTAAATGCGGATL%GGAAGGAGAGGT 901 GGGCAGGTGGAACTGGGCCAGTCACCATCGCAGAGCAACCGTGTCCCTGTGTCTGA~TA 961 CTAGCCCTGATCGGACAGTGCTGATGGCCTGGGGGATTGGGCGTGGAGTGGGCGTGGTCG
CCACTTAGCCAATTTCTACTTGTTTTGCTCCTTTGTCCTTCTCACAGGGACTGCGATTGG GTGTCACTCAGGAATGCAATGCCTTCTGCATCCTAACTTGAGGCACCCAGGATGTAGCAT TGAGCCTATGGGTTCCTAGGTATACATGCCCTATGCCTGGGCTTCAGCAGATCCTGCCTC TGCGCAGTGCTTCCGGGTTTCGAACCTCAGCTCAGCTGTGCTCATTCTCTGCTGGGTACAGGCTG 1261 CTAATTGCTTGCCGCTGTGAAAC~TGGTCCAATTGAGGCGAGGAGGTCTGCCGACTGAC 1321 CTCTGCACCTCCAGCAAACTTTCCTCTTTGTCCTATGTAGTTTGGTGGGGGTG~TGAAT 1381 GAATGGTGTTCATGACAAAAAAAAAAA 1021 1081 1141 1201
FIG. 1. Sequences of cDNA clones 537 and BG21 containing Golli-mbp and MBP exons. The coding sequence is presented in boldfacetype, and the individual exons composing the cDNAs are interrupted by spaces and indicated in italic type.
M B P Gene Is Part of Much Larger Gene, Golli-mbp-We performed a series of pulsed-field gel analyses to determine the distances between the newly discovered exons and those of the MBP gene. The probes used to map the distances of the three new exons are shown in Fig. 3. Sequencing data indicated that the first exon of the Golli-mbp gene contained three BssHII sites. Probes for Golli-mbp exon 1 (probe a), Golli-mbp exon 5/MBP exon 1 (probe f), and Golli-mbp exon 11/MBP exon 7 (probe g) hybridized to a similar 215-kb BssHII band in mouse genomic DNA, thereby defining the maximum length of the transcription unit (Fig. 3 B ) . Restriction enzyme analysis of the X genomic clones placed one SfiI site 7 kb downstream of Golli-mbp exon 2 and another SfiI site -2 kb upstream of MBP exon 1A. Probes for Gollimbp exon 1 (probe a) and Golli-mbp exon 2 (probe b) hybridized to a 24-kb fragment in a BssHIIISfiI digest of genomic DNA (Fig. 3C). This established the physical distance between Golli-mbp exons l and 2 as 17 kb. A Golli-mbp exon 3 probe (probe d) hybridized to a 49-kb SfiIlSfiI fragment (Fig. 3A), while a probe containing the SfiI site region located upstream of Golli-mbp exon 5A (a bridge fragment, probe e) hybridized to both a 49-kb band and a 92-kb band (data not shown). Thus, Golli-mbp exon 3 is located on a 49-kb SfiI fragment that is contiguous with a 92-kb SfiIlSfiI fragment containing the MBP gene (Fig. 3). Probe c, which overlaps the SfiI site downstream of Golli-mbp exon 2, hybridized to a
...
ccagcttcag gctcaagtta cttcagattc cgagcaaagc
”-
CCGCCCTCCC GGGTGCGCGC ACCAGCCTCT AGCGACCCCT TTGGCCTCCG
Exon 2 1 tctcttctct 61 GAGAGAATTA
61 GGAGCAGCCGCTGTCAGTCACCGCCGCTGCGCGCCAGGGAGGACCAGCCTCTGGCTCAGG 121 CGGCCTGAGCTTCACGACCCCGGAACATAGTATCCGCGCCCCAGCGACCCCTAGCCCAGC
421
[I
....
1 61 121 181
BG21
181
AB
ATMCTGGCAAGGCGCCCACCCAGCTGACCCAGGGAACC~CCCCACTTGATCCGCC~T
54 1 TTTCCCGAGATGCCCCGGGAAGGGAGGACAACACCTTCAAAGACAGGCCCTCAGAGTCCG .MBP 1 ~ r7 m p I B 601 ACGAGCTT CAGACCATCCAAGAAGACCCCACAGCAGCTTCCGGAGGCCTGGATGTGATGG 661 CATCACAGAAGAGACCCTCACAGCGATCCAAGTACCTGGCCACAGCAAGTACCATGGACC 121 ATGCCAGOCATGGCTTCCTCCCMGOCACAGAGACACGGGCATCCTTGACTCCATCGGGC “BP 187 r m p 37 8 1 GCTTCTTTAGCGGTGACAGGGGTGCGCCCAAGCGGGGCTCTGGCAAG GACTCACACACGA 841 GAACTACCCATTATGGCTCCCTGCCCCAGAAGTCGCAGCACGGCCGGMPXAAGAT“BP 37 I m p .I901 ACCCAGTAGTCCATTTCTTCMGAACATT GTGACACCTCGAACACCACCTCCATCCCMG .MBP 47 rmp 7961 GGAAG GGAGGAAGAGACAGCCGCTCTGGATCTCCCATGGCGAGACGCTGAGAGCCCTCCC 1 0 2 1 CGCTCAGCCTTCCCGAATCCTGCCCTCGGCTTCTTAATATAACTGCCTTA..
1
.I
789 10
.......... uIJ..[
-Golli 17 r m l i 2241 CACCCCATTGGGTCGCCATGGGAAACCACTCTG-GAGAGAATTATCTGCTGAGAAGG -Golli 27 r c 0 m 3 301. C C A G T M G GATGGAGAGATTCACCGAGGAGA~CTGGA~GAAGAGAA~GTGGGCAAGC -Golli 37 [MBP exon 1A361 TTTCTCAGACGGCCTCAGAGGACAGTGATGTGTTTG GGGAGGCAGATGCGATCCAGAACA 421 ATGGGACCTCGGCTGAGGACACGGCGGTGACAGACTCCAAGCACACAGCAGACCCAAAGA
C
tctagCTCTT GAACACCCCA TTGGGTCGCCA T m A A A C C ACTCTGGAAA TCTGCTGAGA AGGCCAGTAA Ggtaagg Exon 3
1 tgtttttttt ttcagGATGG AGAGATTCAC CGAGGAGAGG CTGGAAAGAA GAGAAGCGTG 61 GGCAAGCTTT CTCAGACGGC CTCAGAGGAC AGTGATGTGT TTGgtatgta
Exon 5c 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141
GTGAGCTCCG AGCCGTAGAG AAGCTGTGGG AGGTGGAACT GGGCCAGTCACCATCGCAGA CCCTGATCGG ACAGTGCTGA TGGCCTGGGG TTAGCCAATT TCTACTTGTT TTGCTCCTTT CACTCAGGAA TGCAATGCCT TCTGCATCCT CCTATGGGTT CCTAGGTATA CATGCCCTAT CAGTGCTTCC GGGTTTCGAA CCTCAGCTGT TTGCTTGCCG CTGTGAAACA ATGGTCCAAT GCACCTCCAG CAAACTTTCC TCTTTGTCCT GGTGTTCATG ACAARRRRRA AAATGTGTGA CAAAAGCAAA TGCCCTGTGT TAAAATGTAT TTCAAGGGCA ACATCTGGGG TGGGTTACTC GTGGCTGCCA CCTTGTAAGT GTATGGCATT CTCTTGGGAG AGATATGGAT GGGCCATTAA GCCTGGGCAA CTTTTCTGCC TGTAAGTTGT AGGCTTTTGA CTGAGATGTA GCAGAGGCCC GTTTTGAAAA TGCTGTAGTCTCCAGCCACA TGTACCTAAT GACGGCTCTCCTGCCCCGTG AGTCTGGCCC TCGCCTTGGA TGTCAGCGCT T G C T A A C A W
TTTAAATGCG GCAACCGTGT GATTGGGCGT GTCCTTCTCA AACTTGAGGC GCCTGGGCTT GCTCATTCTC TGAGGCGAGG ATGTAGTTTG TTCCGTTAAT CAGAAGATAA ATGAGCCTGG TTTCCATCTC AACAAGCCCA GTCTGTTGTC CTCCACGGCT CCTTTGAGCG GCCACCAGAG ACTTGTCATT
GAAAGGAAGG CCCTGTGTCT GGAGTGGGCG CAGGGACTGC ACCCAGGATG CAGCAGATCC TGCTGGGTAC AGGTCTGCCG GTGGGGGTGA TTAAATATAA AATTGTGCAG TCAGTTTCGT TCTGTTGGAT GGTCTTTGAG AAAACTCTGG CTAGGCGCAT TGGGGATGGT CCCCTTGGTG AACACTGGAT
AGAGGTGGGC GAGATACTAG TGGTCGCCAC GATTGGGTGT TAGCATTGAG TGCCTCTGCG AGGCTGCTAA ACTGACCTCT ATGAATGAAT CTTGTGGTTT ATGGTCTAGT CTTGTGGCAG AGCTGAGCAA TACCCTCTCA CGAACGCAAG TGGCAGTGCA TGTGAAATGC TCTCAGCAGC ATGTCTCACT
FIG. 2. Diagrammatic representation of exon structure of Golli-mbp gene (not drawn to scale). Golli-mbp exon 9A/MBP exon 5A was recently reported by Aruga et al. (1991). The s u e d exons have been identified and sequenced in this study. The numbering system of the entire Golli-mbp gene is given abovethe exons, and the numbering system of the MBP gene as it has been known up to now is given belowthe exons to facilitate comparison. Golli-mbp exon 5B/MBP exon 1B is the first exon of the classic MBP transcripts. The nucleotide sequences of the exons (in upper-case letters) and bordering sequences (in lower-case letters) are presented for the four new exons identified in this study. The transcription initiation site for Golli-mbpexon 1is designated by the arrowheads. The translation initiation codon in exon 2 is presented in boldface italic type, with the consensus sequence in italic type. The poly(A)-rich region in exon 5C that marks the 3’-end of BG21 is shown in boldface type. Exon 5C ends at the3”terminus with an EcoRI site, which is underlined.
24-kb band and a49-kb band, linking these two fragments as shown in Fig. 3. Sequence analysis of the DNA surrounding Golli-mbp exon 3 identified a PmlI site 180 bp downstream of the exon. The absolute position of Golli-mbp exon 3 was determined by hybridizing a Southern blot of a SfiIlPmlI double digest of genomic DNAwith genomic probes d and e (data not shown). Both probes hybridized to a common 38-kb DNA fragment, indicating that the total distance from Golli-mbp exon 3 to Golli-mbp exon 5A is -40 kb. Fine mapping studies on a X genomic clone that contained both Golli-mbp exons 3 and 4 demonstrated that Golli-mbp exon 4 (i.e. MBP exon 0 according to Kitamura et al. (1990)) is located -8 kb downstream of Golli-mbp exon 3. The mapping studies then indicate that the total length of the Golli-mbp transcription unit is -105 kb and includes within it the MBPgene.
Structure and Expression of Murine Golli-mbp Gene .+
(BSSHIIL sn I
Probes
a
1
Transcription Initiation Sites 3 2? + . + .
2
3
4933
4
GATC
II
sfi 1
-_
-
6;
-
ef
d
9
38 kb 49 kb
24 kb
92 kb
/M
215 kb
I
B
/A
C
Probes 9
,
Probes a
c c c
d
b
FIG.4. Mapping of Golli-mbp transcription start site 1 by S1 nuclease analysis. RNA (3 pg)washybridized
215Kb-
. L
L
49Kb-
-240-
"
to a singlestranded DNA probe uniformly labeled with [32P]dCTP.Following hybridization, the mixtures were digested with increasing concentrations of S1 nuclease, and the reaction products were electrophoresed next to the corresponding sequencing ladder. Lane 1 , tRNA with 50 units of S1 nuclease; lanes 2-4, PI8 mouse brain poly(A+)RNA with 10,25, and 75 units of SI nuclease, respectively.
located in Golli-mbp exons 1and 11. The PCR products were cloned and identified by sequence analysis. The most abundant product was identical to clone 537, and thesecond most abundant was a cDNA that was much shorter than 537. The FIG.3. A, long distance map of the Golli-mbp gene is shown exon structure of this second clone (TP8)included Golli-mbp indicating rare restriction enzyme sites, positions of the genomic exons 1-3, 7,8, and 11 and a predicted mRNA length of 2.2 probes used for the mapping studies, and sizes of fragments used to kb (see Fig. 5). The largest open reading frame in each of the determine the structure of the gene. Note the map is not drawn to three cDNA clones (TP8,537, and BG21) began with an ATG scale. Three transcription initiation sites are indicated by arrows. initiation codon within Golli-mbp exon 2 (shown in boldface Site 2 has not been unambiguously demonstrated, but is inferred from data presented by Kitamura et al. (1990). This uncertainty is italic type in Fig. 2). This codon was set within a perfect indicated (?). E,Southern blots of mouse genomic DNAdigested with Kozak translation initiation consensus sequence (Kozak, BssHII,separated by pulsed-field gel electrophoresis, and hybridized 1987). to genomic probes a, f, and g. All three probes reacted with a 215-kb The amino acid sequence encoded by TP8 predicts a peptide band. C, Southern blots of mouse genomic DNA digested with a of 69 amino acids that would have no MBP homology because double digest of BssHII and SfiIand hybridized to genomic probes a splicing of Golli-mbp exon 3 to Golli-mbp exon 7 produces a and b (both probes reacted with a 24-kb band) and of genomic DNA digested with SfiIand hybridized to probe d, revealing a 49-kb band. frameshift with respect to MBPpolypeptide coding sequences (Fig. 5). In contrast, for the 537 and BG21 clones, the initiation codon in Golli-mbp exon 2 is in-frame with downstream The transcription initiation site of the Golli-mbp gene was sequences encoding the MBP polypeptide beginning within determined by S1 analysis (Fig. 4) using two independent Golli-mbp exon 5B (Fig. 5). Clone 537 predicts a 250-amino DNA probes of differing lengths. It mapped to a three-nucleo- acid protein, approximately half of which containsMBP tide region (indicated by arrowheads in Fig. 2). There are no amino acid sequence and should be immunoprecipitable with obvious TATA or CAAT boxes in the region immediately MBP antisera. The predicted polypeptide for BG21 contains upstream of the transcription start site. However, there are a 57-amino acid sequence in the COOH-terminal half that is five AP2-binding sites within50 bp upstream of the CAP site, MBP polypeptide sequence encoded by Golli-mbp exon 5B. In with three nested in a GC-rich region 35-50 bp upstream of principle, this product also should be precipitable with MBP the transcription start site. This region also contains anSP1- antisera. Fig. 6 shows the translationproducts of reticulocyte binding site. Thus, the region upstream of the transcription lysates programmed with cRNAs prepared from clones 537 start siteis relatively rich in potential binding sitesfor tran- and BG21 and from a clone encoding the 14-kDa MBP. As scription factors. predicted from the largest open reading frames, the products The expression of 537 includes exons derived from the most of 537 and BG21 cRNA translations were immunoprecipitable upstream new exon we have identified (i.e. Golti-mbp exon 1) with anti-MBP antibodies. The two smaller products in the and themost downstream of the known MBP exons (i.e. Golli- 537 cRNA translation represent initiationat two downstream mbp exon 11).Thus, asshown in Fig. 3, the transcription unit sites in exon 5B corresponding to the usual MBP initiation for 537 encompasses and extends by -73 kb upstream the siteandanother AUG codon 57 nucleotides downstream entire MBPgene as it has been known. (Verdi and Campagnoni, 1990). No internal initiation prodMultiple Alternatively Spliced mRNAsAre Produced in ucts are seen in the BG21 cRNA translation because they are Addition to MBP mRNAs from Golli-mbp Gene-Exons 2,5A, small and would have run off the gel. Like the MBPs (whose 5B, and 6 of the MBP gene are alternatively spliced to produce mobilities are shown by arrows in Fig. 6), the537 and BG21 multiple mRNAs. The fact that the 537 cDNA had MBP translation products migrated with higher apparent molecular exons 2, 5A, 5B, and 6 spliced out of the message raised the masses on SDS-polyacrylamide gel electrophoresis. The unpossibility that other splice products existed. This was ad- usual binding properties of SDS to MBPs and theiranomalous dressed by PCR amplification of brain RNA using primers migration on SDS gels have been observed frequently (Cam-
Structure and Expression of Murine Golli-mbp Gene
4934
I
5 2 3 M 7 8 II
J37
a z c0r
h
Properties Protein Predicted clone cDNA
m
amino acids
sizeTranscript (Kb)
z
kDa
a
250
2.6
9.4
e
27.1
l -
Gm
d
I 2 3 7 8 II
l -
TP8 69
2.2
7.1
7.5
I 2 3 5A.B.C
BG21
5. I
6.4
195
21
-
46
-
30
-
21.5 K MBP
Predicted Protein Sequences 537 8021 TPB 537 8021 TP8 537
8021
IM ~ ~ N B s ~ L s A ? Z A S ~ [ D G E I ~ Q I ~ R . ¶ V ~ S Q T A SWEADI P. ¶ NN ~ GTSAI M D N B S ~ L W ~ N ) O E I B R ~ ~ V O I C G S Q T ~ E W D AS I D Q N~ HGG T M G N ~ s ~ K R E L S A E ~ ~ G E I ~ G ~ ~ V ~ S Qlth-nmlrlpw T ~ E D S D ~ G
61 D T A V T D S N I T A D P X N H W P D P A D P ~ ~ I ~ F S ~ ~ ~ D ~ ~ ~ S D E ~ T I D T A V T D S N I T A D P K N N W P G P A D P ~ ~ I ~ F S R D A L Q T I rvaaepdpr
121 QEDPTWGGLDV QEDPTMSGGLDV
-,,-,,---------MBP antigenic sequence---------~ Q ~ s Q R S K Y P L A T ~ ~ G ~ ~ T G I L D S I M A S Q I ( X P S Q R S K Y ~ T A S ~ ~ G ~ P ~ T G I W S I ~ S ~
8021
ccc---,--,cccccc-----~~ antigenic sequence-----------------181 RGAPRRGSOK D S B T R T T B Y G S L P P ~ Q B G R T Q D E N P ~ ~ I ~ ~ ~ P P S ~ ~ RGAPKRGSGK vsa-p
537
2 4 1 SRSGSPWSR
537
" " " " "
FIG. 5. Exon complement of three Golli-mbp clones isolated in this study with amino acid sequences and physical properties of predicted products they encode. Note that the protein products encoded by clones 537 and BG21 contain MBP protein sequence in the COOH-terminal half of the molecules, designated as MBP antigenic sequence.
pagnoni and Magno, 1974; Smith, 1992). Expression of Alternatively Spliced Golli-mbpmRNAs Is DevelopmentallyRegulated-The expression ofGolli-mbp transcripts was examined between embryonic day 18 and postnatal day 35 by Northern blot analysis of RNA isolated from whole mouse brains. This time frame encompassed the onset (i.e. 7-10 days) and peak (18-24 days) periods of myelination in the developing mouse brain. A series of Golli-mbp gene sequences were used to sequentially probe Northern blots. A probe for Golli-mbp exon 5A hybridized to three mRNAs of 2.3,2.6, and 5.1 kb (Fig. 7A).The 5.1-kb RNA was detected strongly at embryonic day 18and appeared to remain high throughout earlypostnatal development. The expression of the 2.6-kb band appearedto become significant at a slightly later age (postnatal days 2-4), followed by the 2.3-kb band, which was the last tobe expressed. The same blot was stripped and rehybridized to a Golli-mbp exon 1-specific probe (Fig. 7B), and thesame 5.1- and 2.6-kb bands were detected as before; but the 2.3-kb band disappeared, and a new band appeared at 2.2kb. The 2.2-kb transcript had adevelopmental pattern of expression that was similar to that of the 5.1-kb band, but different from that of the 2.3-kb mRNA. We have previously identified the 2.3-kb band as M41-MBP mRNA (Kitamuraet al., 1990). This mRNA is composed of Golli-mbp exons 4, 5A, 5B, 7-9, and 11. It has a developmental pattern of expression identical to that of MBP mRNA (Fig. 70). Northern blot analysis also was performed onbrain poly(A+) RNA from shi mice. This mutation is a deletion of Golli-mbp exons 7-11 from the mouse genome (Roach et al., 1985; Molineaux et al., 1986). When the Northernblots were hybridized to a probe for Golli-mbp exon 1 (Fig. 8, lane shi) and Golli-mbp exon 5A (data not shown),only the 5.1-kb band was present. The presence of the 5.1-kb mRNA in the shi
~
~
~
-
21
x 18.5 K MBP 1 1 4 K MBP
-I4
FIG. 6. Fluorogam of 86S-labeled products ofcell-free translations programmed with cRNAs prepared from 14-kDa MBP, 537, and BG21 cDNA clones. Products of the in uitro translation of the cRNAs were immunoprecipitated with anti-MBP and separated by polyacrylamide gel electrophoresis prior to fluorography.
brain indicatesthat transcription of this product of the Gollimbp gene is not interrupted by the deletion and terminates within the intronbetween Golli-mbp exons 5 and 6. The Northernblot shown in Fig. 7 was stripped again and hybridized to Golli-mbp exon 5C, which is specific for BG21 transcripts; and only the 5.1-kb mRNA hybridized to this probe (Fig. 7C). On the basis of its hybridization to probes specific for Golli-mbp exons 1,5A, and5C and itspresence in shi mice, we conclude that BG21 is a partial-length cDNA clone of the 5.1-kb mRNA. We have isolated overlapping cDNA clones that extend the 3'-portion of BG21 -580 bp to an EcoRI site (see Fig. 2, Exon SC).These and othergenomic clones further downstream of this EcoRI site hybridize to the 5.1-kb mRNA (data not shown). Thus, we conclude that the remaining 3'-portion of BG21 is derived from these intron sequences and represents 3'-untranslated sequence and that BG21 contains the coding region of the 5.1-kb mRNA. Both 537 and TP8 contain exons deleted in the shi mutation, consistent with their identity as the 2.6- and 2.2-kb mRNAs, respectively. Clone 537 is 2.5 kbin length and contains bothGolli-mbp exons 1and 5 4 and clone TP8 is 2.1 kb and contains Golli-mbp exon 1, but not exon 5A. On the basis of these hybridization experiments, we conclude that 1) cDNA clone 537 represents a full-length clone of the 2.6-kb mRNA, 2) TP8 represents a cDNA clone corresponding to the 2.2-kb mRNA, and 3) the 2.2-, 2.6-, and 5.1-kb mRNAs are clones produced from the first transcription start site of the Golli-mbp gene. Fig. 8 also shows a Northern blotof poly(A+)RNA isolated from several tissues and hybridized to a probe specific for
Structure and Expression of Murine Golli-mbp Gene
A
A
P35 P45 .' m
4935 shi
H K S Lu
, u " n , 1 7 -
5.1 Kb-
I
2.62.3-
B
5.1 ~ b -
2.6
5.1 Kb-
-
1
I I
2.62.2-
C
5.1 Kb-
FIG. 8. A, Northern blot of poly(A+) RNA isolated from normal brain (P35 and P45); P18 shi brain; andadult heart ( H ) ,kidney ( K ) , spleen (S),and lung (Lu)hybridized to a probe for Golli-MBP exon 5A. B, the same blot reprobed with cyclophilin.
cerebellar white matter (corpus callosum and external capsule) (Fig. 9a), suggesting that thegene wasexpressed by nonneuronal cells. In situhybridization of this same region to an oligodendroglia-specific marker, proteolipid protein, is shown in Fig. 9b. Expression of Golli-mbp exon 5C could also be detected in cells in the same brain regions in shi mice (Fig. 2.1 Kb9c), consistent with the Northern blots of shi brain RNA showing expression of the 5.1-kb BG21 mRNA (Fig. 8A). These results suggested that glial cells, probably oligodendrocytes and/or their precursors, which compose >90% of the cells in the corpus callosum, were expressing the Golli-mbp transcripts. Since the in vivo data suggested that the Golli-mbp gene was expressed in glial cells, we examined primary mixed glial FIG. 7. Northern blots of mouse brain poly(A+)RNA from cell cultures and enriched oligodendrocytes using combined embryonic day 18 to postnatal day3 5 . 4 pg of RNA were loaded in situ hybridization histochemistry for Golli-mbp mRNAs per lane. The blot was hybridizedsuccessively to probes for Golli-mbp and immunocytochemistry for cell- and stage-specific identiexon 5A ( A ) , Golli-mbp exon 1 ( B ) ,and Golli-mbp exon 5C ( C ) ;with fication. In the primary mixed glial cultures, whichwere a probe containing Golli-mbp exons 7, 8,9B, 10, and 11 (specific for MBP mRNAs) (D); and with cyclophilin, a constitutive probe de- devoid of neurons, we used A2B5 antibodies to identify oligodendrocyte precursors (Raff et al., 1983), antibody A007 to signed to check for uniformity of loading ( E ) . identify immature and mature oligodendrocytes (Knapp, 1991), and anti-galactocerebroside to identify mature oligoGolli-mbp exon 1. The 5.1-kb mRNA was expressed in all the dendrocytes (Raff et al., 1983). Golli-mbp exon 5C was not tissues examined, with highest expression noted in the brain expressed in astrocytes, which form the bed layer of cells in and spleen and with lesser expression in the heart, kidney, these mixed glial cultures. In these newborn cultures mainand lung. tained for 3 days in vitro, a time prior to the expression of Cellular Expression of Golli-mbp Gene in Brain in Vivo and MBP mRNA or protein in these cultures (Amur-Umarjee et in Vitro-We examined the regional and cellular distribution al., 1990a, 1990b), the majority of cells expressing the Golliof Golli-mbp transcripts by nonradioactive (digoxigenin) in mbp transcript colocalized with cells expressing the surface situ hybridization histochemistry ex vivo on tissue sectionsof markers A2B5 (data not shown) and A007 (Fig. 10, a and b). mouse brain and in vitro in mouse brain primary glial cultures. Some cells (indicated by arrows in Fig. 10, a and b ) expressed In vivo, cells that hybridized to a probe for Golli-mbp exon 5C Golli-mbp mRNA, but notA007, probably representing oligowere found only in white matter tracts such as the deep dendrocytes at an earlier stage of differentiation. These re-
D
E
4936
Structure Expression and
FIG. 9. Distribution of Golli-mbp mRNAs in normal and shi mouse brain revealed by in situ hybridization to digoxigenin-labeled riboprobe. Sections from normal mouse brain hybridized to either exon 5C ( a ) or proteolipid protein ( b )antisense riboprobes revealed prominent staining withincells of white matter regions such as the externalcapsule(shown). Brainsections from shi mice hybridized tothe Golli-mbp exon 5C riboprobe were similarly labeled (c). Staining was not observed in control animals hybridized to a sense riboprobe to Golli-mbp exon 5C ( d ) . Bar, 100 pm.
of Murine Golli-mbp Gene
’
are in transition from precursors to immature and mature cells in this cell system (Amur-Umarjee et al., 1990a). The preparation was devoid of neurons, microglia, and astrocytes as determinedby immunocytochemical staining with antineurofilament,F4/80antibody, and anti-glialfibrillary acidic protein, respectively (Amur-Umajee, 1990a). Weexamined this oligodendrocyte population by combined in situhybridization/immunocytochemistry using antibodymarkers incombination with a probe for Golli-mbp exon 1. These results are shown in Fig. 10. Golli-mbp transcripts colocalized with cell types that also expressed A2B5, A007 (Fig. 10, c and d ) , or galactocerebrosides (Fig. 10, e and f). Thus, on the basis of the in vivo regional localization and the i n vitro cell-specific localization, we conclude that, in brain, the Golli-mbp gene products are expressed in cells at several stages in the oligodendrocyte lineage. DISCUSSION
The Golli-mbp transcription unit encompasses the MBP gene. One of the more surprising findings to emerge from this work is that the exons and promoter region composing the MBP gene are included within the Golli-mbp transcription unit and that this transcription unitis -105 kb in length. A 36-kb region of the Golli-mbp gene that containsMBP-encodFIG. 10. Double in situ hybridization/immunocytochemistry analysis of primary glial cell cultures (a and b ) and en- ing exons 5B-11 can function as an independent transcription riched oligodendrocyte cultures ( c - f ) . Immunocytochemical unit. It can be introduced into transgenic mice and expressed within oligodendrocytes of the central nervous system with analyses are shown in a, c, and e, with the corresponding in situ hybridization analyses of the cells in the same field shown in b, d, appropriate developmental specificity (Readhead et al., 1990). and j , respectively. There was colocalization of immunocytochemi- It also can be introduced into shi mice (which have a deletion cally detected A007 ( a ) and Golli-mbp mRNA ( b ) (detected by in situ of exons 7-11), with a subsequent reduction in the clinical hybridization to an exon 5C probe) in primary cultures a t 3 days in vitro. The arrows in a and b indicate two cells that containGolli-mbp signs of the neurological disorder caused by the mutation mRNA, but are not positive for A007. Colli-mbp mRNAs were de- (Readhead et al., 1985). Sequences up to 4 kb upstream of tected in enriched oligodendrocyte cultures (c-j) by in situ hybridi- Golli-mbp exon 5B can be used to drive the expression of zation to a probe for Golli-mbp exon 1 (d and f ) . These mRNAs reporter genes and MBP minigene constructs in transgenic colocalized in most cells with the oligodendrocyte markers A007 ( c ) mice (Kimura et al., 1989; Miskimins et al., 1992). Exon 5A and Ranscht anti-galactocerebroside antibody ( e ) . of the Golli-mbp gene is also capable of driving the expression of reporter genesin transfected cells (Miura et al., 1989; sults were consistent with the i n vivo localization studies Aoyama et al., 1990; Devine-Beach et al., 1990). indicating greatest expression in brain regions rich in oligoAnother interesting featureof the Golli-mbp gene is that it dendrocytes and their precursors. can also be considered to be composed of two overlapping We also prepared enriched oligodendrocyte cultures from genes, those encoding MBP and BG21. In this respect, the primary mixed glial cultures after 7 days i n vitro. This time Golli-mbp gene is similar to the neurofibromatosis gene in was chosen because it is the period when the oligodendrocytes that they are both large transcription units that encompass
S t r u c t u r e and Expression of Murine Golli-mbp Gene
4937
smaller genes (Xu et al., 1990; Viskochil et al., 1991). However, appear to be expressed under a defined temporal regimen in cells at different stages of differentiation in theoligodendrothey are distinctly different in that the smaller MBP and expressed in BG21 portionsof the Golli-mbp gene are not simply included cyte lineage. For example, the BG21 mRNAs are the oligodendrocyte precursor, and theirexpression seems to within an intron of the larger transcription unit, but share continue throughout the immature and mature oligodendroalternatively spliced exons in common. Predicted Products of SomeGolli-mbp Gene Transcripts cyte differentiationstages. While 537mRNAs appear latera t Should Be Recognized by M B P Antibodies-The translation an intermediate stage prior to full differentiation of the oliproducts of both BG21 and 537 cRNAs contain MBP se- godendrocyte, theystill precede the expression of MBP mRNAs from transcription start site3. The productsof these quences that are immunoprecipitable with MBP antisera that correspond to the predicted polypeptides based uponsequence Golli-mbp mRNAs are unlikely to be associated with myelin, analysis. There have been several reports of the presence of but must have some function in the differentiating oligodenMBP-immunoreactive polypeptides in rodent brains and glial drocyte. Their continued expression during postnatal develcell cultures that were not recognized MBP isoforms (Bar- opment may reflect the presence of oligodendrocytes at varybarese andPfeiffer, 1981; Barbarese et al., 1983; Carson et al., ing stages in their lineage rather than substantialexpression of these mRNAs in the terminally differentiated oligodendro1983). Theseimmunoreactive polypeptideshave beenobexpression of the served on Western blots of brain tissue with polyclonal and cyte. Thus,theprogrammatictemporal monoclonal antibodies (Carsonet al., 1983; Bansal et al., 1987) Golli-mbp gene products is likely to play an important role in of the oligodendroand i n vitro translations of brain poly(A+) mRNA (Carson et the proper differentiation and maturation al., 1983). They have been reported in shi brain (Barbarese et cyte in the centralnervous system. In addition to the strong temporal regulation of products al., 1983), and they have been reported to appear earlier in development than the myelin basic proteins (Barbarese and of the gene in the centralnervous system, at least one of the Pfeiffer, 1981; Carson et al., 1983; Bansal et al., 1987). Unfor- gene products, BG21, is expressedin tissuesoutsidethe nervous system. Using in situ hybridization histochemistry, tunately, it has been difficult to assess their relationship to the myelin basic proteins because of the possibility of non- we also have observed its expression in a T-cell and a B-cell expression of BG21 specific cross-immunoreactivity with unrelated proteins. Our line,’ supporting the notion that the tissue of the gene. finding that Golli-mbp gene products contain protein-encod- mRNA is less restricted than other products ing exons of the MBP gene spliced in frame to other Golli- While the significance of this expression is not yet clear, it would suggest that cells in the immune system are exposed to mbp exons would predicttheexistence of otherproteins sequence of MBP encoded expressed earlier in central nervous system development that antigens that contain the primary would contain MBP epitopes. Thus, it is possible that the by exon 5B (i.e. amino acids 1-57 of the mouse). This region MBP-immunoreactive proteins identified in the earlier stud- of the MBP is known to contain immunogenic sites (Bansal ies are products of the Golli-mbp gene produced from tran- et al., 1987; Hruby et al., 1987). It might be expected that animals expressing BG21 would be more tolerant to epitopes scription start site 1. Structure and Expression of Golli-mbp Gene Are Unusual found within this region of the MBP than in other regions and Complex-Virtually every element of the structure and encoded by other exons. Finally, it is worth noting that exon 5 of the Golli-mbp gene expression of the Golli-mbp gene has a precedentinthe is a region of substantial transcriptional complexity: ( a ) it literature, but few (if any) genes appear to possess all the elements of this gene. The entireGolli-mbp gene encompasses possesses three distinct combinatorial regions that appear two transcription units, those of MBP and BG21. It is inter- independently in differentGolli-mbp mRNAs, and parts or all esting that thesetwo transcription units “overlap” each other of it may be alternatively spliced in and out of Golli-mbp in that exons 5A and 5B, which are at the beginning of the transcripts; and( b ) it includes sequences that have independMBP gene, combine with exon 5C to form the end of the ent promoter activity. Exon 5A contains most of what has BG21 gene. Thus, the Golli-mbp gene contains overlapping been described as the “core” promoter activity of the MBP gene in transient transfection assays as well as binding sites transcription units withinit. The splicing pattern of Golli-mbp is verycomplex with for a number of transcription factors (Aoyama et al., 1990; Devine-Beach et al., 1990; Tamura et al., 1990). In view of the respect to both the numbersof products expressed as well as many Golli-mbp gene transcripts that contain this region and their developmentalregulation. The first and third transcripthe high activity of the third transcription start site during tion initiation sites are regulated separately during developmyelination, it seems likely that it must represent a highly ment, as are thesplicing patterns that each undergoes. Golliregulated and important region of the gene. Our findings mbp transcription start site1 appears to express the alternaindicate, however, that it is also likely that the effects of its tively spliced BG21 and TP8 mRNAs during embryonic life regulation extend beyond the expression of myelin basic proand then later expresses 537 mRNA, a third alternatively teins, butalso to transcripts expressed during the differentiaspliced mRNA,duringneonatal life. ThesethreemRNAs tion of the oligodendrocyte prior to the elaboration of the represent a minimum of those thatmay be actually expressed myelin sheath. from this transcription start site since we have observed, but not characterized, other minor PCR productsexpressed from Acknowledgment-We thank Dr. J. Gregor Sutcliffe for providing this start site. The third transcription start site is activitatedthe cyclophilin cDNA probe. later than start site 1, presumably as the oligodendrocyte REFERENCES approaches its terminally differentiated state. Expression of Amur-Umarjee, S., Dasu, R., and Campagnoni, A. T. (1990a) Deu. Neurosci. the approximately seven alternatively spliced forms of MBP 12,251-262 mRNAs generated from the third transcription start site has Amur-Umarjee, S., Hall, L., and Campagnoni A. T. (1990b) Deu. Neorosci. 12, 263-272 been well documented, and the proportions of these change Aoyama, A., Tamura, T., and Mikoshiba, K. (1990) Eiochern. Biophys. Res. Common. 167,648-653 ratherdramaticallywithpostnatal development (CampagAruga, J., Okano, H., and Mikoshiha, K. (1991) J. Neurochern. 56,1222-1226 noni, 1988; Aruga et al., 1991). Ausuhel, F. M., Brent, R., Kingston, R.Moore, E., D. D.,Seidman, J. G., Smith, Another intriguing aspectof the regulation of the Golli-mbp gene is that some of the products we have identified to date * S. Amur-Umarjee and A. T. Campagnoni, unpublished results.
4938
Structure and Expression of Murine Golli-mbp
J. A., and Struhl, K. (eds) (1991) Current Protocols in Moleculur Biology, pp. 4.6.1-4.6.6, Green Publishing Associates/Wiley-Interscience,New York Bansal, G., Martenson, R.E., Leveille, P., and Campagnoni, A. T. (1987) J. Neuroimmunol. 1 6 , 279-294 Barbarese, E., and Pfeiffer, S. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 7 8 ,
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.- - -
7K,21 -2R "
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Roach, A., Takahashi. N.. Pral 42,149-155
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Knapp, P. E. (1991) J. Neurosci. Res. 3 0 , 336-345 Kozak, M. (1987) Nucleic Acids Res. 16,8125-8148
74,5463-5467
""_ ""D: 21nl -21nR
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